U.S. patent application number 12/276776 was filed with the patent office on 2009-06-25 for novel nucleic acid sequences and their use in methods for achieving pathogen resistance in plants.
This patent application is currently assigned to BASF Plant Science GmbH. Invention is credited to Markus Frank, Ralph Hueckelhoven, Karl-Heinz Kogel, Holger Schultheiss.
Application Number | 20090165173 12/276776 |
Document ID | / |
Family ID | 26010034 |
Filed Date | 2009-06-25 |
United States Patent
Application |
20090165173 |
Kind Code |
A1 |
Kogel; Karl-Heinz ; et
al. |
June 25, 2009 |
NOVEL NUCLEIC ACID SEQUENCES AND THEIR USE IN METHODS FOR ACHIEVING
PATHOGEN RESISTANCE IN PLANTS
Abstract
The invention relates to novel RacB cDNA sequences from barley
and to expression cassettes and vectors comprising these promoter
sequences. The invention furthermore relates to transgenic plants
transformed with these expression cassettes or vectors, to
cultures, parts or transgenic propagation material derived from
them, and to their use for the production of foodstuffs, feeding
stuffs, seed, pharmaceuticals or fine chemicals. The invention
furthermore relates to methods of generating or increasing a
pathogen resistance in plants by reducing the expression of an RacB
protein or of a functional equivalent thereof.
Inventors: |
Kogel; Karl-Heinz; (Lollar,
DE) ; Hueckelhoven; Ralph; (Giessen, DE) ;
Schultheiss; Holger; (Friedberg, DE) ; Frank;
Markus; (Neustadt, DE) |
Correspondence
Address: |
CONNOLLY BOVE LODGE & HUTZ, LLP
P O BOX 2207
WILMINGTON
DE
19899
US
|
Assignee: |
BASF Plant Science GmbH
Ludwigshafen
DE
|
Family ID: |
26010034 |
Appl. No.: |
12/276776 |
Filed: |
November 24, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10488222 |
Mar 2, 2004 |
7456335 |
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PCT/EP2002/009719 |
Aug 30, 2002 |
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12276776 |
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Current U.S.
Class: |
800/279 ;
435/252.3; 435/254.2; 435/320.1; 435/419; 536/23.6; 800/298;
800/301; 800/305; 800/306; 800/309; 800/310; 800/312; 800/317;
800/317.1; 800/317.2; 800/317.3; 800/317.4; 800/320; 800/320.1;
800/320.2; 800/320.3; 800/322 |
Current CPC
Class: |
C12N 15/8282 20130101;
C07K 14/415 20130101 |
Class at
Publication: |
800/279 ;
800/298; 800/320.3; 800/320; 800/320.1; 800/320.2; 800/306;
800/312; 800/317.2; 800/317.3; 800/317.4; 800/317.1; 800/317;
800/322; 800/305; 800/309; 800/310; 800/301; 536/23.6; 435/320.1;
435/252.3; 435/254.2; 435/419 |
International
Class: |
C12N 15/82 20060101
C12N015/82; A01H 5/00 20060101 A01H005/00; C12N 15/11 20060101
C12N015/11; C12N 15/00 20060101 C12N015/00; C12N 1/21 20060101
C12N001/21; C12N 1/19 20060101 C12N001/19; C12N 5/04 20060101
C12N005/04 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 3, 2001 |
DE |
10142579.1 |
Jul 2, 2002 |
DE |
10229729.0 |
Claims
1-31. (canceled)
32. A method of generating or increasing a resistance to at least
one pathogen in a plant, which comprises: reducing an amount,
activity or function of an RacB protein in a plant or a tissue,
organ, part or cell thereof, wherein the RacB protein is a
polypeptide comprising the sequence of SEQ ID NO: 4 or a sequence
having at least 90% homology to the sequence of SEQ ID NO: 4;
wherein the reducing comprises introducing into the plant, tissue,
organ, part or cell thereof: a nucleic acid encoding a
double-stranded RacB RNA or an expression cassette comprising said
nucleic acid; and wherein the pathogen is selected from the group
consisting of Plasmodiophoramycota, Oomycota, Ascomycota,
Chytridiomycetes, Zygomycetes, Basidiomycota, Deuteromycetes, and
combinations thereof.
33. The method of claim 32, comprising: stably transforming a plant
cell with a recombinant expression cassette comprising a nucleic
acid encoding a double-stranded RacB RNA; regenerating a plant from
the plant cell; and expressing said nucleic acid in an amount and
over a period of time sufficient to generate or increase said
resistance.
34. The method of claim 32, wherein the plant is a monocot or
dicot.
35. The method of claim 34, wherein the monocot is selected from
the group consisting of wheat oats, millet, barley, rye, maize,
rice, buckwheat, sorghum, triticale, spelt, linseed, sugar cane,
and combinations thereof.
36. An isolated nucleic acid comprising a nucleic acid sequence
encoding a rice RacB protein, wherein the RacB protein is a
polypeptide comprising the sequence of SEQ ID NO: 4.
37. An isolated nucleic acid comprising the sequence of SEQ ID NO:
3 or the complement thereof.
38. A recombinant double-stranded RNA molecule for reducing
expression of an RacB protein, wherein one of the two RNA strands
comprises the sequence of SEQ ID NO: 3 or a sequence having at
least 90% homology to the sequence of SEQ ID NO: 3.
39. A recombinant double-stranded RNA molecule for reducing
expression of an RacB protein, comprising: a sense strand and an
antisense strand, which is essentially complementary to the sense
strand; wherein one of the strands comprises the sequence of SEQ ID
NO: 3 or a sequence having at least 90% homology to the sequence of
SEQ ID NO. 3.
40. The recombinant double-stranded RNA molecule of claim 38,
wherein the two RNA strands are linked covalently to each
other.
41. An expression cassette comprising a nucleic acid encoding the
recombinant double-stranded RNA molecule of claim 38 in operable
linkage with a promoter.
42. The expression cassette of claim 41, wherein the promoter is
functional in a plant.
43. The expression cassette of claim 42, wherein the promoter is a
pathogen-inducible promoter.
44. A vector comprising the expression cassette of claim 41.
45. A transgenic organism comprising the expression cassette of
claim 41, wherein the organism is selected from the group
consisting of bacteria, yeasts, and plants.
46. The transgenic organism of claim 45, wherein the plants are
selected from the group consisting of wheat, oats, millet, barley,
rye, maize, rice, buckwheat, sorghum, triticale, spelt, linseed,
sugar cane, oil seed rape, canola, cress, Arabidopsis, cabbages,
soya, alfalfa, pea, beans, peanut, potato, tobacco, tomato,
eggplant, bell pepper, sunflower, Tagetes, lettuce, Calendula,
melon, pumpkin, squash and zucchini.
47. A transgenic plant or a tissue, organ, part or cell produced by
the method of claim 32.
48. A stably transformed plant produced by the method of claim
33.
49. The method of claim 32, wherein activity comprises GTPase
activity of said RacB protein.
50. The method of claim 32, wherein function comprises a
substrate-binding capacity of said RacB protein.
51. A method of selecting a plant cell with an increased resistance
to a pathogen comprising: introducing into a plant cell a nucleic
acid encoding a double stranded RacB RNA or an expression cassette
comprising said nucleic acid; said double stranded RNA reduces an
amount, activity or function of an RacB protein of the plant cell;
and selecting at least one plant cell wherein resistance to a
pathogen is increased as compared to an untransformed plant cell;
wherein the RacB protein comprises the sequence of SEQ ID NO: 4 or
a sequence having at least 90% homology to the sequence of SEQ ID
NO: 4; and wherein the pathogen is selected from the group
consisting of Plasmodiophoramycota, Oomycota, Ascomycota,
Chytridiomycetes, Zygomycetes, Basidiomycota, Deuteromycetes, and
combinations thereof.
52. The method of claim 51, wherein the activity comprises GTPase
activity of the RacB protein.
53. The method of claim 51, wherein the function comprises a
substrate-binding capacity of the RacB protein.
54. An expression cassette comprising a nucleic acid encoding the
double-stranded RNA molecule of claim 39 in operable linkage with a
promoter.
55. A transgenic organism comprising the expression cassette of
claim 54, wherein the organism is selected from the group of
organisms consisting of bacteria, yeasts, plants.
56. A transgenic organism comprising the vector of claim 44,
wherein the organism is selected from the group of organisms
consisting of bacteria, yeasts, and plants.
57. A recombinant plant cell wherein an expressed amount, activity
or function of an endogenous RacB protein is reduced by stable
transformation with a nucleic acid or an expression cassette
comprising the nucleic acid as compared to an untransformed plant
cell, wherein the nucleic acid comprises a double-stranded RacB
RNA; and wherein the nucleic acid comprises the sequence of SEQ ID
NO: 3 or a sequence having at least 90% homology to the sequence of
SEQ ID NO: 3.
58. The cell of claim 57, wherein the activity comprises GTPase
activity of said RacB protein.
59. The cell of claim 57, wherein the function comprises a
substrate-binding capacity of said RacB protein.
60. The plant cell of claim 57, wherein the endogenous RacB protein
is reduced by at least 60%.
61. The plant cell of claim 57, wherein the endogenous RacB protein
is reduced by at least 90%.
62. The plant cell of claim 57, wherein the double-stranded RNA
molecule comprises a sense strand and an antisense strand which is
essentially complementary to the sense strand.
63. The plant cell of claim 57, wherein the reduction provides the
plant cell with an increased resistance to a pathogen as compared
to the untransformed plant cell.
64. The cell of claim 63, wherein the pathogen is selected from the
group consisting of Plasmodiophoramycota, Oomycota, Ascomycota,
Chytridiomycetes, Zygomycetes, Basidiomycota, Deuteromycetes, and
combinations thereof.
65. The recombinant double-stranded RNA molecule of claim 39,
wherein the two RNA strands are linked covalently to each
other.
66. A transgenic organism comprising the recombinant
double-stranded RNA of claim 39, wherein the organism is selected
from the group of organisms consisting of bacteria, yeasts, and
plants.
67. A transgenic organism comprising a nucleic acid encoding the
double-stranded RNA molecule of claim 38, wherein the organism is
selected from the group consisting of bacteria, yeasts, and
plants.
68. A plant comprising the transformed plant cell of claim 48.
69. A plant comprising the recombinant plant cell of claim 57.
70. A transformed plant cell obtained by the method of claim
51.
71. A plant comprising the transformed plant cell of claim 70.
72. The method of claim 32, wherein the plant tissue, organ, or
part thereof is from a monocot or dicot.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/488,222, filed Mar. 2, 2004 which is a 35 U.S.C. 371
National stage filing of International Application No.
PCT/EP2002/009719, filed Aug. 3, 2002, which claims priority to
German Application No. 10142579.1, filed Sep. 3, 2001 and German
Application No. 10229729.0, filed Jul. 2, 2002. The entire contents
of each of these applications are hereby incorporated by reference
herein.
SUBMISSION OF SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
filed in electronic format via EFS-Web and hereby incorporated by
reference into the specification in its entirety. The name of the
text file containing the Sequence Listing is
Revised_Sequence_List.sub.--12810.sub.--00810_US. The size of the
text file is 171 KB, and the text file was created on Feb. 18,
2009.
FIELD OF THE INVENTION
[0003] The invention relates to novel RacB cDNA sequences from
barley and to expression cassettes and vectors comprising these
sequences. The invention furthermore relates to transgenic plants
transformed with these expression cassettes or vectors, to
cultures, parts or transgenic propagation material derived from
them, and to their use for the production of foodstuffs, feeding
stuffs, seed, pharmaceuticals or fine chemicals. The invention
furthermore relates to methods of generating or increasing a
pathogen resistance in plants by reducing the expression of an RacB
protein or of a functional equivalent thereof.
BACKGROUND OF THE INVENTION
[0004] The aim of plant biotechnology work is the generation of
plants with advantageous novel properties, for example for
increasing agricultural productivity, increasing the quality in the
case of foodstuffs, or for producing specific chemicals or
pharmaceuticals (Dunwell J M (2000) J Exp Bot 51 Spec No: 487-96).
The plant's natural defense mechanisms against pathogens are
frequently insufficient. Fungal diseases alone result in annual
yield losses of many billions of US$. The introduction of foreign
genes from plants, animals or microbial sources can increase the
defenses. Examples are the protection of tobacco against feeding
damage by insects by expressing Bacillus thuringiensis endotoxins
under the control of the 35S CaMV promoter (Vaeck et al. (1987)
Nature 328:33-37) or the protection of tobacco against fungal
infection by expressing a bean chitinase under the control of the
CaMV promoter (Broglie et al. (1991) Science 254:1194-1197).
However, most of the approaches described only offer resistance to
a single pathogen or a narrow spectrum of pathogens.
[0005] Only a few approaches exist which impart a resistance to a
broader spectrum of pathogens, in particular fungal pathogens, to
plants. Systemic acquired resistance (SAR)--a defense mechanism in
a variety of plant/pathogen interactions--can be mediated by the
application of endogenous messenger substances such as jasmonate
(JA) or salicylic acid (SA) (Ward, et al. (1991) Plant Cell
3:1085-1094; Uknes, et al. (1992) Plant Cell 4(6):645-656). Similar
effects can also be achieved by synthetic compounds such as
2,6-dichloroisonicotinic acid (INA) or S-methyl
benzo(1,2,3)thiadiazole-7-thiocarboxylate (BTH; Bion.RTM.)
(Friedrich et al. (1996) Plant J 10(1):61-70; Lawton et al. (1996)
Plant J. 10:71-82). The expression of pathogenesis-related (PR)
proteins, which are highly regulated in the case of an SAR, may
also cause pathogen resistance in some cases.
[0006] In barley, the Mlo locus has been described for some time as
a negative regulator of plant defense. The loss, or loss of
function, of the Mlo gene causes an increased and, above all,
race-unspecific resistance for example against a large number of
mildews (Buschges R et al. (1997) Cell 88:695-705; Jorgensen J H
(1977) Euphytica 26:55-62; Lyngkjaer M F et al. (1995) Plant Pathol
44:786-790). The Mlo phenotype is inherited recessively, which also
suggests a function as a susceptibility gene. Mlo-deficient barley
varieties obtained by traditional breeding are already being widely
used in agriculture. Although these varieties are being grown
intensively, this resistance has proved to be extraordinarily
durable, probably owing to the recessivity. Resistance breakdown
has not been observed as yet. Mlo-like resistances in other plants,
especially in cereal species, have not been described even though
wheat, rye and other cereals are also attacked by comparable mildew
pathogens. The reason in the case of wheat may be, for example, the
existence of a hexaploid genome, which makes the identification of
mutants in which each of the six copies of the gene has been
inactivated extremely difficult.
[0007] The Mlo gene has only recently been cloned (Buschges R et
al. (1997) Cell 88:695-705; WO 98/04586; Schulze-Lefert P, Vogel J
(2000) Trends Plant Sci. 5:343-348). As a consequence, various
homologs have been isolated from other cereal species. Various
methods for obtaining pathogen resistance using these genes have
been described (WO 98/04586; WO 00/01722; WO 99/47552).
[0008] Mlo resistance of a plant to mildew pathogens manifests
itself in two important events, both of which bring about
resistance to penetration: cell wall apposition (CWA) underneath
the penetration site of the pathogen in the epidermal cell wall.
Spreading of this fungal pathogen is almost exclusively restricted
to this subcellular structure (Jorgensen J H and Mortensen K (1977)
Phytopathology 67:678-685; Freialdenhoven A et al. (1996) Plant
Cell 8:5-14). This reaction is caused by the genes Ror1 and Ror2,
which are required for the effect of Mlo (Peterhansel C et al.
(1997) 9:1397-1409).
[0009] The disadvantage in Mlo pathogen resistance is that
Mlo-deficient plants--even in the absence of a pathogen--initiate a
defense mechanism which manifests itself for example in the
spontaneous death of leaf cells (Wolter M et al. (1993) Mol Gen
Genet 239:122-128). A further disadvantage is that the
Mlo-deficient genotypes are hypersusceptible to hemibiotrophic
pathogens such as Magnaporte grisea (M. grisea) and Cochliobolus
sativus (Bipolaris sorokiniana) (Jarosch B et al. (1999) Mol Plant
Microbe Interact 12:508-514; Kumar J et al. (2001) Phytopathology
91:127-133). The Mlo gene therefore appears to be a negative
regulator of cell death. Again, the cause is probably the induction
of cell death in the absence of the Mlo gene, which increases the
susceptibility to these fairly necrotrophic pathogens. This
ambivalent effect, which limits the biotechnological use of Mlo, is
probably due to the fact that necrotrophic fungi are capable of
exploiting the more pronounced HR of the Mlo-deficient host plant
for their infection process. A resistance comparable to Mlo
deficiency, but without the characteristic of inducing cell death,
would be desirable.
[0010] The proteins Rho, Rac and Cdc42 are members of the small GTP
(guanosine triphosphate) binding protein family and regulate a
large number of intracellular processes as "molecular switches",
both in plant and animal organisms. As elements of signal
transduction, they play an important role in the conversion of
extracellular stimuli. For example, they regulate NADPH oxidase and
thus the release of reactive oxygen molecules ("oxidative burst").
Animal or human Rac1 is essential for the formation of the active
NADPH oxidase complex which, in turn, is important for the
formation of superoxide, thus contributing to plant defense (Irani
K and Goldschmidt-Clermont P J (1998) Biochem Pharmacol 55:
1339-1346). The function in plant defense in plants and animals is
largely analogous (Kwong et al. (1995) J Biol Chem 270(34):
19868-19872; Dusi et al. (1996) Biochem J 314:409-412; Diekmann et
al. (1994) Science 265:531-533; Purgin et al. (1997) The Plant Cell
9:2077-2091; Kleinberg et al. (1994) Biochemistry 33:2490-2495;
Prigmore et al. (1995) Journal of Biol Chem 27(18): 10717-10722;
Irani et al. (1997) Science 275:1649-1652; Low et al. (1994)
Advances in Molecular Genetics of Plant-Microbe Interactions
3:361-369 (1994) eds. M J Daniels, Kluwer Acadmic Publishers,
Netherlands; Mehdy et al. (1994) Plant Physiol 105: 467-472;
Sundaresan et al. (1996) Biochem J 318:379-382). Moreover, GTP
binding proteins function in restructuring the cytoskeleton and in
cell transformation (Symon M. (1996) TIBS 21: 178-181), and also in
the activation of transcription (Hill et al. (1995) Cell
81:1159-1170; Chandra et al. (1996) Proc Natl Acad Sci USA
93:13393-13397).
[0011] In plants, there exists a substantial family of Rac-like
proteins (Winge et al. (1997) Plant Mol Biol 35:483-495), which is
also termed Rop family (Lin et al. (1997) The Plant Cell
9:1647-1659). In plants, the Rac proteins appear to have a function
in the release of reactive oxygen molecules as the consequence of
pathogen infection (Groom Q J et al. (1996) Plant J 10: 515-522;
Hassanain H H et al. (2000) Biochem Biophys Res Commun
272(3):783-788; Ono E et al. (2001) Proc Natl Acad Sci USA 98:
759-764). Rac modulates, inter alia, cell wall architecture, signal
transduction in the meristem and the defense against pathogens
(Valster A H et al. (2000) Trends Cell Biol 10(4):141-146). When
the constitutively active form is overexpressed, Rac1 from rice is
capable of inducing a hypersensitive response (HR) at the sites of
M. grisea attack, thus causing pathogen resistance. Analogously,
the expression of a negative dominant form of Rac1 brings about an
increased susceptibility to M. grisea (Kawasaki T et al. (1999)
Proc Natl Acad Sci USA 96:10922-10926; Ono E et al. (2001) Proc
Natl Acad Sci USA 98: 759-764). These findings suggest that an
overexpression of Rac proteins in the plant can bring about
advantageous effects with regard to plant defense.
[0012] WO 00/15815 describes five Rac genes from maize. Although
methods for both an up regulation and a down regulation of Rac
proteins are described and speculatively discussed in connection
with obtaining a resistance to pathogens (p. 55/line 25 et seq.),
the only technical teaching, which describes this use in real
terms, concerns merely an overexpression of the claimed Rac genes
for obtaining pathogen resistance (p. 60/line 21 et seq.). The
author postulates quite unambiguously and in agreement with the
situation described in the prior art (p. 60/line 31 et seq.): "Thus
the present invention is useful in protecting plants from
pathogens. Once a plant is transformed with a polynucleotide
sequence encoding an Rac polypeptide, expression of the polypeptide
in the plant confers resistance to infection by plant pathogens."
The rationale behind this hypothesis (plants defense via reactive
oxygen molecules) is explained hereinbelow and supported by a large
number of references. Beyond this, no differentiation is being made
between the five claimed Rac genes.
[0013] It is an object of the present invention to provide novel
methods of plant defense against pathogens which bring about an
effective defense against as broad a spectrum of pathogens as
possible in as large a number of plant species as possible,
preferably the crop plants used in agriculture. We have found that
this object is achieved by the method according to the
invention.
BRIEF SUMMARY OF THE INVENTION
[0014] The invention firstly comprises a method of generating or
increasing the resistance to at least one pathogen in plants, which
comprises the following steps
[0015] a) reducing the amount, activity or function of an RacB
protein in a plant or a tissue, organ, part or cell thereof,
and
[0016] b) selecting those plants in which, as opposed or as
compared to the original plant, the resistance to at least one
pathogen exists or is increased.
[0017] Surprisingly, the Rac homolog RacB from barley (Hordeum
vulgare) (SEQ ID NO: 1) (hereinbelow: hvRacB), despite a great
similarity with rice Rac1, has a negative control function upon
attack by powdery mildew of barley Blumeria (syn. Erysiphe)
graminis f.sp. hordei (Bgh), as opposed to the former: reducing the
hvRacB expression in the epidermal cell by a sequence-specific RNA
interference approach using double-stranded hvRacB dsRNA ("gene
silencing") significantly prevented the development of haustoria
owing to Bgh infection. Further experiments demonstrated (cf.
Example 7) that this phenotype cannot be observed in an
mlo5-ror1-mutant genotype, namely barley A89. This suggests that
RacB is linked operably to Mlo or Ror1 or both, that is to say they
probably act within a signal cascade.
[0018] Similarly to the loss of function of Mlo, that of HvRacB
confers broad resistance to various Blumeria graminis f.sp. hordei
isolates. In transient gene silencing experiments, HvRacB reduced
the penetration efficiency (development of haustoria) of Bgh by 44%
(cf. Example 7), an effect whose magnitude corresponds to the
effect achieved by Mlo dsRNA (Schweizer P et al. (2000) Plant J
24:895-903). In the wild-type barley variety Ingrid, approximately
60% of the fungal penetrations resulted in the development of
haustoria, while the penetration rate in BCIngrid-mlo5 is virtually
0%. The barley variety A89 (mlo-ror1 dual mutant) shows a
penetration efficiency of approximately 20 to 35%. An altered RacB
expression owing to Bgh inoculation was observed in none of these
variants (cf. Example 7; FIG. 3). The fact that only a penetration
of approximately 50% can be observed even in pathogen-sensitive
wild-type varieties, such as Pallas or Ingrid, can be attributed to
the basal resistance which is always present.
[0019] Interestingly, the gene silencing of hvRacB only enhances
cell wall apposition, but apparently not the spontaneous cell death
of the plant, which is in contrast to Mlo. Thus, HvRacB differs
from OsRac1, a rice homolog of Rac1 (Ono E et al. (2001) Proc Natl
Acad Sci USA 98: 759-764). HvRacB acts predominantly as negative
regulator of cell wall apposition. This difference is of
outstanding importance for its use for obtaining pathogen
resistance in plants. As already described above, Mlo resistance to
biotrophic fungi (for example mildew fungi) is indeed caused, inter
alia, by increased cell wall apposition, but the trade-off is a
higher susceptibility to necrotrophic fungi (Jarosch B et al.
(1999) Mol Plant Microbe Interact 12:508-514; Kumar J et al. (2001)
Phytopathology 91:127-133). Since HvRacB only affects cell all
apposition, this problem of ambivalence can be circumvented.
[0020] Owing to the above findings, RacB must be considered as a
key element for the successful penetration of a pathogen such as
Bgh into the plant cell. Accordingly, the method according to the
invention has all the advantages of Mlo deficiency without
simultaneously showing its biggest shortcoming, namely increased
spontaneous cell death.
[0021] Moreover, the method outperforms all those methods in which
a pathogen-resistant phenotype is realized by overexpressing a
resistance-conferring protein. Switching off a gene can be realized
without expressing a (foreign) protein. In an ideal case, all that
needs doing is to deactivate the endogenous gene. This has not
inconsiderable advantages for approval and acceptance by the
consumer, who is frequently apprehensive toward plants with foreign
proteins. Very especially advantageous in this context is the use
of inducible promoters for reducing the amount, activity or
function of RacB protein, which, for example when using
pathogen-inducible promoters, allows expression only when required
(i.e. pathogen infection).
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1: Alignment of the amino acid sequences of barley RacB
(RACB H.vul.; SEQ ID NO: 2), rice RacB (RAB.sub.--O.sat.; SEQ ID
NO: 4), maize RacB (RACB.sub.--Z.mays; SEQ ID NO: 6), and human
Rac1 (RAC1.sub.--H.sap.; SEQ ID NO: 78) and Rac2 proteins
(RAC2.sub.--H.sap.; SEQ ID NO: 77).
[0023] FIG. 2: Expression of RacB in epidermal tissue.
[0024] FIG. 3: RacB is expressed constitutively in various
resistant barley lines.
[0025] FIG. 4: "RNA interference" with RacB-dsRNA reduces the
penetration efficacy of barley powdery mildew BghA6 in barley.
[0026] FIG. 5: Effect of the genetic background on RacB
function.
[0027] FIG. 6: Overexpression of a constitutively active RacB
mutant in barley cv. Pallas.
[0028] FIG. 7. Plasmid map for expression vector pGY-1 (Schweizer P
et al. (1999) Mol Plant Microbe Interact 12: 647-54; Shinshi H et
al. (1990) Plant Mol Biol 14:357-368).
DETAILED DESCRIPTION OF THE INVENTION
[0029] A partial sequence of the barley RacB cDNA (HvRacB-cDNA)
(GenBank Acc. No.: AJ290240), which is highly conserved relative to
rice RacB (GenBank Acc. No.: AF250327) and maize RacB (GenBank Ace.
No.: AF126053) and very similar to rice Rac1 has been described.
Maize RacB is also one of the five Rac genes in the abovementioned
application WO 00/15815 (Sequence No. 3). The complete coding
sequence of the HvRacB protein has not been described as yet (see
Example 1). Barley RacB has a homology of 95% identity with rice
RacB and maize RacB and is over 55% identical to human RAC1 or RAC2
(Hassanain et al. 2000, FIG. 1). HvRacB is expressed constitutively
in primary leaves of barley (epidermis-specifically) and its
expression level is not affected substantially by Bgh infection.
Expression thus takes place in the tissue which interacts directly
with the Bgh pathogen.
[0030] In principle, the method according to the invention can be
applied to all plant species, preferably to those in which an RacB
protein or a functional equivalent thereof is expressed naturally.
Since the function of RacB is closely connected functionally to the
Mlo gene and the latter has been identified in a large number of
plants, including dicots (Devoto A et al. (1999) J Biol Chem
274(49):34993-5004), it can be assumed that RacB and its homologs
are similarly widely distributed. The sequences from other plants
(for example Arabidopsis thaliana) which are homologous to the RacB
sequences disclosed within the scope of the present invention can
be found readily for example by database searches or by screening
genetic libraries using the RacB sequences as search sequence or
probe.
[0031] The term "plant" as used herein refers to all genera and
species of higher and lower plants of the Plant Kingdom. The term
includes the mature plants, seed, shoots and seedlings and their
derived parts, propagation material, plant organs, tissue,
protoplasts, callus and other cultures, for example cell cultures,
and any other type of plant cell grouping to give functional or
structural units. Mature plants refers to plants at any desired
developmental stage beyond that of the seedling. Seedling refers to
a young immature plant at an early developmental stage. "Plant"
comprises all annual and perennial monocotyledonous and
dicotyledonous plants and includes by way of example but not by
limitation those of the genera Cucurbita, Rosa, Vitis, Juglans,
Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella,
Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis,
Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus,
Lycopersicon, Nicotiana, Solarium, Petunia, Digitalis, Majorana,
Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum,
Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum,
Ranunculus, Senecio, Salpiglossis, Cucumis, Browoalia, Glycine,
Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale,
Triticum, Sorghum, Picea and Populus.
[0032] The term "plant" preferably comprises monocotyledonous crop
plants such as, for example, cereal species such as wheat, barley,
millet rye, triticale, maize, rice, sorghum or oats and also sugar
cane.
[0033] The term furthermore comprises dicotyledonous crop plants
such as, for example,
[0034] Brassicacae such as oilseed rape, canola, cress,
Arabidopsis, cabbages or canola, Leguminosae such as soybean,
alfalfa, pea, beans or peanut
[0035] Solanaceae such as potato, tobacco, tomato, egg plant or
pepper, Asteraceae such as sunflower, Tagetes, lettuce or
Calendula,
[0036] Cucurbitaceae such as melon, pumpkin/squash or zucchini,
[0037] and also linseed, cotton, hemp, clover, spinach, flax, red
pepper, carrot, beet, radish, sugar beet, sweet potato, cucumber,
chicory, cauliflower, broccoli, asparagus, onion, garlic,
celery/celeriac, strawberry, raspberry, blackberry, pineapple,
avocado and the various tree, bush, nut and vine species. Tree
species preferably comprise plum, cherry, peach, nectarine,
apricot, banana, papaya, mango, apple, pear, quince.
[0038] Also comprised are ornamental plants, useful trees,
ornamental trees, flowers, cut flowers, shrubs or lawns such as by
way of example but not by limitation the families of the Rosaceae
such as rose, Ericaceae such as rhododendrons and azaleas,
Euphorbiaceae such as poinsettias and croton, Caryophyllaceae such
as carnations, Solanaceae such as petunias, Gesneriaceae such as
African violet, Balsaminaceae such as touch-me-not, Orchidaceae
such as orchids, Iridaceae such as gladioli, iris, freesia and
crocus, Compositae such as calendula, Geraniaceae such as
geraniums, Liliaceae such as dracaena, Moraceae such as ficus,
Araceae such as philodendron and many others.
[0039] Preferred within the scope of the invention are those plants
which are employed as foodstuffs or feeding stuffs, very especially
preferably monocotyledonous genera and species like the
above-described cereal species.
[0040] The method is applied very especially preferably to
monocotyledonous plants, most preferably to agriculturally
important plants such as wheat, oats, millet, barley, rye, maize,
rice, buckwheat, sorghum, triticale, spelt, linseed or sugar
cane.
[0041] "Pathogen resistance" denotes the reduction or weakening of
disease symptoms of a plant following infection by a pathogen. The
symptoms can be manifold, but preferably comprise those which
directly or indirectly have an adverse effect on the quality of the
plant, the quantity of the yield, the suitability for use as
feeding stuff or foodstuff, or else which make sowing, planting,
harvesting or processing of the crop difficult.
[0042] "Conferring", "existing", "generating" or "increasing" a
pathogen resistance means that the defense mechanisms of a specific
plant species or variety is increasingly resistant to one or more
pathogens due to the use of the method according to the invention
in comparison with the wild type of the plant ("original plant"),
to which the method according to the invention has not been
applied, under otherwise identical conditions (such as, for
example, climatic conditions, growing conditions, pathogen species
and the like). The increased resistance manifests itself preferably
in a reduced manifestation of the disease symptoms, disease
symptoms comprising--in addition to the above-mentioned adverse
effects--for example also the penetration efficiency of a pathogen
into the plant or plant cells or the proliferation efficiency in or
on the same. In this context, the disease symptoms are preferably
reduced by at least 10% or at least 20%, especially preferably by
at least 40% or 60%, very especially preferably by at least 70% or
80% and most preferably by at least 90% or 95%.
[0043] "Selection" with regard to plants in which--as opposed or as
compared to the original plant--resistance to at least one pathogen
exists or is increased means all those methods which a are suitable
for recognizing an existing or increased resistance to pathogens.
These may be symptoms of pathogen infection (for example the
development of haustoria in the case of fungal infection), but may
also comprise the above-described symptoms which relate to the
quality of the plant, the quantity of the yield, the suitability
for use as feeding stuff or foodstuff and the like.
[0044] "Pathogen" within the scope of the invention means by way of
example but not by limitation viruses or viroids, bacteria, fungi,
animal pests such as, for example, insects or nematodes. Especially
preferred are fungi such as, for example, mildew. However, it can
be assumed that a reduced expression of an RacB protein, its
activity or function also brings about resistance to other
pathogens. Changes in the cell wall structure can constitute a
prime mechanism of pathogen resistance.
[0045] The following pathogens may be mentioned by way of example
but not by limitation:
[0046] 1. Fungal Pathogens or Fungus-Like Pathogens:
[0047] Fungal pathogens or fungus-like pathogens (e.g. Chromista)
are preferably from the group comprising the Plasmodiophoramycota,
Oomycota, Ascomycota, Chytridiomycetes, Zygomycetes, Basidiomycota
and Deuteromycetes (Fungi imperfecti). The pathogens mentioned in
Tables 1 and 2 and the diseases with which they are associated may
be mentioned by way of example but not by limitation.
TABLE-US-00001 TABLE 1 Fungal plant diseases Disease Pathogen Leag
rust Puccinia recondita Yellow rust P. striiformis Powdery mildew
Erysiphe graminis/Blumeria graminis Glume blotch Septoria nodorum
Leaf blotch Septoria tritici Ear fusarioses Fusarium spp. Eyespot
Pseudocercosporella herpotrichoides Smut Ustilago spp. Bunt
Tilletia caries Take-all Gaeumannomyces graminis Anthrocnose leaf
blight Colletotrichum graminicola Anthracnose stalk rot
(teleomorph; Glomerella graminicola Politis); Glomerella
tucumanensis (anamorph; Glomerella falcatum Went) Aspergillus ear
and Aspergillus flavus kernel rot Banded leaf and sheath
Rhizoctonia solani Kuhn = Rhizoctonia spot microsclerotia J. Matz
(telomorph: Thanatephorus cucumeris) Black bundle disease
Acremonium strictum W. Gams = Cephalosporium acremonium Auct. non
Corda Black kernel rot Lasiodiplodia theobromae = Botryodiplodia
theobromae Borde blanco Marasmiellus sp. Brown spot (black spot,
Physoderma maydis stalk rot) Cephalosporium kernel Acremonium
strictum = Cephalosporium rot acremonium Charcoal rot Macrophomina
phaseolina Corticium ear rot Thanatephorus cucumeris = Corticium
sasakii Curvularia leaf spot Curvularia clavata, C. eragrostidis, =
C. maculans (teleomorph: Cochliobolus eragrostidis), Curvularia
inaequalis, C. intermedia (teleomorph: Cochliobolus intermedius),
Curvularia lunata (teleomorph: Cochliobolus lunatus), Curvularia
pallescens (teleomorph: Cochliobolus pallescens), Curvularia
senegalensis, C. tuberculata (teleomorph: Cochliobolus
tuberculatus) Didymella leaf spot Didymella exitalis Diplodia ear
rot and Diplodia frumenti (teleomorph: stalk rot Botryosphaeria
festucae) Diplodia ear rot, stalk Diplodia maydis = rot, seed rot
and Stenocarpella maydis seedling blight Diplodia leaf spot or
Stenocarpella macrospora = streak Diplodialeaf macrospora
TABLE-US-00002 TABLE 2 Downy mildew Disease Pathogen Brown stripe
downy Sclerophthora rayssiae var. zeae mildew Crazy top downy
mildew Sclerophthora macrospora = Sclerospora macrospora Green ear
downy mildew Sclerospora graminicola (graminicola downy mildew)
Java downy mildew Peronosclerospora maydis = Sclerospora maydis
Philippine downy mildew Peronosclerospora philippinensis =
Sclerospora philippinensis Sorghum downy mildew Peronosclerospora
sorghi = Sclerospora sorghi Spontaneum downy mildew
Peronosclerospora spontanea = Sclerospora spontanea Sugarcane downy
mildew Peronosclerospora sacchari = Sclerospora sacchari Dry ear
rot (cob, Nigrospora oryzae kernel and stalk rot) (teleomorph:
Khuskia oryzae) Ear rots, minor Alternaria alternata = A. tenuis,
Aspergillus glaucus, A. niger, Aspergillus spp., Botrytis cinerea
(teleomorph: Botryotinia fuckeliana), Cunninghamella sp.,
Curvularia pallescens, Doratomyces stemonitis = Cephalotrichum
stemonitis, Fusarium culmorum, Gonatobotrys simplex, Pithomyces
maydicus, Rhizopus microsporus Tiegh., R. stolonifer = R.
nigricans, Scopulariopsis brumptii Ergot (horse's tooth) Claviceps
gigantea (anamorph: Sphacelia sp.) Eyespot Aureobasidium zeae =
Kabatiella zeae Fusarium ear and stalk Fusarium subglutinans = rot
F. moniliforme var. subglutinans Fusarium kernel, root Fusarium
moniliforme and stalk rot, seed rot (teleomorph: Gibberella
fujikuroi) and seedling blight Fusarium stalk rot, Fusarium
avenaceum seedling root rot (teleomorph: Gibberlla avenacea)
Gibberella ear and stalk Gibberella zeae rot (anamorph: Fusarium
graminearum) Gray ear rot Botryosphaeria zeae = Physalospora zeae
(anamorph: Macrophoma zeae) Gray leaf spot Cercospora sorghi = C.
sorghi var. (Cercospora leaf spot) maydis, C. zeae-maydis
Helminthosporium root Exserohilum pedicellatum = rot
Helminthosporium pedicellatum (teleomorph: Setosphaeria
pedicellata) Hormodendrum ear rot Cladosporium cladosporioides =
(Cladosporium rot) Hormodendrum cladosporioides, C. herbarum
(teleomorph: Mycosphaerella tassiana) Hyalothyridium leaf spot
Hyalothyridium maydis Late wilt Cephalosporium maydis Leaf spots,
minor Alternaria alternata, Ascochyta maydis, A. tritici, A.
zeicola, Bipolaris victoriae = Helminthosporium victoriae
(teleomorph: Cochliobolus victoriae), C. sativus (anamorph:
Bipolaris sorokiniana = H. sorokinianum = H. sativum), Epicoccum
nigrum, Exserohilum prolatum = Drechslera prolata (teleomorph:
Setosphaeria prolata) Graphium penicillioides, Leptosphaeria
maydis, Leptothyrium zeae, Ophiosphaerella herpotricha, (anamorph:
Scolecosporiella sp.) Paraphaeosphaeria michotii, Phoma Sp.,
Septoria zeae, S. zeicola, S. zeina Northern corn leaf Setosphaeria
turcica (anarnorph: blight (white blast, Exserohilum turcicum =
crown stalk rot, stripe) Helminthosporim turcicum) Northern corn
leaf spot Cochliobolus carbonum (anamorph: Helminthosporium ear rot
Bipolaris zeicola = Helminthosporium (race 1) carbonum) Penicillium
ear rot Penicillium spp., P. chrysogenum, (blue eye, blue mold) P.
expansum, P. oxalicum Phaeocytostroma stalk Phaeocytostroma
ambiguum, = rot and root rot Phaeocytosporella zeae Phaeosphaeria
leaf spot Phaeosphaeria maydis = Sphaerulina maydis Physalospora
ear rot Botryosphaeria festucae = Physalospora (Botryosphaeria ear
rot) zeicola (anamorph: Diplodia frumenti) Purple leaf sheath
Hemiparasitic bacteria and fungi Pyrenochaeta stalk rot Phoma
terrestris = and root rot Pyrenochaeta terrestris Pythium root rot
Pythium spp., P. arrhenomanes, P. graminicola Pythium stalk rot
Pythium aphanidermatum = P. butleri L. Red kernel disease (ear
Epicoccum nigrum mold, leaf and seed rot) Rhizoctonia ear rot
Rhizoctonia zeae (teleomorph: Waitea (sclerotial rot) circinata)
Rhizoctonia root rot and Rhizoctonia solani, Rhizoctonia zeae stalk
rot Root rots, minor Alternaria alternata, Cercospora sorghi,
Dictochaeta fertilis, Fusarium acuminatum (teleomorph: Gibberella
acuminata), F. equiseti (teleomorph: G. intricans), F. oxysporum,
F. pallidoroseum, F. poae, F. roseum, G. cyanogena, (anamorph: F.
sulphureum), Microdochium bolleyi,) Mucor sp., Periconia circinata,
Phytophthora cactorum, P. drechsleri, P. nicotianae var.
parasitica, Rhizopus arrhizus Rostratum leaf spot Setosphaeria
rostrata, (anamorph: (Helminthosporium leaf Exserohilum rostratum =
disease, ear and stalk He/minthosporium rostratum) rot) Rust,
common corn Puccinia sorghi Rust, southern corn Puccinia polysora
Rust, tropical corn Physopella pallescens, P. zeae = Angiopsora
zeae Sclerotium ear rot Sclerotium rolfsii Sacc. (teleomorph:
(southern blight) Athelia rolfsii) Seed rot-seedling blight
Bipolaris sorokiniana, P. zeicola = Helminthosporium carbonum,
Diplodia maydis, Exserohilum pedicillatum, Exserohilum turcicum =
Helminthosporium turcicum, Fusarium avenaceum, F. culmorum, F.
moniliforme, Gibberella zeae (anamorph: F. graminearum),
Macrophomina phaseolina, Penicillium spp., Phomopsis sp., Pythium
spp., Rhizoctonia solani, R. zeae, Sclerotium rolfsii, Spicaria sp.
Selenophoma leaf spot Selenophoma sp. Sheath rot Gaeumannomyces
graminis Shuck rot Myrothecium gramineum Silage mold Monascus
purpureus, M ruber Smut, common Ustilago zeae = U. maydis Smut,
false Ustilaginoidea virens Smut, head Sphacelotheca reiliana =
Sporisorium holcisorghi Southern corn leaf Cochliobolus
heterostrophus (anamorph: blight and stalk rot Bipolaris maydis =
Helminthosporium maydis) Southern leaf spot Stenocarpella
macrospora = Diplodia macrospora Stalk rots, minor Cercospora
sorghi, Fusarium episphaeria, F. merismoides, F. oxysporum
Schlechtend, F. poae, F. roseum, F. solani (teleomorph: Nectria
haematococca), F. tricinctum, Mariannaea elegans, Mucor sp.,
Rhopographus zeae, Spicaria sp. Storage rots Aspergillus spp.,
Penicillium spp. and other fungi Tar spot Phyllachora maydis
Trichoderma ear rot and Trichoderma viride = T. lignorum root rot
teleomorph: Hypocrea sp. White ear rot, root and Stenocarpella
maydis = Diplodia zeae stalk rot Yellow leaf blight Ascochyta
ischaemi, Phyllosticta maydis (teleomorph: Mycosphaerella
zeae-maydis) Zonate leaf spot Gloeocercospora sorghi
[0048] The following are especially preferred:
[0049] Plasmodiophoromycota such as Plasmodiophora brassicace
(clubroot of crucifers), Spongospora subterranea (powdery scab of
potato tubers), Polymyxa graminis (root disease of cereals and
grasses),
[0050] Oomycota such as Bremia lactucae (downy mildew of lettuce),
Peronospora (downy mildew) in snapdragon (P. antirrhini), onion (P.
destructor), spinach (P. effusa), soybean (P. manchurica), tobacco
("blue-mold"; P. tabacina) alfalfa and clover (P. trifolium),
Pseudoperonospora humuli (downy mildew of hops), Plasmopara (downy
mildew in grapevines) (P. viticola) and sunflower (P. halstedii),
Sclerophtohra macrospora (downy mildew in cereals and grasses),
Pythlum (seed rot, seedling damping-off, and root rot and all types
of plants, for example damping-off of Beta beet caused by P.
debaryanum), Phytophthora infestans (blight in potato, brown rot in
tomato and the like), Albugo spec. (white rust on cruciferous
plants).
[0051] Ascomycola such as Microdochium nivale (snow mold of rye and
wheat), Fusarium graminearum, Fusarium culmorum (partial ear
sterility mainly in wheat), Fusarium oxysporum (Fusarium wilt of
tomato), Blumeria graminis powdery mildew of barley (f.sp. hordei)
and wheat (f.sp. tritici)), Erysiphe pisi (powdery mildew of pea),
Nectria galligena (Nectria canker of fruit trees), Unicnula necator
(powdery mildew of grapevine), Pseudopeziza tracheiphila (red fire
disease of grapevine), Claviceps purpurea (ergot on, for example,
rye and grasses), Gaeumannomyces graminis (take-all on wheat, rye
and other grasses), Magnaporthe grisea (rice blast disease),
Pyrenophora graminea (leaf stripe of barley), Pyrenophora teres
(net blotch of barley), Pyrenophora tritici-repentis (leaf blight
of wheat), Venturia inaequalis (apple scab), Sclerotinia sclerotium
(stalk break, stem rot), Pseudopeziza medicaginis (leaf spot of
alfalfa, white and red clover).
[0052] Basidiomycetes such as Typhula incarnata (typhula blight on
barley, rye, wheat), Ustilago maydis (blister smut on maize),
Ustilago nuda (loose smut on barley), Ustilago tritici (loose smut
on wheat, spelt), Ustilago avenae (loose smut on oats), Rhizoctonia
solani (rhizoctonia root rot of potato), Sphacelotheca spp. (head
smut of sorghum), Melampsora lini (rust of flax), Puccinia graminis
(stem rust of wheat, barley, rye, oats), Puccinia recondita (leaf
rust on wheat), Puccinia dispersa (brown rust on rye), Puccinia
hordei (leaf rust of barley), Puccinia coronata (crown rust of
oats), Puccinia striiformis (yellow rust of wheat, barley, rye and
a large number of grasses), Uromyces appendiculatus (brown rust of
bean), Sclerotium rolfsii (root and stem rots of many plants).
[0053] Deuteromycetes (Fungi imperfecti) such as Septoria nodorum
(glume blotch) of wheat (Septoria tritici), Pseudocercosporella
herpotrichoides (eyespot of wheat, barley, rye), Rynchosporium
secalis (leaf spot on rye and barley), Alternaria solani (early
blight of potato, tomato), Phoma betae (blackleg on Beta beet),
Cercospora beticola (leaf spot on Beta beet), Alternaria brassicae
(black spot on oilseed rape, cabbage and other crucifers),
Verticillium dahliae (verticillium wilt), Colletotrichum
lindemuthianum (bean anthracnose), Phoma lingam (blackleg of
cabbage and oilseed rape), Botrytis cinerea (grey mold of
grapevine, strawberry, tomato, hops and the like).
[0054] Most preferred are Phytophthora infestans (potato blight,
brown rot in tomato and the like), Microdochium nivale (previously
Fusarium nivale; snow mold of rye and wheat), Fusarium graminearum,
Fusarium culmorum (partial car sterility of wheat), Fusarium
oxysporum (Fusarium wilt of tomato), Blumeria graminis (powdery
mildew of barley (f. sp. hordei) and wheat (f. sp. tritici)),
Magnaporthe grisea (rice blast disease), Sclerotinia sclerotium
(stalk break, stem rot), Septoria nodorum and Septoria tritici
(glume blotch of wheat), Alternaria brassicae (black spot of
oilseed rape, cabbage and other crucifers), Phoma lingam (blackleg
of cabbage and oilseed rape).
[0055] 2. Bacterial Pathogens:
[0056] The pathogens and the diseases associated with them which
are mentioned in Table 3 may be mentioned by way of example but not
by limitation.
TABLE-US-00003 TABLE 3 Bacterial diseases Disease Pathogen
Bacterial leaf blight and Pseudomonas avenae subsp. avenae stalk
rot Bacterial leaf spot Xanthomonas campestris pv. holcicola
Bacterial stalk rot Enterobacter dissolvens = Erwinia dissolvens
Bacterial stalk and top Erwiria carotovora subsp. rot carotovora,
Erwinia chrysanthemi pv. zeae Bacterial stripe Pseudomonas
andropogonis Chocolate spot Pseudomonas syringae pv. coronafaciens
Goss's bacterial wilt and Clavibacter michiganensis subsp. blight
(leaf freckles and nebraskensis = Corynebacterium wilt)
michiganense pv. andnebraskense Holcus spot Pseudomonas syringae
pv. syringae Purple leaf sheath Hemiparasitic bacteria Seed
rot-seedling blight Bacillus subtilis Stewart's disease Pantoea
stewartii = (bacterial wilt) Erwinia stewartii Corn stunt
Spiroplasma kunkelii (achapparramiento, maize stunt, Mesa Central
or Rio Grande maize stunt)
[0057] The following pathogenic bacteria are very especially
preferred: Corynebacterium sepedonicum (bacterial ring rot of
potato), Erwinia carotovora (black leg of potato), Erwinia
amylovora (fire blight of pear, apple, quince), Streptomyces
scabies (potato scab), Pseudomonas syringae pv. tabaci (wildfire of
tobacco), Pseudomonas syringae pv. phaseolicola (grease spot of
dwarf bean), Pseudomonas syringae pv. tomato (bacterial speck of
tomato), Xanthomonas campestris pv. malvacearum (bacterial blight
of cotton) and Xanthomonas campestris pv. oryzae (bacterial leaf
blight of rice and other grasses).
[0058] 3. Viral Pathogens:
[0059] "Viral pathogens" includes all plant viruses such as, for
example, tobacco or cucumber mosaic virus, ringspot virus, necrosis
virus, maize dwarf mosaic virus and the like.
[0060] The pathogens and diseases associated with them which are
mentioned in Table 4 may be mentioned by way of example, but not by
limitation.
TABLE-US-00004 TABLE 4 Viral diseases Disease Pathogen American
wheat striate American wheat striate mosaic virus (wheat striate
mosaic) (AWSMV) Barley stripe mosaic Barley stripe mosaic virus
(BSMV) Barley yellow dwarf Barley yellow dwarf virus (BYDV) Brome
mosaic Brome mosaic virus (BMV) Cereal chlorotic mottle Cereal
chlorotic mottle virus (CCMV) Corn chlorotic vein Corn chlorotic
vein banding virus banding (Brazilian maize (CCVBV) mosaic) Corn
lethal necrosis Virus complex of Maize chlorotic mottle virus
(MCMV) and Maize dwarf mosaic virus (MDMV) A or B or Wheat streak
mosaic virus(WSMV) Cucumber mosaic Cucumber mosaic virus (CMV)
Cynodon chlorotic streak Cynodon chlorotic streak virus (CCSV)
Johnsongrass mosaic Johnsongrass mosaic virus (JGMV) Maize bushy
stunt Mycoplasma-like organism (MLO) associated Maize chlorotic
dwarf Maize chlorotic dwarf virus (MCDV) Maize chlorotic mottle
Maize chlorotic mottle virus (MCMV) Maize dwarf mosaic Maize dwarf
mosaic virus (MDMV) strains A, D, E and F Maize leaf fleck Maize
leaf fleck virus (MLFV) Maize line Maize line virus (MLV) Maize
mosaic (corn leaf Maize mosaic virus (MMV) stripe, enanismo rayado)
Maize mottle and Maize mottle and chlorotic stunt virus chlorotic
stunt Maize pellucid ringspot Maize pellucid ringspot virus (MPRV)
Maize raya gruesa Maize raya gruesa virus (MRGV) Maize rayado fino
(fine Maize rayado fino virus (MRFV) striping disease) Maize red
leaf and red Mollicute stripe Maize red stripe Maize red stripe
virus (MRSV) Maize ring mottle Maize ring mottle virus (MRMV) Maize
rio IV Maize rio cuarto virus (MRCV) Maize rough dwarf Maize rough
dwarf virus (MRDV) (Cereal (nanismo ruvido) tillering disease
virus) Maize sterile stunt Maize sterile stunt virus (strains of
barley yellow striate virus) Maize streak Maize streak virus (MSV)
Maize stripe (maize Maize stripe virus chlorotic stripe, maize hoja
blanca) Maize stunting Maize stunting virus Maize tassel abortion
Maize tassel abortion virus (MTAV) Maize vein enation Maize vein
enation virus (MVEV) Maize wallaby ear Maize wallaby ear virus
(MWEV) Maize white leaf Maize white leaf virus Maize white line
mosaic Maize white line mosaic virus (MWLMV) Millet red leaf Millet
red leaf virus (MRLV) Northern cereal mosaic Northern cereal mosaic
virus (NCMV) Oat pseudorosette Oat pseudorosette virus
(zakuklivanie) Oat sterile dwarf Oat sterile dwarf virus (OSDV)
Rice black-streaked Rice black-streaked dwarf virus dwarf (RBSDV)
Rice stripe Rice stripe virus (RSV) Sorghum mosaic Sorghum mosaic
virus (SrMV) (auch: sugarcane mosaic virus (SCMV) Stamme H, I and
M) Sugarcane Fiji disease Sugarcane Fiji disease virus (FDV)
Sugarcane mosaic Sugarcane mosaic virus (SCMV) strains A, B, D, E,
SC, BC, Sabi and MB (formerly MDMV-B) Wheat spot mosaic Wheat spot
mosaic virus (WSMV)
[0061] 4. Animal Pests
[0062] 4.1 Insect Pathogens:
[0063] The following may be mentioned by way of example, but not by
limitation: insects such as, for example, beetles, caterpillars,
lice or mites. Preferred insects are those of the genera
Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga,
Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera,
Isoptera, Anoplura, Siphonaptera, Trichoptera, etc. Especially
preferred are coleopteran and lepidopteran insects such as, for
example, the European corn borer (ECB), Diabrotica barberi
(northern corn rootworm), Diabrotica undecimpunctata (southern corn
rootworm), Diabrotica virgifera (Western corn rootworm), Agrotis
ipsilon (black cutworm), Crymodes devastator (glassy cutworm),
Feltia ducens (dingy cutworm), Agrotis gladiaria (claybacked
cutworm), Melanotus spp., Aeolus mellillus (wireworm), Aeolus
mancus (wheat wireworm), Horistonotus uhlerii (sand wireworm),
Sphenophorus maidis (maize billbug), Sphenophorus zeae (timothy
billbug), Sphenophorus parvulus (bluegrass billbug), Sphenophorus
callosus (southern corn billbug), Phyllogphaga spp. (white grubs),
Anuraphis maidiradicis (corn root aphid), Delia platura (seedcorn
maggot), Colaspis brunnea (grape colaspis), Stenolophus lecontei
(seedcorn beetle) and Clivinia impressifrons (lender seedcorn
beetle).
[0064] Other examples are: lema (Oulema melanopus), frit fly
(Oscinella frit), wireworms (Agrotis lineatus) and aphids (such as,
for example, the oat grain aphid Rhopalosiphum padi, the blackberry
aphid Sitobion avenae).
[0065] 4.2 Nematodes:
[0066] The pathogens and the diseases associated with them
mentioned in Table 6 may be mentioned by way of example, but not by
limitation.
TABLE-US-00005 TABLE 6 Parasitic nematodes Damage Pathogenic
nematode Awl Dolichodorus spp., D. heterocephalus Bulb and stem
nematode Ditylenchus dipsaci disease; Europe Burrowing Radopholus
similis Cyst nematode disease Heterodera avenae, H. zeae,
Punctodera chalcoensis Dagger Xiphinema spp., X. americanum, X.
mediterraneum False root-knot Nacobbus dorsalis Lance, Columbia
Hoplolaimus columbus Lance Hoplolaimus spp., H. galeatus Lesion
Pratylenchus spp., P. brachyurus, P. crenatus, P. hexincisus, P.
neglectus, P. penetrans, P. scribneri, P. thornei, P. zeae Needle
Longidorus spp., L. breviannulatus Ring Criconemella spp., C.
ornata Root-knot disease Meloidogyne spp., M. chitwoodi, M.
incognita, M. javanica Spiral Helicotylenchus spp. Sting
Belonolaimus spp., B. longicaudatus Stubby-root Paratrichodorus
spp., P. christiei, P. minor, Quinisulcius acutus, Trichodorus spp.
Stunt Tylenchorhynchus dubius
[0067] Very especially preferred are Globodera rostochiensis and G.
pallida (cyst eelworm on potato, tomato and other Solanaceae),
Heterodera schachtii (beet eelworm on sugar and fodder beet,
oilseed rape, cabbage and the like), Heterodera avenae (oat cyst
nematode on oat and other cereal species), Ditylenchus dipsaci
(stem or bulb eelworm, stem eelworm of rye, oats, maize, clover,
tobacco, beet), Anguina tritici (grain nematode, cockle disease of
wheat (spelt, rye), Meloidogyne hapla (root-knot nematode of
carrot, cucumber, lettuce, tomato, potato, sugar beet,
lucerne).
[0068] Examples of preferred fungal or viral pathogens for the
individual varieties are:
[0069] 1. Barley:
[0070] Fungal, bacterial and viral pathogens: Puccinia graminis
f.sp. hordei (barley stem rust), Blumeria (Erysiphe) graminis f.sp.
hordei (barley powdery mildew), barley yellow dwarf virus
(BYDV),
[0071] Pathogenic insects/nematodes: Ostrinia nubilalis (European
corn borer); Agrotis ipsilon (black cutworm); Schizaphis graminum
(greenbug); Blissus leucopterus leucopterus (chinch bug);
Acrosternum hilare (green stink bug); Euschistus servus (brown
stink bug); Deliaplatura (seedcorn maggot); Mayetiola destructor
(Hessian fly); Petrobia latens (brown wheat mite).
[0072] 2. Soybean:
[0073] Fungal, bacterial or viral pathogens: Phytophthora
megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia
solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe
phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum
var. caulivora, Sclerotium rolfsii, Cercospora kikuchii, Cercospora
sojina, Peronospora manshurica, Colletotrichum dematium
(Colletotrichum truncatum), Corynespora cassiicola, Septoria
glycines, Phyllosticta sojicola, Alternaria alternata, Pseudomonas
syringae p.v. glycinea, Xanthomonas campestris p.v. phaseoli,
Microsphaera diffussa, Fusarium semitectum, Phialophora gregata,
soybean mosaic virus, Glomerella glycines, Tobacco Ring spot virus,
Tobacco Streak virus, Phakopsorapachyrhizi, Pythium aphanidermatum,
Pythium ultimun, Pythium debaryanum, Tomato spotted wilt virus,
Heterodera glycines Fusarium solani.
[0074] Pathogenic insects/nematodes: Pseudoplusia includens
(soybean looper); Anticarsia gemmatalis (velvetbean caterpillar);
Plathypena scabra (green cloverworm); Ostrinia nubilalis (European
corn borer); Agrotis ipsilon (black cutworm); Spodoptera exigua
(beet armyworm); Heliothis virescens (cotton budworm); Helicoverpa
zea (cotton bollworm); Epilachna varivestis (Mexican bean beetle);
Myzus persicae (green peach aphid); Empoasca fabae (potato leaf
hopper); Acrosternum hilare (green stink bug); Melanoplus
femurrubrum (redlegged grasshopper); Melanoplus differentialis
(differential grasshopper); Hylemya platura (seedcom maggot);
Sericothrips variabilis (soybean thrips); Thrips tabaci (onion
thrips); Tetranychus turkestani (strawberry spider mite);
Tetranychus urticae (two-spotted spider mite).
[0075] 3. Canola:
[0076] Fungal, bacterial or viral pathogens: Albugo candida,
Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani,
Sclerotinia sclerotiorum, Mycosphaerella brassiccola, Pythium
ultimum, Peronospora parasitica, Fusarium roseum, Alternaria
alternata.
[0077] 4. Alfalfa:
[0078] Fungal, bacterial or viral pathogens: Clavibater michiganese
subsp. insidiosum, Pythium ultimum, Pythium irregulare, Pythium
splendens, Pythium debaryanum, Pythium aphanidermatum, Phytophthora
megasperma, Peronospora trifoliorum, Phoma medicaginis var.
medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis,
Leptotrochila medicaginis, Fusarium, Xanthomonas campestris p.v.
alfalfae, Aphanomyces euteiches, Stemphylium herbarum, Stemphylium
alfalfae.
[0079] 5. Wheat:
[0080] Fungal, bacterial or viral pathogens: Pseudomonas syringae
p.v. atrofaciens, Urocystis agropyri, Xanthomonas campestris p.v.
translucens, Pseudomonas syringae p.v. syringae, Alternaria
alternata, Cladosporium herbarum, Fusarium graminearum, Fusarium
avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici,
Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe
graminis f.sp. tritici, Puccinia graminis f.sp. tritici, Puccinia
recondita f.sp. tritici, Puccinia striiformis, Pyrenophora
tritici-repentis, Septoria nodorum, Septoria tritici, Septoria
avenae, Pseudocercosporella herpotrichoides, Rhizoctonia solani,
Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici; Pythium
aphanidermatum, Pythium arrhenomanes, Pythium ultimum, Bipolaris
sorokiniana, Barley Yellow Dwarf Virus, Brome Mosaic Virus, soil
borne wheat mosaic virus, wheat streak mosaic virus, wheat spindle
streak virus, American wheat striate virus, Claviceps purpurea,
Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia
indica, Rhizoctonia solani, Pythium arrhenomannes, Pythium
gramicola, Pythium aphanidermatum, high plains virus, European
wheat striate virus, Puccinia graminis f.sp. tritici (wheat stem
rust), Blumeria (Erysiphe) graminis f.sp. tritici (wheat powdery
mildew).
[0081] Pathogenic insects/nematodes: Pseudaletia unipunctata
(armyworm); Spodoptera, frugiperda (fall armyworm); Elasmopalpus
lignosellus (lesser cornstalk borer); Agrotis orthogonia (western
cutworm); Elasmopalpus Zignosellus (lesser cornstalk borer); Oulema
melanopus (cereal leaf beetle); Hypera punctata (clover leaf
weevil); Diabrotica undecimpunctata howardi (southern corn
rootworm); Russian wheat aphid; Schizaphis graminum (greenbug);
Macrosiphum avenae (English grain aphid); Melanoplus femurrubrum
(redlegged grasshopper); Melanoplus differentialis (differential
grasshopper); Melanoplus sanguinipes (migratory grasshopper);
Mayetiola destructor (Hessian fly); Sitodiplosis mosellana (wheat
midge); Meromyza americana (wheat stem maggot); Hylemya coarctata
(wheat bulb fly); Frankliniella fusca (tobacco thrips); Cephus
cinctus (wheat stem sawfly); Aceria rulipae (wheat curl mite).
[0082] 6. Sunflower:
[0083] Fungal, bacterial or viral pathogens: Plasmophora halstedii,
Sclerotinia sclerotiorum, aster yellows, Septoria helianthi,
Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae,
Botrytis cinerea, Phoma macdonaldii, Macrophomina phaseolina,
Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus,
Rhizopus stolonifer, Puccinia helianthi, Verticillium dahliae,
Erwinia carotovorum p.v. Carotovora, Cephalosporium acremonium,
Phytophthora cryptogea, Albugo tragopogonis.
[0084] Pathogenic insects/nematodes: Suleima helianthana (sunflower
bud moth); Homoeosoma electellum (sunflower moth); zygogramma
exclamationis (sunflower beetle); Bothyrus gibbosus (carrot
beetle); Neolasioptera murtfeldtiana (sunflower seed midge).
[0085] 7. Maize:
[0086] Fungal, bacterial or viral pathogens: Fusarium moniliforme
var. subglutinans, Erwinia stewartii, Fusarium moniliforme,
Gibberella zeae (Fusarium graminearum), Stenocarpella maydi
(Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium
graminicola, Pythium splendens, Pythium ultimum, Pythium
aphanidermatum, Aspergillus flavus, Bipolaris maydis 0, T
(Cochliobolus heterostrophus), Helminthosporium carbonum I, II
& III (Cochliobolus carbonum), Exserohilum turcicum I, II &
III, Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta
maydis, Kabatiella maydis, Cercospora sorghi, Ustilago maydis,
Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina,
Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum,
Curvularia lunata, Curvularia inaequalis, Curvularia pallescens,
Clavibacter michiganese subsp. nebraskense, Trichoderma viride,
maize dwarf mosaic virus A & B, wheat streak mosaic virus,
maize chlorotic dwarf virus, Claviceps sorghi, Pseudonomas avenae,
Erwinia chrysanthemi p.v. Zea, Erwinia corotovora, cornstunt
spiroplasma, Diplodia macrospora, Sclerophthora macrospora,
Peronosclerospora sorghi, Peronosclerospora philippinesis,
Peronosclerospora maydis, Peronosclerospora sacchari, Spacelotheca
reiliana, Physopella zeae, Cephalosporium maydis, Caphalosporium
acremonium, maize chlorotic mottle virus, high plains virus, maize
mosaic virus, maize rayado fino virus, maize streak virus (MSV),
maize stripe virus, maize rough dwarf virus.
[0087] Pathogenic insects/nematodes: Ostrinia nubilalis (European
corn borer); Agrotis ipsilon (black cutworm); Helicoverpa zea (corn
earworm); Spodoptera frugiperda (fall armyworm); Diatraea
grandiosella (Southwestern corn borer); Elasmopalpus lignosellus
(lesser cornstalk borer); Diatraea saccharalis (sugarcane borer);
Diabrotica virgifera (Western corn rootworm); Diabrotica
longicornis barberi (Northern corn rootworm); Diabrotica
undecimpunctata howardi (Southern corn rootworm); Melanotus spp.
(wireworms); Cyclocephala borealis (Northern masked chafer; white
grub); Cyclocephala immaculata (Southern masked chafer; white
grub); Popillia japonica (Japanese beetle); Chaetocnema pulicaria
(corn flea beetle); Sphenophorus maidis (maize billbug);
Rhopalosiphum maidis (corn leaf aphid); Anuraphis maidiradicis
(corn root aphid); Blissus leucopterus leucopterus (chinch bug);
Melanoplus femur-rubrum (red-legged grasshopper); Melanoplus
sanguinipes (migratory grasshopper); Hylemva platura (seedcom
maggot); Agromyza parvicornis (corn blot leafminer); Anaphothrips
obscrurus (grass thrips); Solenopsis milesta (thief ant);
Tetranychus urticae (two-spotted spider mite).
[0088] 8. Sorghum:
[0089] Fungal, bacterial or viral pathogens: Exserohilum turcicum,
Colletotrichum graminicola (Glomerella graminicola), Cercospora
sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas
syringae p.v. syringae, Xanthomonas campestris p.v. holcicola,
Pseudomonas andropogonis, Puccinia purpurea, Macrophomina
phaseolina, Perconia circinata, Fusarium moniliforme, Alternaria
alternate, Bipolaris sorghicola, Helminthosporium sorghicola,
Curvularia lunata, Phoma insidiasa, Pseudomonas avenae (Pseudomonas
alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola,
Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca
reiliana), Sphacelotheca cruenta, Sporisorium sorghi, sugarcane
mosaic H, maize dwarf mosaic virus A & B, Claviceps sorghi,
Rhizoctonia solani, Acremonium strictum, Sclerophthona macrospora,
Peronosclerospora sorghi, Peronosclerospora philippinensis,
Sclerospora graminicola, Fusarium graminearum, Fusarium oxysporum,
Pythium arrhenomanes, Pythium graminicola.
[0090] Pathogenic insects/nematodes: Chilo partellus (sorghum
borer); Spodoptera frugiperda (fall armyworm); Helicoverpa zea
(corn ear-worm); Elasmopalpus lignosellus (lesser cornstalk borer);
Feltia subterranea (granulate cutworm); Phvllophaga crinita(white
grub); Eleodes, Conoderus and Aceolus spp. (wireworm); Oulema
melanopus (cereal leaf beetle); Chaetocnema pulicaria (corn flea
beetle); Sphenophorus maidis (maize billbug); Rhopalosiphum maidis
(corn leaf aphid); Siphaflava (yellow sugarcane aphid); Blissas
leucopterus leucopterus (chinch bug); Contarinia sorghicola
(sorghum midge); Tetranychus cinnabarinus (carmine spider mite);
Tetranychus urticae (two-spotted spider mite).
[0091] 9. Cotton:
[0092] Pathogenic insects/nematodes: Heliothis virescens (cotton
budworm); Helicoverpa zea (cotton bollworm); Spodoptera exigua
(beet armyworm); Pectinophora gossypiella (pink bollworm);
Anthonomus grandis grandis (boll weevil); Aphis gossypii (cotton
aphid); Pseudatomoscelis seriatus (cotton fleahopper); Trialeurodes
abutilonea (banded-winged whitefly); Lygus lineolaris (tarnished
plant bug); Melanoplus femurrubrum (red-legged grasshopper);
Melanoplus differentialis (differential grasshopper); Thrips tabaci
(onion thrips); Franklinkiella fusca (tobacco thrips); Tetranychus
cinnabarinus (carmine spider mite); Tetranychus urticae
(two-spotted spider mite).
[0093] 10. Rice:
[0094] Pathogenic insects/nematodes: Diatraea saccharalis
(sugarcane borer); Spodoptera frugiperda (fall armyworm);
Helicoverpa zea (corn earworm); Colaspis brunnea (grape colaspis);
Lissorhoptrus oryzophilus (rice water weevil); Sitophilus oryzae
(rice weevil); Nephotettix nigropictus (rice leafhopper); Blissus
leucopterus leucopterus (chinch bug); Acrosternum hilare (green
stink bug).
[0095] 11. Oilseed Rape:
[0096] Pathogenic insects/nematodes: Brevicoryne brassicae (cabbage
aphid); Phyilotreta cruciferae (flea beetle); Mamestra conjgurata
(bertha armyworm); Plutella xylostella (diamond-back moth); Delia
ssp. (root maggots).
[0097] For the purposes of the invention, "RacB protein" is
understood as meaning the RacB protein from barley as shown in SEQ
ID NO: 2, and its homologs from rice (Oryza sative) as shown in SEQ
ID NO: 4 and maize (Zea mays) as shown in SEQ ID NO: 6, and
functional equivalents of the above-mentioned.
[0098] "Amount of protein" is understood as meaning the amount of
an RacB polypeptide in an organism, a tissue, a cell or a cell
compartment. "Reduction" of the amount of protein means the
quantitative reduction of the amount of an RacB protein in an
organism, a tissue, a cell or a cell compartment--for example by
one of the methods described hereinbelow--in comparison with the
wild type of the same genus and species, to which this method had
not been applied, under otherwise identical conditions (such as,
for example, culture conditions, plant age and the like). The
reduction amounts to at least 10%, preferably at least 10% or at
least 20%, especially preferably at least 40% or 60%, very
especially preferably at least 70% or 80%, most preferably at least
90% or 95%.
[0099] "Activity" is preferably understood as meaning the GTPase
activity of an RacB polypeptide in an organism, a tissue, a cell or
a cell compartment. "Reduction" of the activity is understood as
meaning the reduction of the total activity of an RacB protein in
an organism, a tissue, a cell or a cell compartment for example by
one of the methods described hereinbelow--in comparison with the
wild type of the same genus and species, to which this method had
not been applied, under otherwise identical conditions (such as,
for example, culture conditions, plant age and the like). The
reduction amounts to at least 10%, preferably at least 10% or at
least 20%, especially preferably at least 40% or 60%, very
especially preferably at least 70% or 80%, most preferably at least
90% or 95%.
[0100] "Function" is preferably understood as meaning the
substrate-binding capacity of an RacB polypeptide in an organism, a
tissue, a cell or a cell compartment. Suitable substrates are
low-molecular-weight compounds such as GTP, but also the protein
interaction partners of an RacB protein. "Reduction" of the
function is understood as meaning, for example, the quantitative
reduction of the binding capacity or binding strength of an RacB
protein to at least one substrate in an organism, a tissue, a cell
or a cell compartment--for example by one of the methods described
hereinbelow--in comparison with the wild type of the same genus and
species, to which this method had not been applied, under otherwise
identical conditions (such as, for example, culture conditions,
plant age and the like). Reduction is also understood as meaning
the change in substrate specificity as can be expressed for example
by the kcat/Km value. The reduction amounts to at least 10%,
preferably at least 10% or at least 20%, especially preferably at
least 40% or 60%, very especially preferably at least 70% or 80%,
most preferably at least 90% or 95%. Binding partners for RacB can
be identified in a manner with which the skilled worker is
familiar, for example by the yeast-2-hybrid system.
[0101] Methods of determining the amount of protein, the activity
of GTPases or the substrate binding capacity are known to the
skilled worker and have been described on a number of occasions for
GTPases and for Rac proteins from a variety of genera and species
(see, inter alia, Benard V et al. (11999) J Biol Chem
274(19):13198-204; Burstein E S (1998) Oncogene.
17(12):1617-23).
[0102] "Functional equivalents" of an RacB protein is preferably
understood as meaning those sequences which are derived from, or
are homologous to, an RacB protein described by SEQ ID NO: 2, 4 or
6 and which have essentially the same properties.
[0103] "Essentially the same properties" of a functional equivalent
is above all understood as meaning conferring a pathogen-resistant
phenotype or conferring or increasing the resistance to at least
one pathogen while reducing the amount of protein, activity or
function of said functional RacB equivalent in a plant or in a
tissue, part or cells of the same. The absence of a spontaneously
induced cell death in combination with said reduction of the amount
of protein, activity or function of the functional equivalent is
furthermore understood as an essential property.
[0104] In this context, the efficacy of the pathogen resistance can
deviate both downward or upward in comparison with a value obtained
when reducing one of the RacB proteins as shown in SEQ ID NO: 2, 4
or 6. Preferred functional equivalents are those in which the
efficacy of the pathogen resistance--measured, for example, by the
penetration efficacy of a pathogen (formation of
haustoria)--differs by not more than 50%, preferably 25%,
especially preferably 10% from a comparative value obtained by
reducing an RacB protein as shown in NO: 2, 4 or 6. Especially
preferred are those sequences where the reduction increases the
efficacy of pathogen resistance quantitatively by more than 50%,
preferably 100%, especially preferably 500%, very especially
preferably 1000% based on a comparative value obtained by reducing
one of the RacB protein as shown in SEQ ID NO: 2, 4 or 6.
[0105] The comparison is preferably carried out under analogous
conditions. "Analogous conditions" means that all conditions such
as, for example, culture or growing conditions, assay conditions
(such as buffer, temperature, substrates, pathogen concentration
and the like) are kept identical between the experiments to be
compared and that the set-ups differ only by the sequence of the
RacB polypeptides to be compared, their organism of origin and, if
appropriate, the pathogen. When choosing the pathogen, each
comparison requires that the pathogen be chosen which is most
similar to the other equivalent, taking into consideration the
species specificity.
[0106] "Functional equivalents" is understood as meaning, in
particular, natural or artificial mutations of the RacB
polypeptides as shown in SEQ ID NO: 2, 4 or 6 and homologous
polypeptides from other plants which continue to have essentially
the same properties. Homologous polypeptides from the
above-described preferred plants are preferred. The sequences from
other plants (for example Arabidopsis thaliana) which are
homologous to the RacB sequences disclosed within the scope of the
present invention can be found readily for example by database
search or by screening genetic libraries using the RacB sequences
as search sequence or probe.
[0107] Mutations comprise substitutions, additions, deletions,
inversion or insertions of one or more amino acid residues. Thus,
the present invention also comprises those polypeptides which are
obtained by modification of a polypeptide as shown in SEQ ID NO: 2,
4 or 6.
[0108] Homology between two nucleic acid sequences is understood as
meaning the identity of the nucleic acid sequence over in each case
the entire sequence length which is calculated by comparison with
the aid of the program algorithm GAP (Wisconsin Package Version
10.0, University of Wisconsin, Genetics Computer Group (GCG),
Madison, USA; Altschul et al. (1997) Nucleic Acids Res. 25:3389 et
seq.), setting the following parameters:
TABLE-US-00006 Gap weight: 50 Length weight: 3 Average match: 10
Average mismatch: 0
[0109] For example a sequence which has at least 80% homology with
sequence SEQ ID NO: 1 at the nucleic acid level is understood as
meaning a sequence which, upon comparison with the sequence SEQ ID
NO: 1 by the above program algorithm with the above parameter set,
has at least 80% homology.
[0110] Homology between two polypeptides is understood as meaning
the identity of the amino acid sequence over in each case the
entire sequence length which is calculated by comparison with the
aid of the program algorithm GAP (Wisconsin Package Version 10.0,
University of Wisconsin, Genetics Computer Group (GCG), Madison,
USA), setting the following parameters:
TABLE-US-00007 Gap weight: 8 Length weight: 2 Average match: 2,912
Average mismatch: -2,003
[0111] For example a sequence which has at least 80% homology with
sequence SEQ ID NO: 2 at the protein level is understood as meaning
a sequence which, upon comparison with the sequence SEQ ID NO: 2 by
the above program algorithm with the above parameter set, has at
least 80% homology.
[0112] Functional equivalents derived from one of the polypeptide
as shown in SEQ ID NO: 2, 4 or 6 according to the invention by
substitution, insertion or deletion have at least 60%, preferably
at least 80%, by preference at least 90%, especially preferably at
least 95%, very especially preferably at least 98%, homology with
one of the polypeptide as shown in SEQ ID NO: 2, 4 or 6 according
to the invention and are distinguished by essentially the same
properties as the polypeptide as shown in SEQ ID NO: 2, 4 or 6.
[0113] Functional equivalents derived from the nucleic acid
sequence as shown in SEQ ID NO: 1, 3 or 5 according to the
invention by substitution, insertion or deletion have at least 60%,
preferably at least 80%, by preference at least 90%, especially
preferably at least 95%, very especially preferably at least 98%,
homology with one of the polypeptides as shown in SEQ ID NO: 1, 3
or 5 according to the invention and encode polypeptides having
essentially the same properties as the polypeptide as shown in SEQ
ID NO: 2, 4 or 6.
[0114] The RacB proteins comprised as functional equivalents
preferably have at least one of the following sequence motifs:
[0115] a) A G1 element GXXXXGKS/T preferably in the N-terminal
region, very especially preferably an element with the sequence
GDGAVGKT, most preferably an element with the sequence
KCVTVGDGAVGKTC.
[0116] b) A G2 effector region comprising a sequence motif with
PTVFDN, especially preferably NTFPTDYVPTVFDNFSANVV.
[0117] c) A G3 element comprising LWDTAGQ, especially preferably
NLGLWDTAGQEDYN.
[0118] d) A G4 element TKXD, especially preferably TKLD, very
especially preferably LVGTKLDLRDDKQ.
[0119] e) A G5 element EXS, preferably ECSS, very especially
preferably ECSSKTG.
[0120] f) A C-terminal isoprenylation motif (CXXX, Hassanain U H et
al. (2000) Biochem Biophys Res Commun. 272(3):783-8.), especially
preferably CSIL.
[0121] Especially preferably, at least 2 or 3 of these motifs (a to
f) occur in a functionally equivalent RacB protein, very especially
preferably at least 4 or 5 of these motifs, most preferably all
motifs a to f. Further sequence motifs which are typical for RacB,
in particular also motifs which constitute a delimitation against
Rac1 proteins, can be deduced readily by the skilled worker from
the sequence alignment of the known RacB (or Rac1) proteins, as
shown in FIG. 1.
[0122] Examples of the functional equivalents to the RacB proteins
as shown in SEQ ID NO: 2, 4 or 6, which equivalents are to be
reduced in the method according to the invention, can be identified
for example from a variety of organisms whose genomic sequence is
known, such as, for example, from Arabidopsis thaliana, Brassica
napus, Nicotiana tabacum, Solanum tuberosum, or Helianthinum from
databases of homology comparisons.
[0123] The screening of cDNA libraries or genomic libraries of
other organisms, preferably of the plant species which are
mentioned further below as hosts for the transformation, using the
nucleic acid sequences described under SEQ ID NO: 1, 3 or 5 or
parts of these as probe is also a method of identifying homologs in
other species with which the skilled worker is familiar. In this
context, the probes derived from the nucleic acid sequences as
shown in SEQ ID NO: 1, 3 or 5 have a length of at least 20 bp,
preferably 50 bp, particularly preferably 100 bp, very especially
preferably 200 bp, and most preferably 400 bp. A DNA strand which
is complementary to the sequences described under SEQ ID NO: 1, 3
or 5 may also be employed for screening the libraries.
[0124] Functional equivalents, accordingly, comprise DNA sequences
which hybridize under standard conditions with the RacB nucleic
acid sequence described by SEQ ID NO: 1, 3 or 5, with the sequence
complementary thereto or parts of the abovementioned and which, as
complete sequences, encode proteins which have the same properties
as the proteins described under SEQ ID NO: 2, 4 or 6.
[0125] "Standard hybridization conditions" is to be understood in
the broad sense and means stringent or else less stringent
hybridization conditions. Such hybridization conditions are
described, inter alia, by Sambrook J, Fritsch E F, Maniatis T et
al., in Molecular Cloning (A Laboratory Manual), 2nd Edition, Cold
Spring Harbor Laboratory Press, 1989, pages 9.31-9.57) or in
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
(1989), 6.3.1-6.3.6.
[0126] For example, the conditions during the wash step can be
selected from the range of conditions delimited by low-stringency
conditions (approximately 2.times.SSC at 50.degree. C.) and
high-stringency conditions (approximately 0.2.times.SSC at
50.degree. C., preferably at 65.degree. C.): (20.times.SSC: 0.3M
sodium citrate, 3 M NaCl, pH 7.0). In addition, the temperature
during the wash step can be raised from low-stringency conditions
at room temperature, approximately 22.degree. C., to
higher-stringency conditions at approximately 65.degree. C. Both of
the parameters salt concentration and temperature can be varied
simultaneously, or else one of the two parameters can be kept
constant while only the other is varied. Denaturants, for example
formamide or SDS, may also be employed during the
hybridization.
[0127] In the presence of 50% formamide, hybridization is
preferably effected at 42.degree. C. Some examples of conditions
for hybridization and wash step are shown hereinbelow:
[0128] (1) Hybridization conditions be selected, for example, from
the following conditions:
[0129] a) 4.times.SSC at 65.degree. C.,
[0130] b) 6.times.SSC at 45.degree. C.,
[0131] c) 6.times.SSC, 100 .mu.g/ml denatured fragmented fish sperm
DNA at 68.degree. C.,
[0132] d) 6.times.SSC, 0.5% SDS, 100 .mu.g/ml denatured salmon
sperm DNA at 68.degree. C.,
[0133] e) 6.times.SSC, 0.5% SDS, 100 .mu.g/ml denatured fragmented
salmon sperm DNA, 50% formamide at 42.degree. C.,
[0134] f) 50% formamide, 4.times.SSC at 42.degree. C., or
[0135] g) 50% (vol/vol) formamide, 0.1% bovine serum albumin, 0.1%
Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer pH
6.5, 750 mM NaCl, 75 mM sodium citrates at 42.degree. C., or
[0136] h) 2.times. or 4.times.SSC at 50.degree. C. (low-stringency
condition),
[0137] i) 30 to 40% formamide, 2.times. or 4.times.SSC at
42.degree. C. (low-stringency condition).
[0138] (2) Wash steps can be selected, for example, from the
following conditions:
[0139] a) 0.015 M NaCl/0.0015 M sodium citratc/0.1% SDS at
50.degree. C.
[0140] b) 0.1.times.SSC at 65.degree. C.
[0141] c) 0.1.times.SSC, 0.5% SDS at 68.degree. C.
[0142] d) 0.1.times.SSC, 0.5% SDS, 50% formamide, at 42.degree.
C.
[0143] e) 0.2.times.SSC, 0.1% SDS at 42.degree. C.
[0144] f) 2.times.SSC at 65.degree. C. (low-stringency
condition).
[0145] Functional equivalents derived from a polypeptide as shown
in SEQ ID NO: 2, 4 or 6 comprises in particular also the proteins
having the SEQ ID NO: 35, 37, 39, 41, 43, 45, 47, 50, 52, 54, 56,
58, 60, 62, 64, 66, 68 or 70. "Functional equivalents" is to be
understood as meaning, in particular, proteins encoded by a nucleic
acid sequence having the SEQ ID NO: 34, 36, 38, 40, 42, 44, 46, 48,
49, 51, 53, 55, 57, 61, 63, 65, 67 or 69.
[0146] The reduction of the expression of an RacB protein, the RacB
activity or the RacB function can be realized in many ways.
[0147] "Reduction" or "reducing" in connection with an RacB
protein, an RacB activity or an RacB function is to be interpreted
in the wide sense and comprises the partial or essentially complete
inhibition or blocking of the functionality of an RacB protein in a
plant or a part, tissue, organ, cells or seeds thereof, which
inhibition or blocking is based on a variety of cytobiological
mechanisms.
[0148] For the purposes of the invention, a reduction also
comprises a quantitative reduction of an RacB protein down to the
essential complete absence of the RacB protein (i.e. lacking
detectability of RacB activity or RacB function, or lacking
immunological detectability of the RacB protein). In this context,
the expression of a particular RacB protein, or the RacB activity
or RacB function, in a cell or an organism is preferably reduced by
more than 50%, especially preferably by more than 80%, very
especially preferably by more than 90%.
[0149] A variety of strategies for reducing the expression of an
RacB protein, the RacB activity or RacB function are comprised in
accordance with the invention. The skilled worker is aware of a
series of different methods being available for influencing the
expression of an RacB protein, the RacB activity or the RacB
function in the desired manner.
[0150] A reduction of the RacB activity or the RacB function is
preferably achieved by reduced expression of an endogenous RacB
protein.
[0151] A reduction of the amount of RacB protein, the RacB activity
or the RacB function can be effected using the following
methods:
[0152] a) Introduction of a double-stranded RacB RNA nucleic acid
sequence (RacB dsRNA) or an expression cassette(s) ensuring the
expression thereof.
[0153] b) Introduction of an RacB antisense nucleic acid sequences
or an expression cassette ensuring expression thereof comprised are
those methods in which the antisense nucleic acid sequence is
directed against an RacB gene (i.e. genomic DNA sequences) or an
RacB gene transcript (i.e. RNA sequences). .alpha.-Anomeric nucleic
acid sequences are also comprised
[0154] c) Introduction of an RacB antisense nucleic acid sequences
in combination with a ribozyme or an expression cassette ensuring
expression thereof.
[0155] d) Introduction of RacB sense nucleic acid sequences for
inducing cosuppression or an expression cassette ensuring
expression thereof.
[0156] e) Introduction of a nucleic acid sequence encoding
dominant-negative RacB protein or an expression cassette ensuring
expression thereof.
[0157] f) Introduction of DNA- or protein-binding factors against
RacB genes, RacB RNAs or RacB proteins or an expression cassette
ensuring expression thereof.
[0158] g) Introduction of viral nucleic acid sequences and
expression constructs causing RacB RNA degradation or an expression
cassette ensuring expression thereof.
[0159] h) Introduction of constructs for inducing a homologous
recombination on endogenous RacB genes, for example for generating
knock-out mutants
[0160] i) Introduction of mutations into endogenous RacB genes for
generating a loss of function (for example generation of stop
codons, reading-frame shifts and the like).
[0161] In this context, each and every of these methods may bring
about a reduction of the RacB expression, RacB activity or RacB
function for the purposes of the invention. A combined use is also
feasible. Further methods are known to the skilled worker and can
comprise the hindering or prevention of RacB protein processing, of
the RacB protein or RacB mRNA transport, inhibition of ribosome
attachment, inhibition of RNA splicing, induction of an
RacB-RNA-degrading enzyme and/or inhibition of translational
elongation or termination.
[0162] The individual processes which are preferred may be
described in greater detail hereinbelow:
[0163] a) Introduction of a Double-Stranded RacB RNA Nucleic Acid
Sequence (RacB dsRNA)
[0164] The method of regulating genes by means of double-stranded
RNA ("double-stranded RNA interference"; dsRNAi) has been described
repeatedly for animal and plant organisms (for example Matzke M A
et al. (2000) Plant Mol Biol 43:401-415; Fire A. et al (1998)
Nature 391:806-811; WO 99/32619; WO 99/53050; WO 00/68374; WO
00/44914; WO 00/44895; WO 00/49035; WO 00/63364). Express reference
is made to the processes and methods described in the above
references. Effective gene suppression can also be demonstrated
upon transient expression or following transient transformation for
example as the consequence of biolistic transformation (Schweizer P
et al. (2000) Plant J 2000 24: 895-903). dsRNAi methods are based
on the phenomenon that the simultaneous introduction of
complementary strand and counterstrand of a gene transcript causes
the expression of the gene in question to be suppressed in a highly
efficient manner. The phenotype caused greatly resembles a
corresponding knock-out mutant (Waterhouse P M et al. (1998) Proc
Natl Acad Sci USA 95:13959-64).
[0165] The dsRNAi method has proved to be particularly effective
and advantageous for reducing the RacB expression. As described,
inter alia, in WO 99/32619, dsRNAi approaches are markedly superior
to traditional antisense approaches.
[0166] The invention therefore furthermore relates to
double-stranded RNA molecules (dsRNA molecules) which, upon
introduction into a plant (or a cell, tissue, organ or seed derived
therefrom), bring about the reduction of an RacB.
[0167] In the double-stranded RNA molecule for reducing the
expression of an RacB protein,
[0168] a) one of the two RNA strands is essentially identical to at
least a portion of an RacB nucleic acid sequence, and
[0169] b) the corresponding other RNA strand is essentially
identical to at least a portion of the complementary strand of an
RacB nucleic acid sequence.
[0170] In a further preferred embodiment, the double-stranded RNA
molecule for reducing the expression of an RacB protein
comprises
[0171] a) a sense RNA strand comprising at least one ribonucleotide
sequence which is essentially identical to at least part of the
sense RNA transcript of a nucleic acid sequence encoding an RacB
protein, and
[0172] b) an antisense RNA strand which is essentially--preferably
completely--complementary to the RNA sense strand under a).
[0173] With respect to the double-stranded RNA molecules, RacB
nucleic acid sequence is to be understood as meaning, preferably, a
sequence as shown in SEQ ID NO: 1, 3 or 5 or a functional
equivalent thereof as shown in SEQ ID NO: 34, 36, 38, 40, 42, 44,
46, 48, 49, 51, 53, 55, 57, 61, 63, 65, 67 or 69.
[0174] "Essentially identical" means that the dsRNA sequence can
also show insertions, deletions or individual point mutations
compared with the RacB target sequence or a functionally equivalent
target sequence while still bringing about an effective reduction
of the expression. The homology in accordance with the above
definition preferably amounts to at least 75%, preferably at least
80%, very especially preferably at least 90%, most preferably 100%,
between the sense strand of an inhibitory dsRNA and at least part
of the sense RNA transcript of a nucleic acid sequence encoding an
RacB protein or a functional equivalent thereof (or between the
antisense strand of the complementary strand of a nucleic acid
sequence encoding an RacB protein or a functional equivalent
thereof).
[0175] The length of the part-segment amounts to at least 10 bases,
preferably at least 25 bases, especially preferably at least 50
bases, very especially preferably at least 100 bases, most
preferably at least 200 bases or at least 300 bases.
[0176] As an alternative, an "essentially identical" dsRNA can also
be defined as a nucleic acid sequence which is capable of
hybridizing with part of a storage protein gene transcript (for
example in 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50.degree.
C. or 70.degree. C. for 12 to 16 h).
[0177] "Essentially complementary" is to be understood as meaning
that the antisense RNA strand may also contain insertions,
deletions and individual point mutations, compared to the
complement of the sense RNA strand. Preferably, the homology
between the antisense RNA strand and the complement of the sense
RNA strand is at least 80%, preferably at least 90%, very
particularly preferably at least 95%, most preferably 100%.
[0178] "Part of the sense RNA transcript" of a nucleic acid
sequence encoding an RacB protein or a functional equivalent
thereof is to be understood as meaning fragments of an RNA or mRNA
transcribed from a nucleic acid sequence, preferably an RacB gene,
encoding an RacB protein or a functional equivalent thereof. Here,
the fragments preferably have a sequence length of at least 20
bases, preferably at least 50 bases, particularly preferably at
least 100 bases, very particularly preferably at least 200 bases,
most preferably at least 500 bases. Also comprised is the complete
transcribed RNA or mRNA.
[0179] Also comprised is the use of the dsRNA molecules according
to the invention in the methods according to the invention for
generating a pathogen resistance in plants.
[0180] The dsRNA can be composed of one or more strands of
polymerized ribonucleotides. Modifications both of the
sugar-phosphate backbone and of the nucleosides may be present. For
example, the phosphodiester bonds of the natural RNA can be
modified in such a way that they comprise at least one nitrogen or
sulfur hetero atom. Bases can be modified in such a way that the
activity of, for example, adenosine deaminase is restricted. These
and other modifications are described hereinbelow in the methods of
stabilizing antisense RNA.
[0181] To achieve the same purpose, it is, of course, also possible
to introduce a plurality of individual dsRNA molecules each
comprising one of the ribonucleotide sequence sections defined
above into the cell or the organism.
[0182] The dsRNA can be generated enzymatically or fully or
partially synthesized chemically.
[0183] The double-stranded dsRNA structure can be formed starting
from two complementary, separate RNA strands or, preferably,
strands starting from an individual self-complementary RNA
strand.
[0184] In a single self-complementary strand, sense and antisense
sequence may be linked by a linking sequence ("linker") and can
form for example a hairpin structure. The linking sequence can
preferably be an intron which is spliced out after the dsRNA has
been synthesized.
[0185] The nucleic acid sequence encoding a dsRNA can comprise
further elements such as, for example, transcription termination
signals or polyadenylation signals.
[0186] If the two dsRNA strands are to be combined in a cell or
plant, this can be effected in various ways:
[0187] a) transformation of the cell or plant with a vector
comprising both expression cassettes,
[0188] b) cotransformation of the cell or plant with two vectors,
one of them comprising the expression cassettes with the sense
strand and the other comprising the expression cassettes with the
antisense strand,
[0189] c) hybridizing two plants, each of which has been
transformed with one vector, one of the vectors comprising the
expression cassettes with the sense strand and the other comprising
the expression cassettes with the antisense strand.
[0190] The formation of the RNA duplex can be initiated either
outside or within the cell. Like in WO 99/53050, the dsRNA can also
comprise a hairpin structure by linking sense and antisense strand
by means of a linker (for example an intron). The
self-complementary dsRNA structures are preferred since they only
require the expression of one construct and always comprise the
complementary strands in an equimolar ratio.
[0191] The expression cassettes encoding the antisense or sense
strand of a dsRNA or the self-complementary strand of the dsRNA are
preferably inserted into a vector and, using the methods described
hereinbelow, stably inserted into the genome of a plant in order to
ensure permanent expression of the dsRNA, using selection markers
for example.
[0192] The dsRNA can be introduced using a quantity which allows at
least one copy per cell, Greater quantities (for example at least
5, 10, 100, 500 or 1000 copies per cell) may bring about a more
effective reduction.
[0193] As already described, 100% sequence identity between dsRNA
and an RacB gene transcript or the gene transcript of a
functionally equivalent gene is not necessarily required in order
to bring about an effective reduction of the RacB expression.
Accordingly, there is the advantage that the method is tolerant
with regard to sequence deviations as may exist as the consequence
of genetic mutations, polymorphisms or evolutionary divergence.
Thus, for example, it is possible to use the dsRNA generated on the
basis of the RacB sequence of one organism to suppress the RacB
expression in another organism. The high sequence homology between
the RacB sequences from rice, maize and barley allows the
conclusion that this protein is conserved to a high degree within
plants, so that the expression of a dsRNA derived from one of the
disclosed RacB sequences as shown in SEQ ID NO: 1, 3 or 5 appears
to have an advantageous effect in other plant species as well.
[0194] Furthermore, owing to the high homology between the
individual RacB proteins and their functional equivalents, it is
possible using a single dsRNA generated from a certain RacB
sequence of an organism to suppress the expression of further
homologous RacB proteins and/or their functional equivalents of the
same organism or else the expression of RacB proteins in other
related species. For this purpose, the dsRNA preferably comprises
sequence regions of RacB gene transcripts which correspond to
conserved regions. Said conserved regions can easily be found by
comparing sequences.
[0195] The dsRNA can be synthesized either in vivo or in vitro. To
this end, a DNA sequence encoding a dsRNA can be brought into an
expression cassette under the control of at least one genetic
control element (such as, for example, promoter, enhancer,
silencer, splice donor or splice acceptor or polyadenylation
signal). Suitable advantageous constructions are described
hereinbelow. Polyadenylation is not required, nor do elements for
initiating translation have to be present.
[0196] A dsRNA can be synthesized chemically or enzymatically.
[0197] Cellular RNA polymerases or bacteriophage RNA polymerases
(such as, for example, T3, T7 or SP6 RNA polymerase) can be used
for this purpose. Suitable methods for expression of RNA in vitro
are described (WO 97/32016; U.S. Pat. No. 5,593,874; U.S. Pat. No.
5,698,425, U.S. Pat. No. 5,712,135, U.S. Pat. No. 5,789,214, U.S.
Pat. No. 5,804,693). A dsRNA which has been synthesized in vitro
chemically or enzymatically can be isolated completely or to some
degree from the reaction mixture, for example by extraction,
precipitation, electrophoresis, chromatography or combinations of
these methods, before being introduced into a cell, tissue or
organism. The dsRNA can be introduced directly into the cell or
else be applied extracellularly (for example into the interstitial
space).
[0198] However, it is preferred to transform the plant stably with
an expression construct which brings about the expression of the
dsRNA. Suitable methods are described hereinbelow.
[0199] b) Introduction of an RacB Antisense Nucleic Acid
Sequence
[0200] Methods for suppressing a specific protein by preventing its
mRNA from accumulating by means of antisense technology have been
described in many instances, including in the case of plants
(Sheehy et al. (1988) Proc Natl Acad Sci USA 85: 8805-8809; U.S.
Pat. No. 4,801,340; Mol J N et al. (1990) FEDS Lett
268(2):427-430). The antisense nucleic acid molecule hybridizes, or
binds, with the cellular mRNA and/or genomic DNA encoding the RacB
target protein to be suppressed. This suppresses the transcription
and/or translation of the target protein. Hybridization can
originate conventionally by the formation of a stable duplex or--in
the case of genomic DNA--by the antisense nucleic acid molecule
binding to the duplex of the genomic DNA by specific interaction in
the major groove of the DNA helix.
[0201] An antisense nucleic acid sequence suitable for reducing an
RacB protein can be deduced using the nucleic acid sequence
encoding this protein, for example the nucleic acid sequence as
shown in SEQ ID NO: 1, 3 or 5, or the nucleic acid sequence
encoding a functional equivalent thereof, for example a sequence as
shown in SEQ ID NO: 34, 36, 38, 40, 42, 44, 46, 48, 49, 51, 53, 55,
57, 61, 63, 65, 67 or 69, following Watson and Crick's base pairing
rules. The antisense nucleic acid sequence can be complementary to
all of the transcribed mRNA of said protein, be limited to the
coding region, or else only be composed of a nucleotide, which is
partially complementary to the coding or noncoding sequence of the
mRNA. Thus, for example, the oligonucleotide can be complementary
to the region comprising the translation start for said protein.
Antisense nucleic acid sequences can be, for example, 5, 10, 15,
20, 25, 30, 35, 40, 45 or 50 nucleotides in length, but may also be
longer and comprise at least 100, 200, 500, 1000, 2000 or 5000
nucleotides. Antisense nucleic acid sequences can be expressed
recombinantly or synthesized chemically or enzymatically using
methods known to the skilled worker. In the case of chemical
synthesis, natural or modified nucleotides may be used. Modified
nucleotides can impart an increased biochemical stability to the
antisense nucleic acid sequence and lead to an increased physical
stability of the duplex formed of antisense nucleic acid sequence
and sense target sequence. The following can be used: for example
phosphorothioate derivatives and acridine-substituted nucleotides
such as 5-fluorouracil., 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthin, xanthin, 4-acetylcytosine,
5-(carboxyhydroxylmethyl)uracil,
5-carboxymethylaminomethyl-2-thiouridine-,
5-carboxymethylaminomethyluracil, dihydrouracil,
.beta.-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
.beta.-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine,
uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid,
5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil and
2,6-diaminopurine.
[0202] In a further preferred embodiment, the expression of an RacB
protein can be inhibited by nucleotide sequences which are
complementary to the regulatory region of an RacB gene (for example
an RacB promoter and/or enhancer) and which form triple-helical
structures with that DNA double helix so that the transcription of
the RacB gene is reduced. Such methods have been described (Helene
C (1991) Anticancer Drug Res 6(6):569-84; Helene C et al. (1992)
Ann NY Acad Sci 660:27-36; Maher L J (1992) Bioassays
14(12):807-815). In a further embodiment, the antisense nucleic
acid molecule can be an .alpha.-anomeric nucleic acid. Such
.alpha.-anomeric nucleic acid molecules form specific
double-stranded hybrids with complementary RNA in which--as opposed
to the conventional .beta.-nucleic acids--the two strands run
parallel to one another (Gautier C et al. (1987) Nucleic Acids Res
15:6625-6641). The antisense nucleic acid molecule can furthermore
also comprise 2'-O-methylribonucleotides (Inoue et al. (1987)
Nucleic Acids Res 15:6131-6148) or chimeric RNA/DNA analogs (Inoue
et al. (1987) FEDS Lett 215:327-330).
[0203] c) Introduction of an RacB Antisense Nucleic Acid Sequence
in Combination with a Ribozyme
[0204] The above-described antisense strategy can be combined
advantageously with a ribozyme method. Catalytic RNA molecules or
ribozymes can be adapted to suit any target RNA and cleave the
phosphodiester backbone at specific, positions, functionally
deactivating the target RNA (Tanner N K (1999) FEMS Microbiol Rev
23(3):257-275). The ribozyme itself is not modified thereby, but is
capable of cleaving further target RNA molecules analogously,
thereby assuming the qualities of an enzyme. The incorporation of
ribozyme sequences into antisense RNAs confers this enzyme-like
RNA-cleaving quality to precisely these antisense RNAs, thus
increasing their efficacy in inactivating the target RNA. The
generation and the use of such ribozyme antisense RNA molecules is
described, for example, in Haseloff et al. (1988) Nature 334:
585-591.
[0205] In this manner, ribozymes (for example "hammerhead"
ribozymes; Haselhoff and Gerlach (1988) Nature 334:585-591) can be
used catalytically to cleave the mRNA of an enzyme to be
suppressed, for example RacB, and to prevent translation. The
ribozyme technique can increase the efficacy of an antisense
strategy. Methods of expressing ribozymes for reducing specific
proteins are described in (EP 0 291 533, EP 0 321 201, BP 0 360
257). The expression of ribozyme in plant cells has also been
described (Steinecke P et al. (1992) EMBO J. 11(4):1525-1530, due
Feyte R et al. (1996) Mol Gen Genet. 250(3):329-338). Suitable
target sequences and ribozymes can be determined as described for
example by "Steinecke P, Ribozymes, Methods in Cell Biology 50,
Galbraith et al. eds, Academic Press, Inc. (1995), pp. 449-460" by
calculating the secondary structure of ribozyme RNA and target RNA
as well as by their interaction (Bayley C C et al. (1992) Plant
Mol. Biol. 18(2):353-361; Lloyd A M and Davis R W et al. (1994) Mol
Gen Genet. 242(6):653-657). For example, derivatives of the
Tetrahymena L-19 IVS RNA with regions which are complementary to
the mRNA of the RacB protein to be suppressed can be constructed
(see also U.S. Pat. No. 4,987,071 and U.S. Pat. No. 5,116,742). As
an alternative, such ribozymes can also be identified from a
library of diverse ribozymes via a selection process (Bartel D and
Szostak J W (1993) Science 261:1411-1418).
[0206] d) Introduction of an RacB Sense Nucleic Acid Sequence for
Inducing Cosuppression
[0207] The expression of an RacB nucleic acid sequence in sense
orientation can lead to cosuppression of the corresponding
homologous endogenous gene. The expression of sense RNA with
homology with an endogenous gene can reduce or switch off the
expression of the former, similarly to what has been described for
antisense approaches (Jorgensen et al. (1996) Plant Mol Biol
31(5):957-973; Goring et al. (1991) Proc Natl Acad Sci USA
88:1770-1774; Smith et al. (1990) Mol Gen Genet 224:447-481; Napoli
et al. (1990) Plant Cell 2:279-289; Van der Krol et al. (1990)
Plant Cell 2:291-99). In this context, the homologous gene to be
reduced can be represented either fully or only in part by the
construct introduced. The possibility of translation is not
required. The application of this technique to plants is described,
for example, by Napoli et al. (1990) The Plant Cell 2: 279-289 and
in U.S. Pat. No. 5,034,323.
[0208] The cosuppression is preferably realized by using a sequence
essentially identical to at least part of the nucleic acid sequence
encoding an RacB protein or a functional equivalent thereof, for
example the nucleic acid sequence as shown in SEQ ID NO: 1, 3 or 5,
or the nucleic acid sequence encoding a functional equivalent
thereof, for example a sequence as shown in SEQ ID NO: 34, 36, 38,
40, 42, 44, 46, 48, 49, 51, 53, 55, 57, 61, 63, 65, 67 or 69.
[0209] e) Introduction of Nucleic Acid Sequences Encoding a
Dominant-Negative RacB Protein
[0210] The function or activity of an RacB protein can also be
brought about efficiently by expressing a dominant-negative variant
of this RacB protein. Methods of reducing the function or activity
of a protein by coexpressing its dominant-negative form are known
to the skilled worker (Lagna G and Hemmati-Brivanlou A (1998)
Current Topics in Developmental Biology 36:75-98; Perlmutter R M
and Alberola-Ila J (1996) Current Opinion in Immunology
8(2):285-90; Sheppard D (1994) American Journal of Respiratory Cell
& Molecular Biology. 11(1):1-6; Herskowitz I (1987) Nature
329(6136):219-22).
[0211] For example, a dominant-negative RacB variant can be brought
about by modifying the amino acid threonine at position 20 in the
RacB proteins from maize, rice or barley to, preferably, aspartic
acid. The threonine which is preferably to be mutated, or, if
appropriate, also serine (similarly to threonine at position 20 in
RacB from maize, rice or barley) in RacB homologs from other
species can be determined for example by computer-aided comparison
("alignment"). These mutations for achieving a dominant-negative
RacB variant are preferably carried out at the level of the nucleic
acid sequence encoding RacB proteins. A corresponding mutation can
be brought about for example by PCR-mediated in-vitro mutagenesis
using suitable oligonucleotide primers, by which the desired
mutation is introduced. This is done using methods known to the
skilled worker; for example, the "LA PCR in vitro Mutagenesis Kit"
(Takara Shuzo, Kyoto) may be used for this purpose. A method of
generating a dominant-negative variant of the maize RacB protein is
also described in WO 00/15815 (Example 4, p. 69).
[0212] Especially preferred are the dominant-negative variants of
the RacB proteins from barley, rice or maize described under SEQ ID
NO: 7, 8 and 9.
[0213] f) Introduction of DNA--or Protein-Binding Factors Against
RacB Genes, RacB RNAs or RacB Proteins
[0214] RacB gene expression may also be reduced using specific
DNA-binding factors, for example factors of the zinc finger
transcription factor type. These factors attach to the genomic
sequence of the endogenous target gene, preferably in the
regulatory regions, and bring about repression of the endogenous
gene. The use of such a method makes possible the reduction of the
expression of an endogenous RacB gene without it being necessary to
recombinantly manipulate its sequence. Suitable methods for the
preparation of suitable factors have been described (Dreier B et
al. (2001) J Biol Chem 276(31):29466-78; Dreier B et al. (2000) J
Mol Biol 303(4):489-502; Beerli R R et al. (2000) Proc Natl Acad
Sci USA 97 (4):1495-1500; Beerli R R et al. (2000) J Biol Chem
275(42):32617-32627; Segal D J and Barbas C F 3rd. (2000) Curr Opin
Chem Biol 4(1):34-39; Kang J S and Kim J S (2000) J Biol Chem
275(12):8742-8748; Beerli R R et al. (1998) Proc Natl Acad Sci USA
95(25):14628-14633; Kim J S et al. (1997) Proc Natl Acad Sci USA
94(8):3616-3620; Klug A (1999) J Mol Biol 293(2):215-218; Tsai S Y
et al. (1998) Adv Drug Deliv Rev 30(1-3):23-31; Mapp A K et al.
(2000) Proc Natl Acad Sci USA 97(8):3930-3935; Sharrocks A D et
al., (1997) Int J Biochem Cell Biol 29(12):1371-1387; Zhang L et
al. (2000) J Biol Chem 275(43):33850-33860).
[0215] These factors can be selected using any desired portion of
an RacB gene. This segment is preferably located in the promoter
region. For gene suppression, however, it may also be in the region
of the coding exons or introns. The segments in question can be
obtained by the skilled worker from Genbank by database search or,
starting from an RacB cDNA whose gene is not present in Genbank, by
screening a genomic library for corresponding genomic clones. The
skilled worker is familiar with the methods required therefor.
[0216] Furthermore, it is possible to introduce, into cells,
factors which inhibit the RacB target protein itself. The
protein-binding factors can be, for example, aptamers (Famulok M
and Mayer G (1999) Curr Top Microbiol Immunol 243:123-36) or
antibodies or antibody fragments or single-chain antibodies.
Methods for obtaining these factors have been described and are
known to the skilled worker. For example, a cytoplasmic scFv
antibody was employed to modulate the activity of the phytochrome A
protein in genetically modified tobacco plants (Owen M et al.
(1992) Biotechnology (N Y) 10(7):790-794; Franken F et al. (1997)
Curr Opin Biotechnol 8(4):411-416; Whitelam (1996) Trend Plant Sci
1:286-272).
[0217] Gene expression may also be suppressed by tailor-made
low-molecular-weight synthetic compounds, for example of the
polyamide type (Dervan P B and Burli R W (1999) Current Opinion in
Chemical Biology 3:688-693; Gottesfeld J M et al. (2000) Gene Expr
9(1-2):77-91). These oligormers are composed of the units
3-(dimethylamino)propylamine, N-methyl-3-hydroxypyrrole,
N-methylimidazole and N-methylpyrrole and can be adapted to any
piece of double-stranded DNA in such a way that they bind into the
major groove in a sequence-specific manner and block the expression
of these gene sequences. Suitable methods have been described (see,
inter alia, Bremer R E et al. (2001) Bioorg Med Chem.
9(8):2093-103; Ansari A Z et al. (2001) Chem Biol. 8(6):583-92;
Gottesfeld J M et al. (2001) J. Mol. Biol. 309(3):615-29; Wurtz N R
et al. (2001) Org Lett 3(8):1201-3; Wang C C et al. (2001) Bioorg
Med Chem 9(3):653-7; Urbach A R and Dervan P B (2001) Proc Natl
Acad Sci USA 98(8):4343-8; Chiang S Y et al. (2000) J. Biol. Chem.
275(32):24246-54).
[0218] g) Introduction of Viral Nucleic Acid Sequences and
Expression Constructs which Cause RacB RNA Degradation
[0219] RacB expression can also be brought about efficiently by
inducing the specific RacB RNA degradation by the plant with the
aid of a viral expression system (amplicon) (Angell, S M et al.
(1999) Plant J. 20(3):357-362). These systems--also termed "VIGS"
(viral induced gene silencing)--introduce, into the plant, nucleic
acid sequences with homology to the transcripts to be suppressed,
with the aid of viral vectors. Then, transcription is switched off,
probably mediated by plant defense mechanisms against viruses.
Suitable techniques and methods have been described (Ratcliff F et
al. (2001) Plant J 25(2):237-45; Fagard M and Vaucheret H (2000)
Plant Mol Biol 43(2-3):285-93; Anandalakshmi R et al. (1998) Proc
Natl Acad Sci USA 95(22):13079-84; Ruiz M T (1998) Plant Cell
10(6): 937-46).
[0220] h) Introduction of Constructs for Inducing a Homologous
Recombination on Endogenous RacB Genes, for Example for Generating
Knock-Out Mutants
[0221] An example of what is used for generating a homologously
recombinant organism with reduced RacB activity is a nucleic acid
construct comprising at least part of an endogenous RacB gene which
is modified by a deletion, addition or substitution of at least one
nucleotide in such a way that its functionality is reduced or fully
destroyed. The modification may also relate to the regulatory
elements (for example the promoter) of the gene, so that the coding
sequence remains unmodified, but expression (transcription and/or
translation) does not take place and is reduced.
[0222] In the case of conventional homologous recombination, the
modified region is flanked at its 5' and 3' end by further nucleic
acid sequences which must be sufficient in length for making
possible recombination. They are, as a rule, in the range of
several hundred bases to several kilobases in length (Thomas K R
and Capecchi M R (1987). Cell 51:503; Strepp et al. (1998) Proc
Natl Acad Sci USA 95(8):4368-4373). For homologous recombination,
the host organism--for example a plant--is transformed with the
recombination construct using the methods described hereinbelow,
and clones which have successfully undergone recombination are
selected, for example using a resistance to antibiotics or
herbicides.
[0223] Homologous recombination is a relatively rare event in
higher eukaryotes, especially in plants. Random integrations into
the host genome predominate. One possibility of eliminating the
randomly integrated sequences and thus increasing the number of
cell clones with a correct homologous recombination is the use of a
sequence-specific recombination system as described in U.S. Pat.
No. 6,110,736, by which unspecifically integrated sequences can be
deleted again, which simplifies the selection of events which have
integrated successfully via homologous recombination. A large
number of sequence-specific recombination systems can be used,
examples being the Cre/lox system of bacteriophage P1, the FLP/FRT
system of yeast, the Gin recombinase of phage Mu, the Pin
recombinase from E. coli, and the R/RS system of the pSR1 plasmid.
The bacteriophage P1 Cre/10.times. and the yeast FLP/FRT system are
preferred. The FLP/FRT and cre/lox recombinase system has already
been applied to plant systems (Odell et al. (1990) Mol Gen Genet
223: 369-378).
[0224] i) Introduction of Mutations into Endogenous RacB Genes for
Generating a Loss of Function (For Example Generation of Stop
Codons, Reading-Frame Shifts and the Like)
[0225] Further suitable methods for reducing the RacB activity are
the introduction of nonsense mutations into endogenous RacB genes,
for example by introducing RNA/DNA oligonucleotides into the plant
(Zhu et al. (2000) Nat Biotechnol 18(5):555-558) and the generation
of knock-out mutants with the aid of, for example, T-DNA
mutagenesis (Koncz et al. (1992) Plant Mol Biol 20(5):963-976), ENU
(N-ethyl-N-nitrosourea) mutagenesis or homologous recombination
(Hohn B and Puchta (1999) H Proc Natl Acad Sci USA 96:8321-8323.).
Point mutations can also be generated by means of DNA-RNA hybrids
also known under the name "chimeraplasty" (Cole-Strauss-et al.
(1999) Nucl Acids Res. 27(5):1323-1330; Kmiec (1999) Gene therapy
American Scientist 87(3):240-247).
[0226] The methods of dsRNAi, cosuppression by means of sense RNA
and "VIGS" ("virus induced gene silencing") are also termed
"post-transcriptional gene silencing" (PTGS). PTGS methods, like
the reduction of the RacB function or activity with
dominant-negative RacB variants, are especially advantageous
because the demands regarding the homology between the endogenous
gene to be suppressed and the sense or dsRNA nucleic acid sequence
expressed recombinantly (or between the endogenous gene and its
dominant-negative variant) are lower than, for example, in the case
of a traditional antisense approach. Such criteria with regard to
homology are mentioned in the description of the dsRNAI method and
can generally be applied to PTGS methods or dominant-negative
approaches. Owing to the high degree of homology between the RacB
proteins from maize, rice and barley, a high degree of conservation
of this protein in plants can be assumed. Thus, using the RacB
nucleic acid sequences from barley, maize or rice, it is presumably
also possible efficiently to suppress the expression of homologous
RacB proteins in other species without the isolation and structure
elucidation of the RacB homologs occurring therein being required.
Considerably less labor is therefore required. Similarly, the use
of dominant-negative variants of an RacB protein from rice, maize
or barley can presumably also efficiently reduce or suppress the
function/activity of its homolog in other plant species.
[0227] All of the substances and compounds which directly or
indirectly bring about a reduction in protein quantity, RNA
quantity, gene activity or protein activity of an RacB protein
shall subsequently be combined in the term "anti-RacB" compounds.
The term "anti-RacB" compound explicitly includes the nucleic acid
sequences, peptides, proteins or other factors employed in the
above-described methods.
[0228] For the purposes of the invention, "introduction" comprises
all of the methods which are capable of directly or indirectly
introducing an "anti-RacB" compound into a plant or a cell,
compartment, tissue, organ or seed thereof, or of generating such a
compound there. Direct and indirect methods are comprised. The
introduction can lead to a transient presence of an "anti-RacB"
compound (for example a dsRNA) or else to its stable presence.
[0229] Owing to the different nature of the above-described
approaches, the "anti-RacB" compound can exert its function
directly (for example by insertion into an endogenous RacB gene).
However, its function can also be exerted indirectly following
transcription into all RNA (for example in the case of antisense
approaches) or following transcription and translation into a
protein (for example in the case of binding factors). The invention
comprises both directly and indirectly acting "anti-RacB"
compounds.
[0230] The term "introducing" comprises for example methods such as
transfection, transduction or transformation.
[0231] "Anti-RacB" compounds therefore also comprises recombinant
expression constructs which bring about expression (i.e.
transcription and, if appropriate, translation), for example of an
RacB dsRNA or an RacB "antisense" RNA, preferably in a plant or a
part, tissue, organ or seed thereof.
[0232] In said expression constructs, a nucleic acid molecule whose
expression (transcription and, if appropriate, translation)
generates an "anti-RacB" compound is preferably operably linked to
at least one genetic control element (for example a promoter) which
ensures expression in an organism, preferably in plants. If the
expression construct is to be introduced directly into a plant and
the "anti-RacB" compound (for example the RacB dsRNA) is to be
generated therein in plantae, plant-specific genetic control
elements (for example promoters) are preferred. However, the
"anti-RacB" compound may also be generated in other organisms or in
vitro and then be introduced into the plant (as described in
Examples 6 and 7). Preferred in this context are all of the
prokaryotic or eukaryotic genetic control elements (for example
promoters) which permit the expression in the organism chosen in
each case for the preparation.
[0233] Operable linkage is to be understood as meaning, for
example, the sequential arrangement of a promoter with the nucleic
acid sequence to be expressed (for example an "anti-RacB" compound)
and, if appropriate, further regulatory elements such as, for
example, a terminator in such a way that each of the regulatory
elements can fulfil its function when the nucleic acid sequence is
expressed recombinantly, depending on the arrangement of the
nucleic acid sequences in relation to sense or antisense RNA. To
this end, direct linkage in the chemical sense is not necessarily
required. Genetic control sequences such as, for example, enhancer
sequences, can also exert their function on the target sequence
from positions which are further away, or indeed from other DNA
molecules. Preferred arrangements are those in which the nucleic
acid sequence to be expressed recombinantly is positioned behind
the sequence acting as promoter, so that the two sequences are
linked covalently to each other. The distance between the promoter
sequence and the nucleic acid sequence to be expressed
recombinantly is preferably less than 200 base pairs, especially
preferably less than 100 base pairs, very especially preferably
less than 50 base pairs.
[0234] Operable linkage, and an expression cassette, can be
generated by means of customary recombination and cloning
techniques as are described, for example, in Maniatis T, Fritsch E
F and Sambrook J (1989) Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor (NY), in Silhavy
T J, Berman M L and Enquist L W (1984) Experiments with Gene
Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y.),
in Ausubel F M et al. (1987) Current Protocols in Molecular
Biology, Greene Publishing Assoc. and Wiley Interscience and in
Gelvin et al. (1990) In: Plant Molecular Biology Manual. However,
further sequences which, for example, act as a linker with specific
cleavage sites for restriction enzymes, or as a signal peptide, may
also be positioned between the two sequences. The insertion of
sequences may also lead to the expression of fusion proteins.
Preferably, the expression cassette, consisting of a linkage of
promoter and nucleic acid sequence to be expressed, can exist in a
vector-integrated form and be inserted into a plant genome, for
example by transformation.
[0235] However, expression cassette also denotes those
constructions in which a promoter is positioned behind an
endogenous RacB gene, for example by means of homologous
recombination, and the reduction according to the invention of an
RacB protein is brought about by the expression of an antisense
RacB RNA.
[0236] Analogously, an "anti-RacB" compound (for example a nucleic
acid sequence encoding an RacB dsRNA or an RacB antisense RNA) can
be positioned behind an endogenous promoter in such a way that the
same effect is manifested. Both approaches lead to inventive
expression cassettes.
[0237] The term plant-specific promoters is understood as meaning,
in principle, any promoter which is capable of governing the
expression of genes, in particular foreign genes, in plants or
plant parts, plant cells, plant tissues or plant cultures. In this
context, expression can be, for example, constitutive, inducible or
development-dependent.
[0238] The following are preferred:
[0239] a) Constitutive Promoters
[0240] Preferred vectors are those which make possible constitutive
expression in plants (Benfey et al. (1989) EMBO J. 8:2195-2202).
"Constitutive" promoter is understood as meaning those promoters
which ensure expression in a large number of, preferably all,
tissues over a substantial period of plant development, preferably
at all stages of plant development. In particular a plant promoter
or a promoter derived from a plant virus are preferably used.
Particularly preferred is the promoter of the CaMV cauliflower
mosaic virus .sup..sup.35S transcript (Franck et al. (1980) Cell
21:285-294; Odell et al. (1985) Nature 313:810-812; Shewmaker et
al. (1985) Virology 140:281-288; Gardner et al. (1986) Plant Mol
Biol 6:221-228) or the 19S CaMV promoter (U.S. Pat. No. 5,352,605;
WO 84/02913; Benfey et al. (1989) EMBO J. 8:2195-2202). Another
suitable constitutive promoter is the "Rubisco small subunit (SSU)"
promoter (U.S. Pat. No. 4,962,028), the leguminB promoter (GenBank
Acc. No. X03677), the Agrobacterium nopaline synthase promoter, the
TR dual promoter, the Agrobacterium OCS (octopine synthase)
promoter, the ubiquitin promoter (Holtorf S et al. (1995) Plant Mol
Biol 29:637-649), the ubiquitin 1 promoter (Christensen et al.
(1992) Plant Mol Biol 18:675-689; Bruce et al. (1989) Proc Natl
Acad Sci USA 86:9692-9696), the Smas promoter, the cinnamyl alcohol
dehydrogenase promoter (U.S. Pat. No. 5,683,439), the promoters of
the vacuolar ATPase subunits or the promoter of a proline-rich
protein from wheat (WO 91/13991), and further promoters of genes
whose constitutive expression in plants is known to the skilled
worker. A particularly preferred constitutive promoter is the
promoter of the nitrilase-1 (nit1) gene from A. thaliana (GenBank
Acc.-No.: Y07648.2, nucleotides 2456-4340, Hillebrand et al. (1996)
Gene 170:197-200).
[0241] b) Tissue-Specific Promoters
[0242] Preferred are furthermore promoters with specificity for the
anthers, ovaries, flowers, leaves, stems, roots and seeds.
[0243] Seed-Specific Promoters
[0244] such as, for example, the phaseolin promoter (U.S. Pat. No.
5,504,200; Bustos M M et al. (1989) Plant Cell 1(9):839-53), the 2S
albumin gene promoter (Joseffson L G et al. (1987) J Biol Chem
262:12196-12201), the legumin promoter (Shirsat A et al. (1989) Mol
Gen Genet 215(2): 326-331), the USP (unknown seed protein) promoter
(Baumlein H et al. (1991) Mol Gen Genet 225(3):459-67), the napin
gene promoter (U.S. Pat. No. 5,608,152; Stalberg K et al. (1996) L
Planta 199:515-519), the sucrose binding protein promoter (WO
00/26388) or the legumin B4 promoter (LeB4; Bumlein H et al. (1991)
Mol Gen Genet 225: 121-128; Baeumlein et al. (1992) Plant Journal
2(2):233-9; Fiedler U et al. (1995) Biotechnology (NY)
13(10):1090f), the Arabidopsis oleosin promoter (WO 98/45461), the
Brassica Bce4 promoter (WO 91/13980). Further suitable
seed-specific promoters are those of the genes encoding the
high-molecular-weight glutenin (HMWG), gliadin, branching enzyme,
ADP glucose pyrophosphatase (AGPase) or starch synthase.
Furthermore preferred are promoters which permit seed-specific
expression in monocots such as maize, barley, wheat, rye, rice and
the like. The following can be employed advantageously: the
promoter of the lpt2 or lpt1 gene (WO 95/15389, WO 95/23230) or the
promoters described in WO 99/16890 (promoters of the hordein gene,
the glutelin gene, the oryzin gene, the prolamin gene, the gliadin
gene, the glutelin gene, the zein gene, the kasirin gene or the
secalin gene).
[0245] Tuber-, storage-root- or root-specific promoters such as,
for example, the patatin promoter class I (B33), the potato
cathepsin D inhibitor promoter.
[0246] Leaf-Specific Promoters
[0247] such as the potato cytosolic FBPase promoter (WO 97/05900),
the Rubisco (ribulose-1,5-bisphosphate carboxylase) SSU (small
subunit) promoter or the ST-LSI promoter from potato (Stockhaus et
al. (1989) EMBO J. 8:2445-2451). Very especially preferred are
epidermis-specific promoters such as, for example, the OXLP gene
(oxalate-oxidase-like protein) promoter (Wei et al. (1998) Plant
Mol. Biol. 36:101-112).
[0248] Flower-Specific Promoters
[0249] [0245] such as, for example, the phytoene synthase promoter
(WO 92/16635) or the promoter of the P-rr gene (WO 98/22593).
[0250] Anther-Specific Promoters
[0251] such as the 5126 promoter (U.S. Pat. No. 5,689,049, U.S.
Pat. No. 5,689,051), the glob-1 promoter and the .gamma.-zein
promoter.
[0252] c) Chemically Inducible Promoters
[0253] The expression cassettes can also comprise a chemically
inducible promoter (review article: Gatz et al. (1997) Annu Rev
Plant Physiol Plant Mol Biol 48:89-108), by which the expression of
the exogenous gene in the plant at a particular point in time can
be controlled. Such promoters such as, for example, the PRP1
promoter (Ward et al. (1993) Plant Mol Biol 22:361-366), a
salicylic-acid-inducible promoter (WO 95/19443), a
benzenesulfonamide-inducible promoter (EP 0 388.186), a
tetracyclin-inducible promoter (Gatz et al. (1992) Plant J
2:397-404)., an abscisic-acid-inducible promoter (EP 0 335 528) or
an ethanol- or cyclohexylnone-inducible promoter (WO 93/21334) can
likewise be used.
[0254] d) Stress- or Pathogen-Inducible Promoters
[0255] Further preferred promoters are those which are induced by
biotic or abiotic stress such as, for example, the
pathogen-inducible promoter of the PRP1 gene (or gst1 promoter)
e.g. from potato (WO 96/28561; Ward et al. (1993) Plant Mol Biol
22:361-366), the tomato high-temperature-inducible hsp70 or hsp80
promoter (U.S. Pat. No. 5,187,267), the potato
low-temperature-inducible alpha-amylase promoter (WO 96/12814), the
light-inducible PPDK promoter, or the wounding-induced pinII
promoter (EP-A 0 375 091).
[0256] Further pathogen-inducible promoters comprise the Fis1
promoter from flax (WO 96/34949), the Vst1 promoter (Schubert et
al., (1997) Plant Mol Biol 34:417-426) and the EAS4 sesquiterpene
cyclase promoter from tobacco (U.S. Pat. No. 6,100,451).
[0257] Pathogen-inducible promoters comprise those of genes which
are induced as a consequence of infection by pathogens, such as,
for example, genes of PR proteins, SAR proteins,
.beta.-1,3-glucanase, chitinase and the like (for example Redolfi
et al. (1983) Neth J Plant Pathol 89:245-254; Uknes, et al. (1992)
The Plant Cell 4:645-656; Van Loon (1985) Plant Mol Viral
4:111-116; Marineau et al. (1987) Plant Mol Biol 9:335-342; Matton
et al. (1987) Molecular Plant-Microbe Interactions 2:325-342;
Somssich et al. (1986) Proc Natl Acad Sci USA 83:2427-2430;
Somssich et al. (1988) Mol Gen Genetics 2:93-98; Chen et al. (1996)
Plant J 10:955-966; Zhang and Sing (1994) Proc Natl Acad Sci USA
91:2507-2511; Warner, et al. (1993) Plant J 3:191-201; Siebertz et
al. (1989) Plant Cell 1:961-968 (1989).
[0258] Also comprised are wounding-inducible promoters such as that
of the pinII gene (Ryan (1990) Ann Rev Phytopath 28:425-449; Duan
et al. (1996) Nat Biotech 14:494-498), of the wun1 and wun2 gene
(U.S. Pat. No. 5,428,148), of the win1 and win2 gene (Stanford et
al. (1989) Mol Gen Genet 215:200-208), of system in (McGurl et al.
(1992) Science 225:1570-1573), of the WIP1 gene (Rohmeier et al.
(1993) Plant Mol Biol 22:783-792; Eckelkamp et al. (1993), FEBS
Letters 323:73-76), of the MPI gene (Corderok et al. (1994) Plant J
6(2):141-150) and the like.
[0259] A source of further pathogen-inducible promoters is the PR
gene family. A number of elements in these promoters have been
found to be advantageous. Thus, the region -364 to -288 in the
promoter of PR-2d provides salicylate specificity (Buchel et al.
(1996) Plant Mol Biol 30, 493-504). The sequence 5'-TCATCTTCTT-3'
is encountered repeatedly in the promoter of barley
.beta..-1,3-glucanase and more than 30 further stress-induced
genes. In tobacco, this region binds a nuclear protein whose
abundance is increased by salicylate. The PR-1 promoters from
tobacco and Arabidopsis (EP-A 0 332 104, WO 98/03536) are likewise
suitable for use as pathogen-inducible promoters. "Acidic
PR-5'-(aPR5)-promoters from barley (Schweizer et al. (1997) Plant
Physiol 114:79-88) and wheat (Rebmann et al. (1991) Plant Mol Biol
16:329-331) are preferred, since they are particularly specifically
pathogen-induced. aPR5 proteins accumulate within about 4 to 6
hours after pathogen attack and have only very limited background
expression (WO 99/66057). One approach to achieve higher
pathogen-induced specificity is the preparation of synthetic
promoters from combinations of known pathogen-responsive elements
(Rushton et al. (2002) Plant Cell 14, 749-762; WO 00/01830; WO
99/66057). Further pathogen-inducible promoters from different
species are known to the person skilled in the art (EP-A 1 165 794;
EP-A 1 062 356; EP-A 1 041 148; EP-A 1 032 684;
[0260] e) Development-Dependent Promoters
[0261] Further suitable promoters are, for example,
fruit-maturation-specific promoters such as, for example, the
tomato fruit-maturation-specific promoter (WO 94/21794, EP 409
625). Development-dependent promoters comprise partly the
tissue-specific promoters, since individual tissues develop by
nature in a development-dependent fashion.
[0262] Especially preferred are constitutive promoters and leaf-
and/or stem-specific, pathogen-inducible and epidermis-specific
promoters, with pathogen-inducible and epidermis-specific promoters
being most preferred.
[0263] Furthermore, further promoters may be linked operably to the
nucleic acid sequence to be expressed, which promoters make
possible the expression in further plant tissues or in other
organisms, such as, for example, E. coli bacteria. Suitable plant
promoters are, in principle, all of the above-described
promoters.
[0264] The nucleic acid sequences present in the expression
cassettes or vectors according to the invention can be linked
operably to further genetic control sequences in addition to a
promoter. The term "genetic control sequences" is to be understood
in the broad sense and refers to all those sequences which have an
effect on the materialization or the function of the expression
cassette according to the invention. For example, genetic control
sequences modify the transcription and translation in prokaryotic
or eukaryotic organisms. Preferably, the expression cassettes
according to the invention comprise the promoter with specificity
for the embryonal epidermis and/or the flower 5'-upstream of the
nucleic acid sequence in question to be expressed recombinantly,
and 3'-downstream a terminator sequence as additional genetic
control sequence and, if appropriate, further customary regulatory
elements, in each case linked operably to the nucleic acid sequence
to be expressed recombinantly.
[0265] Genetic control sequences also comprise further promoters,
promoter elements or minimal promoters, all of which can modify the
expression-governing properties. Thus, for example, the
tissue-specific expression may additionally depend on certain
stressors, owing to genetic control sequences. Such elements have
been described, for example, for water stress, abscisic acid (Lam E
and Chua N H, J Biol Chem 1991; 266(26): 17131-17135) and heat
stress (Schoffl F et al., Molecular & General Genetics
217(2-3):246-53, 1989).
[0266] Further advantageous control sequences are, for example, in
the Gram-positive promoters amy and SPO2, and in the yeast or
fungal promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28,
ADH.
[0267] In principle, all natural promoters with their regulatory
sequences like those mentioned above may be used for the method
according to the invention. In addition, synthetic promoters may
also be used advantageously.
[0268] Genetic control sequences furthermore also comprise the
5'-untranslated regions, introns or noncoding 3'-region of genes,
such as, for example, the actin-1 intron, or the Adh1-S introns 1,
2 and 6 (general reference: The Maize Handbook, Chapter 116,
Freeling and Walbot, Eds., Springer, New York (1994)). It has been
demonstrated that they may play a significant roles in the
regulation of gene expression. Thus, it has been demonstrated that
5'-untranslated sequences can enhance the transient expression of
heterologous genes. Examples of translation enhancers which may be
mentioned are the tobacco mosaic virus 5' leader sequence (Gallie
et al. (1987) Nucl Acids Res 15:8693-8711) and the like.
Furthermore, they may promote tissue specificity (Rouster J et al.
(1998) Plant J 15:435-440).
[0269] The expression cassette may advantageously comprise one or
more of what are known as enhancer sequences, linked operably to
the promoter, which make possible an increased recombinant
expression of the nucleic acid sequence. Additional advantageous
sequences, such as further regulatory elements or terminators, may
also be inserted at the 3' end of the nucleic acid sequences to be
expressed recombinantly. One or more copies of the nucleic acid
sequences to be expressed recombinantly may be present in the gene
construct.
[0270] Polyadenylation signals which are suitable as control
sequences are plant polyadenylation signals, preferably those which
essentially correspond to T-DNA polyadenylation signals from
Agrobacterium tumefaciens, in particular gene 3 of the T-DNA
(octopin synthase) of the Ti plasmid pTiACHS (Gielen et al. (1984)
EMBO J. 3:835 et seq.) or functional equivalents thereof. Examples
of terminator sequences which are especially suitable are the OCS
(octopin synthase) terminator and the NOS (nopalin synthase)
terminator.
[0271] Control sequences are furthermore to be understood as those
which make possible homologous recombination or insertion into the
genome of a host organism or which permit removal from the genome.
In the case of homologous recombination, for example the natural
promoter of a particular gene may be exchanged for a promoter with
specificity for the embryonal epidermis and/or the flower. Methods
such as the cre/lox technology permit a tissue specific, if
appropriate inducible, removal of the expression cassette from the
genome of the host organism (Sauer B (1998) Methods. 14(4):381-92).
In this method, specific flanking sequences (lox sequences), which
later allow removal by means of cre recombinase, are attached to
the target gene.
[0272] An expression cassettes and the vectors derived from it may
comprise further functional elements. The term functional element
is to be understood in the broad sense and refers to all those
elements which have an effect on the generation, amplification or
function of the expression cassettes, vectors or transgenic
organisms according to the invention. The following may be
mentioned by way of example, but not by limitation:
[0273] a) Selection markers which confer a resistance to a
metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO
98/45456), antibiotics or biocides, preferably herbicides, such as,
for example, kanamycin, G 418, bleomycin or hygromycin, or else
phosphinothricin and the like. Especially preferred selection
markers are those which confer resistance to herbicides. Examples
which may be mentioned are: DNA sequences which encode
phosphinothricin acetyl transferases (PAT) and which inactivate
glutamin synthase inhibitors (bar and pat genes),
5-enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase
genes), which confer resistance to Glyphosate.RTM.
(N-(phosphonomethyl)glycine), the gox gene, which encodes
Glyphosate.RTM..-degrading enzymes (Glyphosate oxidoreductase), the
deh gene (encoding a dehalogenase which inactivates dalapon),
sulfonylurea- and imidazolinone-inactivating acetolactate
synthases, and bxn genes, which encode bromoxynil-degrading
nitrilase enzymes, the aasa gene, which confers resistance to the
antibiotic apectinomycin, the streptomycin phosphotransferase (SPT)
gene, which allows resistance to streptomycin, the neomycin
phosphotransferase (NPTII) gene, which confers resistance to
kanamycin or geneticidin, the hygromycin phosphotransferase (HPT)
gene, which mediates resistance to hygromycin, the acetolactate
synthase gene (ALS), which confers resistance to sulfonylurea
herbicides (for example mutated ALS variants with, for example, the
S4 and/or Hra mutation).
[0274] b) Reporter genes which encode readily quantifiable proteins
and, via their color or enzyme activity, make possible an
assessment of the transformation efficacy, the site of expression
or the time of expression. Very especially preferred in this
context are genes encoding reporter proteins (Schenborn E,
Groskreutz D. Mol Biotechnol. 1999; 13(1):29-44) such as the green
fluorescent protein (GFP). (Sheen et al. (1995) Plant Journal
8(5):777-784; Haseloff et al. (1997) Proc Natl Acad Sci USA
94(6):2122-2127; Reichel et al. (1996) Proc Natl Acad Sci USA
93(12):5888-5893; Tian et al. (1997) Plant Cell Rep 16:267-271; WO
97/41228; Chui W L et al. (1996) Curr Biol 6:325-330; Leffel S M et
al. (1997) Biotechniques. 23(5):912-8), chloramphenicol
transferase, a luciferase (Ow et al. (1986) Science 234:856-859;
Millar et al. (1992) Plant Mol Biol Rep 10:324-414), the aequorin
gene (Prasher et al. (1985) Biochem Biophys Res Commun
126(3):1259-1268), .beta..-galactosidase, R locus gene (encoding a
protein which regulates the production of anthocyanin pigments (red
coloring) in plant tissue and thus makes possible the direct
analysis of the promoter activity without addition of further
auxiliary substances or chromogenic substrates; Dellaporta et al.
In: Chromosome Structure and Function: Impact of New Concepts, 18th
Stadler Genetics Symposium, 11:263-282, 1988), with
.beta.-glucuronidase being very especially preferred (Jefferson et
al., EMBO J. 1987, 6, 3901-3907).
[0275] c) Origins of replication, which ensure amplification of the
expression cassettes or vectors according to the invention in, for
example, E. coli. Examples which may be mentioned are ORI (origin
of DNA replication), the pBR322 ori or the P15A ori (Sambrook et
al.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
[0276] d) Elements which are necessary for Agrobacterium-mediated
plant transformation, such as, for example, the right or left
border of the T-DNA or the vir region.
[0277] To select cells which have successfully undergone homologous
recombination, or else to select transformed cells, it is, as a
rule, necessary additionally to introduce a selectable marker,
which confers resistance to a biocide (for example herbicide), a
metabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO
98/45456) or an antibiotic to the cells which have successfully
undergone recombination. The selection marker permits the selection
of the transformed cells from untransformed ones (McCormick et al.
(1986) Plant Cell Reports 5:81-84).
[0278] The introduction of an expression cassette according to the
invention into an organism or cells, tissues, organs, parts or
seeds thereof (preferably into plants or plant cells, tissue,
organs, parts or seeds) can be effected advantageously using
vectors which comprise the expression cassettes. The expression
cassette can be introduced into the vector (for example a plasmid)
via a suitable restriction cleavage site. The plasmid formed is
first introduced into E. coli. Correctly transformed E. coli are
selected, grown, and the recombinant plasmid is obtained by the
methods familiar to the skilled worker. Restriction analysis and
sequencing may serve to verify the cloning step.
[0279] Examples of vectors may be plasmids, cosmids, phages,
viruses or else agrobacteria. In an advantageous embodiment, the
expression cassette is introduced by means of plasmid vectors.
Preferred vectors are those which make possible stable integration
of the expression cassette into the host genome.
[0280] The generation of a transformed organism (or of a
transformed cell or tissue) requires introducing the DNA, RNA or
protein in question into the relevant host cell.
[0281] A multiplicity of methods are available for this procedure,
which is termed transformation (or transduction or transfection)
(Keown et al. (1990) Methods in Enzymology 185:527-537). For
example, the DNA or RNA can be introduced directly by
microinjection or by bombardment with DNA-coated microparticles.
Also, the cell can be permeabilized chemically, for example using
polyethylene glycol, so that DNA can enter the cell by diffusion.
The DNA can also be introduced by protoplast fusion with other
DNA-containing units such as minicells, cells, lysosomes or
liposomes. Another suitable method of introducing DNA is
electroporation, where the cells are permeabilized reversibly by an
electrical pulse. Suitable methods have been described (for example
by Bilang et al. (1991) Gene 100:247-250; Scheid et al. (1991) Mol
Gen Genet 228:104-112; Guerche et al. (1987) Plant Science
52:111-116; Neuhause et al. (1987) Theor Appl Genet 75:30-36; Klein
et al. (1987) Nature 327:70-73; Howell et al. (1980) Science
208:1265; Horsch et al. (1985) Science 227:1229-1231; DeBlock et
al. (1989) Plant Physiology 91:694-701; Methods for Plant Molecular
Biology (Weissbach and Weissbach, eds.) Academic Press Inc. (1988);
and Methods in Plant Molecular Biology (Schuler and Zielinski,
eds.) Academic Press Inc. (1989)).
[0282] In plants, the above-described methods of transforming and
regenerating plants from plant tissues or plant cells are exploited
for transient or stable transformation. Suitable methods are
especially protoplast transformation by polyethylene-glycol-induced
DNA uptake, the biolistic method with the gene gun, what is known
as the particle bombardment method, electroporation, incubation of
dry embryos in DNA-containing solution, and microinjection.
[0283] In addition to these "direct" transformation techniques,
transformation can also be effected by bacterial infection by means
of Agrobacterium tumefaciens or Agrobacterium rhizogenes. The
Agrobacterium-mediated transformation is best suited to
dicotyledonous plant cells. The methods are described, for example,
by Horsch R B et al. (1985) Science 225: 1229f.
[0284] When agrobacteria are used, the expression cassette must be
integrated into specific plasmids, either into a shuttle or
intermediate vector, or into a binary vector. If a Ti or Ri plasmid
is to be used for the transformation, at least the right border,
but in most cases the right and left border, of the Ti or Ri
plasmid T-DNA is linked to the expression cassette to be introduced
in the form of a flanking region.
[0285] Binary vectors are preferably used. Binary vectors are
capable of replication both in E. coli and in Agrobacterium. As a
rule, they comprise a selection marker gene and a linker or
polylinker flanked by the right and left T-DNA border sequence.
They can be transferred directly into Agrobacterium (Holsters et
al. (1978) Mol Gen Genet 163:181-187). The selection marker gene
permits the selection of transformed agrobacteria and is, for
example, the nptII gene, which confers resistance to kanamycin. The
Agrobacterium which acts as host organism in this case should
already contain a plasmid with the vir region. The latter is
required for transferring the T-DNA to the plant cell. An
Agrobacterium transformed in this way can be used for transforming
plant cells. The use of T-DNA for transforming plant cells has been
studied and described intensively (EP 120 516, Hoekema, In: The
Binary Plant Vector System, Offsetdrukkerij Kanters B. V.,
Alblasserdam, Chapter V; An et al. (1985) EMBO J. 4:277-287).
Various binary vectors are known, some of which are commercially
available such as, for example, pBI101.2 or pBIN19 (Clontech
Laboratories, Inc. USA).
[0286] Further promoters which are suitable for expression in
plants have been described (Rogers et al. (1987) Meth in Enzymol
153:253-277; Schardl et al. (1987) Gene 61:1-11; Berger et al.
(1989) Proc Natl Acad Sci USA 86:8402-8406).
[0287] Direct transformation techniques are suitable for any
organism and cell type.
[0288] The plasmid used need not meet any particular requirements
in the case of the injection or electroporation of DNA or RNA into
plant cells. Simple plasmids such as those of the pUC series can be
used. If complete plants are to be regenerated from the transformed
cells, it is necessary for an additional selectable marker gene to
be located on the plasmid.
[0289] Stably transformed cells, i.e. those which contain the
introduced DNA integrated into the DNA of the host cell, can be
selected from untransformed cells when a selectable marker is part
of the DNA introduced. Examples of genes which can act as markers
are all those which are capable of conferring resistance to
antibiotics or herbicides (such as kanamycin, G 418, bleomycin,
hygromycin or phosphinothricin) (see above). Transformed cells
which express such marker genes are capable of surviving in the
presence of concentrations of a corresponding antibiotic or
herbicide which kill an untransformed wild type. Examples are
mentioned above and preferably comprise the bar gene, which confers
resistance to the herbicide phosphinothricin (Rathore K S et al.
(1993) Plant Mol Biol 21(5):871-884), the nptII gene, which confers
resistance to kanamycin, the hpt gene, which confers resistance to
hygromycin, or the EPSP gene, which confers resistance to the
herbicide Glyphosate. The selection marker permits the selection of
transformed cells from untransformed cells (McCormick et al. (1986)
Plant Cell Reports 5:81-84). The resulting plants can be bred and
hybridized in the customary fashion. Two or more generations should
be grown in order to ensure that the genomic integration is stable
and hereditary.
[0290] The abovementioned methods are described, for example, in
Jenes B et al. (1993) Techniques for Gene Transfer, in: Transgenic
Plants, Vol. 1, Engineering and Utilization, edited by S D Kung and
R Wu, Academic Press, pp. 128-143 and in Potrykus (1991) Annu Rev
Plant Physiol Plant Molec Biol 42:205-225). The construct to be
expressed is preferably cloned into a vector which is suitable for
the transformation of Agrobacterium tumefaciens, for example pBin19
(Bevan et al. (1984) Nucl Acids Res 12:8711f).
[0291] As soon as a transformed plant cell has been generated, a
complete plant can be obtained using methods known to the skilled
worker. For example, callus cultures are used as starting material.
The development of shoot and root can be induced in this as yet
undifferentiated cell biomass in a known fashion. The shoots
obtained can be planted out and bred.
[0292] The skilled worker is familiar with such methods of
regenerating plant parts and intact plants from plant cells.
Methods to do so are described, for example, by Fennell et al.
(1992) Plant Cell Rep. 11: 567-570; Stoeger et al (1995) Plant Cell
Rep. 14:273-278; Jahne et al. (1994) Theor Appl Genet
89:525-533.
[0293] The method according to the invention can advantageously be
combined with further methods which bring about pathogen resistance
(for example to insects, fungi, bacteria, nematodes and the like),
stress resistance or another improvement of the plant properties.
Examples are mentioned, inter alia, by Dunwell J M, Transgenic
approaches to crop improvement, J Exp Bot. 2000; 51 Spec No; pages
487-96.
[0294] The invention furthermore relates to the barley RacB protein
as shown in SEQ ID NO: 2, and to dominant-negative variant thereof,
for example described by SEQ ID NO: 7.
[0295] The invention furthermore relates to nucleic acid sequences
encoding the barley RacB protein, preferably the nucleic acid
sequence as shown in SEQ ID NO: 1, the nucleic acid sequence
complementary thereto, and the sequences derived owing to
degeneracy of the genetic code.
[0296] The invention furthermore relates to the polypeptide
encoding functional equivalents of the RacB protein from barley as
shown in SEQ ID NO: 35, 37 or 39.
[0297] The invention furthermore relates to nucleic acid sequences
encoding functional equivalents of the RacB protein from barley,
preferably the nucleic acid sequence as shown in SEQ ID NO: 34, 36
or 38, the nucleic acid sequence complementary thereto and the
sequences derived by degeneration of the genetic code.
[0298] The invention furthermore relates to transgenic expression
cassettes comprising one of the nucleic acid sequences according to
the invention. In the transgenic expression cassettes according to
the invention, the nucleic acid sequence encoding the barley RacB
protein is linked to at least one genetic control element as
defined above in such a manner that it is capable of expression
(transcription and, if appropriate, translation) in any organism,
preferably in plants. Suitable genetic control elements are
described above. The transgenic expression cassettes may also
comprise further functional elements in accordance with the above
definition. The inserted nucleic acid sequence encoding a barley
RacB protein can be inserted in the expression cassette in sense or
antisense orientation and thus lead to the expression of sense or
antisense RNA. Transgenic vectors comprising the transgenic
expression cassettes are also in accordance with the invention.
[0299] "Transgenic", for example regarding a nucleic acid sequence,
an expression cassette or a vector comprising said nucleic acid
sequence or an organism transformed with said nucleic acid
sequence, expression cassette or vector, refers to all those
constructs originating by recombinant methods in which either
[0300] a) the RacB nucleic acid sequence, or
[0301] b) a genetic control sequence linked operably to the RacB
nucleic acid sequence, for example a promoter, or
[0302] c) (a) and (b) are not located in their natural genetic
environment or have been modified by recombinant methods, an
example of a modification being a substitutions, additions,
deletions, inversion or insertions of one or more nucleotide
residues. Natural genetic environment refers to the natural
chromosomal locus in the organism of origin, or to the presence in
a genomic library. In the case of a genomic library, the natural
genetic environment of the nucleic acid sequence is preferably
retained, at least in part. The environment flanks the nucleic acid
sequence at least at one side and has a sequence of at least 50 bp,
preferably at least 500 bp, especially preferably at least 1000 bp,
very especially preferably at least 5000 bp, in length. A naturally
occurring expression cassette--for example the naturally occurring
combination of the RacB promoter with the corresponding RacB
gene--becomes a transgenic expression cassette when it is modified
by non-natural, synthetic "artificial" methods such as, for
example, mutagenization. Such methods have been described (U.S.
Pat. No. 5,565,350; WO 00/15815; also see above).
[0303] The invention also relates to transgenic organisms
transformed with at least one of the nucleic acid sequences
according to the invention, expression cassette according to the
invention or vector according to the invention, and to cells, cell
cultures, tissues, parts--such as, for example, leaves, roots and
the like in the case of plant organisms--or propagation material
derived from such organisms. The term organism is to be understood
in the broad sense and refers to prokaryotic and eukaryotic
organisms, preferably bacteria, yeasts, fungi, animal organisms and
plant organisms.
[0304] The following are preferred:
[0305] a) fungi such as Aspergillus, Eremothecium, Trichoderma,
Ashbya, Neurospora, Fusarium, Beauveria or other fungi described in
Indian Chem Eng. Section B. Vol 37, No. 1, 2 (1995) on page 15,
Table 6. Especially preferred is the filamentous hemiascomycete
Ashbya gossypii or Eremothecium ashbyii,
[0306] b) yeasts such as Candida, Saccharomyces, Hansenula or
Pichia, with Saccharomyces cerevisiae or Pichia pastoris (ATCC
Accession No. 201178) being especially preferred,
[0307] c) plants in accordance with the above definition of
"plants",
[0308] d) vertebrates and invertebrates. Especially preferred
vertebrates are nonhuman mammals such as in dog, cat, sheep, goat,
chicken, mouse, rat, cattle or horse. Preferred animal cells
comprise CHO, COS and HEK293 cells. Preferred invertebrates
comprise insect cells such as Drosophila S2 and Spodoptera Sf9 or
Sf21 cells,
[0309] e) prokaryotic organisms such as Gram-positive or
Gram-negative bacteria such as Acetobacter, Gluconobacter,
Corynebacterium, Brevibacterium, Bacillus, Clostridium,
Cyanobacter, Escherichia (mainly Escherichia coli), Serratia,
Staphylococcus, Aerobacter, Alcaligenes, Penicillium or
Klebsiella.
[0310] Host or starting organisms which are preferred as transgenic
organisms are mainly plants in accordance with the above
definition. Included within the scope of the invention are all
genera and species of higher and lower plants of the Plant Kingdom.
Furthermore included are the mature plants, seed, shoots and
seedlings, and parts, propagation material and cultures derived
therefrom, for example cell cultures. Mature plants refers to
plants at any developmental stage beyond that of the seedling. The
term seedling refers to a young immature plant in an early
developmental stage. Plants preferred as host organisms are in
particular plants which can be used for the process according to
the invention to obtain a pathogen resistance according to the
criteria mentioned above. Very particularly preferred are
monocotyledonous plants, such as wheat, oats, millet, barley, rye,
maize, rice, buckwheat, sorghum, triticale, spelt, linseed, sugar
cane, as dicotyledonous crop plants, such as oil seed rape, canola,
cress, Arabidopsis, cabbages, soya, alfalfa, pea, beans, peanut,
potato, tobacco, tomato, eggplant, bell pepper, sunflower, Tagetes,
lettuce, Calendula, melon, pumpkin/squash or zucchini.
[0311] The transgenic organisms can be generated with the
above-described methods for the transformation or transfection of
organisms.
[0312] The invention furthermore relates to the use of the
transgenic organisms according to the invention and of the cells,
cell cultures, parts such as, for example, roots, leaves and the
like in the case of transgenic plant organisms--derived from them,
and to transgenic propagation material such as seeds or fruits, for
the production of foodstuffs or feeding stuffs, pharmaceuticals or
fine chemicals.
[0313] Furthermore preferred is a method for the recombinant
production of pharmaceuticals or fine chemicals in host organisms,
wherein a host organism is transformed with one of the
above-described expression cassettes and this expression cassette
comprises one or more structural genes which encode the desired
fine chemical or catalyze the biosynthesis of the desired fine
chemical, the transformed host organism is cultured, and the
desired fine chemical is isolated from the culture medium. This
method can be applied widely to fine chemicals such as enzymes,
vitamins, amino acids, sugars, fatty acids, and natural and
synthetic flavorings, aroma substances and colorants. Especially
preferred is the production of tocopherols and tocotrienols and
carotenoids. The transformed host organisms are cultured and the
products are isolated from the host organisms or the culture medium
by methods known to the skilled worker. The production of
pharmaceuticals such as, for example, antibodies or vaccines, is
described by Hood E E, Jilka J M. (1999) Curr Opin Biotechnol.
10(4):382-6; Ma J K, Vine N D. (1999) Curr Top Microbiol Immunol.
236:275-92.
[0314] Sequences [0315] 1. SEQ ID NO: 1 Nucleic acid sequence
encoding the barley (Hordeum vulgare) RacB protein. [0316] 2. SEQ
ID NO: 2 Amino acid sequence encoding the barley (Hordeum vulgare)
RacB protein. [0317] 3. SEQ ID NO: 3 Nucleic acid sequence encoding
the rice (Oryza sativa) RacB protein. [0318] 4. SEQ ID NO: 4 Amino
acid sequence encoding the rice (Oryza sativa) RacB protein. [0319]
5. SEQ ID NO: 5 Nucleic acid sequence encoding the maize (Zea mays)
RacB protein. [0320] 6. SEQ ID NO: 6 Amino acid sequence encoding
the maize (Zea mays) RacB protein. [0321] 7. SEQ ID NO: 7 Amino
acid sequence encoding a dominant-negative variant of the RacB
protein (Hordeum vulgare). [0322] 8. SEQ ID NO: 8 Amino acid
sequence encoding a dominant-negative variant of the rice (Oryza
sativa) RacB protein. [0323] 9. SEQ ID NO: 9 Amino acid sequence
encoding a dominant-negative variant of the maize (Zea mays) RacB
protein. [0324] 10. SEQ ID NO, 10 Oligonucleotide primer ONP-1
TABLE-US-00008 [0324] 5'-GGATCCGATGAGCGCGTCCAGGTT-3'
[0325] 11. SEQ ID NO: 11 Oligonucleotide primer ONP-2
TABLE-US-00009 [0325] 5'-TCGACCTTCGCCCTTGTTCTTTGTC-3'
[0326] 12. SEQ ID NO: 12 RACE-RacB primer
TABLE-US-00010 [0326] 5'-gtgggcacatagtcggtggggaaggt-3'
[0327] 13. SEQ ID NO: 13 GeneRacer.TM. 5' primer:
TABLE-US-00011 [0327] 5'-CGACTGGAGCACGAGGACACTGA-3
[0328] 14. SEQ ID NO: 14 GeneRacer.TM. 5' nested primer:
TABLE-US-00012 [0328] 5'-GGACACTGACATGGACTGAAGGAGTA-3
[0329] 15. SEQ ID NO: 15 RacB sense primer
TABLE-US-00013 [0329] 5'-gttcatcaagtgcgtcaccgtg-3'
[0330] 16. SEQ ID NO: 16 RacB antisense primer
TABLE-US-00014 [0330] 5'-ttagcttcctcagttcttccctg-3'
[0331] 17. SEQ ID NO: 17 BAS sense primer
TABLE-US-00015 [0331] 5'-cgcgccgcagccgagtacgac-3'
[0332] 18. SEQ ID NO: 18 BAS antisense primer
TABLE-US-00016 [0332] 5'-gtcacaaaaacacatgtaacc-3'
[0333] 19. SEQ ID NO: 19 OXLP sense primer
TABLE-US-00017 [0333] 5'-ggccgacatgcattcaccag-3'
[0334] 20. SEQ ID NO: 20 OXLP antisense primer
TABLE-US-00018 [0334] 5'-catctgatattgctgggtctg-3'
[0335] 21. SEQ ID NO: 21 UBI sense primer
TABLE-US-00019 [0335] 5'-ccaagatgcagatcttcgtga--3'
[0336] 22. SEQ ID NO: 22 UBI antisense primer
TABLE-US-00020 [0336] 5'-ttcgcgataggtaaaagagca-3'
[0337] 23. SEQ ID NO: 23 M13 fwd primer
TABLE-US-00021 [0337] 5'-GTAAAACGACGGCCAGTG-3'
[0338] 24. SEQ ID NO: 24 M13 rev primer
TABLE-US-00022 [0338] 5'-GGAAACAGCTATGACCATG-3'
[0339] 25. SEQ ID NO: 25 HvRop6 LEFT PRIMER
TABLE-US-00023 [0339] 5'-GTGGAGGCGCGGCGAGA-3'
[0340] 26. SEQ ID NO: 26 HvRop6 RIGHT PRIMER
TABLE-US-00024 [0340] 5'-CCATGCTTCATCTCCATAGTCA-3'
[0341] 27. SEQ ID NO: 27 HvRacD LEFT PRIMER
TABLE-US-00025 [0341] 5'-ggatccCGATTCCATCAGGAAAGCAT-3'
[0342] 28. SEQ ID NO: 28 HvRacD RIGHT PRIMER
TABLE-US-00026 [0342] 5'-gtcgacGCGAGACACTGCAAAACAAA--3'
[0343] 29. SEQ ID NO: 29 HvRop4 LEFT PRIMER
TABLE-US-00027 [0343] 5'-GGATCCttctcgtccatttagccggc-3'
[0344] 30. SEQ ID NO: 30 HvRop4 RIGHT PRIMER
TABLE-US-00028 [0344] 5'-GTCGACtgatcacttgaagcatgccag-3'
[0345] 31. SEQ ID NO: 31 RacB5' BamHI Primer
TABLE-US-00029 [0345] 5'-GGATCCGATGAGCGCGTCCAGGT-T-3'
[0346] 32. SEQ ID NO: 32 RacB3' SalI Primer
TABLE-US-00030 [0346] 5'-GTCGACCTTCGCCCTTGTTCTTTGTC-3'
[0347] 33. SEQ ID NO 33 V15 mutagenesis Primer
TABLE-US-00031 [0347] 5'-ACCGTGGGGGACGTCGCCGTCGGCAAGAC-3'
[0348] 34. SEQ ID NO: 34 Nucleic acid sequence encoding the RacB
homolog HvRop6 from barley (Hordeum vulgare). [0349] 35. SEQ ID NO:
35 Amino acid sequence encoding the RacB homolog HvRop6 from barley
(1Hordeum vulgare). [0350] 36. SEQ ID NO: 36 Nucleic acid sequence
encoding the RacB homolog HvRacD from barley (Hordeum vulgare).
[0351] 37. SEQ ID NO: 37 Amino acid sequence encoding the RacB
homolog HvRacD from barley (Hordeum vulgare). [0352] 38. SEQ ID NO:
38 Nucleic acid sequence encoding the RacB homolog HvRop4 from
barley (Hordeum vulgare). [0353] 39. SEQ ID NO: 39 Nucleic acid
sequence encoding the RacB homolog HvRop4 from barley (Hordeum
vulgare). [0354] 40. SEQ ID NO: 40 Nucleic acid sequence encoding
the RacB homolog Zea mays ROP6 (GenBank Acc.-No.: AJ278665) [0355]
41. SEQ ID NO: 41 Amino acid sequence encoding the RacB homolog Zea
mays ROP6 [0356] 42. SEQ ID NO: 42 Nucleic acid sequence encoding
the RacB homolog Oryza saliva subsp. japonica PACDP (RACD) (GenBank
Acc.-No.: AF218381) [0357] 43. SEQ ID NO: 43 Amino acid sequence
encoding the RacB homolog Oryza saliva subsp. japonica RACDP [0358]
44 SEQ ID NO: 44 Nucleic acid sequence encoding the RacB homolog
Oryza saliva ROP4 (GenBank Acc.-No.: AF380335) [0359] 45. SEQ ID
NO: 45 Amino acid sequence encoding the RacB) homolog Oryza sativa
ROP4 [0360] 46. SEQ ID NO: 46 Nucleic acid sequence encoding the
RacB homolog Zea mays RACA (GenBank Acc.-No.: AF126052) [0361] 47.
SEQ ID NO: 47 Amino acid sequence encoding the RacB homolog Zea
mays RACA [0362] 48. SEQ ID NO: 48 Nucleic acid sequence encoding
an RacB homolog from Hordeum vulgare (GenBank Acc.-No.: BM816965)
[0363] 49. SEQ ID NO: 49 Nucleic acid sequence encoding an RacB
homolog from Arabidopsis thaliana (At3g51300) [0364] 50. SEQ ID NO:
50 Amino acid sequence encoding an RacB homolog from Arabidopsis
thaliana (At3g51300) [0365] 51. SEQ ID NO: 51 Nucleic acid sequence
encoding an RacB homolog from Arabidopsis thaliana (At2g17800)
[0366] 52. SEQ ID NO: 52 Amino acid sequence encoding an RacB
homolog from Arabidopsis thaliana (At2g17800) [0367] 53. SEQ ID NO:
53 Nucleic acid sequence encoding an RacB homolog from Arabidopsis
thaliana (At4g35950) [0368] 54. SEQ ID NO: 54 Amino acid sequence
encoding an RacB homolog from Arabidopsis thaliana (At4g35950)
[0369] 55. SEQ ID NO: 55 Nucleic acid sequence encoding an RacB
homolog from Arabidopsis thaliana (At1g75840) [0370] 56. SEQ ID NO:
56 Amino acid sequence encoding an RacB homolog from Arabidopsis
thaliana (At1g75840) [0371] 57. SEQ ID NO: 57 Nucleic acid sequence
encoding an RacB homolog from Arabidopsis thaliana (At4g35020)
[0372] 58. SEQ ID NO: 58 Amino acid sequence encoding an RacB
homolog from Arabidopsis thaliana (At4g35020) [0373] 59. SEQ ID NO:
59 Nucleic acid sequence encoding an RacB homolog from Arabidopsis
thaliana (At1g20090) [0374] 60. SEQ ID NO: 60 Amino acid sequence
encoding an RacB homolog from Arabidopsis thaliana (At1g20090)
[0375] 61. SEQ ID NO: 61 Nucleic acid sequence encoding an RacB
homolog from Arabidopsis thaliana (At5g45970) [0376] 62. SEQ ID NO:
62 Amino acid sequence encoding an RacB homolog from Arabidopsis
thaliana (At5g4S970) [0377] 63. SEQ ID NO: 63 Nucleic acid sequence
encoding an RacB homolog from Arabidopsis thaliana (At3g48040)
[0378] 64. SEQ ID NO: 64 Amino acid sequence encoding an RacB
homolog from Arabidopsis thaliana (At3g48040) [0379] 65. SEQ ID NO:
65 Nucleic acid sequence encoding an RacB homolog from Arabidopsis
thaliana (At5g62880) [0380] 66. SEQ ID NO; 66 Amino acid sequence
encoding an RacB homolog from Arabidopsis thaliana (At5g62880)
[0381] 67. SEQ ID NO: 67 Nucleic acid sequence encoding an RacB
homolog from Arabidopsis thaliana (At4g28950) [0382] 68. SEQ ID NO:
68 Amino acid sequence encoding an RacB homolog from Arabidopsis
thaliana (At4g28950) [0383] 69. SEQ ID NO: 79 Nucleic acid sequence
encoding an RacB homolog from Arabidopsis thaliana (At7g44690)
[0384] 70. SEQ ID NO: 70 Amino acid sequence encoding an RacB
homolog from Arabidopsis thaliana (At2g44690) [0385] 71. SEQ ID NO:
71 Oligonucleotide primer Fra 186
TABLE-US-00032 [0385] 5'-ATGAGCGCGTCCAGGTTCATA-3'
[0386] 72. SEQ ID NO: 72 Oligonucleotide primer Fra 187
TABLE-US-00033 [0386] 5'-ATCAAACACGCCCTTCACGTT-3'
[0387] 73. SEQ ID NO: 73 Transgenic expression vector
pSUN3NIT_AtRacB_s for expression of Arbidopsis thalianan RacB in
sense orientation [0388] 74. SEQ ID NO: 74 Transgenic expression
vector pSUN3NIT_AtRacB_as for expression of Arbidopsis thalianan
RacB in antisense orientation [0389] 75. SEQ ID NO: 75 Transgenic
expression vector pSUN3NIT_HvRacB_s for expression of a barley RacB
fragment in sense orientation [0390] 76. SEQ ID NO: 76 Transgenic
expression vector pSUN3NIT_HvRacB_as for expression of a barley
RacB fragment in antisense orientation
FIGURES
[0391] 1. FIG. 1: Alignment of the amino acid sequences of barley
RacB, rice RacB, maize RacB, and human Rac1 and Rac2 proteins.
[0392] Regions in boxes show the position of the G1 element
(GXXXXGKS/T; amino acid 13 to 20), of the 62 effector region (amino
acid 29 to 45), of the G3 element (LWDTAGQ; amino acid 58 to 64),
of the G4 element (TKXD, amino acid 118 to 121), of the G5 element
(EXS) and of the C-terminal isoprenylation motif (CXXX, Hassanain H
H et al. (2000) Biochem Biophys Res Commun. 272(3):783-8.). Hyphens
indicate sequence gaps. Asterisks denote amino acids which are
identical in all homologs. Amino acids which differ between barley
on the one hand and maize and rice on the other hand are shown
white against black. The position which is advantageously modified
to obtain a dominant-negative RacB variant is marked by a black
triangle above the sequence.
[0393] 2. FIG. 2: Expression of RacB in epidermal tissue
[0394] RT-PCR of RNA from the barley lines Pallas and BCPMla12
(P10) 24 h post-inoculation ("hai" hours after inoculation") with
BghA6. To extract the RNA, strips of the abaxial epidermis (E, from
inoculated locations of leaves) were removed from the mesophyll and
the adaxial epidermis (M). Ubiquitin 1 (Ubi) acted as marker for
tissue-unspecific expression, OXLP as positive control for gene
expression in the epidermis, and Bas as positive control for gene
expression in mesophyll cells. The RT-PCR was carried out over 25
amplification cycles as described hereinbelow. RT-PCR products were
denatured in the gel, blotted and detected by means of antisense
RNA probes under stringent conditions.
[0395] 3. FIG. 3: RacB is expressed constitutively in various
resistant barley lines.
[0396] RNA was isolated from the variety Ingrid (Mlo, Ror1,
Bgh-susceptible), BCIngrid-mlo5 (mlo5, Ror1, Bgh-resistant) and A
89 (mlo5, ror1, moderately susceptible to BghA6) immediately prior
to inoculation (0 O) or 8, 15 or 24 h post-inoculation with Bgh and
24 h thereafter from noninoculated control plants (24 O). Ubiquitin
1 (Ubi) was used as marker for constitutive expression, OXLP as
positive control for Bgh-induced gene expression in the epidermal
layer. OXLP expression was detected via Northern blot. The RT-PCR
for RacB and Ubi was carried out as described over 25 amplification
cycles. The PCR products were denatured in the gel, blotted and
detected by means of antisense RNA samples under stringent
conditions.
[0397] 4. FIG. 4: "RNA interference" with RacB-dsRNA reduces the
penetration efficacy of barley powdery mildew BghA6 in barley.
[0398] The relative penetration efficacy (RPE) was determined in
six individual experiments with inoculation with Bgh from barley
cv. Pallas. The RPE was calculated as the difference between the
penetration efficacy of cells transformed with RacB-dsRNA and the
penetration efficacy of cells transformed with control dsRNA (here:
average penetration efficacy 57%). The RPE percentage (%-RPE) is
calculated from the RPE minus 1, multiplied by 100.
RPE = [ PE of cells transformed with RacB - dsRNA ] [ PE of cells
transformed with control dsRNA ] ##EQU00001## % - RPE = 100 * ( RPE
- 1 ) ##EQU00001.2##
[0399] The black columns represent the %-RPE upon evaluation of at
least 100 interaction sites for in each case one independent
experiment. The white column represents the average %-RPE of the
experiments with the RacB-dsRNA ("RACB-dsRNA"). The error bar
indicates the standard error.
[0400] "control" represents the parallel experiments with a control
dsRNA.
[0401] In cells which had been bombarded with RacB-dsRNA, the %-RPE
was markedly reduced in comparison with cells which had been
bombarded with a control dsRNA (TR: human thyroid receptor
dsRNA).
[0402] 5. FIG. 5: Effect of the genetic background on RacB
function
[0403] The %-RPE was studied in 5 independent experiments by
inoculating barley cv. Pallas, Ingrid or A89, which had previously
been transformed with RacB-dsRNA, with BghA6.
[0404] The %-RPE is markedly reduced in Pallas (Mlo Ror1, black
bars, experiments 1 and 2) or Ingrid (Mlo Ror1, black bars,
experiments 3, 4 and 5). The %-RPE of the susceptible mutant A89
(mlo5 ror1, black bars, experiments 1 to 5), however, was not
reduced. White bars indicate the mean, error bars the standard
error.
[0405] 6. FIG. 6: Overexpression of a constitutively active RacB
mutant in barley cv. Pallas
[0406] A constitutively active mutant of barley RACE (exchange
G->V in position 15; RacB-V15) was transiently overexpressed in
the barley variety Pallas in 5 independent experiments using the
expression construct pGY-RacBV15. For comparison, corresponding
experiments were carried out using the vector alone, without RacB
insert (pGY).
[0407] The expression of a constitutive RacB mutant results in
significantly higher susceptibility to pathogen attack by mildew of
barley, compared to the controls (FIG. 6-A). In all cases, the
relative susceptibility to the fungal pathogen is increased (FIG.
6-B). These results, too, demonstrate the key function of racB in
the defense of pathogens.
[0408] 7. FIG. 7: Plasmid map for expression vector pGY-1
(Schweizer P et al. (1999) Mol Plant Microbe Interact 12: 647-54;
Shinshi H et al. (1990) Plant Mol Biol 14:357-368).
EXAMPLES
General Methods
[0409] The chemical synthesis of oligonucleotides can be effected,
for example, in the known fashion using the phosphoamidite method
(Voet, Voet, 2nd Edition, Wiley Press New York, pages 896-897). The
cloning steps carried out for the purposes of the present invention
such as, for example, restriction cleavages, agarose gel
electrophoresis, purification of DNA fragments, transfer of nucleic
acids to nitrocellulose and nylon membranes, linking DNA fragments,
transformation of E. coli cells, growing bacteria, multiplying
phages and sequence analysis of recombinant DNA, are carried out as
described by Sambrook et al. (1989) Cold Spring Harbor Laboratory
Press; ISBN 0-87969-309-6. The sequencing of recombinant DNA
molecules is carried out with an MWG-Licor laser fluorescence DNA
sequencer following the method of Sanger (Sanger et al. (1977) Proc
Natl Acad Sci USA 74:5463-5467).
Example 1
Plants, Pathogens and Inoculation
[0410] The barley variety Ingrid is from James McKey, University of
Uppsala, Sweden. The variety Pallas and backcrossed line
BCIngrid-mlo5 was donated by Lisa Munk, Department of Plant
Pathology, Royal Veterinary and Agricultural University,
Copenhagen, Denmark. Its production has been described (K.o
slashed.lster P et al. (1986) Crop Sci 26: 903-907). Line A89 was
provided by Paul Schulze-Lefert (Max-Plank-Institut fur
Zuchtungsforschung, Cologne, Germany).
[0411] Unless otherwise specified, the seed which had been
pregerminated on moist filter paper for 12 to 36 hours in the dark
was sown along the edge of a square pot (8.times.8 cm; 5 kernels
per pot) in Fruhstorfer soil, type P, covered with soil and watered
regularly with tap water. All of the plants were cultured in
controlled-environment cabinets or chambers for 5 to 8 days at 16
to 18.degree. C., 50 to 60% relative atmospheric humidity and a
16-hr-light/8-hr-dark rhythm at 3000 or 5000 lux (photon flow
density 50 and 60 .mu.mols-.sup.1 m-.sup.2, respectively) and used
in the experiments during the seedling stage. In experiments in
which applications to the primary leaves were carried out, the
latter were developed fully.
[0412] Before the transient transfection experiments were carried
out, the plants were grown in controlled-environment cabinets or
chambers at a daytime temperature of 24.degree. C., a nighttime
temperature of 20.degree. C., 50 to 60% relative atmospheric
humidity and a 16-hr light/8-hr-dark rhythm at 30 000 lux.
[0413] Barley powdery mildew Blumeria graminis (DC) Speer f.sp.
hordei Em. Marchal race A6 (Wiberg A (1974) Hereditas 77: 89-148)
(BghA6) was used for the inoculation of barley plants. The fungus
was provided by the Department of Biometry, JLU Gie.beta.en.
Inoculum was growl in controlled-environment cabinets under
identical conditions to those described above for the plants by
transferring the conidia of infected plant material at a rate of
100 conidia/mm.sup.2 to 7-day-old barley plants cv. Golden Promise,
which were grown regularly.
[0414] The inoculation with BghA6 was carried out using 7-day-old
seedlings by shaking off the conidia of already infected plants in
an inoculation tower at a rate of approx. 100 conidia/mm.sup.2
(unless otherwise specified).
Example 2
RNA Extraction
[0415] Total RNA was extracted from 8 to 10 primary leaf segments
(length 5 cm) by means of "RNA extraction buffer" (AGS, Heidelberg,
Germany).
[0416] To this end, the central primary leaf segments 5 cm in
length were harvested and homogenized in liquid nitrogen in
mortars. The homogenate was stored at -7.degree. C. until the RNA
was extracted.
[0417] Total RNA was extracted from the deep-frozen leaf material
with the aid of an RNA extraction kit (AGS, Heidelberg). To this
end, 200 mg of the deep-frozen leaf material were covered with 1.7
ml RNA extraction buffer (AGS) in a microcentrifuge tube (2 ml) and
immediately mixed thoroughly. After addition of 200 .mu.l of
chloroform, the mixture was again mixed thoroughly and shaken for
45 minutes on a horizontal shaker at 200 rpm at room temperature.
To separate the phases, the tubes were subsequently centrifuged for
15 minutes at 20 000 g and 4.degree. C., and the upper, aqueous
phase was transferred into a fresh microcentrifuge tube, while the
bottom phase was discarded. The aqueous phase was repurified with
900 .mu.l of chloroform by homogenizing for 10 seconds and
recentrifuging (see above) and removing the aqueous phase (3
times). Then, 850 .mu.l of 2-propanol were added and the mixture
was homogenized and placed on ice for 30 to 60 minutes in order to
precipitate the RNA. Thereafter, the mixture was centrifuged for 20
minutes (see above), the supernatant was carefully decanted off, 2
ml of 70% strength ethanol (-20.degree. C.) were pipetted in, and
the mixture was mixed and recentrifuged for 10 minutes.
[0418] Then, the supernatant was again decanted off, and the pellet
was carefully freed from residual fluid, using a pipette, and then
dried in a stream of clean air in a clean bench. Then, the RNA was
dissolved in 50 .mu.l of DEPC water on ice, mixed and centrifuged
for 5 minutes (see above). 40 .mu.l of the supernatant,
constituting the RNA solution, were transferred into a fresh
microcentrifuge tube and stored at -70.degree. C.
[0419] The RNA concentration was determined photometrically. To
this end, the RNA solution was diluted 1:99 (v/v) with distilled
water, and the absorption was measured at 260 nm (Beckman
Photometer DU 7400); (E.sub.260 1 at 40 .mu.g RNA/ml). The
concentrations of the RNA solutions were subsequently adjusted to 1
.mu.g/.mu.l with DEPC water to match the calculated RNA contents
and verified in an agarose gel.
[0420] To verify the RNA concentrations in a horizontal agarose gel
(1% agarose in 1.times.MOPS buffer with 0.2 .mu.g/ml ethidium
bromide), 1 .mu.l of RNA solution was treated with 1 .mu.l of
10.times.MOPS, 1 .mu.l of color marker and 7 .mu.l of DEPC water,
separated according to size in 1.times.MOPS running buffer over 1.5
hours at a voltage of 120 V in the gel, and photographed under UV
light. Any differences in concentration of the RNA extracts were
adjusted with DEPC water, and the adjustment was rechecked in the
gel.
Example 3
Cloning the Barley RacB cDNA Sequence
[0421] The cDNA fragments required for isolating the HvRacB cDNA,
cloning it, sequencing it and generating probes were obtained by
means of RT-PCR using the "One Step RT-PCR Kit" (Life Technologies,
Karlsruhe, Germany or Qiagen, Hilden, Germany). To this end, total
RNA from barley seedlings was used as template.
[0422] The RNA was isolated from Pallas 3, 5 and 0.7/days after
germination. Moreover, RNA was isolated from Pallas and the
backcrossed lines with mlo5, Mlg or Mla12 1, 2 and 5 days after
inoculation with BghA6 on day 7 after germination. The following
primers are used for the RT-PCR:
TABLE-US-00034 ONP-1 5'-GGATCCGATGAGCGCGTCCAGGTT-3' (SEQ ID NO: 10)
and ONP-2 5'-GTCGACCTTCGCCCTTGTTCTTTGTC-3' (SEQ ID NO: 11)
[0423] 1000 ng of total RNA, 0.4 mM dNTPs, in each case 0.6 mM
OPN-1 and OPN-2 primer, 10 .mu.l of RNase inhibitor and 1 .mu.l of
enzyme mix in 1.times.RT buffer (One Step RT-PCR Kit, Qiagen,
Hilden) were employed for each reaction (25 .mu.l batch).
[0424] The following temperature program is used (PTC-100TM model
96V; MJ Research, Inc., Watertown, Mass.):
TABLE-US-00035 1 cycle of 30 min at 50.degree. C. 1 cycle of 150
sec at 94.degree. C. 30 cycles of 45 sec at 94.degree. C., 1 min at
55.degree. C. and 2 min at 72.degree. C. 1 cycle of 7 min at
72.degree. C.
[0425] The PCR product was separated by 2% w/v agarose gel
electrophoresis. This gave an RT-PCR product of in total 642 bp
which was composed of the RacB sequence (SEQ ID NO: 1) and terminal
sequences encoding restriction endonuclease restriction sites. The
fragment encodes a 591 bp open reading frame encoding a polypeptide
of 197 amino acids. The corresponding cDNA was isolated from an
agarose gel and cloned into vector pGEM-T (Promega, Mannheim,
Germany) by means of T-overhang ligation. The cDNAs were sequenced
starting from the plasmid DNA using the "Thermo Sequenase
Fluorescent Labeled Primer Cycle Sequencing Kit" (Amersham,
Freiburg, Germany).
[0426] Since a primer has been deduced from the rice RacB sequence
as starting primer OPN-1 (GenBank Acc. No.: AF250327), this region
(i.e. the 5'-end) of the barley RacB cDNA was reverified by means
of RACE technology using the "GeneRacer Kit" (INVITROGENE Life
Technologies). To this end, 100 ng of poly-A mRNA, 1 .mu.l of
10.times.CIP buffer, 10 units of RNAse inhibitor, 10 units of CIP
("calf intestinal phosphatase") and DEPC-treated water were treated
for 1 hour at 50.degree. C. in a total volume of 10 .mu.l. To
precipitate the RNA, a further 90 .mu.l of DEPC water and 100 .mu.l
of phenol:chloroform were added and the mixture was mixed
thoroughly for approximately 30 seconds. After centrifugation for 5
minutes at 20 000 g, the top phase was treated with 2 .mu.l of 10
mg/ml mussel glycogen, 10 .mu.l of 3 M sodium acetate (pH 5.2) in a
fresh micro reaction vessel. 220 .mu.l of 95% ethanol were added
and the mixture was incubated on ice. The RNA was subsequently
precipitated by centrifugation for 20 minutes at 20 000 g and
4.degree. C. The supernatant was discarded, 500 .mu.l of 75%
ethanol were added, and the mixture was vortexed briefly and
recentrifuged for 2 minutes (20 000 g). Again, the supernatant was
discarded, and the precipitate was dried in the air for 0.2 minutes
at room temperature and subsequently suspended in 6 .mu.l of DEPC
water. mRNA CAP structures were removed by adding 1 .mu.l of
10.times.TAP buffer, 10 units of RNAsin and 1 unit of TAP (tobacco
acid pyrophosphatase). The mixture was incubated for 1 h at
37.degree. C. and subsequently cooled on ice. Again, the RNA was
precipitated as described above and transferred into a reaction
vessel with 0.25 .mu.g of GeneRacer oligonucleotide primer. The
oligonucleotide primer was resuspended in the RNA solution, and the
mixture was incubated for 5 minutes at 70.degree. C. and then
ice-cooled. 1 .mu.l of 10.times. ligase buffer, 10 mM ATP, 1 unit
of RNAsin and 5 units of T4 RNA ligase were added, and the batch
was incubated for 1 h at 37.degree. C. Again, the RNA was
precipitated as described above and resuspended in 13 .mu.l of DEPC
water. 10 pmol of oligo-dT primer were added to the RNA, and the
mixture was immediately heated at 70.degree. C. and again cooled on
ice. 1 .mu.l of each dNTP solution (25 mM), 2 .mu.l of 10.times.RT
buffer, 5 u (1 .mu.l) of AMV reverse transcriptase and 20 units of
RNAsin were added, and the reaction solution was incubated for 1
hour at 42.degree. C. and subsequently for 15 minutes at 85.degree.
C. The first-strand cDNA thus prepared was stored at -20.degree.
C.
[0427] The following primer was used to amplify the 5'-cDNA
ends:
TABLE-US-00036 RACE RacB primer: 5'-gtgggcacatagtcggtggggaaggt-3'
(SEQ ID NO: 12) GeneRacer .TM. 5'-primer:
5'-CGACTGGAGCACGAGGACACTGA-3 (SEQ ID NO: 13) GeneRacer .TM.
5'-nested primer: 5'-GGACACTGACATGGACTGAAGGAGTA-3 (SEQ ID NO:
14)
[0428] The batch (total volume 25 .mu.l) was composed as follows:
[0429] 1 .mu.l primer RACE-RacB (5 pmol/.mu.l), [0430] 0.5 .mu.l
GeneRacer 5'-primer (10 pmol/.mu.l) [0431] 2.5 .mu.l 10.times.
buffer Qiagen, [0432] 2.5 .mu.l dNTPs (2 mM) [0433] 0.5 .mu.l cDNA
[0434] 0.2 .mu.l QiagenTAG (5 u/.mu.l) [0435] 17.8 .mu.l H2O
[0436] The PCR conditions were:
TABLE-US-00037 94.degree. C. denaturation for 5 minutes 5 cycles of
30 seconds at 70.degree. C. (annealing), 1 min at 72.degree. C.
(extension), 30 seconds at 94.degree. C. (denaturation) 5 cycles of
30 seconds at 68.degree. C. (annealing), 1 min at 72.degree. C.
(extension), 30 seconds at 94.degree. C. (denaturation) 28 cycles
of 30 seconds at 66.degree. C. (annealing), 1 min at 72.degree. C.
(extension), 30 seconds at 94.degree. C. (denaturation) 72.degree.
C. final extension for 10 minutes 4.degree. C. cooling until
further use
[0437] The PCR gave a product of approx. 400 bp product. Starting
from this product, a nested PCR with the RacB-specific
oligonucleotide primer and the "GeneRacer nested 5'-primer" was
carried out:
TABLE-US-00038 94.degree. C. denaturation for 5 minutes 30 cycles
of 30 sec at 64.degree. C. (annealing), 1 min at 72.degree. C.
(extension), 30 sec at 94.degree. C. (denaturation) 72.degree. C.
final extension for 10 minutes 4.degree. C. cooling until further
use
[0438] The PCR product obtained was isolated via a gel, extracted
from the gel, cloned into PGEM-T by means of T-overhang ligation,
and sequenced. The sequence in the region of the primer OPN-1 was
absolutely identical to the sequence of rice racB, so that no point
mutations could be generated by means of primers. Thus, the
sequence shown under SEQ ID NO: 1 is identical to the barley RacB
sequence.
Example 4
Reverse Transcription Polymerase Chain Reaction (RT-PCR)
[0439] The "One Step RT-PCR Kit" (Qiagen, Hilden, Germany) was used
for the semi-quantitative RT-PCR. In doing so, RNA (prepared as
above) was first translated into cDNA (reverse transcription) and
the sought cDNA was amplified in a subsequent PCR reaction using
specific primers. To estimate the initial amount of template RNA,
the amplification was interrupted during the exponential phase in
order to reflect differences in the target RNA. The PCR products
were separated by means of an agarose gel, denatured, blotted onto
nylon membranes, and detected with specific non-radiolabeled probes
under stringent standard conditions. Hybridization, wash steps and
immunodetection were carried out as described under "Northern
blot".
[0440] The following components were combined for the individual
reactions (25 .mu.l batch) using the "One Step RT-PCR Kit" (Qiagen,
Hilden, Germany):
[0441] 1000 ng total RNA of a specific sample
[0442] 0.4 mM dNTPs
[0443] 0.6 .mu.M of each sense and antisense primer
[0444] 0.1 .mu.l RNase inhibitor
[0445] 1 .mu.l enzyme mix in 1.times.RT buffer
[0446] cDNA synthesis (reverse transcription) was carried out for
30 minutes at 50.degree. C. The reverse transcriptase was
subsequently inactivated for 15 minutes at 95.degree. C., which
simultaneously causes activation of DNA polymerase and denaturation
of cDNA. A PCR was subsequently carried out with the following
program:
TABLE-US-00039 denaturation for 1 minute at 94.degree. C. 25 cycles
of 1 minute at 54.degree. C. primer annealing 1 minute at
72.degree. C. primer extension 10 minutes at 72.degree. C.
completion of the DNA duplexes then: termination of the reaction at
4.degree. C.
[0447] The PCR products were separated in a 1.times.TBE agarose gel
using ethidium bromide.
[0448] The following oligonucleotide primer pairs were used for the
amplifications in the individual batches:
[0449] a) amplification of a 387 bp fragment of the barley RacB
cDNA
TABLE-US-00040 RacB sense 5'-gttcatcaagtgcgtcaccgtg-3' (SEQ ID NO:
15) RacB antisense 5'-ttagcttcctcagttcttccctg-3' (SEQ ID NO:
16)
[0450] b) amplification of a 674 bp fragment of barley BAS cDNA
(GenBank Acc. No. Z34917)
TABLE-US-00041 BAS sense 5'-cgcgccgcagccgagtacgac-3' (SEQ ID NO:
17) BAS antisense 5'-gtcacaaaaacacatgtaacc-3' (SEQ ID NO: 18)
[0451] c) amplification of a 506 bp OXLP cDNA fragment (GenBank
Acc. No. X93171)
TABLE-US-00042 OXLP sense 5'-ggccgacatgcattcaccag-3' (SEQ ID NO:
19) OXLP antisense 5'-catctgatattgctgggtctg-3' (SEQ ID NO: 20)
[0452] d) amplification of a 513 bp Ubi cDNA fragment (GenBank
accession M60175)
TABLE-US-00043 UBI sense 5'-ccaagatgcagatcttcgtga-3' (SEQ ID NO:
21) UBI antisense 5'-ttcgcgataggtaaaagagca-3' (SEQ ID NO: 22)
[0453] All of the fragments obtained were additionally ligated into
the vector PGEM-T by means of T-overhang ligation and were used as
starting plasmids for the generation of probes (for example for
Northern blots) or dsRNA. The individual constructs were named
pGEMT-RAC1, PGEMT-BAS, pGEMT-OXLP, pGEMT-UBI.
Example 5
Northern Blot Analysis
[0454] To prepare the Northern blotting, the RNA was separated in
an agarose gel under denaturing conditions. To this end, part of
the RNA solution (corresponding to 5 .mu.g of RNA) was mixed with
an identical volume of sample buffer (with ethidium bromide),
denatured for 5 minutes at 94.degree. C., placed on ice for 5
minutes, centrifuged briefly and applied to the gel. The
1.times.MOPS gel (1.5% agarose, ultra pure grade) contained 5
percent by volume of concentrated formaldehyde solution (36.5%
[v/v]). The R(NA was separated for 2 hours at 100 V and
subsequently blotted.
[0455] Northern blotting was done as an upward capillary RNA
transfer. To this end, the gel was first agitated gently for 30
minutes in 25 mM sodium hydrogen/dihydrogen phosphate buffer (pH
6.5) and cut to size. A piece of Whatman paper was prepared in such
a way that it rested on a horizontal support and extended on 2
sides into a trough with 25 mM sodium hydrogen/dihydrogen phosphate
buffer (pH 6.5). This piece of paper was covered with the gel,
uncovered parts of the piece of Whatman paper being covered with a
plastic film. The gel was then covered with a positively charged
nylon membrane (Boehringer-Mannheim), avoiding air bubbles,
whereupon the membrane was recovered to a height of approximately 5
cm with a stack of blotting paper. The blotting paper was
additionally weighed down with a sheet of glass and with a 100 g
weight. Blotting was carried out overnight at room temperature. The
membrane was rinsed briefly in twice-distilled water and irradiated
with UV light in a crosslinking apparatus (Biorad) with a light
energy of 125 mJ in order to immobilize the RNA. The uniformity of
the RNA transfer to the membrane was checked on a UV light
bench.
[0456] To detect barley mRNA, 10 .mu.g of total RNA from each
sample were resolved in an agarose gel and blotted onto a
positively charged nylon membrane by capillary transfer. Detection
was effected using the DIG system.
[0457] Probe preparation: Digogygenin- or fluorescein-labeled RNA
probes were prepared for hybridization with the mRNAS to be
detected. The probes were generated by in-vitro transcription of a
PCR product by means of a T7 or SP6 RNA polymerase, using labeled
UTPs. The template for the PCR-aided amplification was provided by
the above-described plasmid vectors pGEMT-RAC1, pGEMT-BAS,
pGEMT-OXLP, PGEMT-UBI.
[0458] Depending on the orientation of the insert, different RNA
polymerases were used for generating the antisense strand. T7 RNA
polymerase was used for PGEMT-BAS and PGEMT-OXLP, while SP6-RNA
polymerase was used for pGEMT-RAC1 and pGEMT-UBI.
[0459] The insert of the individual vector was amplified via PCR
using flanking standard primers (M13 fwd and rev). The reaction
proceeded with the following end concentrations in a total volume
of 50.mu.l of PCR buffer (Silverstar):
TABLE-US-00044 M13-fwd: 5'-GTAAAACGACGGCCAGTG-3' (SEQ ID NO: 23)
M13-rev: 5'-GGAAACAGCTATGACCATG-3' (SEQ ID NO: 24)
[0460] 10% dimethyl sulfoxide (v/v)
[0461] 2 ng/.mu.l of each primer (M13 forward and reversed)
[0462] 1.5 mM MgCl.sub.2,
[0463] 0.2 mM dNTPs,
[0464] 4 units Taq polymerase (Silverstar),
[0465] 2 ng/.mu.l plasmid DNA.
[0466] The amplification was carried out in a Thermocycler
(Perkin-Elmar 2400) with the following temperature program:
TABLE-US-00045 94.degree. C. denaturing for 3 minutes 30 cycles of
30 seconds at 94.degree. C. (denaturing) 30 seconds at 58.degree.
C. (annealing), 1.2 minutes at. 72.degree. C. (extension),
72.degree. C. final extension for 5 minutes 4.degree. C. cooling
until further use
[0467] The success of the reaction was verified in a 1% agarose
gel. The products were subsequently purified using a "High Pure
PCR-Product Purification Kit" (Boehringer-Mannheim). This gave
approximately 40 .mu.l of column eluate, which was again verified
in the gel and stored at -20.degree. C.
[0468] The RNA polymerization, the hybridization and the
immunodetection were carried out largely following the kit
manufacturer's instructions regarding the nonradioactive RNA
detection (DIG System User's Guide, DIG-Luminescence detection Kit,
Boehringer-Mannheim, Kogel et al. (1994) Plant Physiol
106:1264-1277). 4 .mu.l of purified PCR product were treated with 2
.mu.l of transcription buffer, 2 .mu.l of NTP labeling mix, 2 .mu.l
of NTP mix and 10 .mu.l of DEPC water. Then, 2 .mu.l of the T7 RNA
polymerase solution were pipetted in. The reaction was then carried
out for 2 hours at 37.degree. C. and then made up to 100 .mu.l with
DEPC water. The RNA probe was detected in an ethidium bromide gel
and stored at -20.degree. C.
[0469] To prepare the hybridization, the membranes were first
agitated gently for 1 hour at 68.degree. C. in 2.times.SSC (salt,
sodium citrate), 0.1% SDS buffer (sodium dodecyl sulfate), the
buffer being renewed twice or 3 times. The membranes were
subsequently applied to the internal wall of hybridization tubes
preheated at 68.degree. C. and incubated for 30 minutes with 10 ml
of Dig-Easy hybridization buffer in a preheated hybridization oven.
In the meantime, 10 .mu.l of probe solution were denatured for 5
minutes at 94.degree. C. in 80 .mu.l of hybridization buffer, and
the mixture was subsequently placed on ice and centrifuged briefly.
For the hybridization, the probe was then transferred into 10 ml of
hybridization buffer at a temperature of 68.degree. C., and the
buffer in the hybridization tube was replaced by this probe buffer.
Hybridization was then carried out overnight, likewise at
68.degree. C.
[0470] Prior to the immunodetection of RNA-RNA hybrids, the blots
were washed twice under stringent conditions for in each case 20
minutes in 0.1% (w/v) SDS, 0.1.times.SSC at 68.degree. C.
[0471] For the immunodetection, the blots were first agitated twice
for 5 minutes in 2.times.SSC, 0.1% SDS at RT. 2 stringent wash
steps were subsequently carried out for in each case 15 minutes at
68.degree. C. in 0.1.times.SSC, 0.1% SDS. The solution was then
replaced by wash buffer without Tween. The reaction mix was shaken
for 1 minute and the solution was exchanged for blocking reagent.
After a further 30 minutes' shaking, 10 .mu.l of anti-fluorescein
antibody solution were added, and shaking was continued for 60
minutes. This was followed by two 15-minute wash steps in
Tween-containing wash buffer. The membrane was subsequently
equilibrated for 2 minutes in substrate buffer and, after being
left to drain, transferred to a sheet of acetate paper. A mixture
of 20 .mu.l CDP-Star.TM. and 2 ml of substrate buffer was then
divided uniformly on the "RNA side" of the membrane. The membrane
was subsequently covered with a second sheet of acetate paper and
the edges were heat-sealed to provide a water-tight seal, avoiding
air bubbles. In a dark room, the membrane was then covered for 10
minutes with an X-ray film and the film was subsequently developed.
The exposure time was varied as a function of the luminescence
reaction.
[0472] Unless otherwise specified, the solutions were part of the
kit as delivered (DIG-Luminescence detection Kit,
Boehringer-Mannheim). All the others were prepared from the
following stock solutions by dilution with autoclaved distilled
water. Unless otherwise specified, all the stock solutions were
made with DEPC (like DEPC water) and subsequently autoclaved.
[0473] DEPC water: distilled water is treated overnight at
37.degree. C. with diethyl pyrocarbonate (DEPC, 0.1%, w/v) and
subsequently autoclaved. [0474] 10.times.MOPS buffer: 0.2 M MOPS
(morpholine-3-propanesulfonic acic), 0.05 M sodium acetate, 0.01 M
EDTA, pH brought to 7.0 with 10 M NaOH. [0475] 20.times.SSC (sodium
chloride/sodium citrate, salt/sodium citrate): 3 M NaClo, 0.3 M
trisodium citrate.times.2H.sub.2O, pH brought to 7.0 with 4 M HCl.
[0476] 1% SDS (sodium dodecyl sulfate) sodium dodecyl sulfate
(w/v), without DEPC. [0477] RNA sample buffer: 760 .mu.l formamide,
260 .mu.l formaldehyde, 100 .mu.l ethidium bromide (10 mg/ml), 80
.mu.l glycerol, 80 .mu.l bromophenol blue (saturated), 160 .mu.l
10.times.MOPS, 100 .mu.l water. [0478] 10.times. wash buffer
without Tween: 1.0 M maleic acid, 1.5 M NaCl; without DEPC, bring
to pH 7.5 with NaOH (solid, approx. 77 g) and 10 M NaOH. [0479]
Tween-containing wash buffer: made by adding Tween to wash buffer
without Tween (0.3%, v/v). [0480] 10.times. blocking reagent:
suspend 50 g of blocking powder (Boehringer-Mannheim) in 500 ml of
wash buffer without Tween. [0481] Substrate buffer: bring 100 mM
Tris (trishydroxymethylaminomethane)-, 150 mM NaCl to pH 9.5 with 4
M HCl. [0482] 10.times. color marker: 50% glycerol (v/v), 1.0 mM
EDTA pH 8.0, 0.25% bromophenol blue (w/v), 0.25% xylene cyanole
(w/v).
Example 6
In Vitro Synthesis of the RacB dsRNA
[0483] All of the plasmids (pGEMT-RAC1, pGEMT-BAS, pGEMT-OXLP,
pGEMT-UBI) which were employed for in-vitro transcription comprise
the T7 and SP6 promoters (pGEM-T, Promega) at the respective ends
of the nucleic acid sequence inserted, which makes possible the
synthesis of sense or antisense RNA. The plasmids can be linearized
with suitable restriction enzymes in order to ensure correct
transcription of the nucleic acid sequence inserted and to prevent
reading being continued into vectorial sequences.
[0484] To this end, 10 .mu.g of plasmid DNA were cleaved with in
each case at the side of the insert which was located distally from
the promoter. The cleaved plasmids are extracted in 200.mu.l of
water with an identical volume of phenol/chloroform/isoamyl
alcohol, transferred into a new Eppendorf reaction vessel
(RNAse-free) and centrifuged for 5 minutes at 20 000 g. 180 .mu.l
of the plasmid solution were treated with 420 .mu.l of ethanol, and
the mixture was placed on ice and subsequently precipitated by
centrifugation for 30 minutes at 20 000 g and -4.degree. C. The
precipitate was taken up in 10 .mu.l of TE buffer.
[0485] To prepare the RacB dsRNA, the plasmid pGEMT-Rac1 was
digested with SpeI, and sense RNA was transcribed using T7 RNA
polymerase.
[0486] Furthermore, pGEMT-Rac1 was digested with NcoI, and
antisense RNA was transcribed using SP6 RNA polymerase. RNA
polymerases were obtained from Roche Molecular Biology, Mannheim,
Germany.
[0487] Each transcription reaction contained the following in a
volume of 40 .mu.l: [0488] 2 .mu.l of linearized plasmid DNA (1
.mu.g) [0489] 2 .mu.l of NTPs (25 mM) (1.25 mM of each NTP) [0490]
4 .mu.l of 10.times. reaction buffer (Roche Molecular Biology),
[0491] 1 .mu.l of RNAsin RNAsin (27 units; Roche Molecular
Biology), [0492] 2 .mu.l of RNA polymerase (40 units) [0493] 29
.mu.l of DEPC water
[0494] Following incubation for 2 hours at 37.degree. C., part of
the reactions from the transcription of the sense and antisense
strand, respectively, were mixed, denatured for 5 minutes at
95.degree. C. and subsequently hybridized (annealed) by cooling to
a final temperature of 37.degree. C. over 30 minutes.
Alternatively, it is also possible first to denature the mixture of
sense and antisense strand and then to cool it for 30 minutes at
-20.degree. C. The protein precipitate which formed during
denaturing and hybridization was removed by briefly centrifuging
the reaction at 20 800 g, and the supernatant was used directly for
coating tungsten particles (see hereinbelow). For analysis, 1 .mu.l
of each RNA strand and of the dsRNA were resolved on a
nondenaturing agarose gel. Successful hybridization manifested
itself by a band shift toward higher molecular weight in comparison
with the single strands.
[0495] 4 .mu.l of the dsRNA were ethanol-precipitated (by adding 6
.mu.l of water, 1 .mu.l of 3M sodium acetate solution and 25 .mu.l
of ethanol, and centrifugation for at least 5 minutes at 20 000 g
and 4.degree. C.) and the pellet was resuspended in 500 .mu.l of
water. The absorption spectrum between 230 and 300 nm was measured
or the absorption at 280 and 260 nm was determined in order to
determine the purity and concentration of the dsRNA. As a rule, 80
to 100 .mu.l of dsRNA with an OD.sub.260/OD.sub.280 ratio of 1.80
to 1.95 were obtained. Digestion with DNase I can optionally be
carried out, but has no significant effect on the results which
follow.
[0496] The control dsRNA used was the human thyroid receptor dsRNA
(starting vector pT7betaSal (Norman C et al. (1988) Cell
55(6):989-1003), provided by Dr. Baniahmad, Department of Genetics,
Gie.beta.en, Germany; the sequence of the insert is described under
the GenBank Ace. No.: NM.sub.--000461). To generate the sense RNA,
the plasmid was digested with PvuII, to generate the antisense RNA,
it was digested with HindIII, and the RNA was then transcribed with
T7 and SP6 RNA polymerase, respectively. The individual process
steps for generating the control dsRNA are carried out analogously
to those described above for the RacB dsRNA.
Example 7
Transient Transformation, RNAi, and Evaluation of the Development
of the Fungal Pathogen
[0497] Barley cv. Pallas leaf segments were transformed with an
RacB dsRNA together with a GFP expression vector. The leaves were
subsequently inoculated with Bgh, and the result was analyzed after
48 hours by light and fluorescence microscopy. The penetration into
GFP-expressing cells was assessed by detecting haustoria in live
cells and by assessing the fungal development on precisely these
cells. In all six experiments, bombardment of barley cv. Pallas
with RacB dsRNA resulted in a reduced number of successfully
Bgh-penetrated cells in comparison with cells which had been
bombarded with a foreign control dsRNA (human thyroid hormone
receptor dsRNA, TR). The resistance-inducing effect of the Racy
dsRNA caused an average reduction in Bgh penetration efficacy by
44% (FIG. 4).
[0498] A method was employed for the transient transformation which
had already been described for the biolistic introduction of dsRNA
into epidermal cells of barley leaves (Schweizer P et al. (1999)
Mol Plant Microbe Interact 12:647-54; Schweizer P et al. (2000)
Plant J 2000 24: 895-903). Tungsten particles with a diameter of
1.1 .mu.m (particle density 25 mg/ml) were coated with dsRNA
(preparation see above) together with plasmid DNA of the vector
pGFP (GFP under the control of the CaMV 35S promoter) as
transformation marker. For each bombardment, the following amounts
of dsRNA and reporter plasmid were used for coating: 1 .mu.g of
pGFP and 2 .mu.g of dsRNA. Double-stranded RNA was synthesized in
vitro by annealing sense and antisense RNA (see above).
[0499] To prepare microcarriers, 55 mg of tungsten particles (M 17,
diameter 1.1 .mu.m; Bio-Rad, Munich) were washed twice with 1 ml of
autoclaved distilled water and once with 1 ml of absolute ethanol,
dried and taken up in 1 ml of 50% strength glycerol (approx. 50
mg/ml stock solution). The solution was diluted to 25 mg/ml with
50% strength glycerol, mixed thoroughly prior to use, and suspended
in an ultrasonic bath. To coat the microcarriers for each
bombardment, 1 .mu.g of plasmid, 2 .mu.g of dsRNA (1 .mu.l), 12.5
.mu.l of tungsten particle suspension (25 mg/ml), 12.5 .mu.l of 1 M
Ca(NO.sub.3).sub.2 solution (pH 10) were combined dropwise with
constant mixing, the mixture was left to stand for 10 minutes at RT
and then briefly centrifuged, and 20 .mu.l of the supernatant were
drawn off. The remainder with the tungsten particles is resuspended
(ultrasonic bath) and employed in the experiment.
[0500] Segments (approx. 4 cm in length) of barley primary leaves
were used. The tissue was placed on 0.5% Phytagar (GibcoBRL.TM.
Life Technologies.TM., Karlsruhe) supplemented with 20 .mu.g/ml
benzimidazole in Petri dishes (diameter 6.5 cm), and the edges were
coveted directly prior to particle bombardment with a stencil
provided with a rectangular opening of 2.2 cm.times.2.3 cm. One
after the other, the dishes were placed on the bottom of the vacuum
chamber (Schweizer P et al. (1999) Mol Plant Microbe Interact
12:647-54) over which a nylon mesh (mesh size 0.2 mm, Millipore,
Eschborn) on an apertured plate had been inserted (5 cm above the
bottom, 11 cm underneath the macrocarrier, see hereinbelow) to act
as diffuser in order to disperse particle aggregates and to slow
down the particle stream. For each bombardment, the macrocarrier
(plastic syringe filter holder, 13 mm, Gelman Sciences, Swinney,
UK), which was attached at the top of the chamber, was loaded with
5.8 .mu.l of DNA-coated tungsten particles (microcarrier, see
hereinbelow). The pressure in the chamber was reduced by 0.9 bar
using a diaphragm vacuum pump (Vacuubrand, Wertheim), and the
surface of the plant tissue was bombarded with the tungsten
particles at a helium gas pressure of 9 bar. The chamber was
aerated immediately thereafter. To label transformed cells, the
leaves were bombarded with the plasmid (pGFP; vector pUC18-based,
CaMV 35S promoter/terminator cassette with inserted GFP gene;
Schweizer P et al. (1999) Mol Plant Microbe Interact 12:647-54;
provided by Dr. P. Schweizer Schweizer P, Institut fur
Pflanzengenetik [Department of Plant Genetics] IPK, Gatersleben,
Germany). Each time before another plasmid was used for the
bombardment, the macrocarrier was cleaned thoroughly with water.
Following incubation for four hours after the bombardment with
slightly open Petri dishes, RT and daylight, the leaves were
inoculated with 100 conidia/mm.sup.2 of the barley powdery mildew
fungus (race A6) and incubated for a further 36 to 48 hours under
identical conditions.
[0501] Leaf segments were bombarded with the coated particles using
a particle inflow gun. 312 .mu.g of tungsten particles were applied
per bombardment. 4 hours after bombardment, the leaf segments were
inoculation inoculated with Blumeria graminis f.sp. hordei mildew
(race A6) and, after a further 40 hours, evaluated with regard to
infection symptoms. The result (for example the penetration
efficacy, defined as percentage of attacked cells with a mature
haustorium and a secondary hypha (secondary elongating hyphae) was
analyzed by fluorescence and light microscopy. Inoculation with 100
conidia/mm.sup.2 results in an attack frequency of approximately
50% of the transformed cells. A minimum of 100 interaction sites
were evaluated for each individual experiment. Transformed
(GFP-expressing) cells were identified under excitation with blue
light. Three different categories of transformed cells were
distinguished:
[0502] 1. Penetrated cells comprising a readily recognizable
haustorium. A cell with more than one haustorium counted as one
cell.
[0503] 2. Cells which were attacked by a fungal appressorium, but
comprise no haustorium. A cell which was attacked repeatedly by
Bgh, but contains no haustorium, counted as one cell.
[0504] 3. Cells which are not attacked by Bgh.
[0505] Stomatal cells and subsidiary cells were excluded from the
evaluation. Surface structures of Bgh were analyzed by light
microscopy or fluorescent staining of the fungus with 0.1%
Calcofluor (w/v in water) for 30 sec. The fungal development can be
evaluated readily by staining with Calcofluor followed by
fluorescence microscopy. While the fungus develops a primary germ
tube and an appressorial germ tube in cells transformed with RacB
dsRNA, it fails to develop a haustorium. The development of
haustoria is a precondition for the development of a secondary
hyphae.
[0506] The relative penetration efficacies (RPEs) were calculated
as the difference between the penetration efficacies in transformed
cells (transformation with RacB dsRNA or control dsRNA) and the
penetration efficacies of untransformed cells (here: average
penetration efficacy 57%). The percentage RPE (%-RPE) is calculated
from the RPE minus 1, multiplied by 100.
RPE = [ PE of cells transformed with RacB - dsRNA ] [ PE of cells
transformed with control dsRNA ] ##EQU00002## % - RPE = 100 * ( RPE
- 1 ) ##EQU00002.2##
[0507] The %-RPE value (deviation from the average penetration
efficacy of the control) serves to determine the susceptibility of
cells transfected with RacB dsRNA (FIG. 4).
[0508] In the case of the control dsRNA, no difference between the
transfection with the control dsRNA and with water was found in
five independent experiments with regard to the penetration
efficacy of Bgh.
[0509] The deviation of the PE in various genotypes was also
studied. To demonstrate the operable linkage with the Mlo gene, an
mlo5 genotype (A89, mlo5 ror1, background: Ingrid), which owing to
a mutation of the Ror1 gene has only moderate susceptibility to Bgh
attack (Freialdenhoven et al. (1996) Plant cell 8:5-14), was
employed. In this doubly-mutant genotype, the efficacy of RacB
dsRNA was studied in comparison with a wild-type Mlo genotype.
However, no prevention of the development of haustoria in A89 was
observed in five independent experiments, while in parallel
experiments with Pallas and Ingrid the PE was markedly reduced
(FIG. 5). Interestingly, the effect of the RacB dsRNA was more
pronounced in Pallas than in Ingrid (FIG. 5, Experiments 1 and 2 in
comparison with 3, 4 and 5).
[0510] To rule out an effect of the dsRNA on the transformation
rate or survival rate of the attacked cells, the number of
GFP-expressing cells was compared between control and RacB dsRNA
experiments (Table 7). The RacB dsRNA had no effect on the total
number, or the number of attacked, GFP-expressing cells.
TABLE-US-00046 TABLE 7 Transformation rates of barley leaves
following bombardment with dsRNA Number of GFP - expressing cells
per bombardment.sup.a Total Attacked Total Attacked (Control
(Control (RacB (RacB Line dsRNA) dsRNA) dsRNA) dsRNA) n.sup.c
Pallas 34.3 .+-. 4.6 16.0 .+-. 2.2 33.9 .+-. 4.8 15.5 .+-. 1.4 6
(21) (Mlo Ror1) Ingrid 51.0 .+-. 8.9 27.6 .+-. 8.7 49.9 .+-. 5.6
31.5 .+-. 7.8 3 (11) (Mlo Ror1) A 89 34.3 .+-. 5.4 18.1 .+-. 4.0
34.1 .+-. 5.5 16.7 .+-. 3.8 5 (22) (mlo5 ror1) .sup.a4 leaves were
bombarded per bombardment. The data shown are means and standard
error. .sup.cNumber of independent experiments (bombardments n in
each case for control and RacB dsRNA).
Example 8
Constitutively Active RACB Mutant
[0511] A putatively constitutively active barley RACB mutant
(substitution G->V at position 15; RacB-V15) was generated and
overexpressed in the barley variety Pallas in order to positively
identify RACB as susceptibility factor. First, full-length RACB was
synthesized via RT-PCR. The following oligonucleotide primers were
employed for this purpose:
TABLE-US-00047 RacB5'BamHI: 5'-GGATCCGATGAGCGCGTCCAGGTT-3' (SEQ ID
NO: 31) RacB3'SalI: 5'-GTCGACCTTCGCCCTTGTTCTTTGTC-3' (SEQ ID NO:
32)
[0512] The cDNA was cloned into PGEM-T and subsequently excised via
the primer cleavage sites and cloned into pGY-1 (Schweizer P et al.
(1999) Mol Plant Microbe Interact 12: 647-54; FIG. 7) via
BamHI/SalI cleavage sites. The construct is referred to as
pGY1-RacB.
[0513] The nucleic acid sequence encoding the constitutively active
RacB mutant RACB-V15 was generated using the
"Transformer.TM.Site-Directed Mutagenesis Kit" (Clonetech,
Heidelberg), following the manufacturer's instructions. The
starting vector employed was pGY1-RacB. The following
oligonucleotide was used as mutagenesis primer:
TABLE-US-00048 (SEQ ID NO: 33) V15:
5'-ACCGTGGGGGACGTCGCCGTCGG-CAAGAC-3'
[0514] Then, RACB-V15 was then overexpressed transiently in 5
independent experiments in the barley variety Pallas under the
control of the .sup.35S CamV promoter. The experiments were carried
out as described by Schultheiss et al. (Schultheiss H et al. (2002)
Plant Physiol 128:1447-1454), except that, after the particle
bombardment, 24 hours instead of 4 hours elapsed prior to
inoculation. The particles were coated as described in Schweizer et
al. (Schweizer P et al. (1999) Mol Plant Microbe Interact
12:647-54).
[0515] The expression of a constitutive RacB mutant brings about a
significantly increased susceptibility to pathogen attack by
powdery mildew of barley in comparison with the controls. Again,
these results confirm the key function of RacB in the defense of
pathogens. The RACB-V15 effects. (see FIG. 6-A/B) are significant
in the t-test when a two-tail paired test is carried out. The
relative susceptibility to the fungal pathogen is increased in all
cases (FIG. 6-B).
Example 9
Further HvRac Homologs
[0516] All full-length sequences were isolated from RNA using
specific primers and cloned into PGEM-T and sequenced (Huckelhoven
et al. (2001) Plant Mol Biol; Schultheiss et al. (2002) Plant
Physiol 128:1447-1454). In some cases, the sequences are very
similar to RacB.
TABLE-US-00049 a) HvRop6: LEFT PRIMER 5'-GTGGAGGCGCGGCGAGA-3' (SEQ
ID NO: 25) RIGHT PRIMER 5'-CCATGCTTCATCTCCATAGTCA-3' (SEQ ID NO:
26) b) HvRacD: LEFT PRIMER 5'-ggatccCGATTCCATCAGGAAAGCAT-3' (SEQ ID
NO: 27) RIGHT PRIMER 5'-gtcgacGCGAGACACTGCAAAACAAA-3' (SEQ ID NO:
28) c) HvRop4: LEFT PRIMER 5'-GGATCCttctcgtccatttagccggc-3' (SEQ ID
NO: 29) RIGHT PRIMER 5'-GTCGACtgatcacttgaagcatgccag-3' (SEQ ID NO:
30)
Example 10
Generation of Sense and Antisense Constructs with the Gene AtRacB
for Expression in Arabidopsis thaliana
[0517] A fragment of the Arabidopsis RacB homolog (MIPS-Code:
AT4g35950; SEQ ID NO: 53; hereinbelow AtRacB) is isolated via PCR
from an Arabidopsis thaliana cDNA library. The primer sequences
used are:
TABLE-US-00050 Fra 186: 5'-ATGAGCGCGTCCAGGTTCATA-3' (SEQ ID NO: 71)
Fra 187: 5'-ATCAAACACGCCCTTCACGTT-3' (SEQ ID NO: 72)
[0518] The amplification proceeds in a T3 thermocycler from
Biometra with the following temperature profile.
[0519] 35 cycles of 1 min at 95.degree. C., 0.5 min at 59.degree.
C. and 3 min at 72.degree. C. Subsequent extension for 5 min at
72.degree. C.
[0520] The PCR products is cloned into the vector pCR2.1 (in
accordance with the pCR Script Cloning Kit, Stratagene, Heidelberg)
following the manufacturer's instructions. A fragment is excised
from the vector construct via the restriction enzyme EcoRI (Roche,
Mannheim). The fragment can be isolated via gel electrophoresis and
subsequent purification using anion exchanger columns (QIAex
Purification Kit, Qiagen, Hilden). Accordingly, the binary vector
pSUN3-Nit is opened up via the enzymes XmaI and EcoRI and subjected
to purification by means of gel electrophoresis followed by elution
over anion exchanger columns (QIAex Purification Kit, Qiagen,
Hilden).
[0521] Since blunt ends have to be generated for the cloning of
insert and vector with 5'-overhangs, both the eluted AtRacB
fragment and the eluted pSUN3-Nit fragment are treated with 2 .mu.l
of dNTP mix I (10 mM each of dATP, dCTP, dCTP, dTTP; Pharmacia,
Freiburg) and 1.6 .mu.l of Klenow fragment (USB/Amersham,
Braunschweig, 2 U/.mu.l) for filling up the overhang and incubated
for 30 min at 37.degree. C. To prevent religation, the vectors are
first purified via a QIAquick Spin Column (Qiagen, Hilden), treated
with CIAP (Calf Intestinal Alkaline Phosphatase, GibcoBRL,
Eggenstein, 1 U/.mu.l) and finally purified via a 0.8% strength
agarose gel.
[0522] To prepare the following ligation mix in a total volume of
50 .mu.l, 34 .mu.l of H.sub.2O, 5 .mu.l of ligation buffer and 1
.mu.l of T4 ligase (Roche, Mannheim) are additionally added to the
10 .mu.l of cut DNA. This mixture is incubated overnight at
16.degree. C. The ligase is subsequently inactivated for 10 min at
65.degree. C. The ligation is now followed by precipitation with
0.1 volume of sodium acetate (pH 5.2) and 2.5 volumes of ethanol.
After centrifugation (30 min, 15 000 g, 4.degree. C.), the pellet
is dried in 70% ethanol and resuspended in 10 .mu.l of H.sub.2O.
2.mu.l of this pellet are transformed by electroporation (E. coli
Pulser, Bio-Rad) into Escherichia coli bacteria, strain DH5.alpha..
The DNA-treated bacteria are plated onto LB plates supplemented
with the antibiotic ampicillin (50 mg/l). After incubation for 16 h
at 37.degree. C., bacteria are scraped from the colonies which have
grown and transferred into tubes containing 3 ml of LB-Amp liquid
medium each. After incubation for 16 h at 37.degree. C., the
cultures, which have grown to great density, are centrifuged.
Plasmid DNA is isolated from the bacterial pellets by means of the
QIAprep DNA miniprep kit (Qiagen, Hilden) following the
manufacturer's instructions and subjected to analytic digests with
various enzyme combinations. These control digests allow the
isolation of constructs in which the AtRacB gene is cloned in sense
or antisense orientation behind the of the A. thaliana nitrilase-1
(nit1) gene, which is constitutively active in plants (GenBank
Acc.-No.: Y07648.2, nucleotides 2456-4340, Hillebrand et al. (1996)
Gene 170:197-200). These constructs are named pSUN3NIT_AtRacB_s
(SEQ ID NO: 73) and pSUN3NIT atRacB_as (SEQ ID NO: 74) and used for
the transformation of Arabidopsis plants. The constructs comprise
the complete sequence of AtRacB, so that the expression vector
pSUN3NIT_HvRacB_s, which comprises the fragment in sense
orientation, is capable of expressing a functional AtRacB protein.
The vector acts primarily as negative control and leads in most
cases to reduced pathogen resistance, but in some cases (see
hereinbelow) also to an increase in pathogen resistance, presumably
via a cosuppression effect.
Example 11
Generation of Sense and Antisense Constructs with the Gene HvRacB
for Expression in Arabidopsis thaliana
[0523] Various nonfunctional fragments of the gene HvRacB are to be
prepared for expression in Arabidopsis plants. To this end, the
plasmid, which contains the HvRacB gene subcloned into the
bacterial vector PGEM-T, is digested with the enzyme combinations
BamHI/HindIII (Roche, Mannheim). Overhanging 5'-single strands are
filled up by treatment with Klenow polymerase in the presence of a
mixture of nucleotides (see above). The resulting HvRacB fragment
with these blunt ends is cloned directly into a pSUN3NIT vector,
which is opened up with the enzymes BglII and SpeI (Roche,
Mannheim) in its multiple cloning site and whose 5'-overhangs are
filled up by means of treatment with Klenow polymerase (as
described above). For the ligation batches, each of which has a
total volume of 50 .mu.l, 34 .mu.l of H.sub.2O, 5 .mu.l of ligation
buffer and 1 .mu.l of T4 ligase (Roche, Mannheim) are additionally
added to the 10 .mu.l of cut DNA. This mixture is incubated
overnight at 16.degree. C. The ligase is subsequently inactivated
for 10 min at 65.degree. C. The ligation is now followed by
precipitation with 0.1 volume of sodium acetate (pH 5.2) and 2.5
volumes of ethanol. After centrifugation (30 min, 15 000 g,
4.degree. C.), the pellet is dried in 70% ethanol and resuspended
in 10 .mu.l of H.sub.2O. 2 .mu.l of this pellet are transformed by
electroporation (E. coli Pulser, Bio-Rad) into Escherichia coli
bacteria, strain DH5.alpha.. The DNA-treated bacteria are plated
onto LB plates supplemented with the antibiotic ampicillin (50
mg/l). After incubation for 16 h at 37.degree. C., bacteria are
scraped from the colonies which have grown and transferred into
tubes containing 3 ml of LB-Amp liquid medium each. After
incubation for 16 h at 37.degree. C., the cultures, which have
grown to great density, are centrifuged. Plasmid DNA is isolated
from the bacterial pellets by means of the QIAprep DNA miniprep kit
(Qiagen, Hilden) following the manufacturer's instructions and
subjected to analytic digests with various enzyme combinations.
These control digests allow the identification of constructs in
which the appropriate gene construct is cloned in sense or
antisense orientation behind the promoter of the A. thaliana
nitrilase-1 gene, which is constitutively active in plants (see
above). These constructs are named pSUN3NIT_HvRacB_s (SEQ ID NO:
75) and pSUN3NIT_HvRacB_as (SEQ ID NO: 76) and used for the
transformation of Arabidopsis plants. The constructs comprise a
truncated fragment of hvRacB, so that not even the expression
vector pSUN3NIT_HvRacB_s, which comprises the fragment in sense
orientation, is capable of expressing a functional HvRacB protein.
The vector acts primarily as negative control but leads in some
cases (see hereinbelow) also to an increase in pathogen resistance,
presumably via a cosuppression effect.
Example 12
Transformation of Arabidopsis thaliana, and Analysis of the Fungal
Resistance
[0524] Wild-type A. thaliana plants (Columbia) are with the
Agrabacterium tumefaciens strain (EHA105) based on a modified
method (Steve Clough and Andrew Bent (1998) Plant J 16(6):735-743)
of the vacuum infiltration method of Bechtold et al. (Bechtold N et
al. (1993) CR Acad Sci Paris, Life Sciences 316:1194-1199).
[0525] The A. tumefaciens cells used are previously transformed
with the plasmids pSUN3NIT AtRacB_s (SEQ ID NO: 73),
pSUN3NIT_atRacB_as (SEQ ID NO: 74), pSUN3NIT_HvRacB_s (SEQ ID NO:
75) and pSUN3NIT_HvRacB_as (SEQ ID NO: 76).
[0526] Seeds of the Agrobacterium-transformed primary transformants
are selected on the basis of their kanamycin resistance.
Antibiotic-resistant seedlings are planted in soil and, when grown
into fully developed plants, used for biochemical analysis.
[0527] To analyze the resistance of the transgenic Arabidopsis
plants to pathogenic fungi, inoculations with the biotrophic fungi
Peronospora parasitica and Erysiphe cichoracearum are
performed.
[0528] a) Peronospora parasitica
[0529] Plants aged 5 to 8 weeks are sprayed with a conidia spore
suspension (approx. 106 spores/ml). The inoculated plants are kept
overnight in a refrigerator at approximately 16.degree. C. under
dark and damp conditions, being covered with a plastic bag. After
one day, the plastic bag is opened slightly and later removed
completely. Six days after inoculation, the plants are again
covered with the plastic bag overnight, whereby sporulation is
induced. On the next day, the leaves are examined for the
appearance of conidiophores. Over the next days, the intercellular
growth of the fungus leads to the induction of weak chloroses up to
severe necroses in the leaves. These symptoms are quantified and
tested for significance.
[0530] b) Erysiphe cichoracearum
[0531] The biotrophic mildew fungus is grown on Arabidopsis plants.
To infect the 4-week-old transgenic RacB-expressing Arabidopsis
plants, conidiophores are removed from the leaf surface with a fine
brush and applied to the leaves of the transgenic plants. The
plants are incubated for 7 days at 20.degree. C. 7 days after the
inoculation, the conidiophores appear on the leaves, and chloroses
and necroses can be observed over the following days. These
symptoms are quantified and tested for significance.
[0532] c) Results
[0533] The transgenic Arabidopsis plants which express antisense
sequences for AtRacB or HvRacB are significantly more resistant to
Peronospora parasitica and to Erysiphe cichoracearum than
nontransgenic wild-type plants.
[0534] The transgenic Arabidopsis plants which express sense
sequences for the complete AtRacB are in are in most cases
significantly more susceptible to both Peronospora parasitica and
Erysiphe cichoracearum than nontransgenic wildtype plants. In some
cases, however, an increased resistance can also be observed
(presumably via a cosuppression effect).
[0535] Transgenic Arabidopsis plants which express sense sequences
for the an HvRacB fragment are in are in some cases significantly
more resistant to both Peronospora parasitica and Erysiphe
cichoracearum than nontransgenic wildtype plants.
Sequence CWU 1
1
761857DNAHordeum vulgareCDS(90)..(680)misc_feature(780)n is a, c, g
or t 1atcttaacca gctccctacc tccccttctt cttcctcctc ctcccctgtc
tcgcccgcag 60cttcaccggc agcgagggaa agagggagg atg agc gcg tcc agg
ttc ata aag 113Met Ser Ala Ser Arg Phe Ile Lys1 5tgc gtc acc gtg
ggg gac ggc gcc gtc ggc aag acc tgc atg ctc atc 161Cys Val Thr Val
Gly Asp Gly Ala Val Gly Lys Thr Cys Met Leu Ile10 15 20tcc tac acc
tcc aac acc ttc ccc acc gac tat gtg ccc acg gtg ttt 209Ser Tyr Thr
Ser Asn Thr Phe Pro Thr Asp Tyr Val Pro Thr Val Phe25 30 35 40gac
aac ttc agt gct aat gtt gtg gtt gat ggc aac act gtc aac ctt 257Asp
Asn Phe Ser Ala Asn Val Val Val Asp Gly Asn Thr Val Asn Leu45 50
55ggg cta tgg gat act gca ggt cag gaa gac tac aac aga ctg aga ccg
305Gly Leu Trp Asp Thr Ala Gly Gln Glu Asp Tyr Asn Arg Leu Arg
Pro60 65 70ctg agt tat cgt gga gct gat gtc ttc ctt ctg gcc ttc tcg
ctt atc 353Leu Ser Tyr Arg Gly Ala Asp Val Phe Leu Leu Ala Phe Ser
Leu Ile75 80 85agc aag gct agc tat gag aat gtt tca aag aag tgg ata
cct gaa ctg 401Ser Lys Ala Ser Tyr Glu Asn Val Ser Lys Lys Trp Ile
Pro Glu Leu90 95 100aag cat tat gca cca ggt gtg cct att atc ctc gtg
gga aca aag ctt 449Lys His Tyr Ala Pro Gly Val Pro Ile Ile Leu Val
Gly Thr Lys Leu105 110 115 120gat ctt cga gat gac aag cag ttc ttt
gtg gac cat cct ggt gct gtt 497Asp Leu Arg Asp Asp Lys Gln Phe Phe
Val Asp His Pro Gly Ala Val125 130 135cct atc act act gct cag ggg
gag gaa cta aaa aag tta ata ggc gca 545Pro Ile Thr Thr Ala Gln Gly
Glu Glu Leu Lys Lys Leu Ile Gly Ala140 145 150ccc tac tac atc gaa
tgc agc tcg aag acc caa cta aat gtc aag ggt 593Pro Tyr Tyr Ile Glu
Cys Ser Ser Lys Thr Gln Leu Asn Val Lys Gly155 160 165gta ttt gat
gcg gca ata aag gtg gta ctg cag cca cca aag gca aag 641Val Phe Asp
Ala Ala Ile Lys Val Val Leu Gln Pro Pro Lys Ala Lys170 175 180aag
aag aaa aag gcg cag agg ggg gct tgc tcc atc ttg tgatctaatc 690Lys
Lys Lys Lys Ala Gln Arg Gly Ala Cys Ser Ile Leu185 190
195aatcggtaga caaagaacaa gggcgaagtt gccgccatgc tatattattg
ttacgtcttg 750cttcagcgga gctgcactct catggtcgtn ctnccttccc
tcacccccac cccaccctag 810gttacccacc ggcagctgca acaaggtctc
tttgtcgagg catcggg 8572197PRTHordeum vulgare 2Met Ser Ala Ser Arg
Phe Ile Lys Cys Val Thr Val Gly Asp Gly Ala1 5 10 15Val Gly Lys Thr
Cys Met Leu Ile Ser Tyr Thr Ser Asn Thr Phe Pro20 25 30Thr Asp Tyr
Val Pro Thr Val Phe Asp Asn Phe Ser Ala Asn Val Val35 40 45Val Asp
Gly Asn Thr Val Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln50 55 60Glu
Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala Asp Val65 70 75
80Phe Leu Leu Ala Phe Ser Leu Ile Ser Lys Ala Ser Tyr Glu Asn Val85
90 95Ser Lys Lys Trp Ile Pro Glu Leu Lys His Tyr Ala Pro Gly Val
Pro100 105 110Ile Ile Leu Val Gly Thr Lys Leu Asp Leu Arg Asp Asp
Lys Gln Phe115 120 125Phe Val Asp His Pro Gly Ala Val Pro Ile Thr
Thr Ala Gln Gly Glu130 135 140Glu Leu Lys Lys Leu Ile Gly Ala Pro
Tyr Tyr Ile Glu Cys Ser Ser145 150 155 160Lys Thr Gln Leu Asn Val
Lys Gly Val Phe Asp Ala Ala Ile Lys Val165 170 175Val Leu Gln Pro
Pro Lys Ala Lys Lys Lys Lys Lys Ala Gln Arg Gly180 185 190Ala Cys
Ser Ile Leu19531113DNAOryza sativaCDS(158)..(748) 3ccttgctttg
ctcctccttc aaccttcttc tttcttggag tttcttgaga gagagagaga 60gagagagaga
gagagagaga gagagagaga ggggggggag cggtcgcagg aggaggagga
120cggcggcgtc tgctgcgacc gacggggagc ggcgagg atg agc gcg tcc agg ttc
175Met Ser Ala Ser Arg Phe1 5ata aag tgc gtc acc gtc ggg gac ggc
gcc gtc ggc aag acc tgc atg 223Ile Lys Cys Val Thr Val Gly Asp Gly
Ala Val Gly Lys Thr Cys Met10 15 20ctc atc tcc tac acc tcc aac acc
ttc ccc act gat tat gtt ccg acg 271Leu Ile Ser Tyr Thr Ser Asn Thr
Phe Pro Thr Asp Tyr Val Pro Thr25 30 35gtg ttt gac aac ttc agt gcc
aac gtc gtg gtt gat ggt aac acc gtc 319Val Phe Asp Asn Phe Ser Ala
Asn Val Val Val Asp Gly Asn Thr Val40 45 50aac ctc ggg cta tgg gac
act gca ggt cag gag gat tac aac aga ctg 367Asn Leu Gly Leu Trp Asp
Thr Ala Gly Gln Glu Asp Tyr Asn Arg Leu55 60 65 70aga cca ctg agt
tat cgt gga gct gat gtt ttc ctt ctg gcc ttc tcg 415Arg Pro Leu Ser
Tyr Arg Gly Ala Asp Val Phe Leu Leu Ala Phe Ser75 80 85cta atc agc
aag gcc agc tat gag aat gtt tca aag aag tgg ata cct 463Leu Ile Ser
Lys Ala Ser Tyr Glu Asn Val Ser Lys Lys Trp Ile Pro90 95 100gag ctg
aag cat tat gca cct ggt gtt cct atc atc ctt gtg gga aca 511Glu Leu
Lys His Tyr Ala Pro Gly Val Pro Ile Ile Leu Val Gly Thr105 110
115aag ctt gat ctt cga gat gac aag cag ttt ttt gtg gac cat cct ggt
559Lys Leu Asp Leu Arg Asp Asp Lys Gln Phe Phe Val Asp His Pro
Gly120 125 130gct gtt cct atc acc act gct cag gga gag gaa cta aga
aag caa ata 607Ala Val Pro Ile Thr Thr Ala Gln Gly Glu Glu Leu Arg
Lys Gln Ile135 140 145 150ggc gcc cca tac tac atc gaa tgc agc tca
aag acc caa cta aac gtc 655Gly Ala Pro Tyr Tyr Ile Glu Cys Ser Ser
Lys Thr Gln Leu Asn Val155 160 165aag ggc gtt ttc gat gcg gca ata
aag gtg gtg ctg cag cca ccc aag 703Lys Gly Val Phe Asp Ala Ala Ile
Lys Val Val Leu Gln Pro Pro Lys170 175 180gcg aag aag aag aaa aag
gcg caa agg ggg gcg tgc tcc att ttg 748Ala Lys Lys Lys Lys Lys Ala
Gln Arg Gly Ala Cys Ser Ile Leu185 190 195tgatctaatc atcagtagac
gacgaagaag aagaacgatg aagttgccag gctttattat 808tgttgcgtct
tgcttcagcg aaacagcatt catggtccgg ggatcctagt ttactggcag
868ctgcagcaag gcctctttgt cgaggcaatg agcgatccgt ttgtttcatt
ttctcctttc 928tgccttgtga ttatctcgtg tgactgacaa gtcgtggcaa
ttaggtaact ttcctagatg 988gtatttcctg tgtttgagaa aaaaaattct
tgttatccct gtttcataag tagacatgat 1048gtaatcgcac tcagtttatt
cttttccttc ttatttcact tcaatggaaa attatgtttc 1108ccttc
11134197PRTOryza sativa 4Met Ser Ala Ser Arg Phe Ile Lys Cys Val
Thr Val Gly Asp Gly Ala1 5 10 15Val Gly Lys Thr Cys Met Leu Ile Ser
Tyr Thr Ser Asn Thr Phe Pro20 25 30Thr Asp Tyr Val Pro Thr Val Phe
Asp Asn Phe Ser Ala Asn Val Val35 40 45Val Asp Gly Asn Thr Val Asn
Leu Gly Leu Trp Asp Thr Ala Gly Gln50 55 60Glu Asp Tyr Asn Arg Leu
Arg Pro Leu Ser Tyr Arg Gly Ala Asp Val65 70 75 80Phe Leu Leu Ala
Phe Ser Leu Ile Ser Lys Ala Ser Tyr Glu Asn Val85 90 95Ser Lys Lys
Trp Ile Pro Glu Leu Lys His Tyr Ala Pro Gly Val Pro100 105 110Ile
Ile Leu Val Gly Thr Lys Leu Asp Leu Arg Asp Asp Lys Gln Phe115 120
125Phe Val Asp His Pro Gly Ala Val Pro Ile Thr Thr Ala Gln Gly
Glu130 135 140Glu Leu Arg Lys Gln Ile Gly Ala Pro Tyr Tyr Ile Glu
Cys Ser Ser145 150 155 160Lys Thr Gln Leu Asn Val Lys Gly Val Phe
Asp Ala Ala Ile Lys Val165 170 175Val Leu Gln Pro Pro Lys Ala Lys
Lys Lys Lys Lys Ala Gln Arg Gly180 185 190Ala Cys Ser Ile
Leu19551393DNAZea maysCDS(398)..(988) 5gtcgacccac gcgtccgcgg
acgcgtgggc ggacgcgtgg gtccccaccc accaccgcgc 60cgggccacca ccacccactc
taccctcccc tccccaccac cactagcacc caccgtcccg 120gcgcggagac
cgcttccctc cctccgcctc cgcaaccctc tcccgcctcg cccgcgcctc
180cctccatttg tccgcggctc ccctccctcc cgatcttaac cacccgccac
ccggcttcct 240ctcccccttc ttcctccctc aaaccagacg ctcgcccccc
tttcctccac gcctatcttc 300ttcagacgac cagcaggagg tacgaggaag
accacctagg aggcctctct ctctctctcc 360ccagccaccc ccgtagcgag
agggagggcg gaagagg atg agc gcg tcc agg ttc 415Met Ser Ala Ser Arg
Phe1 5ata aag tgc gtc acg gtc ggg gac ggc gcc gtc ggc aag acc tgc
atg 463Ile Lys Cys Val Thr Val Gly Asp Gly Ala Val Gly Lys Thr Cys
Met10 15 20ctc atc tcc tac acc tcc aac acc ttc ccc acc gac tat gtt
ccg aca 511Leu Ile Ser Tyr Thr Ser Asn Thr Phe Pro Thr Asp Tyr Val
Pro Thr25 30 35gtg ttt gat aac ttc agt gcc aac gtt gtg gtt gat ggt
aat act gtc 559Val Phe Asp Asn Phe Ser Ala Asn Val Val Val Asp Gly
Asn Thr Val40 45 50aac ctc ggc ctc tgg gac act gca ggt caa gag gat
tac aac aga ctg 607Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln Glu Asp
Tyr Asn Arg Leu55 60 65 70aga cca ctg agc tat cgt gga gct gat gtt
ttt ctt ctg gct ttc tca 655Arg Pro Leu Ser Tyr Arg Gly Ala Asp Val
Phe Leu Leu Ala Phe Ser75 80 85ctg atc agt aag gcc agc tat gag aat
gtt tcg aag aag tgg ata cct 703Leu Ile Ser Lys Ala Ser Tyr Glu Asn
Val Ser Lys Lys Trp Ile Pro90 95 100gaa ctg aag cat tat gca cct ggt
gtg cca att att ctc gta ggg aca 751Glu Leu Lys His Tyr Ala Pro Gly
Val Pro Ile Ile Leu Val Gly Thr105 110 115aag ctt gat ctt cga gac
gac aag cag ttc ttt gtg gac cat cct ggt 799Lys Leu Asp Leu Arg Asp
Asp Lys Gln Phe Phe Val Asp His Pro Gly120 125 130gct gtc cct atc
act act gct cag gga gag gag cta aga aag caa ata 847Ala Val Pro Ile
Thr Thr Ala Gln Gly Glu Glu Leu Arg Lys Gln Ile135 140 145 150ggc
gct cca tac tac atc gaa tgc agc tcg aag acc caa cta aac gtg 895Gly
Ala Pro Tyr Tyr Ile Glu Cys Ser Ser Lys Thr Gln Leu Asn Val155 160
165aag ggc gtc ttc gat gcg gcg ata aag gtt gtg ctg cag ccg cct aag
943Lys Gly Val Phe Asp Ala Ala Ile Lys Val Val Leu Gln Pro Pro
Lys170 175 180gcg aag aag aag aaa aag gtg cag agg ggg gcg tgc tcc
att ttg 988Ala Lys Lys Lys Lys Lys Val Gln Arg Gly Ala Cys Ser Ile
Leu185 190 195tgatctaatc atcggtagat gaagaaacaa gggcgaaggt
gccatggctt tatcatcgtc 1048gcgtcttgct tcagtggaac agcatgaatg
gtccccaccc cctctaggtt tactggcggc 1108tcggctgcag cgagttctca
tctctttgtc gaggcattga gcgatatgtt tgtttcattt 1168tcctccttcc
tgccttgtga ttatctggtg tgtgtgtgtg tgtgactgac gaagtcgcgg
1228cgattaggta actcgcttag aaggtatttc ccgtgtttga gcaaaagaaa
gtatccctgt 1288tatctctgtt ccataagtta gacatgatgt aatcgtacta
agtttatttt tacttatttc 1348acttgaatgg aaaagtatgc ttcccattta
aaaaaaaaaa aaaaa 13936197PRTZea mays 6Met Ser Ala Ser Arg Phe Ile
Lys Cys Val Thr Val Gly Asp Gly Ala1 5 10 15Val Gly Lys Thr Cys Met
Leu Ile Ser Tyr Thr Ser Asn Thr Phe Pro20 25 30Thr Asp Tyr Val Pro
Thr Val Phe Asp Asn Phe Ser Ala Asn Val Val35 40 45Val Asp Gly Asn
Thr Val Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln50 55 60Glu Asp Tyr
Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala Asp Val65 70 75 80Phe
Leu Leu Ala Phe Ser Leu Ile Ser Lys Ala Ser Tyr Glu Asn Val85 90
95Ser Lys Lys Trp Ile Pro Glu Leu Lys His Tyr Ala Pro Gly Val
Pro100 105 110Ile Ile Leu Val Gly Thr Lys Leu Asp Leu Arg Asp Asp
Lys Gln Phe115 120 125Phe Val Asp His Pro Gly Ala Val Pro Ile Thr
Thr Ala Gln Gly Glu130 135 140Glu Leu Arg Lys Gln Ile Gly Ala Pro
Tyr Tyr Ile Glu Cys Ser Ser145 150 155 160Lys Thr Gln Leu Asn Val
Lys Gly Val Phe Asp Ala Ala Ile Lys Val165 170 175Val Leu Gln Pro
Pro Lys Ala Lys Lys Lys Lys Lys Val Gln Arg Gly180 185 190Ala Cys
Ser Ile Leu1957197PRTArtificial sequenceDescription of the
artificial sequence dominant-negative mutant of barley RacB protein
7Met Ser Ala Ser Arg Phe Ile Lys Cys Val Thr Val Gly Asp Gly Ala1 5
10 15Val Gly Lys Asn Cys Met Leu Ile Ser Tyr Thr Ser Asn Thr Phe
Pro20 25 30Thr Asp Tyr Val Pro Thr Val Phe Asp Asn Phe Ser Ala Asn
Val Val35 40 45Val Asp Gly Asn Thr Val Asn Leu Gly Leu Trp Asp Thr
Ala Gly Gln50 55 60Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg
Gly Ala Asp Val65 70 75 80Phe Leu Leu Ala Phe Ser Leu Ile Ser Lys
Ala Ser Tyr Glu Asn Val85 90 95Ser Lys Lys Trp Ile Pro Glu Leu Lys
His Tyr Ala Pro Gly Val Pro100 105 110Ile Ile Leu Val Gly Thr Lys
Leu Asp Leu Arg Asp Asp Lys Gln Phe115 120 125Phe Val Asp His Pro
Gly Ala Val Pro Ile Thr Thr Ala Gln Gly Glu130 135 140Glu Leu Lys
Lys Leu Ile Gly Ala Pro Tyr Tyr Ile Glu Cys Ser Ser145 150 155
160Lys Thr Gln Leu Asn Val Lys Gly Val Phe Asp Ala Ala Ile Lys
Val165 170 175Val Leu Gln Pro Pro Lys Ala Lys Lys Lys Lys Lys Ala
Gln Arg Gly180 185 190Ala Cys Ser Ile Leu1958197PRTArtificial
sequenceDescription of the artificial sequence dominant-negative
mutant of rice RacB protein 8Met Ser Ala Ser Arg Phe Ile Lys Cys
Val Thr Val Gly Asp Gly Ala1 5 10 15Val Gly Lys Asn Cys Met Leu Ile
Ser Tyr Thr Ser Asn Thr Phe Pro20 25 30Thr Asp Tyr Val Pro Thr Val
Phe Asp Asn Phe Ser Ala Asn Val Val35 40 45Val Asp Gly Asn Thr Val
Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln50 55 60Glu Asp Tyr Asn Arg
Leu Arg Pro Leu Ser Tyr Arg Gly Ala Asp Val65 70 75 80Phe Leu Leu
Ala Phe Ser Leu Ile Ser Lys Ala Ser Tyr Glu Asn Val85 90 95Ser Lys
Lys Trp Ile Pro Glu Leu Lys His Tyr Ala Pro Gly Val Pro100 105
110Ile Ile Leu Val Gly Thr Lys Leu Asp Leu Arg Asp Asp Lys Gln
Phe115 120 125Phe Val Asp His Pro Gly Ala Val Pro Ile Thr Thr Ala
Gln Gly Glu130 135 140Glu Leu Arg Lys Gln Ile Gly Ala Pro Tyr Tyr
Ile Glu Cys Ser Ser145 150 155 160Lys Thr Gln Leu Asn Val Lys Gly
Val Phe Asp Ala Ala Ile Lys Val165 170 175Val Leu Gln Pro Pro Lys
Ala Lys Lys Lys Lys Lys Ala Gln Arg Gly180 185 190Ala Cys Ser Ile
Leu1959197PRTArtificial sequenceSITE(20)Thr/Asn mutation for
dominant-negative phenotype 9Met Ser Ala Ser Arg Phe Ile Lys Cys
Val Thr Val Gly Asp Gly Ala1 5 10 15Val Gly Lys Asn Cys Met Leu Ile
Ser Tyr Thr Ser Asn Thr Phe Pro20 25 30Thr Asp Tyr Val Pro Thr Val
Phe Asp Asn Phe Ser Ala Asn Val Val35 40 45Val Asp Gly Asn Thr Val
Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln50 55 60Glu Asp Tyr Asn Arg
Leu Arg Pro Leu Ser Tyr Arg Gly Ala Asp Val65 70 75 80Phe Leu Leu
Ala Phe Ser Leu Ile Ser Lys Ala Ser Tyr Glu Asn Val85 90 95Ser Lys
Lys Trp Ile Pro Glu Leu Lys His Tyr Ala Pro Gly Val Pro100 105
110Ile Ile Leu Val Gly Thr Lys Leu Asp Leu Arg Asp Asp Lys Gln
Phe115 120 125Phe Val Asp His Pro Gly Ala Val Pro Ile Thr Thr Ala
Gln Gly Glu130 135 140Glu Leu Arg Lys Gln Ile Gly Ala Pro Tyr Tyr
Ile Glu Cys Ser Ser145 150 155 160Lys Thr Gln Leu Asn Val Lys Gly
Val Phe Asp Ala Ala Ile Lys Val165 170 175Val Leu Gln Pro Pro Lys
Ala Lys Lys Lys Lys Lys Val Gln Arg Gly180 185 190Ala Cys Ser Ile
Leu1951024DNAArtificial sequenceDescription of the artificial
sequence oligonucleotide primer 10ggatccgatg agcgcgtcca ggtt
241126DNAArtificial sequenceDescription of the artificial sequence
oligonucleotide primer 11gtcgaccttc gcccttgttc tttgtc
261226DNAArtificial sequenceDescription of the artificial sequence
oligonucleotide primer 12gtgggcacat agtcggtggg gaaggt
261323DNAArtificial sequenceDescription of the artificial sequence
oligonucleotide primer 13cgactggagc acgaggacac tga
231426DNAArtificial sequenceDescription of the artificial sequence
oligonucleotide primer 14ggacactgac atggactgaa ggagta
261522DNAArtificial sequenceDescription of the
artificial sequence oligonucleotide primer 15gttcatcaag tgcgtcaccg
tg 221623DNAArtificial sequenceDescription of the artificial
sequence oligonucleotide primer 16ttagcttcct cagttcttcc ctg
231721DNAArtificial sequenceDescription of the artificial sequence
oligonucleotide primer 17cgcgccgcag ccgagtacga c
211821DNAArtificial sequenceDescription of the artificial sequence
oligonucleotide primer 18gtcacaaaaa cacatgtaac c
211920DNAArtificial sequenceDescription of the artificial sequence
oligonucleotide primer 19ggccgacatg cattcaccag 202021DNAArtificial
sequenceDescription of the artificial sequence oligonucleotide
primer 20catctgatat tgctgggtct g 212121DNAArtificial
sequenceDescription of the artificial sequence oligonucleotide
primer 21ccaagatgca gatcttcgtg a 212221DNAArtificial
sequenceDescription of the artificial sequence oligonucleotide
primer 22ttcgcgatag gtaaaagagc a 212318DNAArtificial
sequenceDescription of the artificial sequence oligonucleotide
primer 23gtaaaacgac ggccagtg 182419DNAArtificial
sequenceDescription of the artificial sequence oligonucleotide
primer 24ggaaacagct atgaccatg 192517DNAArtificial
sequenceDescription of the artificial sequence oligonucleotide
primer 25gtggaggcgc ggcgaga 172622DNAArtificial sequenceDescription
of the artificial sequence oligonucleotide primer 26ccatgcttca
tctccatagt ca 222726DNAArtificial sequenceDescription of the
artificial sequence oligonucleotide primer 27ggatcccgat tccatcagga
aagcat 262826DNAArtificial sequenceDescription of the artificial
sequence oligonucleotide primer 28gtcgacgcga gacactgcaa aacaaa
262926DNAArtificial sequenceDescription of the artificial sequence
oligonucleotide primer 29ggatccttct cgtccattta gccggc
263027DNAArtificial sequenceDescription of the artificial sequence
oligonucleotide primer 30gtcgactgat cacttgaagc atgccag
273124DNAArtificial sequenceDescription of the artificial sequence
oligonucleotide primer 31ggatccgatg agcgcgtcca ggtt
243226DNAArtificial sequenceDescription of the artificial sequence
oligonucleotide primer 32gtcgaccttc gcccttgttc tttgtc
263329DNAArtificial sequenceDescription of the artificial sequence
oligonucleotide primer 33accgtggggg acgtcgccgt cggcaagac
2934721DNAHordeum vulgareCDS(38)..(673)coding for RacB homologue
(Rop6) 34gtggaggcgc ggcgagagcg gcggaggcgg aggagag atg agc gtg acc
aag ttc 55Met Ser Val Thr Lys Phe1 5atc aag tgc gtc acg gtg ggg gac
ggc gcc gtc ggc aag acc tgc atg 103Ile Lys Cys Val Thr Val Gly Asp
Gly Ala Val Gly Lys Thr Cys Met10 15 20ctc atc tgc tac acc agc aac
agg ttc ccc agt gat tac atc ccc acg 151Leu Ile Cys Tyr Thr Ser Asn
Arg Phe Pro Ser Asp Tyr Ile Pro Thr25 30 35gtg ttc gac aac ttc agc
gcc aac gtc tcc gtc gac ggc aac atc gtc 199Val Phe Asp Asn Phe Ser
Ala Asn Val Ser Val Asp Gly Asn Ile Val40 45 50aac ctc ggc cta tgg
gac acc gcc ggg caa gaa gac tac agc cgg ctg 247Asn Leu Gly Leu Trp
Asp Thr Ala Gly Gln Glu Asp Tyr Ser Arg Leu55 60 65 70agg ccg ctg
agc tac aga ggc gcc gac gtg ttc gtg ctc gcc ttc tcc 295Arg Pro Leu
Ser Tyr Arg Gly Ala Asp Val Phe Val Leu Ala Phe Ser75 80 85ctc atc
agc agc gcc agc tac gag aat gtt ctt aag aag tgg atg cca 343Leu Ile
Ser Ser Ala Ser Tyr Glu Asn Val Leu Lys Lys Trp Met Pro90 95 100gag
ctc cgc cgg ttc gcg ccg aat gtc ccc att gtt ctt gtt ggg acc 391Glu
Leu Arg Arg Phe Ala Pro Asn Val Pro Ile Val Leu Val Gly Thr105 110
115aag cta gat ctg cgt gac cac aga gcc tac ctc gcc gac cac ccc ggt
439Lys Leu Asp Leu Arg Asp His Arg Ala Tyr Leu Ala Asp His Pro
Gly120 125 130gct tca gca atc aca act gca cag ggt gaa gaa ctt agg
aag cag atc 487Ala Ser Ala Ile Thr Thr Ala Gln Gly Glu Glu Leu Arg
Lys Gln Ile135 140 145 150ggc gcc gcg gct tac atc gag tgc agc tcc
aag aca cag cag aac gtc 535Gly Ala Ala Ala Tyr Ile Glu Cys Ser Ser
Lys Thr Gln Gln Asn Val155 160 165aag gct gtg ttt gac acc gcc ata
aag gtg gtc ctc cag ccg ccg agg 583Lys Ala Val Phe Asp Thr Ala Ile
Lys Val Val Leu Gln Pro Pro Arg170 175 180aga agg gag gtg atg tcc
gcc agg aag aaa acc agg cga agc tct gga 631Arg Arg Glu Val Met Ser
Ala Arg Lys Lys Thr Arg Arg Ser Ser Gly185 190 195tgc tcc atc aag
cac ttg atc tgc ggg agt acg tgc gct gct 673Cys Ser Ile Lys His Leu
Ile Cys Gly Ser Thr Cys Ala Ala200 205 210tgaattagca ccatggaggc
ctggactgac tatggagatg aagcatgg 72135212PRTHordeum vulgare 35Met Ser
Val Thr Lys Phe Ile Lys Cys Val Thr Val Gly Asp Gly Ala1 5 10 15Val
Gly Lys Thr Cys Met Leu Ile Cys Tyr Thr Ser Asn Arg Phe Pro20 25
30Ser Asp Tyr Ile Pro Thr Val Phe Asp Asn Phe Ser Ala Asn Val Ser35
40 45Val Asp Gly Asn Ile Val Asn Leu Gly Leu Trp Asp Thr Ala Gly
Gln50 55 60Glu Asp Tyr Ser Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala
Asp Val65 70 75 80Phe Val Leu Ala Phe Ser Leu Ile Ser Ser Ala Ser
Tyr Glu Asn Val85 90 95Leu Lys Lys Trp Met Pro Glu Leu Arg Arg Phe
Ala Pro Asn Val Pro100 105 110Ile Val Leu Val Gly Thr Lys Leu Asp
Leu Arg Asp His Arg Ala Tyr115 120 125Leu Ala Asp His Pro Gly Ala
Ser Ala Ile Thr Thr Ala Gln Gly Glu130 135 140Glu Leu Arg Lys Gln
Ile Gly Ala Ala Ala Tyr Ile Glu Cys Ser Ser145 150 155 160Lys Thr
Gln Gln Asn Val Lys Ala Val Phe Asp Thr Ala Ile Lys Val165 170
175Val Leu Gln Pro Pro Arg Arg Arg Glu Val Met Ser Ala Arg Lys
Lys180 185 190Thr Arg Arg Ser Ser Gly Cys Ser Ile Lys His Leu Ile
Cys Gly Ser195 200 205Thr Cys Ala Ala21036699DNAHordeum
vulgareCDS(67)..(657)coding for RacB homologue (RacD) 36ggatcccgat
tccatcagga aagcatatag actagcccag taaatagaaa taagnaaaga 60tcggcg atg
agc gca tct cgg ttc atc aag tgc gtg acg gtg ggg gac 108Met Ser Ala
Ser Arg Phe Ile Lys Cys Val Thr Val Gly Asp1 5 10ggc gcc gtg gga
aag aca tgc ctc ctc atc tca tac aca tcc aac acc 156Gly Ala Val Gly
Lys Thr Cys Leu Leu Ile Ser Tyr Thr Ser Asn Thr15 20 25 30ttc ccc
aca gac tat gtc cca aca gtt ttc gac aac ttc agc gct aac 204Phe Pro
Thr Asp Tyr Val Pro Thr Val Phe Asp Asn Phe Ser Ala Asn35 40 45gtc
gtg gtt gac ggc agc acc gtc aac ctc gga tta tgg gat act gca 252Val
Val Val Asp Gly Ser Thr Val Asn Leu Gly Leu Trp Asp Thr Ala50 55
60gga caa gaa gac tat aat cga cta cgc cca cta agc tac cgt ggt gcc
300Gly Gln Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly
Ala65 70 75gat gtc ttc ctg ctc gcc ttt tct ctc atc agc aaa gca agc
tac gag 348Asp Val Phe Leu Leu Ala Phe Ser Leu Ile Ser Lys Ala Ser
Tyr Glu80 85 90aat gtc act aag aag tgg att cca gag tta cgg cac tat
gct cct ggc 396Asn Val Thr Lys Lys Trp Ile Pro Glu Leu Arg His Tyr
Ala Pro Gly95 100 105 110gtg ccc ata att ctt gtt gga aca aag ctt
gat ctg cgg gat gac aag 444Val Pro Ile Ile Leu Val Gly Thr Lys Leu
Asp Leu Arg Asp Asp Lys115 120 125cag ttt ttt gtg gat cac cct ggg
gcg gtt cct att tcc act gct cag 492Gln Phe Phe Val Asp His Pro Gly
Ala Val Pro Ile Ser Thr Ala Gln130 135 140ggt gaa gag ctg aag aag
gtg att ggc gcg act gcc tac atc gag tgc 540Gly Glu Glu Leu Lys Lys
Val Ile Gly Ala Thr Ala Tyr Ile Glu Cys145 150 155agc tca aaa aca
cag cag aac atc aag gcg gtg ttt gat gcg gcg atc 588Ser Ser Lys Thr
Gln Gln Asn Ile Lys Ala Val Phe Asp Ala Ala Ile160 165 170aag gtg
gtc ctc cag cct ccg aag cag aag cgg aag aag agg aag tca 636Lys Val
Val Leu Gln Pro Pro Lys Gln Lys Arg Lys Lys Arg Lys Ser175 180 185
190cag aaa gga tgc agc atc ttg taaagctaaa atcccttttg ttttgcagtg
687Gln Lys Gly Cys Ser Ile Leu195tctcgcgtcg ac 69937197PRTHordeum
vulgare 37Met Ser Ala Ser Arg Phe Ile Lys Cys Val Thr Val Gly Asp
Gly Ala1 5 10 15Val Gly Lys Thr Cys Leu Leu Ile Ser Tyr Thr Ser Asn
Thr Phe Pro20 25 30Thr Asp Tyr Val Pro Thr Val Phe Asp Asn Phe Ser
Ala Asn Val Val35 40 45Val Asp Gly Ser Thr Val Asn Leu Gly Leu Trp
Asp Thr Ala Gly Gln50 55 60Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser
Tyr Arg Gly Ala Asp Val65 70 75 80Phe Leu Leu Ala Phe Ser Leu Ile
Ser Lys Ala Ser Tyr Glu Asn Val85 90 95Thr Lys Lys Trp Ile Pro Glu
Leu Arg His Tyr Ala Pro Gly Val Pro100 105 110Ile Ile Leu Val Gly
Thr Lys Leu Asp Leu Arg Asp Asp Lys Gln Phe115 120 125Phe Val Asp
His Pro Gly Ala Val Pro Ile Ser Thr Ala Gln Gly Glu130 135 140Glu
Leu Lys Lys Val Ile Gly Ala Thr Ala Tyr Ile Glu Cys Ser Ser145 150
155 160Lys Thr Gln Gln Asn Ile Lys Ala Val Phe Asp Ala Ala Ile Lys
Val165 170 175Val Leu Gln Pro Pro Lys Gln Lys Arg Lys Lys Arg Lys
Ser Gln Lys180 185 190Gly Cys Ser Ile Leu19538677DNAHordeum
vulgareCDS(27)..(665)coding for RacB homologue (Rop4) 38ggatccttct
cgtccattta gccggc atg gcg tcc agc gcc tcc cgg ttc atc 53Met Ala Ser
Ser Ala Ser Arg Phe Ile1 5aag tgc gtc acc gtc ggg gac ggc gcc gtc
ggc aag acc tgc atg ctc 101Lys Cys Val Thr Val Gly Asp Gly Ala Val
Gly Lys Thr Cys Met Leu10 15 20 25atc tgc tac acc agc aac aag ttc
ccc acc gac tac gtg ccc acc gtg 149Ile Cys Tyr Thr Ser Asn Lys Phe
Pro Thr Asp Tyr Val Pro Thr Val30 35 40ttc gac aat ttc agc gcg aac
gtg gtg gtg gac ggc acc acc gtg aac 197Phe Asp Asn Phe Ser Ala Asn
Val Val Val Asp Gly Thr Thr Val Asn45 50 55ctg ggc ctc tgg gac act
gca ggg cag gag gac tac aac aga ttg aga 245Leu Gly Leu Trp Asp Thr
Ala Gly Gln Glu Asp Tyr Asn Arg Leu Arg60 65 70ccg ctg agc tac cgg
gga gcc gac gtc ttc gtg ctc tcc ttc tcg ctc 293Pro Leu Ser Tyr Arg
Gly Ala Asp Val Phe Val Leu Ser Phe Ser Leu75 80 85gtc agc cga gcc
agc tac gag aat gtc atg aag aag tgg cta ccg gag 341Val Ser Arg Ala
Ser Tyr Glu Asn Val Met Lys Lys Trp Leu Pro Glu90 95 100 105ctt cag
cac cat gca ccc ggc gtg cca aca gtg ctg gtt ggt aca aag 389Leu Gln
His His Ala Pro Gly Val Pro Thr Val Leu Val Gly Thr Lys110 115
120cta gat cta cgt gaa gac aag caa tac tta ctt gac cac ccc ggc gtg
437Leu Asp Leu Arg Glu Asp Lys Gln Tyr Leu Leu Asp His Pro Gly
Val125 130 135gtg cct gtt act aca gct cag ggg gag gaa ctc cgc aag
cac atc ggt 485Val Pro Val Thr Thr Ala Gln Gly Glu Glu Leu Arg Lys
His Ile Gly140 145 150gca act tgt tat gtc gaa tgc agc tca aag aca
cag cag aat gtc aaa 533Ala Thr Cys Tyr Val Glu Cys Ser Ser Lys Thr
Gln Gln Asn Val Lys155 160 165gct gtg ttt gat gct gcc atc aag gta
gtg atc aaa cct cca aca aag 581Ala Val Phe Asp Ala Ala Ile Lys Val
Val Ile Lys Pro Pro Thr Lys170 175 180 185cag agg gaa agg agg aag
aag aaa gca cgg caa gga tgt gca tca ttg 629Gln Arg Glu Arg Arg Lys
Lys Lys Ala Arg Gln Gly Cys Ala Ser Leu190 195 200ggt acc ctg tca
aga agg aag ctg gca tgc ttc aag tgatcagtcg ac 677Gly Thr Leu Ser
Arg Arg Lys Leu Ala Cys Phe Lys205 21039213PRTHordeum vulgare 39Met
Ala Ser Ser Ala Ser Arg Phe Ile Lys Cys Val Thr Val Gly Asp1 5 10
15Gly Ala Val Gly Lys Thr Cys Met Leu Ile Cys Tyr Thr Ser Asn Lys20
25 30Phe Pro Thr Asp Tyr Val Pro Thr Val Phe Asp Asn Phe Ser Ala
Asn35 40 45Val Val Val Asp Gly Thr Thr Val Asn Leu Gly Leu Trp Asp
Thr Ala50 55 60Gly Gln Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr
Arg Gly Ala65 70 75 80Asp Val Phe Val Leu Ser Phe Ser Leu Val Ser
Arg Ala Ser Tyr Glu85 90 95Asn Val Met Lys Lys Trp Leu Pro Glu Leu
Gln His His Ala Pro Gly100 105 110Val Pro Thr Val Leu Val Gly Thr
Lys Leu Asp Leu Arg Glu Asp Lys115 120 125Gln Tyr Leu Leu Asp His
Pro Gly Val Val Pro Val Thr Thr Ala Gln130 135 140Gly Glu Glu Leu
Arg Lys His Ile Gly Ala Thr Cys Tyr Val Glu Cys145 150 155 160Ser
Ser Lys Thr Gln Gln Asn Val Lys Ala Val Phe Asp Ala Ala Ile165 170
175Lys Val Val Ile Lys Pro Pro Thr Lys Gln Arg Glu Arg Arg Lys
Lys180 185 190Lys Ala Arg Gln Gly Cys Ala Ser Leu Gly Thr Leu Ser
Arg Arg Lys195 200 205Leu Ala Cys Phe Lys21040945DNAZea
maysCDS(37)..(672)coding for RacB homologue (Rop6) 40gagaagaagg
gggcctgccg gccggggctg ggagac atg agc gtg acc aag ttc 54Met Ser Val
Thr Lys Phe1 5atc aag tgc gtc acg gtg ggc gac ggc gcg gtg ggc aag
acc tgc atg 102Ile Lys Cys Val Thr Val Gly Asp Gly Ala Val Gly Lys
Thr Cys Met10 15 20ctc atc tgc tac acc agc aac aag ttc ccc acg gac
tac atc ccc acg 150Leu Ile Cys Tyr Thr Ser Asn Lys Phe Pro Thr Asp
Tyr Ile Pro Thr25 30 35gtg ttc gac aac ttc agc gcc aac gtc tcc gtg
gac ggc agc atc gtc 198Val Phe Asp Asn Phe Ser Ala Asn Val Ser Val
Asp Gly Ser Ile Val40 45 50aac ctg ggc ctc tgg gac acc gcg ggg caa
gag gac tac agc agg ctg 246Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln
Glu Asp Tyr Ser Arg Leu55 60 65 70cgg ccg ctg agc tac agg ggc gcg
gac gtg ttc gtg ctg gcc ttc tcc 294Arg Pro Leu Ser Tyr Arg Gly Ala
Asp Val Phe Val Leu Ala Phe Ser75 80 85ctg atc agc agg gcg agc tac
gag aac gtt ctt aag aag tgg gtg cca 342Leu Ile Ser Arg Ala Ser Tyr
Glu Asn Val Leu Lys Lys Trp Val Pro90 95 100gag ctt cgc aga ttc gcg
ccc aac gtc ccg gtc gtt ctt gtt ggg acc 390Glu Leu Arg Arg Phe Ala
Pro Asn Val Pro Val Val Leu Val Gly Thr105 110 115aag tta gat ctc
cgc gac cac aga gcc tac ctc gcc gac cat cct gga 438Lys Leu Asp Leu
Arg Asp His Arg Ala Tyr Leu Ala Asp His Pro Gly120 125 130gct tca
gca gtc acc acg gcg cag ggt gag gaa ctg agg aag cag atc 486Ala Ser
Ala Val Thr Thr Ala Gln Gly Glu Glu Leu Arg Lys Gln Ile135 140 145
150ggc gct gcg gcc tac atc gag tgc agt tcc aaa acc cag cag aac gtc
534Gly Ala Ala Ala Tyr Ile Glu Cys Ser Ser Lys Thr Gln Gln Asn
Val155 160 165aag tct gtc ttc gat acg gcc atc aaa gtg gtc ctt cag
ccc cca cgg 582Lys Ser Val Phe Asp Thr Ala Ile Lys Val Val Leu Gln
Pro Pro Arg170 175 180agg agg gag gca gtg cct gcc agg aag aag aac
agg cgt ggc tcc gga 630Arg Arg Glu Ala Val Pro Ala Arg Lys Lys Asn
Arg Arg Gly Ser Gly185 190 195tgc tct ata atg aac ctt gtg tgt ggc
agc aca tgt gct gct 672Cys Ser Ile Met Asn Leu Val Cys Gly Ser Thr
Cys Ala Ala200 205 210tagggagtct actagaacac tgaaccggaa gggaggtgaa
ggcgtgattc atggtgtgta 732atgtgctgtg gcaactggca agttagtttg
ctatagatga ggatgactgc tgcttttgtt 792ttccttggcc catctgctgt
agttcgtcag gctcttcaag ggctgacttt ttaccagact 852gcagtgttgt
gtaagaagtt tgctagacgc tgtaactgta atgttctccg ctgatgtggt
912ataactaaga tacgagtaag cttgagcgtg ttc 94541212PRTZea mays 41Met
Ser Val Thr Lys Phe Ile Lys Cys Val Thr Val Gly Asp Gly Ala1 5 10
15Val Gly Lys Thr Cys Met Leu Ile Cys Tyr Thr Ser Asn Lys Phe Pro20
25
30Thr Asp Tyr Ile Pro Thr Val Phe Asp Asn Phe Ser Ala Asn Val Ser35
40 45Val Asp Gly Ser Ile Val Asn Leu Gly Leu Trp Asp Thr Ala Gly
Gln50 55 60Glu Asp Tyr Ser Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala
Asp Val65 70 75 80Phe Val Leu Ala Phe Ser Leu Ile Ser Arg Ala Ser
Tyr Glu Asn Val85 90 95Leu Lys Lys Trp Val Pro Glu Leu Arg Arg Phe
Ala Pro Asn Val Pro100 105 110Val Val Leu Val Gly Thr Lys Leu Asp
Leu Arg Asp His Arg Ala Tyr115 120 125Leu Ala Asp His Pro Gly Ala
Ser Ala Val Thr Thr Ala Gln Gly Glu130 135 140Glu Leu Arg Lys Gln
Ile Gly Ala Ala Ala Tyr Ile Glu Cys Ser Ser145 150 155 160Lys Thr
Gln Gln Asn Val Lys Ser Val Phe Asp Thr Ala Ile Lys Val165 170
175Val Leu Gln Pro Pro Arg Arg Arg Glu Ala Val Pro Ala Arg Lys
Lys180 185 190Asn Arg Arg Gly Ser Gly Cys Ser Ile Met Asn Leu Val
Cys Gly Ser195 200 205Thr Cys Ala Ala21042850DNAOryza
sativaCDS(112)..(702)coding for RacB homologue (RACDP / RACD)
42agcaagcagc agctgaggtg aggtccgtgg cgttggagtg aggactgagg aggaagaaga
60gggcgggatc tagggtaccg gatgcgctgg ctgtgctgag tgagagtaga g atg agc
117Met Ser1gcg tct cgg ttc atc aag tgc gtc acc gtg ggg gac ggc gcc
gtg ggc 165Ala Ser Arg Phe Ile Lys Cys Val Thr Val Gly Asp Gly Ala
Val Gly5 10 15aag acc tgc atg ctc atc tcc tac acc tcc aac acc ttc
ccc acg gac 213Lys Thr Cys Met Leu Ile Ser Tyr Thr Ser Asn Thr Phe
Pro Thr Asp20 25 30tat gtt cca act gtt ttt gat aac ttc agt gca aat
gtt gtg gtc gat 261Tyr Val Pro Thr Val Phe Asp Asn Phe Ser Ala Asn
Val Val Val Asp35 40 45 50ggg agc act gtg aac ttg ggg ttg tgg gat
aca gca gga caa gag gac 309Gly Ser Thr Val Asn Leu Gly Leu Trp Asp
Thr Ala Gly Gln Glu Asp55 60 65tac aat agg cta cgc ccg ttg agc tat
cgt ggc gct gat gtt ttc ctg 357Tyr Asn Arg Leu Arg Pro Leu Ser Tyr
Arg Gly Ala Asp Val Phe Leu70 75 80ctg gcc ttt tct ctg atc agc aaa
gca agc tat gag aat gtt tct aaa 405Leu Ala Phe Ser Leu Ile Ser Lys
Ala Ser Tyr Glu Asn Val Ser Lys85 90 95aag tgg ata cct gaa tta agg
cat tat gct cct ggt gtg cca ata att 453Lys Trp Ile Pro Glu Leu Arg
His Tyr Ala Pro Gly Val Pro Ile Ile100 105 110ctc gtt gga aca aag
ctt gat ctg cgg gat gat aag caa ttt ttc gta 501Leu Val Gly Thr Lys
Leu Asp Leu Arg Asp Asp Lys Gln Phe Phe Val115 120 125 130gat cac
cct ggt gct gta cct att tcc act gct cag ggc gaa gag ctg 549Asp His
Pro Gly Ala Val Pro Ile Ser Thr Ala Gln Gly Glu Glu Leu135 140
145agg aaa ctc att ggt gca gcg gca tac att gaa tgc agt tca aaa aca
597Arg Lys Leu Ile Gly Ala Ala Ala Tyr Ile Glu Cys Ser Ser Lys
Thr150 155 160cag caa aac atc aag gca gtt ttc gat gct gcg att aag
gtg gtt ctc 645Gln Gln Asn Ile Lys Ala Val Phe Asp Ala Ala Ile Lys
Val Val Leu165 170 175cag cct cca aag caa aag aag aag aag aaa aag
gcg cag aaa gga tgt 693Gln Pro Pro Lys Gln Lys Lys Lys Lys Lys Lys
Ala Gln Lys Gly Cys180 185 190gcc atc ttg taattaaatg gtagacagtg
cagtgcagat cgatgtatcc 742Ala Ile Leu195cttcatttgt agcctctggc
ttcaatcgtc gcttgtttgt ataattacgc tagatgccac 802cggcagaaga
tataatatag tcctcctgcc tttgtggtgt tggtctct 85043197PRTOryza sativa
43Met Ser Ala Ser Arg Phe Ile Lys Cys Val Thr Val Gly Asp Gly Ala1
5 10 15Val Gly Lys Thr Cys Met Leu Ile Ser Tyr Thr Ser Asn Thr Phe
Pro20 25 30Thr Asp Tyr Val Pro Thr Val Phe Asp Asn Phe Ser Ala Asn
Val Val35 40 45Val Asp Gly Ser Thr Val Asn Leu Gly Leu Trp Asp Thr
Ala Gly Gln50 55 60Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg
Gly Ala Asp Val65 70 75 80Phe Leu Leu Ala Phe Ser Leu Ile Ser Lys
Ala Ser Tyr Glu Asn Val85 90 95Ser Lys Lys Trp Ile Pro Glu Leu Arg
His Tyr Ala Pro Gly Val Pro100 105 110Ile Ile Leu Val Gly Thr Lys
Leu Asp Leu Arg Asp Asp Lys Gln Phe115 120 125Phe Val Asp His Pro
Gly Ala Val Pro Ile Ser Thr Ala Gln Gly Glu130 135 140Glu Leu Arg
Lys Leu Ile Gly Ala Ala Ala Tyr Ile Glu Cys Ser Ser145 150 155
160Lys Thr Gln Gln Asn Ile Lys Ala Val Phe Asp Ala Ala Ile Lys
Val165 170 175Val Leu Gln Pro Pro Lys Gln Lys Lys Lys Lys Lys Lys
Ala Gln Lys180 185 190Gly Cys Ala Ile Leu195441169DNAOryza
sativaCDS(169)..(813)coding for RacB homologue (Rop 4) 44aacagttcag
agaggaagca tgtgacactt ccctctgtcc ctctctctct ctctagcctc 60caaccatcgc
ttcaccaaga agccatcacc tcctcctctc tatcaagttc tctcccctct
120cttgctgtct ctgcttgctg ctgctgctgc tcgattcggc cggcggcc atg gcg tcc
177Met Ala Ser1agc gcg tcg cgg ttc atc aag tgc gtc acg gtc ggg gac
ggc gcc gtc 225Ser Ala Ser Arg Phe Ile Lys Cys Val Thr Val Gly Asp
Gly Ala Val5 10 15ggc aag acc tgc atg ctc atc tgc tac acc agc aac
aag ttc ccc act 273Gly Lys Thr Cys Met Leu Ile Cys Tyr Thr Ser Asn
Lys Phe Pro Thr20 25 30 35gat tac gta ccc act gtt ttt gac aat ttc
agt gca aac gtg gtg gtc 321Asp Tyr Val Pro Thr Val Phe Asp Asn Phe
Ser Ala Asn Val Val Val40 45 50gac ggc acc acg gtg aat ttg ggt ctc
tgg gat act gca ggg cag gaa 369Asp Gly Thr Thr Val Asn Leu Gly Leu
Trp Asp Thr Ala Gly Gln Glu55 60 65gat tac aac aga ttg agg ccg cta
agc tac cgt ggc gcc gat gtc ttt 417Asp Tyr Asn Arg Leu Arg Pro Leu
Ser Tyr Arg Gly Ala Asp Val Phe70 75 80gtg ctt gcc ttc tcc cta gtg
agc cga gct agc tat gag aat gtc atg 465Val Leu Ala Phe Ser Leu Val
Ser Arg Ala Ser Tyr Glu Asn Val Met85 90 95aag aag tgg tta cca gag
ctt cag cat tat gca cca ggg gtg cca att 513Lys Lys Trp Leu Pro Glu
Leu Gln His Tyr Ala Pro Gly Val Pro Ile100 105 110 115gtg ttg gtt
ggg acc aaa ttg gat ctt cgt gaa gat aaa cac tac tta 561Val Leu Val
Gly Thr Lys Leu Asp Leu Arg Glu Asp Lys His Tyr Leu120 125 130ctt
gac cat cct agc ttg gtg cct gtg act aca gca cag gga gag gaa 609Leu
Asp His Pro Ser Leu Val Pro Val Thr Thr Ala Gln Gly Glu Glu135 140
145ctc cgc aag cac att ggc gca acg tgt tac atc gaa tgc agc tca aag
657Leu Arg Lys His Ile Gly Ala Thr Cys Tyr Ile Glu Cys Ser Ser
Lys150 155 160aca cag cag aat gta aaa gct gtg ttt gat gct gcc atc
aag gta gta 705Thr Gln Gln Asn Val Lys Ala Val Phe Asp Ala Ala Ile
Lys Val Val165 170 175atc aag cct cca aca aag cag agg gac agg aag
aag aag aaa aca cgg 753Ile Lys Pro Pro Thr Lys Gln Arg Asp Arg Lys
Lys Lys Lys Thr Arg180 185 190 195cgc gga tgt tct ttc ttc tgc aag
ggt gtc atg tcc aga aga agg cta 801Arg Gly Cys Ser Phe Phe Cys Lys
Gly Val Met Ser Arg Arg Arg Leu200 205 210gta tgc ttc aag
tgaacaagag gggttctttg atgagcagag cagaggtctg 853Val Cys Phe
Lys215tgagacaaaa tgatgtcttg tgtttgataa ttgctttatc tcaaaagttc
cagtttgata 913gttgcatttc caacctatat atatcctgtt tggcaattaa
ctactacctc cgtcccaaaa 973tataacaact tttggctatg aatctgaacg
cacagttatc cagattcata gctaaaaata 1033cttatatttt gggacggagg
gagtactagt agtagattac tatcctgtcc atgtaatgta 1093ttaggaaggt
taatagcact ccctacatct cagaatggaa gttgttttgg ttttaaaaaa
1153aaaaaaaaaa aaaaaa 116945215PRTOryza sativa 45Met Ala Ser Ser
Ala Ser Arg Phe Ile Lys Cys Val Thr Val Gly Asp1 5 10 15Gly Ala Val
Gly Lys Thr Cys Met Leu Ile Cys Tyr Thr Ser Asn Lys20 25 30Phe Pro
Thr Asp Tyr Val Pro Thr Val Phe Asp Asn Phe Ser Ala Asn35 40 45Val
Val Val Asp Gly Thr Thr Val Asn Leu Gly Leu Trp Asp Thr Ala50 55
60Gly Gln Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala65
70 75 80Asp Val Phe Val Leu Ala Phe Ser Leu Val Ser Arg Ala Ser Tyr
Glu85 90 95Asn Val Met Lys Lys Trp Leu Pro Glu Leu Gln His Tyr Ala
Pro Gly100 105 110Val Pro Ile Val Leu Val Gly Thr Lys Leu Asp Leu
Arg Glu Asp Lys115 120 125His Tyr Leu Leu Asp His Pro Ser Leu Val
Pro Val Thr Thr Ala Gln130 135 140Gly Glu Glu Leu Arg Lys His Ile
Gly Ala Thr Cys Tyr Ile Glu Cys145 150 155 160Ser Ser Lys Thr Gln
Gln Asn Val Lys Ala Val Phe Asp Ala Ala Ile165 170 175Lys Val Val
Ile Lys Pro Pro Thr Lys Gln Arg Asp Arg Lys Lys Lys180 185 190Lys
Thr Arg Arg Gly Cys Ser Phe Phe Cys Lys Gly Val Met Ser Arg195 200
205Arg Arg Leu Val Cys Phe Lys210 215461127DNAZea
maysCDS(190)..(831)coding for RacB homologue (RacA) 46gtcgacccac
gcgtccgccc agaagtcacg caccaaacac caccaccaaa gaaggcgaga 60acgtactccg
tccctcccct cccctcccct ccccttcccc tcgaggctcc aggaccgtct
120cctcgcctgc tcatccgccg ctgcttccct tctctgggct cggagaaccg
gagagaagcg 180cgcgcggcc atg gcg tcc agc gcc tct cgg ttc atc aag tgc
gtc acg gtc 231Met Ala Ser Ser Ala Ser Arg Phe Ile Lys Cys Val Thr
Val1 5 10ggc gac ggt gcc gtg ggc aag aca tgt atg ctc atc tgc tac
acc agc 279Gly Asp Gly Ala Val Gly Lys Thr Cys Met Leu Ile Cys Tyr
Thr Ser15 20 25 30aac aag ttc ccc act gac tac ata cct acg gtg ttc
gac aat ttc agt 327Asn Lys Phe Pro Thr Asp Tyr Ile Pro Thr Val Phe
Asp Asn Phe Ser35 40 45gca aat gta gtt gtg gat ggc acc act gtg aat
ttg ggc ctt tgg gat 375Ala Asn Val Val Val Asp Gly Thr Thr Val Asn
Leu Gly Leu Trp Asp50 55 60acc gct ggg cag gaa gat tac aac cgc ctg
agg cct cta agc tac cga 423Thr Ala Gly Gln Glu Asp Tyr Asn Arg Leu
Arg Pro Leu Ser Tyr Arg65 70 75ggt gca gat gtt ttc gtg ctt gca ttc
tca ctt gtg agc cga gct agc 471Gly Ala Asp Val Phe Val Leu Ala Phe
Ser Leu Val Ser Arg Ala Ser80 85 90tat gag aat atc atg aag aag tgg
ata cca gag ctt caa cat tat gca 519Tyr Glu Asn Ile Met Lys Lys Trp
Ile Pro Glu Leu Gln His Tyr Ala95 100 105 110cct ggg gtg ccc gtt
gtt ttg gca ggc aca aaa ttg gat ctt cgt gaa 567Pro Gly Val Pro Val
Val Leu Ala Gly Thr Lys Leu Asp Leu Arg Glu115 120 125gac aag cac
tac ttg atg gac cat cct gga ttg gtg cct gtt acc act 615Asp Lys His
Tyr Leu Met Asp His Pro Gly Leu Val Pro Val Thr Thr130 135 140gca
cag ggg gag gaa ctt cgt aga caa att ggt gct atg tat tac att 663Ala
Gln Gly Glu Glu Leu Arg Arg Gln Ile Gly Ala Met Tyr Tyr Ile145 150
155gaa tgc agc tca aag aca cag cag aat gtc aaa gct gtg ttc gat gct
711Glu Cys Ser Ser Lys Thr Gln Gln Asn Val Lys Ala Val Phe Asp
Ala160 165 170gcc atc aag gta gta atc cag cct cca act aaa ata aga
gaa aag aag 759Ala Ile Lys Val Val Ile Gln Pro Pro Thr Lys Ile Arg
Glu Lys Lys175 180 185 190aag aaa aaa tca cgc aaa gga tgt tct atg
atg aac atc ttc ggt gga 807Lys Lys Lys Ser Arg Lys Gly Cys Ser Met
Met Asn Ile Phe Gly Gly195 200 205aga aaa atg cta tgc ttc aag tcc
tgaatggttc aagggggtct tacatggact 861Arg Lys Met Leu Cys Phe Lys
Ser210gataccacga gtgtgacccc gagtttgcga agcttgaaat cttgatgtgc
tcgttgcgca 921tgtgtatatt tgcacctttg gttattaatg actagaggta
ggtaattgaa actagtctgc 981ttaagcgttc tgcactgctg gtgtggttag
ctctatgagt taagcagttc gacagaggcc 1041aaaccgacag tgagattttg
ttctttcatg gaaatgtgcc aatgtcacag ctttttcgtg 1101aaaaaaaaaa
aaaaaaaaaa aaaaaa 112747214PRTZea mays 47Met Ala Ser Ser Ala Ser
Arg Phe Ile Lys Cys Val Thr Val Gly Asp1 5 10 15Gly Ala Val Gly Lys
Thr Cys Met Leu Ile Cys Tyr Thr Ser Asn Lys20 25 30Phe Pro Thr Asp
Tyr Ile Pro Thr Val Phe Asp Asn Phe Ser Ala Asn35 40 45Val Val Val
Asp Gly Thr Thr Val Asn Leu Gly Leu Trp Asp Thr Ala50 55 60Gly Gln
Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala65 70 75
80Asp Val Phe Val Leu Ala Phe Ser Leu Val Ser Arg Ala Ser Tyr Glu85
90 95Asn Ile Met Lys Lys Trp Ile Pro Glu Leu Gln His Tyr Ala Pro
Gly100 105 110Val Pro Val Val Leu Ala Gly Thr Lys Leu Asp Leu Arg
Glu Asp Lys115 120 125His Tyr Leu Met Asp His Pro Gly Leu Val Pro
Val Thr Thr Ala Gln130 135 140Gly Glu Glu Leu Arg Arg Gln Ile Gly
Ala Met Tyr Tyr Ile Glu Cys145 150 155 160Ser Ser Lys Thr Gln Gln
Asn Val Lys Ala Val Phe Asp Ala Ala Ile165 170 175Lys Val Val Ile
Gln Pro Pro Thr Lys Ile Arg Glu Lys Lys Lys Lys180 185 190Lys Ser
Arg Lys Gly Cys Ser Met Met Asn Ile Phe Gly Gly Arg Lys195 200
205Met Leu Cys Phe Lys Ser21048901DNAHordeum
vulgaremisc_feature(1)..(901)coding for RacB homologue (RacA)
48cccgggctgc aggaattcgg cacgaggcaa gaagtcacgc accaaacacc accccccatc
60accgccgctc cgctccccag tcccccaccc ctcctccgcc cccttcctcg agccgagctc
120cgggggaagg aatcggagag gccggcgcgc ggcgagccat ggcgtccagc
gcctcccggt 180tcatcaagtg cgtcacggtg ggcgacggcg ccgtcggcaa
gacctgcatg ctcatctgct 240acaccagcaa caagttcccc accgactaca
tacccacggt gttcgacaat ttcagcgcga 300acgtggtggc ggacggcacc
acggtgaatt tgggcctttg ggacaccgcc gggcaggagg 360attacaaccg
gctgaggcct ctaagctacc gcggcgccga cgttttcgtg cttgccttct
420cccttgtgag ccgagctagc tatgagaata tcatgaagaa gtggataccg
gagcttcagc 480attacgcgcc cggcgtacct gttgtgctgg taggcacaaa
actggatctt cgtgaagata 540agcactattt gctggaccac cctgggatga
tacccgttac cacagcacag ggggaggaac 600ttcgtaagca agttggtgct
ttatattaca tagagtgcag ctcaaagaca caacagaatg 660tcaaagctgt
gtttgatgct gctatcaagg tagtaatcca gccccccact aaacaaagag
720aaaagaagaa aaagaaacag cgtcggggat gttctatgat gaacttcagc
ggaaggaaat 780gctatgcttc aaatcctgaa tgatgaaaga gaaggttcct
tgcctngaac gattgtcacg 840gttgcgctgc accaatttga caacacctcc
aaaccggttg aatgtgctgg attgcaccgt 900c 90149594DNAArabidopsis
thalianaCDS(1)..(591)coding for RacB homologue 49atg agc gct tcg
agg ttc gta aag tgc gtg acg gtt ggt gat gga gct 48Met Ser Ala Ser
Arg Phe Val Lys Cys Val Thr Val Gly Asp Gly Ala1 5 10 15gtc gga aaa
act tgt ttg ttg att tct tac aca agc aac act ttc cct 96Val Gly Lys
Thr Cys Leu Leu Ile Ser Tyr Thr Ser Asn Thr Phe Pro20 25 30acg gat
tat gtg cct acc gtt ttc gat aat ttc agt gcc aat gtt gtg 144Thr Asp
Tyr Val Pro Thr Val Phe Asp Asn Phe Ser Ala Asn Val Val35 40 45gtt
aat gga agc act gtg aat ctt gga ttg tgg gac act gca ggg caa 192Val
Asn Gly Ser Thr Val Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln50 55
60gag gat tac aat aga tta aga cca ctg agt tac cgt gga gca gat gtt
240Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala Asp
Val65 70 75 80ttc att ttg gcc ttc tct ctt atc agt aaa gcc agt tat
gaa aac gtc 288Phe Ile Leu Ala Phe Ser Leu Ile Ser Lys Ala Ser Tyr
Glu Asn Val85 90 95tcc aaa aag tgg atc ccg gag ttg aaa cat tac gcg
cct ggt gtc ccc 336Ser Lys Lys Trp Ile Pro Glu Leu Lys His Tyr Ala
Pro Gly Val Pro100 105 110atc gtc ctt gtt gga aca aag ctt gat ctt
cga gat gat aaa cag ttc 384Ile Val Leu Val Gly Thr Lys Leu Asp Leu
Arg Asp Asp Lys Gln Phe115 120 125ttt atc gac cat cct ggt gct gtt
ccg att act act gct cag gga gag 432Phe Ile Asp His Pro Gly Ala Val
Pro Ile Thr Thr Ala Gln Gly Glu130 135 140gag ctg agg aag caa ata
gga gca cct act tac atc gaa tgc agt tcc 480Glu Leu Arg Lys Gln Ile
Gly Ala Pro Thr Tyr Ile Glu Cys Ser Ser145 150 155 160aaa act caa
gag aat gtg aag gcg gtg ttt gac gca gcc atc cga gtg 528Lys Thr Gln
Glu Asn Val Lys Ala Val Phe Asp Ala Ala Ile Arg Val165 170 175gtg
ttg caa ccg cca aag cag aag aag aag aag agc aaa gcg cag aag 576Val
Leu Gln Pro Pro Lys Gln Lys Lys Lys Lys Ser Lys Ala Gln Lys180 185
190gca tgc tcc att cta tga 594Ala Cys Ser Ile Leu195
50197PRTArabidopsis thaliana 50Met Ser Ala Ser Arg Phe Val Lys Cys
Val Thr Val Gly Asp Gly
Ala1 5 10 15Val Gly Lys Thr Cys Leu Leu Ile Ser Tyr Thr Ser Asn Thr
Phe Pro20 25 30Thr Asp Tyr Val Pro Thr Val Phe Asp Asn Phe Ser Ala
Asn Val Val35 40 45Val Asn Gly Ser Thr Val Asn Leu Gly Leu Trp Asp
Thr Ala Gly Gln50 55 60Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr
Arg Gly Ala Asp Val65 70 75 80Phe Ile Leu Ala Phe Ser Leu Ile Ser
Lys Ala Ser Tyr Glu Asn Val85 90 95Ser Lys Lys Trp Ile Pro Glu Leu
Lys His Tyr Ala Pro Gly Val Pro100 105 110Ile Val Leu Val Gly Thr
Lys Leu Asp Leu Arg Asp Asp Lys Gln Phe115 120 125Phe Ile Asp His
Pro Gly Ala Val Pro Ile Thr Thr Ala Gln Gly Glu130 135 140Glu Leu
Arg Lys Gln Ile Gly Ala Pro Thr Tyr Ile Glu Cys Ser Ser145 150 155
160Lys Thr Gln Glu Asn Val Lys Ala Val Phe Asp Ala Ala Ile Arg
Val165 170 175Val Leu Gln Pro Pro Lys Gln Lys Lys Lys Lys Ser Lys
Ala Gln Lys180 185 190Ala Cys Ser Ile Leu19551594DNAArabidopsis
thalianaCDS(1)..(591)coding for RacB homologue 51atg agc gct tcg
agg ttc ata aag tgt gtc acc gtt ggc gac gga gct 48Met Ser Ala Ser
Arg Phe Ile Lys Cys Val Thr Val Gly Asp Gly Ala1 5 10 15gtt ggt aaa
acc tgt ttg ctg att tct tac acc agc aac act ttt cct 96Val Gly Lys
Thr Cys Leu Leu Ile Ser Tyr Thr Ser Asn Thr Phe Pro20 25 30acg gat
tat gta ccg act gtt ttc gat aac ttt agc gca aat gtg gtt 144Thr Asp
Tyr Val Pro Thr Val Phe Asp Asn Phe Ser Ala Asn Val Val35 40 45gtt
aat gga gcc act gtg aat ctt ggg cta tgg gat acc gca ggg cag 192Val
Asn Gly Ala Thr Val Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln50 55
60gag gat tat aac aga tta aga cct ttg agt tac cgc ggt gct gat gtt
240Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala Asp
Val65 70 75 80ttc atc tta gca ttc tct ctt atc agt aag gct agt tat
gag aat gtc 288Phe Ile Leu Ala Phe Ser Leu Ile Ser Lys Ala Ser Tyr
Glu Asn Val85 90 95tcc aag aag tgg atc cca gag ctg aag cat tat gcc
cct ggt gtc cct 336Ser Lys Lys Trp Ile Pro Glu Leu Lys His Tyr Ala
Pro Gly Val Pro100 105 110ata gtt ctt gtt gga acc aaa cta gat ctt
cgg gat gac aaa cag ttc 384Ile Val Leu Val Gly Thr Lys Leu Asp Leu
Arg Asp Asp Lys Gln Phe115 120 125ttc att gac cac cct ggc gct gta
cca att act act gct cag gga gag 432Phe Ile Asp His Pro Gly Ala Val
Pro Ile Thr Thr Ala Gln Gly Glu130 135 140gaa ctg aag aaa cta att
gga gct ccc gca tac atc gag tgc agt tca 480Glu Leu Lys Lys Leu Ile
Gly Ala Pro Ala Tyr Ile Glu Cys Ser Ser145 150 155 160aaa aca caa
gag aac gtg aaa gga gta ttt gat gca gcg atc cga gtg 528Lys Thr Gln
Glu Asn Val Lys Gly Val Phe Asp Ala Ala Ile Arg Val165 170 175gtt
ctt caa cct cca aag cag aag aaa aag aaa agc aaa gca caa aaa 576Val
Leu Gln Pro Pro Lys Gln Lys Lys Lys Lys Ser Lys Ala Gln Lys180 185
190gcc tgc tcc att ttg taa 594Ala Cys Ser Ile Leu195
52197PRTArabidopsis thaliana 52Met Ser Ala Ser Arg Phe Ile Lys Cys
Val Thr Val Gly Asp Gly Ala1 5 10 15Val Gly Lys Thr Cys Leu Leu Ile
Ser Tyr Thr Ser Asn Thr Phe Pro20 25 30Thr Asp Tyr Val Pro Thr Val
Phe Asp Asn Phe Ser Ala Asn Val Val35 40 45Val Asn Gly Ala Thr Val
Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln50 55 60Glu Asp Tyr Asn Arg
Leu Arg Pro Leu Ser Tyr Arg Gly Ala Asp Val65 70 75 80Phe Ile Leu
Ala Phe Ser Leu Ile Ser Lys Ala Ser Tyr Glu Asn Val85 90 95Ser Lys
Lys Trp Ile Pro Glu Leu Lys His Tyr Ala Pro Gly Val Pro100 105
110Ile Val Leu Val Gly Thr Lys Leu Asp Leu Arg Asp Asp Lys Gln
Phe115 120 125Phe Ile Asp His Pro Gly Ala Val Pro Ile Thr Thr Ala
Gln Gly Glu130 135 140Glu Leu Lys Lys Leu Ile Gly Ala Pro Ala Tyr
Ile Glu Cys Ser Ser145 150 155 160Lys Thr Gln Glu Asn Val Lys Gly
Val Phe Asp Ala Ala Ile Arg Val165 170 175Val Leu Gln Pro Pro Lys
Gln Lys Lys Lys Lys Ser Lys Ala Gln Lys180 185 190Ala Cys Ser Ile
Leu19553594DNAArabidopsis thalianaCDS(1)..(591)coding for RacB
homologue 53atg agc gca tca agg ttc ata aag tgc gtc acc gtt ggt gat
gga gct 48Met Ser Ala Ser Arg Phe Ile Lys Cys Val Thr Val Gly Asp
Gly Ala1 5 10 15gtt ggt aaa acc tgt ttg ctg att tct tat acc agc aac
acc ttt ccc 96Val Gly Lys Thr Cys Leu Leu Ile Ser Tyr Thr Ser Asn
Thr Phe Pro20 25 30acg gat tat gtt ccg act gtt ttc gat aac ttt agt
gca aat gtg gtt 144Thr Asp Tyr Val Pro Thr Val Phe Asp Asn Phe Ser
Ala Asn Val Val35 40 45gtt aat gga gcc acg gtg aat ctt gga ttg tgg
gat act gca ggg caa 192Val Asn Gly Ala Thr Val Asn Leu Gly Leu Trp
Asp Thr Ala Gly Gln50 55 60gag gac tat aac aga tta aga cct ttg agt
tac cgt ggt gct gat gtt 240Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser
Tyr Arg Gly Ala Asp Val65 70 75 80ttc att ctt gcc ttc tct ctc att
agt aag gct agt tat gag aat gtt 288Phe Ile Leu Ala Phe Ser Leu Ile
Ser Lys Ala Ser Tyr Glu Asn Val85 90 95tcc aag aag tgg att cct gag
ttg aag cac tat gct cct ggt gtc cca 336Ser Lys Lys Trp Ile Pro Glu
Leu Lys His Tyr Ala Pro Gly Val Pro100 105 110att gtc ctt gtt gga
acc aaa cta gat ctt cga gat gac aaa cag ttt 384Ile Val Leu Val Gly
Thr Lys Leu Asp Leu Arg Asp Asp Lys Gln Phe115 120 125ttc atc gac
cat cct ggt gct gtc cct att acc act gtt cag gga gag 432Phe Ile Asp
His Pro Gly Ala Val Pro Ile Thr Thr Val Gln Gly Glu130 135 140gag
ctg aag aag cta att gga gcg cca gct tac atc gag tgc agt tca 480Glu
Leu Lys Lys Leu Ile Gly Ala Pro Ala Tyr Ile Glu Cys Ser Ser145 150
155 160aaa tca caa gag aac gtg aag ggc gtg ttt gat gca gcg atc aga
gtg 528Lys Ser Gln Glu Asn Val Lys Gly Val Phe Asp Ala Ala Ile Arg
Val165 170 175gtc ctt caa cct cca aag cag aag aaa aag aag aac aaa
gca caa aag 576Val Leu Gln Pro Pro Lys Gln Lys Lys Lys Lys Asn Lys
Ala Gln Lys180 185 190gcc tgc tcc atc ttg taa 594Ala Cys Ser Ile
Leu19554197PRTArabidopsis thaliana 54Met Ser Ala Ser Arg Phe Ile
Lys Cys Val Thr Val Gly Asp Gly Ala1 5 10 15Val Gly Lys Thr Cys Leu
Leu Ile Ser Tyr Thr Ser Asn Thr Phe Pro20 25 30Thr Asp Tyr Val Pro
Thr Val Phe Asp Asn Phe Ser Ala Asn Val Val35 40 45Val Asn Gly Ala
Thr Val Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln50 55 60Glu Asp Tyr
Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala Asp Val65 70 75 80Phe
Ile Leu Ala Phe Ser Leu Ile Ser Lys Ala Ser Tyr Glu Asn Val85 90
95Ser Lys Lys Trp Ile Pro Glu Leu Lys His Tyr Ala Pro Gly Val
Pro100 105 110Ile Val Leu Val Gly Thr Lys Leu Asp Leu Arg Asp Asp
Lys Gln Phe115 120 125Phe Ile Asp His Pro Gly Ala Val Pro Ile Thr
Thr Val Gln Gly Glu130 135 140Glu Leu Lys Lys Leu Ile Gly Ala Pro
Ala Tyr Ile Glu Cys Ser Ser145 150 155 160Lys Ser Gln Glu Asn Val
Lys Gly Val Phe Asp Ala Ala Ile Arg Val165 170 175Val Leu Gln Pro
Pro Lys Gln Lys Lys Lys Lys Asn Lys Ala Gln Lys180 185 190Ala Cys
Ser Ile Leu19555591DNAArabidopsis thalianaCDS(1)..(588)coding for
RacB homologue 55atg agt gct tcg agg ttt ata aag tgt gtc acc gtc
ggc gat ggt gcc 48Met Ser Ala Ser Arg Phe Ile Lys Cys Val Thr Val
Gly Asp Gly Ala1 5 10 15gtc gga aaa act tgt atg ctg att tct tac aca
agc aac act ttc cct 96Val Gly Lys Thr Cys Met Leu Ile Ser Tyr Thr
Ser Asn Thr Phe Pro20 25 30acg gac tat gtt cca act gtt ttc gac aac
ttc agt gct aat gtg gtt 144Thr Asp Tyr Val Pro Thr Val Phe Asp Asn
Phe Ser Ala Asn Val Val35 40 45gta gat ggg aac acg gtg aat ctt gga
ttg tgg gat aca gct ggt caa 192Val Asp Gly Asn Thr Val Asn Leu Gly
Leu Trp Asp Thr Ala Gly Gln50 55 60gaa gac tat aac agg tta aga ccg
ttg agt tac cgt ggt gcc gat gtc 240Glu Asp Tyr Asn Arg Leu Arg Pro
Leu Ser Tyr Arg Gly Ala Asp Val65 70 75 80ttc att ctt gca ttc tcg
ctt att agc aaa gct agc tac gag aat gta 288Phe Ile Leu Ala Phe Ser
Leu Ile Ser Lys Ala Ser Tyr Glu Asn Val85 90 95gcc aag aag tgg att
cct gag ctt agg cat tat gcc cct ggt gtt cct 336Ala Lys Lys Trp Ile
Pro Glu Leu Arg His Tyr Ala Pro Gly Val Pro100 105 110ata atc ctc
gtt gga acg aaa ctc gat ctt cga gat gac aag caa ttc 384Ile Ile Leu
Val Gly Thr Lys Leu Asp Leu Arg Asp Asp Lys Gln Phe115 120 125ttc
ata gac cat cct ggt gca gtg cct att act aca aac cag gga gag 432Phe
Ile Asp His Pro Gly Ala Val Pro Ile Thr Thr Asn Gln Gly Glu130 135
140gaa cta aag aaa ctg ata gga tca cca atc tac att gaa tgt agt tca
480Glu Leu Lys Lys Leu Ile Gly Ser Pro Ile Tyr Ile Glu Cys Ser
Ser145 150 155 160aag act cag cag aat gtg aaa gca gtc ttt gac gca
gcc ata aaa gtg 528Lys Thr Gln Gln Asn Val Lys Ala Val Phe Asp Ala
Ala Ile Lys Val165 170 175gtg ctt cag cca ccg aaa cag aag aag aag
aaa aag aac aag aac cgc 576Val Leu Gln Pro Pro Lys Gln Lys Lys Lys
Lys Lys Asn Lys Asn Arg180 185 190tgc gtg ttc ttg tga 591Cys Val
Phe Leu19556196PRTArabidopsis thaliana 56Met Ser Ala Ser Arg Phe
Ile Lys Cys Val Thr Val Gly Asp Gly Ala1 5 10 15Val Gly Lys Thr Cys
Met Leu Ile Ser Tyr Thr Ser Asn Thr Phe Pro20 25 30Thr Asp Tyr Val
Pro Thr Val Phe Asp Asn Phe Ser Ala Asn Val Val35 40 45Val Asp Gly
Asn Thr Val Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln50 55 60Glu Asp
Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala Asp Val65 70 75
80Phe Ile Leu Ala Phe Ser Leu Ile Ser Lys Ala Ser Tyr Glu Asn Val85
90 95Ala Lys Lys Trp Ile Pro Glu Leu Arg His Tyr Ala Pro Gly Val
Pro100 105 110Ile Ile Leu Val Gly Thr Lys Leu Asp Leu Arg Asp Asp
Lys Gln Phe115 120 125Phe Ile Asp His Pro Gly Ala Val Pro Ile Thr
Thr Asn Gln Gly Glu130 135 140Glu Leu Lys Lys Leu Ile Gly Ser Pro
Ile Tyr Ile Glu Cys Ser Ser145 150 155 160Lys Thr Gln Gln Asn Val
Lys Ala Val Phe Asp Ala Ala Ile Lys Val165 170 175Val Leu Gln Pro
Pro Lys Gln Lys Lys Lys Lys Lys Asn Lys Asn Arg180 185 190Cys Val
Phe Leu19557597DNAArabidopsis thalianaCDS(1)..(594)coding for RacB
homologue 57atg agt gct tca agg ttt atc aag tgt gtc act gtc ggc gac
ggt gct 48Met Ser Ala Ser Arg Phe Ile Lys Cys Val Thr Val Gly Asp
Gly Ala1 5 10 15gtt gga aag act tgt ctt ctc atc tcc tac act agc aac
act ttc ccc 96Val Gly Lys Thr Cys Leu Leu Ile Ser Tyr Thr Ser Asn
Thr Phe Pro20 25 30acg gat tat gtg cca act gtg ttc gat aat ttc agt
gcc aat gtg att 144Thr Asp Tyr Val Pro Thr Val Phe Asp Asn Phe Ser
Ala Asn Val Ile35 40 45gtt gat ggc aac act atc aac ttg gga ttg tgg
gat act gca ggg caa 192Val Asp Gly Asn Thr Ile Asn Leu Gly Leu Trp
Asp Thr Ala Gly Gln50 55 60gag gac tac aat aga cta aga cct ttg agc
tat cgc ggt gca gat gtc 240Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser
Tyr Arg Gly Ala Asp Val65 70 75 80ttc tta ctt gca ttc tca ctt gtc
agc aaa gct agc tat gaa aat gtt 288Phe Leu Leu Ala Phe Ser Leu Val
Ser Lys Ala Ser Tyr Glu Asn Val85 90 95tct aaa aag tgg gtt cct gaa
ctg aga cat tat gct cct ggt gtt ccc 336Ser Lys Lys Trp Val Pro Glu
Leu Arg His Tyr Ala Pro Gly Val Pro100 105 110atc atc ctc gtt gga
aca aag ctt gat ctt cga gat gat aag caa ttc 384Ile Ile Leu Val Gly
Thr Lys Leu Asp Leu Arg Asp Asp Lys Gln Phe115 120 125ttt gcc gag
cac cct ggt gct gtg cct atc tct acc gct cag ggt gaa 432Phe Ala Glu
His Pro Gly Ala Val Pro Ile Ser Thr Ala Gln Gly Glu130 135 140gaa
cta aag aag ctg att ggg gcg cct gct tat atc gaa tgc agt gca 480Glu
Leu Lys Lys Leu Ile Gly Ala Pro Ala Tyr Ile Glu Cys Ser Ala145 150
155 160aaa act caa cag aat gtg aaa gca gtg ttt gat gcg gct atc aag
gtc 528Lys Thr Gln Gln Asn Val Lys Ala Val Phe Asp Ala Ala Ile Lys
Val165 170 175gtt ctc cag cca cca aaa aac aag aag aag aag aag aga
aaa tct cag 576Val Leu Gln Pro Pro Lys Asn Lys Lys Lys Lys Lys Arg
Lys Ser Gln180 185 190aaa ggt tgt tct ata ctc tga 597Lys Gly Cys
Ser Ile Leu19558198PRTArabidopsis thaliana 58Met Ser Ala Ser Arg
Phe Ile Lys Cys Val Thr Val Gly Asp Gly Ala1 5 10 15Val Gly Lys Thr
Cys Leu Leu Ile Ser Tyr Thr Ser Asn Thr Phe Pro20 25 30Thr Asp Tyr
Val Pro Thr Val Phe Asp Asn Phe Ser Ala Asn Val Ile35 40 45Val Asp
Gly Asn Thr Ile Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln50 55 60Glu
Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala Asp Val65 70 75
80Phe Leu Leu Ala Phe Ser Leu Val Ser Lys Ala Ser Tyr Glu Asn Val85
90 95Ser Lys Lys Trp Val Pro Glu Leu Arg His Tyr Ala Pro Gly Val
Pro100 105 110Ile Ile Leu Val Gly Thr Lys Leu Asp Leu Arg Asp Asp
Lys Gln Phe115 120 125Phe Ala Glu His Pro Gly Ala Val Pro Ile Ser
Thr Ala Gln Gly Glu130 135 140Glu Leu Lys Lys Leu Ile Gly Ala Pro
Ala Tyr Ile Glu Cys Ser Ala145 150 155 160Lys Thr Gln Gln Asn Val
Lys Ala Val Phe Asp Ala Ala Ile Lys Val165 170 175Val Leu Gln Pro
Pro Lys Asn Lys Lys Lys Lys Lys Arg Lys Ser Gln180 185 190Lys Gly
Cys Ser Ile Leu19559588DNAArabidopsis thalianaCDS(1)..(585)coding
for RacB homologue 59atg gcg tca agg ttt ata aag tgt gtg acc gtc
gga gat ggt gcc gtc 48Met Ala Ser Arg Phe Ile Lys Cys Val Thr Val
Gly Asp Gly Ala Val1 5 10 15gga aaa act tgc atg ctc att tct tac act
agc aat act ttt cct act 96Gly Lys Thr Cys Met Leu Ile Ser Tyr Thr
Ser Asn Thr Phe Pro Thr20 25 30gat tat gtg cca act gtt ttc gac aac
ttc agt gct aat gtg gtt gtt 144Asp Tyr Val Pro Thr Val Phe Asp Asn
Phe Ser Ala Asn Val Val Val35 40 45gat ggc aac act gtc aat ctt gga
ttg tgg gat act gct ggt caa gag 192Asp Gly Asn Thr Val Asn Leu Gly
Leu Trp Asp Thr Ala Gly Gln Glu50 55 60gac tac aac agg tta cga cct
ttg agt tac cgt ggt gct gat gtt ttc 240Asp Tyr Asn Arg Leu Arg Pro
Leu Ser Tyr Arg Gly Ala Asp Val Phe65 70 75 80att ctt gct ttc tct
ctt att agc aag gct agc tat gag aat ata gcc 288Ile Leu Ala Phe Ser
Leu Ile Ser Lys Ala Ser Tyr Glu Asn Ile Ala85 90 95aag aag tgg att
cct gag ctc agg cat tat gct cct ggt gtt ccc att 336Lys Lys Trp Ile
Pro Glu Leu Arg His Tyr Ala Pro Gly Val Pro Ile100 105 110atc ctt
gtt ggg aca aaa ctc gat ctt cga gat gac aag caa ttc ttt 384Ile Leu
Val Gly Thr Lys Leu Asp Leu Arg Asp Asp Lys Gln Phe Phe115 120
125ata gat cat cct ggt gct gtg cca att act aca aac cag gga gag gaa
432Ile Asp His Pro Gly Ala Val Pro Ile Thr Thr Asn Gln Gly Glu
Glu130 135 140ctg aag aaa ctg att gga tct gct gtc tac att gaa tgt
agt tca aag 480Leu Lys Lys Leu Ile Gly Ser Ala Val Tyr Ile Glu Cys
Ser Ser Lys145 150 155 160aca cag cag aac gtg aag gca gtg ttt gat
gca gct ata aaa gtg gtg 528Thr Gln Gln Asn Val Lys Ala Val Phe Asp
Ala Ala Ile Lys Val Val165 170 175ctt cag cca
cca aag cag aag aag aag aaa aag aat aag aac cgt tgc 576Leu Gln Pro
Pro Lys Gln Lys Lys Lys Lys Lys Asn Lys Asn Arg Cys180 185 190gcg
ttc ttg tga 588Ala Phe Leu19560195PRTArabidopsis thaliana 60Met Ala
Ser Arg Phe Ile Lys Cys Val Thr Val Gly Asp Gly Ala Val1 5 10 15Gly
Lys Thr Cys Met Leu Ile Ser Tyr Thr Ser Asn Thr Phe Pro Thr20 25
30Asp Tyr Val Pro Thr Val Phe Asp Asn Phe Ser Ala Asn Val Val Val35
40 45Asp Gly Asn Thr Val Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln
Glu50 55 60Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala Asp
Val Phe65 70 75 80Ile Leu Ala Phe Ser Leu Ile Ser Lys Ala Ser Tyr
Glu Asn Ile Ala85 90 95Lys Lys Trp Ile Pro Glu Leu Arg His Tyr Ala
Pro Gly Val Pro Ile100 105 110Ile Leu Val Gly Thr Lys Leu Asp Leu
Arg Asp Asp Lys Gln Phe Phe115 120 125Ile Asp His Pro Gly Ala Val
Pro Ile Thr Thr Asn Gln Gly Glu Glu130 135 140Leu Lys Lys Leu Ile
Gly Ser Ala Val Tyr Ile Glu Cys Ser Ser Lys145 150 155 160Thr Gln
Gln Asn Val Lys Ala Val Phe Asp Ala Ala Ile Lys Val Val165 170
175Leu Gln Pro Pro Lys Gln Lys Lys Lys Lys Lys Asn Lys Asn Arg
Cys180 185 190Ala Phe Leu19561606DNAArabidopsis
thalianaCDS(1)..(603)coding for RacB homologue 61atg agc aca gca
aga ttc att aag tgt gtg act gtc gga gat gga gca 48Met Ser Thr Ala
Arg Phe Ile Lys Cys Val Thr Val Gly Asp Gly Ala1 5 10 15gtg gga aag
act tgt atg ctc att tca tat acc agc aat acg ttt cct 96Val Gly Lys
Thr Cys Met Leu Ile Ser Tyr Thr Ser Asn Thr Phe Pro20 25 30acg gat
tat gtt cca aca gtt ttc gac aac ttc agc gca aat gtg gtg 144Thr Asp
Tyr Val Pro Thr Val Phe Asp Asn Phe Ser Ala Asn Val Val35 40 45gtc
gac ggg agt acc gtg aac ctt ggc ctg tgg gat act gcc ggt cag 192Val
Asp Gly Ser Thr Val Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln50 55
60gaa gat tat aat agg ctt agg cct ttg agt tac aga gga gca gat gtc
240Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala Asp
Val65 70 75 80ttc tta tta gca ttt tcc ctt ata agc aag gcc agt tac
gag aat att 288Phe Leu Leu Ala Phe Ser Leu Ile Ser Lys Ala Ser Tyr
Glu Asn Ile85 90 95cac aaa aag tgg ctt ccg gag ctg aaa cat tat gct
cct ggc atc ccc 336His Lys Lys Trp Leu Pro Glu Leu Lys His Tyr Ala
Pro Gly Ile Pro100 105 110att gtg ctc gtc gga aca aaa tta gat ttg
agg gat gac aag cag ttc 384Ile Val Leu Val Gly Thr Lys Leu Asp Leu
Arg Asp Asp Lys Gln Phe115 120 125ttg aag gat cat cca gga gca gct
tct ata aca act gct cag gga gaa 432Leu Lys Asp His Pro Gly Ala Ala
Ser Ile Thr Thr Ala Gln Gly Glu130 135 140gaa tta agg aaa atg att
gga gct gtt agg tac tta gag tgc agc tcc 480Glu Leu Arg Lys Met Ile
Gly Ala Val Arg Tyr Leu Glu Cys Ser Ser145 150 155 160aaa acc caa
cag aat gtg aag gca gtg ttt gat aca gcg ata agg gta 528Lys Thr Gln
Gln Asn Val Lys Ala Val Phe Asp Thr Ala Ile Arg Val165 170 175gct
ttg agg cca cca aag gca aag aaa aag ata aaa cca ttg aag act 576Ala
Leu Arg Pro Pro Lys Ala Lys Lys Lys Ile Lys Pro Leu Lys Thr180 185
190aag aga tca aga ata tgc ttt ttc cta taa 606Lys Arg Ser Arg Ile
Cys Phe Phe Leu195 20062201PRTArabidopsis thaliana 62Met Ser Thr
Ala Arg Phe Ile Lys Cys Val Thr Val Gly Asp Gly Ala1 5 10 15Val Gly
Lys Thr Cys Met Leu Ile Ser Tyr Thr Ser Asn Thr Phe Pro20 25 30Thr
Asp Tyr Val Pro Thr Val Phe Asp Asn Phe Ser Ala Asn Val Val35 40
45Val Asp Gly Ser Thr Val Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln50
55 60Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala Asp
Val65 70 75 80Phe Leu Leu Ala Phe Ser Leu Ile Ser Lys Ala Ser Tyr
Glu Asn Ile85 90 95His Lys Lys Trp Leu Pro Glu Leu Lys His Tyr Ala
Pro Gly Ile Pro100 105 110Ile Val Leu Val Gly Thr Lys Leu Asp Leu
Arg Asp Asp Lys Gln Phe115 120 125Leu Lys Asp His Pro Gly Ala Ala
Ser Ile Thr Thr Ala Gln Gly Glu130 135 140Glu Leu Arg Lys Met Ile
Gly Ala Val Arg Tyr Leu Glu Cys Ser Ser145 150 155 160Lys Thr Gln
Gln Asn Val Lys Ala Val Phe Asp Thr Ala Ile Arg Val165 170 175Ala
Leu Arg Pro Pro Lys Ala Lys Lys Lys Ile Lys Pro Leu Lys Thr180 185
190Lys Arg Ser Arg Ile Cys Phe Phe Leu195 20063606DNAArabidopsis
thalianaCDS(1)..(603)coding for RacB homologue 63atg gct tcg agt
gct tca aaa ttc atc aaa tgt gtg act gtt gga gat 48Met Ala Ser Ser
Ala Ser Lys Phe Ile Lys Cys Val Thr Val Gly Asp1 5 10 15ggt gcc gtt
gga aaa act tgt atg ctc atc tgc tac act agc aac aaa 96Gly Ala Val
Gly Lys Thr Cys Met Leu Ile Cys Tyr Thr Ser Asn Lys20 25 30ttc cct
act gac tac ata cca aca gtt ttt gac aac ttt agt gtt aat 144Phe Pro
Thr Asp Tyr Ile Pro Thr Val Phe Asp Asn Phe Ser Val Asn35 40 45gtt
gtg gtt gaa ggc atc act gtg aac tta ggc ctt tgg gac act gcc 192Val
Val Val Glu Gly Ile Thr Val Asn Leu Gly Leu Trp Asp Thr Ala50 55
60ggg caa gaa gac tat aac aga cta agg cct tta agt tac aga gga gca
240Gly Gln Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly
Ala65 70 75 80gat gtt ttt gtg ttg gct ttc tca ttg atc agc cga gct
agc tat gag 288Asp Val Phe Val Leu Ala Phe Ser Leu Ile Ser Arg Ala
Ser Tyr Glu85 90 95aat gtg ttt aaa aag tgg atc cct gaa ctc caa cac
ttt gca cca gga 336Asn Val Phe Lys Lys Trp Ile Pro Glu Leu Gln His
Phe Ala Pro Gly100 105 110gtc ccc att gtg ctt gtt ggt acc aaa atg
gat ctt cgt gaa gat aga 384Val Pro Ile Val Leu Val Gly Thr Lys Met
Asp Leu Arg Glu Asp Arg115 120 125cat tac ttg tct gat cat cct gga
ctg tcc ccg gta act aca tca cag 432His Tyr Leu Ser Asp His Pro Gly
Leu Ser Pro Val Thr Thr Ser Gln130 135 140gga gag gaa ctc cgc aag
cat atc gga gcg act tat tac att gaa tgt 480Gly Glu Glu Leu Arg Lys
His Ile Gly Ala Thr Tyr Tyr Ile Glu Cys145 150 155 160agc tca aaa
act caa cag aat gtg aaa gcc gta ttt gat gct gct att 528Ser Ser Lys
Thr Gln Gln Asn Val Lys Ala Val Phe Asp Ala Ala Ile165 170 175aaa
gta gta att aaa cca gca gtg aaa caa aag gag aag aag aag aag 576Lys
Val Val Ile Lys Pro Ala Val Lys Gln Lys Glu Lys Lys Lys Lys180 185
190cag aag cct cgc agc gga tgt ctc tcg taa 606Gln Lys Pro Arg Ser
Gly Cys Leu Ser195 20064201PRTArabidopsis thaliana 64Met Ala Ser
Ser Ala Ser Lys Phe Ile Lys Cys Val Thr Val Gly Asp1 5 10 15Gly Ala
Val Gly Lys Thr Cys Met Leu Ile Cys Tyr Thr Ser Asn Lys20 25 30Phe
Pro Thr Asp Tyr Ile Pro Thr Val Phe Asp Asn Phe Ser Val Asn35 40
45Val Val Val Glu Gly Ile Thr Val Asn Leu Gly Leu Trp Asp Thr Ala50
55 60Gly Gln Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly
Ala65 70 75 80Asp Val Phe Val Leu Ala Phe Ser Leu Ile Ser Arg Ala
Ser Tyr Glu85 90 95Asn Val Phe Lys Lys Trp Ile Pro Glu Leu Gln His
Phe Ala Pro Gly100 105 110Val Pro Ile Val Leu Val Gly Thr Lys Met
Asp Leu Arg Glu Asp Arg115 120 125His Tyr Leu Ser Asp His Pro Gly
Leu Ser Pro Val Thr Thr Ser Gln130 135 140Gly Glu Glu Leu Arg Lys
His Ile Gly Ala Thr Tyr Tyr Ile Glu Cys145 150 155 160Ser Ser Lys
Thr Gln Gln Asn Val Lys Ala Val Phe Asp Ala Ala Ile165 170 175Lys
Val Val Ile Lys Pro Ala Val Lys Gln Lys Glu Lys Lys Lys Lys180 185
190Gln Lys Pro Arg Ser Gly Cys Leu Ser195 20065648DNAArabidopsis
thalianaCDS(1)..(645)coding for RacB homologue 65atg gct tca agt
gct tca aag ttc atc aag tgt gtg act gtt ggt gat 48Met Ala Ser Ser
Ala Ser Lys Phe Ile Lys Cys Val Thr Val Gly Asp1 5 10 15ggt gct gtt
ggt aaa acc tgt atg ctc atc tgc tac acc agc aat aaa 96Gly Ala Val
Gly Lys Thr Cys Met Leu Ile Cys Tyr Thr Ser Asn Lys20 25 30ttc ccc
act gac tac ata cca aca gtt ttt gac aac ttt agt gca aat 144Phe Pro
Thr Asp Tyr Ile Pro Thr Val Phe Asp Asn Phe Ser Ala Asn35 40 45gtt
gtt gtt gaa ggc acc act gtc aat ttg ggg ctt tgg gac act gct 192Val
Val Val Glu Gly Thr Thr Val Asn Leu Gly Leu Trp Asp Thr Ala50 55
60ggg caa gaa gac tat aac aga tta agg cct tta agt tac agg gga gca
240Gly Gln Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly
Ala65 70 75 80gat gtt ttc gtc ttg tct ttc tca tta gtc agc cga gct
agc tac gag 288Asp Val Phe Val Leu Ser Phe Ser Leu Val Ser Arg Ala
Ser Tyr Glu85 90 95aat gtt ttt aaa aag tgg atc cct gaa ctc caa cac
ttt gct cca gga 336Asn Val Phe Lys Lys Trp Ile Pro Glu Leu Gln His
Phe Ala Pro Gly100 105 110gtt ccc ctt gtc ctt gtt ggt acc aaa tta
gat ctt cgt gaa gat aag 384Val Pro Leu Val Leu Val Gly Thr Lys Leu
Asp Leu Arg Glu Asp Lys115 120 125cat tat ttg gct gat cat cct gga
cta tcc cct gta act act gca cag 432His Tyr Leu Ala Asp His Pro Gly
Leu Ser Pro Val Thr Thr Ala Gln130 135 140gga gag gag ttg cgt aag
cta att ggt gcg acg tat tac att gag tgt 480Gly Glu Glu Leu Arg Lys
Leu Ile Gly Ala Thr Tyr Tyr Ile Glu Cys145 150 155 160agt tca aaa
act caa cag aat gtg aaa gca gtt ttt gat tct gcg ata 528Ser Ser Lys
Thr Gln Gln Asn Val Lys Ala Val Phe Asp Ser Ala Ile165 170 175aag
gaa gtg atc aaa cct ctg gtt aaa caa aag gag aag act aag aag 576Lys
Glu Val Ile Lys Pro Leu Val Lys Gln Lys Glu Lys Thr Lys Lys180 185
190aag aag aag caa aag tcg aat cac ggc tgt tta tca aat gtt ctg tgt
624Lys Lys Lys Gln Lys Ser Asn His Gly Cys Leu Ser Asn Val Leu
Cys195 200 205ggg agg ata gtg act cgg cat tga 648Gly Arg Ile Val
Thr Arg His210 21566215PRTArabidopsis thaliana 66Met Ala Ser Ser
Ala Ser Lys Phe Ile Lys Cys Val Thr Val Gly Asp1 5 10 15Gly Ala Val
Gly Lys Thr Cys Met Leu Ile Cys Tyr Thr Ser Asn Lys20 25 30Phe Pro
Thr Asp Tyr Ile Pro Thr Val Phe Asp Asn Phe Ser Ala Asn35 40 45Val
Val Val Glu Gly Thr Thr Val Asn Leu Gly Leu Trp Asp Thr Ala50 55
60Gly Gln Glu Asp Tyr Asn Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala65
70 75 80Asp Val Phe Val Leu Ser Phe Ser Leu Val Ser Arg Ala Ser Tyr
Glu85 90 95Asn Val Phe Lys Lys Trp Ile Pro Glu Leu Gln His Phe Ala
Pro Gly100 105 110Val Pro Leu Val Leu Val Gly Thr Lys Leu Asp Leu
Arg Glu Asp Lys115 120 125His Tyr Leu Ala Asp His Pro Gly Leu Ser
Pro Val Thr Thr Ala Gln130 135 140Gly Glu Glu Leu Arg Lys Leu Ile
Gly Ala Thr Tyr Tyr Ile Glu Cys145 150 155 160Ser Ser Lys Thr Gln
Gln Asn Val Lys Ala Val Phe Asp Ser Ala Ile165 170 175Lys Glu Val
Ile Lys Pro Leu Val Lys Gln Lys Glu Lys Thr Lys Lys180 185 190Lys
Lys Lys Gln Lys Ser Asn His Gly Cys Leu Ser Asn Val Leu Cys195 200
205Gly Arg Ile Val Thr Arg His210 21567630DNAArabidopsis
thalianaCDS(1)..(627)coding for RacB homologue 67atg agt gct tcg
aag ttc ata aaa tgt gtt act gtt gga gat ggg gct 48Met Ser Ala Ser
Lys Phe Ile Lys Cys Val Thr Val Gly Asp Gly Ala1 5 10 15gtt ggg aag
aca tgt atg ctt atc tgt tac act agc aac aag ttt cct 96Val Gly Lys
Thr Cys Met Leu Ile Cys Tyr Thr Ser Asn Lys Phe Pro20 25 30act gat
tat ata ccg act gtg ttc gac aat ttc agt gcc aat gta gct 144Thr Asp
Tyr Ile Pro Thr Val Phe Asp Asn Phe Ser Ala Asn Val Ala35 40 45gtg
gat gga caa atc gtt aat tta ggg cta tgg gac act gcc ggt caa 192Val
Asp Gly Gln Ile Val Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln50 55
60gaa gat tac agt agg tta aga cca ttg agt tat aga gga gct gat atc
240Glu Asp Tyr Ser Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala Asp
Ile65 70 75 80ttc gtc tta gcc ttt tcg ctt att agc aag gcg agt tac
gaa aat gta 288Phe Val Leu Ala Phe Ser Leu Ile Ser Lys Ala Ser Tyr
Glu Asn Val85 90 95ctc aag aag tgg atg cct gaa ctt cgt cgg ttt gcg
cca aat gtt ccc 336Leu Lys Lys Trp Met Pro Glu Leu Arg Arg Phe Ala
Pro Asn Val Pro100 105 110ata gtt ctt gtt ggt aca aag cta gat ctc
cgg gat gac aag gga tac 384Ile Val Leu Val Gly Thr Lys Leu Asp Leu
Arg Asp Asp Lys Gly Tyr115 120 125ctc gcg gat cac acc aat gtc att
acc tct act cag gga gag gaa ttg 432Leu Ala Asp His Thr Asn Val Ile
Thr Ser Thr Gln Gly Glu Glu Leu130 135 140agg aag caa att ggt gca
gct gct tat att gag tgt agt tcc aag act 480Arg Lys Gln Ile Gly Ala
Ala Ala Tyr Ile Glu Cys Ser Ser Lys Thr145 150 155 160caa caa aat
gtg aaa gca gtg ttt gat aca gcg atc aag gtg gtt ctt 528Gln Gln Asn
Val Lys Ala Val Phe Asp Thr Ala Ile Lys Val Val Leu165 170 175cag
cct cca agg agg aaa gag gtc ccg agg agg agg aag aat cat aga 576Gln
Pro Pro Arg Arg Lys Glu Val Pro Arg Arg Arg Lys Asn His Arg180 185
190aga tcc ggt tgc tcc att gcg agt att gtc tgt gga ggt tgc acc gct
624Arg Ser Gly Cys Ser Ile Ala Ser Ile Val Cys Gly Gly Cys Thr
Ala195 200 205gct taa 630Ala68209PRTArabidopsis thaliana 68Met Ser
Ala Ser Lys Phe Ile Lys Cys Val Thr Val Gly Asp Gly Ala1 5 10 15Val
Gly Lys Thr Cys Met Leu Ile Cys Tyr Thr Ser Asn Lys Phe Pro20 25
30Thr Asp Tyr Ile Pro Thr Val Phe Asp Asn Phe Ser Ala Asn Val Ala35
40 45Val Asp Gly Gln Ile Val Asn Leu Gly Leu Trp Asp Thr Ala Gly
Gln50 55 60Glu Asp Tyr Ser Arg Leu Arg Pro Leu Ser Tyr Arg Gly Ala
Asp Ile65 70 75 80Phe Val Leu Ala Phe Ser Leu Ile Ser Lys Ala Ser
Tyr Glu Asn Val85 90 95Leu Lys Lys Trp Met Pro Glu Leu Arg Arg Phe
Ala Pro Asn Val Pro100 105 110Ile Val Leu Val Gly Thr Lys Leu Asp
Leu Arg Asp Asp Lys Gly Tyr115 120 125Leu Ala Asp His Thr Asn Val
Ile Thr Ser Thr Gln Gly Glu Glu Leu130 135 140Arg Lys Gln Ile Gly
Ala Ala Ala Tyr Ile Glu Cys Ser Ser Lys Thr145 150 155 160Gln Gln
Asn Val Lys Ala Val Phe Asp Thr Ala Ile Lys Val Val Leu165 170
175Gln Pro Pro Arg Arg Lys Glu Val Pro Arg Arg Arg Lys Asn His
Arg180 185 190Arg Ser Gly Cys Ser Ile Ala Ser Ile Val Cys Gly Gly
Cys Thr Ala195 200 205Ala69600DNAArabidopsis
thalianaCDS(1)..(597)coding for RacB homologue 69atg gct gca aca
tca aca tca tca gca aca gct aca acg ttt ata aag 48Met Ala Ala Thr
Ser Thr Ser Ser Ala Thr Ala Thr Thr Phe Ile Lys1 5 10 15tgt gtc act
gtt ggc gat gga gct ctt ttg gtg act gtt gag atc ttg 96Cys Val Thr
Val Gly Asp Gly Ala Leu Leu Val Thr Val Glu Ile Leu20 25 30tta tta
cag gat tat gtt cca aca gtg ttc gac aat ttc aat gct aat 144Leu Leu
Gln Asp Tyr Val Pro Thr Val Phe Asp Asn Phe Asn Ala Asn35 40 45gtt
tta gtc gat ggt aaa act gtc aat ctg ggt ctc tgg gat act gct 192Val
Leu Val Asp Gly Lys Thr Val Asn Leu Gly Leu Trp Asp Thr Ala50 55
60ggt caa gaa gac tac aat agg gtt aga cca ttg agt tac aga gga gca
240Gly Gln Glu Asp Tyr Asn Arg Val Arg Pro Leu Ser Tyr Arg Gly
Ala65 70 75 80gat gtt ttc att ctt gcc
ttc tca ctt att agc agg cct agc ttt gag 288Asp Val Phe Ile Leu Ala
Phe Ser Leu Ile Ser Arg Pro Ser Phe Glu85 90 95aac att gct aaa aag
tgg gta ccc gag ctg aga cat tat gcc ccg act 336Asn Ile Ala Lys Lys
Trp Val Pro Glu Leu Arg His Tyr Ala Pro Thr100 105 110gtg cct att
gtt ctt gtg gga acc aaa tca gat cta aga gac aac atg 384Val Pro Ile
Val Leu Val Gly Thr Lys Ser Asp Leu Arg Asp Asn Met115 120 125cag
ttc cca aag aat tat cca ggt gct tgc aca atc ttc cca gaa cag 432Gln
Phe Pro Lys Asn Tyr Pro Gly Ala Cys Thr Ile Phe Pro Glu Gln130 135
140ggt caa gaa cta aga aag gaa ata gga gca tta gca tac ata gag tgc
480Gly Gln Glu Leu Arg Lys Glu Ile Gly Ala Leu Ala Tyr Ile Glu
Cys145 150 155 160agc tca aaa gca caa atg aac gta aaa gcc gtg ttt
gat gaa gcg atc 528Ser Ser Lys Ala Gln Met Asn Val Lys Ala Val Phe
Asp Glu Ala Ile165 170 175aaa gta gtt tta cat cct cct tca aag act
aag aag cga aag aga aag 576Lys Val Val Leu His Pro Pro Ser Lys Thr
Lys Lys Arg Lys Arg Lys180 185 190atc ggt tta tgc cat gtt ctt tga
600Ile Gly Leu Cys His Val Leu19570199PRTArabidopsis thaliana 70Met
Ala Ala Thr Ser Thr Ser Ser Ala Thr Ala Thr Thr Phe Ile Lys1 5 10
15Cys Val Thr Val Gly Asp Gly Ala Leu Leu Val Thr Val Glu Ile Leu20
25 30Leu Leu Gln Asp Tyr Val Pro Thr Val Phe Asp Asn Phe Asn Ala
Asn35 40 45Val Leu Val Asp Gly Lys Thr Val Asn Leu Gly Leu Trp Asp
Thr Ala50 55 60Gly Gln Glu Asp Tyr Asn Arg Val Arg Pro Leu Ser Tyr
Arg Gly Ala65 70 75 80Asp Val Phe Ile Leu Ala Phe Ser Leu Ile Ser
Arg Pro Ser Phe Glu85 90 95Asn Ile Ala Lys Lys Trp Val Pro Glu Leu
Arg His Tyr Ala Pro Thr100 105 110Val Pro Ile Val Leu Val Gly Thr
Lys Ser Asp Leu Arg Asp Asn Met115 120 125Gln Phe Pro Lys Asn Tyr
Pro Gly Ala Cys Thr Ile Phe Pro Glu Gln130 135 140Gly Gln Glu Leu
Arg Lys Glu Ile Gly Ala Leu Ala Tyr Ile Glu Cys145 150 155 160Ser
Ser Lys Ala Gln Met Asn Val Lys Ala Val Phe Asp Glu Ala Ile165 170
175Lys Val Val Leu His Pro Pro Ser Lys Thr Lys Lys Arg Lys Arg
Lys180 185 190Ile Gly Leu Cys His Val Leu1957121DNAArtificial
sequenceDescription of the artificial sequence oligonucleotide
primer 71atgagcgcgt ccaggttcat a 217221DNAArtificial
sequenceDescription of the artificial sequence oligonucleotide
primer 72atcaaacacg cccttcacgt t 217310828DNAArtificial
sequenceDescription of the artificial sequence transgenic
expression vector pSUN3NIT_AtRacB_s for expression of A-thalianan
RacB sense RNA 73ttccatggac atacaaatgg acgaacggat aaaccttttc
acgccctttt aaatatccga 60ttattctaat aaacgctctt ttctcttagg tttacccgcc
aatatatcct gtcaaacact 120gatagtttaa actgaaggcg ggaaacgaca
atcagatcta gtaggaaaca gctatgacca 180tgattacgcc aagcttgcat
gcctgcaggt cgactctaga ggatccccca tcaagatctt 240ggtgatgtag
caagagctaa gttgtacttc gatycggttg gacattactc gagaccagat
300gttttacact tgaccgtaaa tgagcacccg aagaaaccgg taacattcat
ttcgaaggta 360gagaaagcgg aagatgactc aaacaagtaa tcggttgtga
ttcgtcagtt catgtcactc 420ctatgaagga gtcaagttca aaatgttatg
ttgagtttca aacttttatg ctaaactttt 480tttcttaatt ttcgttaata
atggaagaga accaattctc ttgtatctaa agattatcca 540tctatcatcc
aatttgagtg ttcaattctg gatgttgtgt taccctacat tctacaacca
600tgtagccaat tattatgaat ctggctttga tttcagttgt gttcttttct
ttttttcttt 660gcatatttgc atttagaatg tttaataatt aagttactgt
atttccacat acattagttc 720caagaatata catatattaa tttatttttc
ttaaaaatgt tttggaatga ctaatattga 780caacgaaaat agaagctatg
ctaaaccatt acgtatatgt gacttcacat gttgttgttt 840tacattccct
atatatatgg atggctgtca caatcagaaa cgtgatcgaa aaaagacaaa
900cagtgtttgc ataaaaagac tatttcgttt cattgacaat ttgtgtttat
ttgtaaagaa 960aagtggcaaa gtggaatttg agttcctgca agtaagaaag
atgaaataaa agacttgagt 1020gtgtgttttt ttcttttatc tgaaagctgc
aatgaaatat tcctaccaag cccgtttgat 1080tattaattgg ggtttggttt
tcttgatgcg aactaattgg ttatataaga aactatacaa 1140tccatgttaa
ttcaaaaatt ttgatttctc ttgtaggaat atgatttact atatgagact
1200ttcttttcgc caataatagt aaatccaaag atatttgacc ggaccaaaac
acattgatct 1260attttttagt ttatttaatc cagtttctct gagataattc
attaaggaaa acttagtatt 1320aacccatcct aagattaaat aggagccaaa
ctcacatttc aaatattaaa taacataaaa 1380tggatttaaa aaatctatac
gtcaaatttt atttatgaca tttcttattt aaatttatat 1440ttaatgaaat
acagctaaga caaaccaaaa aaaaaatact ttctaagtgg tccaaaacat
1500caattccgtt caatattatt aggtagaatc gtacgaccaa aaaaggtagg
ttaatacgaa 1560attacaaaca tatctatata catagtatat attattacct
attatgagga atcaaaatgc 1620atcaaatatg gatttaagga atccataaaa
gaataaattc tacggaaaaa aaaaaaagaa 1680taaattcttt taagttttta
atttgttttt tatttggtag ttctccattt tgttttattt 1740cgtttggatt
tattgtgtcc aaatactttg taaaccaccg ttgtaattct taaacggggt
1800tttcacttct tttttatatt cagacataaa gcatcggctg gtttaatcaa
tcaatagatt 1860ttatttttct tctcaattat tagtaggttt gatgtgaact
ttacaaaaaa aacaaaaaca 1920aatcaatgca gagaaaagaa accacgtggg
ctagtcccac cttgtttcat ttccaccaca 1980ggttcgatct tcgttaccgt
ctccaatagg aaaataaacg tgaccacaaa aaaaaaacaa 2040aaaaaaagtc
tatatattgc ttctctcaag tctctgagtg tcatgaacca aagtaaaaaa
2100caaagactcg agtggatccc cggaattcgc ccttatgagc gcatcaaggt
tcataaagtg 2160cgtcaccgtt ggtgatggag ctgttggtaa aacctgtttg
ctgatttctt ataccagcaa 2220cacctttccc acggntattc atcaatcatt
ctccctcctt tttttgatat ctgattcatt 2280tgattctgat tgtgccatat
atgaattggg atccatacat actaaaaatg ttgtatacat 2340ttccattgga
acaggattat gttccgactg ttttcgataa ctttagtgca aatgtggttg
2400tcaatggggc cacggtgaat cttggattgt gggatactgc aggtaaatga
atgatgatcc 2460aattcataat ccttggtgag agagctttcc gtgatgaatc
agaggtcgaa attatgtttt 2520tggtttatgc agggcaagag gactataaca
gattaagacc tttgagttac cgtggtgctg 2580atgttttcat tcttgccttc
tctctcatta gtaaggctag ttatgagaat gtttccaaga 2640aggtcagttt
cgtccgaact ggtcgactat ttaacaattg agagttccaa attttgatgc
2700ttcttttctt ttacagtgga ttcctgagtt gaagcactat gctcctggtg
tcccaattgt 2760ccttgttgga accaaactag gttacttcct cctctcacat
ttgtccttgt ttatgcattt 2820atttatatat atgtctgatt cccatgctta
cactgccatt ttccttttca ctttattaag 2880ctgctctggt ataatatata
tcatgacatt agtggacata aaacttcacc ttctcttgat 2940tgtggttaaa
acttgtacat gttcaagatg tattcgttag gtgaaactga gggtagtttt
3000cagagaatat cattggtcaa caggcttctt cttgtatctt gcacttcttg
tgataaagca 3060accgtatcct ataacacacg cctttaagca tcctccaatg
aaatagctac tgtatagcaa 3120gtgtatacct ttataaaaga cacttgcaag
atcttcagtc aactcatgat cctggccttt 3180ttgattgtct aaccttggtt
gttgtcagat cttcgagatg acaaacagtt tttcatcgac 3240catcctggtg
ctgtccctat taccactgtt caggtaagaa tacagttatt tcctcagtgc
3300aattttatca gctttaccac cgttaagcat tttccctctc tgcatggaag
ggagaggagc 3360tgaagaagct aattggagcg ccagcttaca tcgagtgcag
ttcaaaatca caagaggtaa 3420acgaataaaa gacatctcat gaatcatctt
ttcggtgtta gattcttctt tttttgatga 3480aaacaatgtg actataactg
cagaacgtga agggcgtgtt tgataagggc gaattaattc 3540actggccgtc
gttttacaac gactcagagc ttgacaggag gcccgatcta gtaacataga
3600tgacaccgcg cgcgataatt tatcctagtt tgcgcgctat attttgtttt
ctatcgcgta 3660ttaaatgtat aattgcggga ctctaatcat aaaaacccat
ctcataaata acgtcatgca 3720ttacatgtta attattacat gcttaacgta
attcaacaga aattatatga taatcatcgc 3780aagaccggca acaggattca
atcttaagaa actttattgc caaatgtttg aacgatcggg 3840gatcatccgg
gtctgtggcg ggaactccac gaaaatatcc gaacgcagca agatctagag
3900cttgggtccc gctcagaaga actcgtcaag aaggcgatag aaggcgatgc
gctgcgaatc 3960gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc
cattcgccgc caagctcttc 4020agcaatatca cgggtagcca acgctatgtc
ctgatagcgg tccgccacac ccagccggcc 4080acagtcgatg aatccagaaa
agcggccatt ttccaccatg atattcggca agcaggcatc 4140gccatgggtc
acgacgagat cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag
4200ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga
caagaccggc 4260ttccatccga gtacgtgctc gctcgatgcg atgtttcgct
tggtggtcga atgggcaggt 4320agccggatca agcgtatgca gccgccgcat
tgcatcagcc atgatggata ctttctcggc 4380aggagcaagg tgagatgaca
ggagatcctg ccccggcact tcgcccaata gcagccagtc 4440ccttcccgct
tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag
4500ccacgatagc cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca
ggtcggtctt 4560gacaaaaaga accgggcgcc cctgcgctga cagccggaac
acggcggcat cagagcagcc 4620gattgtctgt tgtgcccagt catagccgaa
tagcctctcc acccaagcgg ccggagaacc 4680tgcgtgcaat ccatcttgtt
caatcatgcg aaacgatcca gatccggtgc agattatttg 4740gattgagagt
gaatatgaga ctctaattgg ataccgaggg gaatttatgg aacgtcagtg
4800gagcattttt gacaagaaat atttgctagc tgatagtgac cttaggcgac
ttttgaacgc 4860gcaataatgg tttctgacgt atgtgcttag ctcattaaac
tccagaaacc cgcggctgag 4920tggctccttc aacgttgcgg ttctgtcagt
tccaaacgta aaacggcttg tcccgcgtca 4980tcggcggggg tcataacgtg
actcccttaa ttctccgctc atgatcagat tgtcgtttcc 5040cgccttcagt
ttaaactatc agtgtttgac aggatcctgc ttggtaataa ttgtcattag
5100attgttttta tgcatagatg cactcgaaat cagccaattt tagacaagta
tcaaacggat 5160gttaattcag tacattaaag acgtccgcaa tgtgttatta
agttgtctaa gcgtcaattt 5220gtttacacca caatatatcc tgccaccagc
cagccaacag ctccccgacc ggcagctcgg 5280cacaaaatca ccacgcgtta
ccaccacgcc ggccggccgc atggtgttga ccgtgttcgc 5340cggcattgcc
gagttcgagc gttccctaat catcgaccgc acccggagcg ggcgcgaggc
5400cgccaaggcc cgaggcgtga agtttggccc ccgccctacc ctcaccccgg
cacagatcgc 5460gcacgcccgc gagctgatcg accaggaagg ccgcaccgtg
aaagaggcgg ctgcactgct 5520tggcgtgcat cgctcgaccc tgtaccgcgc
acttgagcgc agcgaggaag tgacgcccac 5580cgaggccagg cggcgcggtg
ccttccgtga ggacgcattg accgaggccg acgccctggc 5640ggccgccgag
aatgaacgcc aagaggaaca agcatgaaac cgcaccagga cggccaggac
5700gaaccgtttt tcattaccga agagatcgag gcggagatga tcgcggccgg
gtacgtgttc 5760gagccgcccg cgcacgtctc aaccgtgcgg ctgcatgaaa
tcctggccgg tttgtctgat 5820gccaagctgg cggcctggcc ggccagcttg
gccgctgaag aaaccgagcg ccgccgtcta 5880aaaaggtgat gtgtatttga
gtaaaacagc ttgcgtcatg cggtcgctgc gtatatgatg 5940cgatgagtaa
ataaacaaat acgcaagggg aacgcatgaa ggttatcgct gtacttaacc
6000agaaaggcgg gtcaggcaag acgaccatcg caacccatct agcccgcgcc
ctgcaactcg 6060ccggggccga tgttctgtta gtcgattccg atccccaggg
cagtgcccgc gattgggcgg 6120ccgtgcggga agatcaaccg ctaaccgttg
tcggcatcga ccgcccgacg attgaccgcg 6180acgtgaaggc catcggccgg
cgcgacttcg tagtgatcga cggagcgccc caggcggcgg 6240acttggctgt
gtccgcgatc aaggcagccg acttcgtgct gattccggtg cagccaagcc
6300cttacgacat atgggccacc gccgacctgg tggagctggt taagcagcgc
attgaggtca 6360cggatggaag gctacaagcg gcctttgtcg tgtcgcgggc
gatcaaaggc acgcgcatcg 6420gcggtgaggt tgccgaggcg ctggccgggt
acgagctgcc cattcttgag tcccgtatca 6480cgcagcgcgt gagctaccca
ggcactgccg ccgccggcac aaccgttctt gaatcagaac 6540ccgagggcga
cgctgcccgc gaggtccagg cgctggccgc tgaaattaaa tcaaaactca
6600tttgagttaa tgaggtaaag agaaaatgag caaaagcaca aacacgctaa
gtgccggccg 6660tccgagcgca cgcagcagca aggctgcaac gttggccagc
ctggcagaca cgccagccat 6720gaagcgggtc aactttcagt tgccggcgga
ggatcacacc aagctgaaga tgtacgcggt 6780acgccaaggc aagaccatta
ccgagctgct atctgaatac atcgcgcagc taccagagta 6840aatgagcaaa
tgaataaatg agtagatgaa ttttagcggc taaaggaggc ggcatggaaa
6900atcaagaaca accaggcacc gacgccgtgg aatgccccat gtgtggagga
acgggcggtt 6960ggccaggcgt aagcggctgg gttgtctgcc ggccctgcaa
tggcactgga acccccaagc 7020ccgaggaatc ggcgtgagcg gtcgcaaacc
atccggcccg gtacaaatcg gcgcggcgct 7080gggtgatgac ctggtggaga
agttgaaggc cgcgcaggcc gcccagcggc aacgcatcga 7140ggcagaagca
cgccccggtg aatcgtggca agcggccgct gatcgaatcc gcaaagaatc
7200ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg
gcgacgagca 7260accagatttt ttcgttccga tgctctatga cgtgggcacc
cgcgatagtc gcagcatcat 7320ggacgtggcc gttttccgtc tgtcgaagcg
tgaccgacga gctggcgagg tgatccgcta 7380cgagcttcca gacgggcacg
tagaggtttc cgcagggccg gccggcatgg ccagtgtgtg 7440ggattacgac
ctggtactga tggcggtttc ccatctaacc gaatccatga accgataccg
7500ggaagggaag ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg
acgtactcaa 7560gttctgccgg cgagccgatg gcggaaagca gaaagacgac
ctggtagaaa cctgcattcg 7620gttaaacacc acgcacgttg ccatgcagcg
tacgaagaag gccaagaacg gccgcctggt 7680gacggtatcc gagggtgaag
ccttgattag ccgctacaag atcgtaaaga gcgaaaccgg 7740gcggccggag
tacatcgaga tcgagctagc tgattggatg taccgcgaga tcacagaagg
7800caagaacccg gacgtgctga cggttcaccc cgattacttt ttgatcgatc
ccggcatcgg 7860ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag
gcagaagcca gatggttgtt 7920caagacgatc tacgaacgca gtggcagcgc
cggagagttc aagaagttct gtttcaccgt 7980gcgcaagctg atcgggtcaa
atgacctgcc ggagtacgat ttgaaggagg aggcggggca 8040ggctggcccg
atcctagtca tgcgctaccg caacctgatc gagggcgaag catccgccgg
8100ttcctaatgt acggagcaga tgctagggca aattgcccta gcaggggaaa
aaggtcgaaa 8160aggtctcttt cctgtggata gcacgtacat tgggaaccca
aagccgtaca ttgggaaccg 8220gaacccgtac attgggaacc caaagccgta
cattgggaac cggtcacaca tgtaagtgac 8280tgatataaaa gagaaaaaag
gcgatttttc cgcctaaaac tctttaaaac ttattaaaac 8340tcttaaaacc
cgcctggcct gtgcataact gtctggccag cgcacagccg aagagctgca
8400aaaagcgcct acccttcggt cgctgcgctc cctacgcccc gccgcttcgc
gtcggcctat 8460cgcggccgct ggccgctcaa aaatggctgg cctacggcca
ggcaatctac cagggcgcgg 8520acaagccgcg ccgtcgccac tcgaccgccg
gcgcccacat caaggcaccc tgcctcgcgc 8580gtttcggtga tgacggtgaa
aacctctgac acatgcagct cccggagacg gtcacagctt 8640gtctgtaagc
ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
8700ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat
actggcttaa 8760ctatgcggca tcagagcaga ttgtactgag agtgcaccat
atgcggtgtg aaataccgca 8820cagatgcgta aggagaaaat accgcatcag
gcgctcttcc gcttcctcgc tcactgactc 8880gctgcgctcg gtcgttcggc
tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 8940gttatccaca
gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
9000ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
gcccccctga 9060cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
aacccgacag gactataaag 9120ataccaggcg tttccccctg gaagctccct
cgtgcgctct cctgttccga ccctgccgct 9180taccggatac ctgtccgcct
ttctcccttc gggaagcgtg gcgctttctc atagctcacg 9240ctgtaggtat
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
9300ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
ccaacccggt 9360aagacacgac ttatcgccac tggcagcagc cactggtaac
aggattagca gagcgaggta 9420tgtaggcggt gctacagagt tcttgaagtg
gtggcctaac tacggctaca ctagaaggac 9480agtatttggt atctgcgctc
tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 9540ttgatccggc
aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
9600tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
ggtctgacgc 9660tcagtggaac gaaaactcac gttaagggat tttggtcatg
catgatatat ctcccaattt 9720gtgtagggct tattatgcac gcttaaaaat
aataaaagca gacttgacct gatagtttgg 9780ctgtgagcaa ttatgtgctt
agtgcatcta acgcttgagt taagccgcgc cgcgaagcgg 9840cgtcggcttg
aacgaatttc tagctagaca ttatttgccg actaccttgg tgatctcgcc
9900tttcacgtag tggacaaatt cttccaactg atctgcgcgc gaggccaagc
gatcttcttc 9960ttgtccaaga taagcctgtc tagcttcaag tatgacgggc
tgatactggg ccggcaggcg 10020ctccattgcc cagtcggcag cgacatcctt
cggcgcgatt ttgccggtta ctgcgctgta 10080ccaaatgcgg gacaacgtaa
gcactacatt tcgctcatcg ccagcccagt cgggcggcga 10140gttccatagc
gttaaggttt catttagcgc ctcaaataga tcctgttcag gaaccggatc
10200aaagagttcc tccgccgctg gacctaccaa ggcaacgcta tgttctcttg
cttttgtcag 10260caagatagcc agatcaatgt cgatcgtggc tggctcgaag
atacctgcaa gaatgtcatt 10320gcgctgccat tctccaaatt gcagttcgcg
cttagctgga taacgccacg gaatgatgtc 10380gtcgtgcaca acaatggtga
cttctacagc gcggagaatc tcgctctctc caggggaagc 10440cgaagtttcc
aaaaggtcgt tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt
10500caccgtaacc agcaaatcaa tatcactgtg tggcttcagg ccgccatcca
ctgcggagcc 10560gtacaaatgt acggccagca acgtcggttc gagatggcgc
tcgatgacgc caactacctc 10620tgatagttga gtcgatactt cggcgatcac
cgcttccccc atgatgttta actttgtttt 10680agggcgactg ccctgctgcg
taacatcgtt gctgctccat aacatcaaac atcgacccac 10740ggcgtaacgc
gcttgctgct tggatgcccg aggcatagac tgtaccccaa aaaaacagtc
10800ataacaagcc atgaaaaccg ccactgcg 108287410828DNAArtificial
sequenceDescription of the artificial sequence transgenic
expression vector pSUN3NIT_AtRacB_as for expression of A-thalianan
RacB antisense RNA 74ttccatggac atacaaatgg acgaacggat aaaccttttc
acgccctttt aaatatccga 60ttattctaat aaacgctctt ttctcttagg tttacccgcc
aatatatcct gtcaaacact 120gatagtttaa actgaaggcg ggaaacgaca
atcagatcta gtaggaaaca gctatgacca 180tgattacgcc aagcttgcat
gcctgcaggt cgactctaga ggatccccca tcaagatctt 240ggtgatgtag
caagagctaa gttgtacttc gatycggttg gacattactc gagaccagat
300gttttacact tgaccgtaaa tgagcacccg aagaaaccgg taacattcat
ttcgaaggta 360gagaaagcgg aagatgactc aaacaagtaa tcggttgtga
ttcgtcagtt catgtcactc 420ctatgaagga gtcaagttca aaatgttatg
ttgagtttca aacttttatg ctaaactttt 480tttcttaatt ttcgttaata
atggaagaga accaattctc ttgtatctaa agattatcca 540tctatcatcc
aatttgagtg ttcaattctg gatgttgtgt taccctacat tctacaacca
600tgtagccaat tattatgaat ctggctttga tttcagttgt gttcttttct
ttttttcttt 660gcatatttgc atttagaatg tttaataatt aagttactgt
atttccacat acattagttc 720caagaatata catatattaa tttatttttc
ttaaaaatgt tttggaatga ctaatattga 780caacgaaaat agaagctatg
ctaaaccatt acgtatatgt gacttcacat gttgttgttt 840tacattccct
atatatatgg atggctgtca caatcagaaa cgtgatcgaa aaaagacaaa
900cagtgtttgc ataaaaagac tatttcgttt cattgacaat ttgtgtttat
ttgtaaagaa 960aagtggcaaa gtggaatttg agttcctgca agtaagaaag
atgaaataaa agacttgagt 1020gtgtgttttt ttcttttatc tgaaagctgc
aatgaaatat tcctaccaag cccgtttgat 1080tattaattgg ggtttggttt
tcttgatgcg aactaattgg ttatataaga aactatacaa 1140tccatgttaa
ttcaaaaatt ttgatttctc ttgtaggaat atgatttact atatgagact
1200ttcttttcgc caataatagt aaatccaaag atatttgacc ggaccaaaac
acattgatct 1260attttttagt ttatttaatc cagtttctct gagataattc
attaaggaaa acttagtatt 1320aacccatcct aagattaaat aggagccaaa
ctcacatttc
aaatattaaa taacataaaa 1380tggatttaaa aaatctatac gtcaaatttt
atttatgaca tttcttattt aaatttatat 1440ttaatgaaat acagctaaga
caaaccaaaa aaaaaatact ttctaagtgg tccaaaacat 1500caattccgtt
caatattatt aggtagaatc gtacgaccaa aaaaggtagg ttaatacgaa
1560attacaaaca tatctatata catagtatat attattacct attatgagga
atcaaaatgc 1620atcaaatatg gatttaagga atccataaaa gaataaattc
tacggaaaaa aaaaaaagaa 1680taaattcttt taagttttta atttgttttt
tatttggtag ttctccattt tgttttattt 1740cgtttggatt tattgtgtcc
aaatactttg taaaccaccg ttgtaattct taaacggggt 1800tttcacttct
tttttatatt cagacataaa gcatcggctg gtttaatcaa tcaatagatt
1860ttatttttct tctcaattat tagtaggttt gatgtgaact ttacaaaaaa
aacaaaaaca 1920aatcaatgca gagaaaagaa accacgtggg ctagtcccac
cttgtttcat ttccaccaca 1980ggttcgatct tcgttaccgt ctccaatagg
aaaataaacg tgaccacaaa aaaaaaacaa 2040aaaaaaagtc tatatattgc
ttctctcaag tctctgagtg tcatgaacca aagtaaaaaa 2100caaagactcg
agtggatccc cggaattcgc ccttatcaaa cacgcccttc acgttctgca
2160gttatagtca cattgttttc atcaaaaaaa gaagaatcta acaccgaaaa
gatgattcat 2220gagatgtctt ttattcgttt acctcttgtg attttgaact
gcactcgatg taagctggcg 2280ctccaattag cttcttcagc tcctctccct
tccatgcaga gagggaaaat gcttaacggt 2340ggtaaagctg ataaaattgc
actgaggaaa taactgtatt cttacctgaa cagtggtaat 2400agggacagca
ccaggatggt cgatgaaaaa ctgtttgtca tctcgaagat ctgacaacaa
2460ccaaggttag acaatcaaaa aggccaggat catgagttga ctgaagatct
tgcaagtgtc 2520ttttataaag gtatacactt gctatacagt agctatttca
ttggaggatg cttaaaggcg 2580tgtgttatag gatacggttg ctttatcaca
agaagtgcaa gatacaagaa gaagcctgtt 2640gaccaatgat attctctgaa
aactaccctc agtttcacct aacgaataca tcttgaacat 2700gtacaagttt
taaccacaat caagagaagg tgaagtttta tgtccactaa tgtcatgata
2760tatattatac cagagcagct taataaagtg aaaaggaaaa tggcagtgta
agcatgggaa 2820tcagacatat atataaataa atgcataaac aaggacaaat
gtgagaggag gaagtaacct 2880agtttggttc caacaaggac aattgggaca
ccaggagcat agtgcttcaa ctcaggaatc 2940cactgtaaaa gaaaagaagc
atcaaaattt ggaactctca attgttaaat agtcgaccag 3000ttcggacgaa
actgaccttc ttggaaacat tctcataact agccttacta atgagagaga
3060aggcaagaat gaaaacatca gcaccacggt aactcaaagg tcttaatctg
ttatagtcct 3120cttgccctgc ataaaccaaa aacataattt cgacctctga
ttcatcacgg aaagctctct 3180caccaaggat tatgaattgg atcatcattc
atttacctgc agtatcccac aatccaagat 3240tcaccgtggc cccattgaca
accacatttg cactaaagtt atcgaaaaca gtcggaacat 3300aatcctgttc
caatggaaat gtatacaaca tttttagtat gtatggatcc caattcatat
3360atggcacaat cagaatcaaa tgaatcagat atcaaaaaaa ggagggagaa
tgattgatga 3420atanccgtgg gaaaggtgtt gctggtataa gaaatcagca
aacaggtttt accaacagct 3480ccatcaccaa cggtgacgca ctttatgaac
cttgatgcgc tcataagggc gaattaattc 3540actggccgtc gttttacaac
gactcagagc ttgacaggag gcccgatcta gtaacataga 3600tgacaccgcg
cgcgataatt tatcctagtt tgcgcgctat attttgtttt ctatcgcgta
3660ttaaatgtat aattgcggga ctctaatcat aaaaacccat ctcataaata
acgtcatgca 3720ttacatgtta attattacat gcttaacgta attcaacaga
aattatatga taatcatcgc 3780aagaccggca acaggattca atcttaagaa
actttattgc caaatgtttg aacgatcggg 3840gatcatccgg gtctgtggcg
ggaactccac gaaaatatcc gaacgcagca agatctagag 3900cttgggtccc
gctcagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc
3960gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc
caagctcttc 4020agcaatatca cgggtagcca acgctatgtc ctgatagcgg
tccgccacac ccagccggcc 4080acagtcgatg aatccagaaa agcggccatt
ttccaccatg atattcggca agcaggcatc 4140gccatgggtc acgacgagat
cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag 4200ttcggctggc
gcgagcccct gatgctcttc gtccagatca tcctgatcga caagaccggc
4260ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga
atgggcaggt 4320agccggatca agcgtatgca gccgccgcat tgcatcagcc
atgatggata ctttctcggc 4380aggagcaagg tgagatgaca ggagatcctg
ccccggcact tcgcccaata gcagccagtc 4440ccttcccgct tcagtgacaa
cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag 4500ccacgatagc
cgcgctgcct cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt
4560gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat
cagagcagcc 4620gattgtctgt tgtgcccagt catagccgaa tagcctctcc
acccaagcgg ccggagaacc 4680tgcgtgcaat ccatcttgtt caatcatgcg
aaacgatcca gatccggtgc agattatttg 4740gattgagagt gaatatgaga
ctctaattgg ataccgaggg gaatttatgg aacgtcagtg 4800gagcattttt
gacaagaaat atttgctagc tgatagtgac cttaggcgac ttttgaacgc
4860gcaataatgg tttctgacgt atgtgcttag ctcattaaac tccagaaacc
cgcggctgag 4920tggctccttc aacgttgcgg ttctgtcagt tccaaacgta
aaacggcttg tcccgcgtca 4980tcggcggggg tcataacgtg actcccttaa
ttctccgctc atgatcagat tgtcgtttcc 5040cgccttcagt ttaaactatc
agtgtttgac aggatcctgc ttggtaataa ttgtcattag 5100attgttttta
tgcatagatg cactcgaaat cagccaattt tagacaagta tcaaacggat
5160gttaattcag tacattaaag acgtccgcaa tgtgttatta agttgtctaa
gcgtcaattt 5220gtttacacca caatatatcc tgccaccagc cagccaacag
ctccccgacc ggcagctcgg 5280cacaaaatca ccacgcgtta ccaccacgcc
ggccggccgc atggtgttga ccgtgttcgc 5340cggcattgcc gagttcgagc
gttccctaat catcgaccgc acccggagcg ggcgcgaggc 5400cgccaaggcc
cgaggcgtga agtttggccc ccgccctacc ctcaccccgg cacagatcgc
5460gcacgcccgc gagctgatcg accaggaagg ccgcaccgtg aaagaggcgg
ctgcactgct 5520tggcgtgcat cgctcgaccc tgtaccgcgc acttgagcgc
agcgaggaag tgacgcccac 5580cgaggccagg cggcgcggtg ccttccgtga
ggacgcattg accgaggccg acgccctggc 5640ggccgccgag aatgaacgcc
aagaggaaca agcatgaaac cgcaccagga cggccaggac 5700gaaccgtttt
tcattaccga agagatcgag gcggagatga tcgcggccgg gtacgtgttc
5760gagccgcccg cgcacgtctc aaccgtgcgg ctgcatgaaa tcctggccgg
tttgtctgat 5820gccaagctgg cggcctggcc ggccagcttg gccgctgaag
aaaccgagcg ccgccgtcta 5880aaaaggtgat gtgtatttga gtaaaacagc
ttgcgtcatg cggtcgctgc gtatatgatg 5940cgatgagtaa ataaacaaat
acgcaagggg aacgcatgaa ggttatcgct gtacttaacc 6000agaaaggcgg
gtcaggcaag acgaccatcg caacccatct agcccgcgcc ctgcaactcg
6060ccggggccga tgttctgtta gtcgattccg atccccaggg cagtgcccgc
gattgggcgg 6120ccgtgcggga agatcaaccg ctaaccgttg tcggcatcga
ccgcccgacg attgaccgcg 6180acgtgaaggc catcggccgg cgcgacttcg
tagtgatcga cggagcgccc caggcggcgg 6240acttggctgt gtccgcgatc
aaggcagccg acttcgtgct gattccggtg cagccaagcc 6300cttacgacat
atgggccacc gccgacctgg tggagctggt taagcagcgc attgaggtca
6360cggatggaag gctacaagcg gcctttgtcg tgtcgcgggc gatcaaaggc
acgcgcatcg 6420gcggtgaggt tgccgaggcg ctggccgggt acgagctgcc
cattcttgag tcccgtatca 6480cgcagcgcgt gagctaccca ggcactgccg
ccgccggcac aaccgttctt gaatcagaac 6540ccgagggcga cgctgcccgc
gaggtccagg cgctggccgc tgaaattaaa tcaaaactca 6600tttgagttaa
tgaggtaaag agaaaatgag caaaagcaca aacacgctaa gtgccggccg
6660tccgagcgca cgcagcagca aggctgcaac gttggccagc ctggcagaca
cgccagccat 6720gaagcgggtc aactttcagt tgccggcgga ggatcacacc
aagctgaaga tgtacgcggt 6780acgccaaggc aagaccatta ccgagctgct
atctgaatac atcgcgcagc taccagagta 6840aatgagcaaa tgaataaatg
agtagatgaa ttttagcggc taaaggaggc ggcatggaaa 6900atcaagaaca
accaggcacc gacgccgtgg aatgccccat gtgtggagga acgggcggtt
6960ggccaggcgt aagcggctgg gttgtctgcc ggccctgcaa tggcactgga
acccccaagc 7020ccgaggaatc ggcgtgagcg gtcgcaaacc atccggcccg
gtacaaatcg gcgcggcgct 7080gggtgatgac ctggtggaga agttgaaggc
cgcgcaggcc gcccagcggc aacgcatcga 7140ggcagaagca cgccccggtg
aatcgtggca agcggccgct gatcgaatcc gcaaagaatc 7200ccggcaaccg
ccggcagccg gtgcgccgtc gattaggaag ccgcccaagg gcgacgagca
7260accagatttt ttcgttccga tgctctatga cgtgggcacc cgcgatagtc
gcagcatcat 7320ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga
gctggcgagg tgatccgcta 7380cgagcttcca gacgggcacg tagaggtttc
cgcagggccg gccggcatgg ccagtgtgtg 7440ggattacgac ctggtactga
tggcggtttc ccatctaacc gaatccatga accgataccg 7500ggaagggaag
ggagacaagc ccggccgcgt gttccgtcca cacgttgcgg acgtactcaa
7560gttctgccgg cgagccgatg gcggaaagca gaaagacgac ctggtagaaa
cctgcattcg 7620gttaaacacc acgcacgttg ccatgcagcg tacgaagaag
gccaagaacg gccgcctggt 7680gacggtatcc gagggtgaag ccttgattag
ccgctacaag atcgtaaaga gcgaaaccgg 7740gcggccggag tacatcgaga
tcgagctagc tgattggatg taccgcgaga tcacagaagg 7800caagaacccg
gacgtgctga cggttcaccc cgattacttt ttgatcgatc ccggcatcgg
7860ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag gcagaagcca
gatggttgtt 7920caagacgatc tacgaacgca gtggcagcgc cggagagttc
aagaagttct gtttcaccgt 7980gcgcaagctg atcgggtcaa atgacctgcc
ggagtacgat ttgaaggagg aggcggggca 8040ggctggcccg atcctagtca
tgcgctaccg caacctgatc gagggcgaag catccgccgg 8100ttcctaatgt
acggagcaga tgctagggca aattgcccta gcaggggaaa aaggtcgaaa
8160aggtctcttt cctgtggata gcacgtacat tgggaaccca aagccgtaca
ttgggaaccg 8220gaacccgtac attgggaacc caaagccgta cattgggaac
cggtcacaca tgtaagtgac 8280tgatataaaa gagaaaaaag gcgatttttc
cgcctaaaac tctttaaaac ttattaaaac 8340tcttaaaacc cgcctggcct
gtgcataact gtctggccag cgcacagccg aagagctgca 8400aaaagcgcct
acccttcggt cgctgcgctc cctacgcccc gccgcttcgc gtcggcctat
8460cgcggccgct ggccgctcaa aaatggctgg cctacggcca ggcaatctac
cagggcgcgg 8520acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat
caaggcaccc tgcctcgcgc 8580gtttcggtga tgacggtgaa aacctctgac
acatgcagct cccggagacg gtcacagctt 8640gtctgtaagc ggatgccggg
agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg 8700ggtgtcgggg
cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa
8760ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
aaataccgca 8820cagatgcgta aggagaaaat accgcatcag gcgctcttcc
gcttcctcgc tcactgactc 8880gctgcgctcg gtcgttcggc tgcggcgagc
ggtatcagct cactcaaagg cggtaatacg 8940gttatccaca gaatcagggg
ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 9000ggccaggaac
cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
9060cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
gactataaag 9120ataccaggcg tttccccctg gaagctccct cgtgcgctct
cctgttccga ccctgccgct 9180taccggatac ctgtccgcct ttctcccttc
gggaagcgtg gcgctttctc atagctcacg 9240ctgtaggtat ctcagttcgg
tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 9300ccccgttcag
cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
9360aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
gagcgaggta 9420tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
tacggctaca ctagaaggac 9480agtatttggt atctgcgctc tgctgaagcc
agttaccttc ggaaaaagag ttggtagctc 9540ttgatccggc aaacaaacca
ccgctggtag cggtggtttt tttgtttgca agcagcagat 9600tacgcgcaga
aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
9660tcagtggaac gaaaactcac gttaagggat tttggtcatg catgatatat
ctcccaattt 9720gtgtagggct tattatgcac gcttaaaaat aataaaagca
gacttgacct gatagtttgg 9780ctgtgagcaa ttatgtgctt agtgcatcta
acgcttgagt taagccgcgc cgcgaagcgg 9840cgtcggcttg aacgaatttc
tagctagaca ttatttgccg actaccttgg tgatctcgcc 9900tttcacgtag
tggacaaatt cttccaactg atctgcgcgc gaggccaagc gatcttcttc
9960ttgtccaaga taagcctgtc tagcttcaag tatgacgggc tgatactggg
ccggcaggcg 10020ctccattgcc cagtcggcag cgacatcctt cggcgcgatt
ttgccggtta ctgcgctgta 10080ccaaatgcgg gacaacgtaa gcactacatt
tcgctcatcg ccagcccagt cgggcggcga 10140gttccatagc gttaaggttt
catttagcgc ctcaaataga tcctgttcag gaaccggatc 10200aaagagttcc
tccgccgctg gacctaccaa ggcaacgcta tgttctcttg cttttgtcag
10260caagatagcc agatcaatgt cgatcgtggc tggctcgaag atacctgcaa
gaatgtcatt 10320gcgctgccat tctccaaatt gcagttcgcg cttagctgga
taacgccacg gaatgatgtc 10380gtcgtgcaca acaatggtga cttctacagc
gcggagaatc tcgctctctc caggggaagc 10440cgaagtttcc aaaaggtcgt
tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt 10500caccgtaacc
agcaaatcaa tatcactgtg tggcttcagg ccgccatcca ctgcggagcc
10560gtacaaatgt acggccagca acgtcggttc gagatggcgc tcgatgacgc
caactacctc 10620tgatagttga gtcgatactt cggcgatcac cgcttccccc
atgatgttta actttgtttt 10680agggcgactg ccctgctgcg taacatcgtt
gctgctccat aacatcaaac atcgacccac 10740ggcgtaacgc gcttgctgct
tggatgcccg aggcatagac tgtaccccaa aaaaacagtc 10800ataacaagcc
atgaaaaccg ccactgcg 108287510036DNAArtificial sequenceDescription
of the artificial sequence transgenic expression vector
pSUN3NIT_HvRacB_s for expression of barley RacB sense RNA fragment
75ttccatggac atacaaatgg acgaacggat aaaccttttc acgccctttt aaatatccga
60ttattctaat aaacgctctt ttctcttagg tttacccgcc aatatatcct gtcaaacact
120gatagtttaa actgaaggcg ggaaacgaca atcagatcta gtaggaaaca
gctatgacca 180tgattacgcc aagcttgcat gcctgcaggt cgactctaga
ggatccccca tcaagatctt 240ggtgatgtag caagagctaa gttgtacttc
gatycggttg gacattactc gagaccagat 300gttttacact tgaccgtaaa
tgagcacccg aagaaaccgg taacattcat ttcgaaggta 360gagaaagcgg
aagatgactc aaacaagtaa tcggttgtga ttcgtcagtt catgtcactc
420ctatgaagga gtcaagttca aaatgttatg ttgagtttca aacttttatg
ctaaactttt 480tttcttaatt ttcgttaata atggaagaga accaattctc
ttgtatctaa agattatcca 540tctatcatcc aatttgagtg ttcaattctg
gatgttgtgt taccctacat tctacaacca 600tgtagccaat tattatgaat
ctggctttga tttcagttgt gttcttttct ttttttcttt 660gcatatttgc
atttagaatg tttaataatt aagttactgt atttccacat acattagttc
720caagaatata catatattaa tttatttttc ttaaaaatgt tttggaatga
ctaatattga 780caacgaaaat agaagctatg ctaaaccatt acgtatatgt
gacttcacat gttgttgttt 840tacattccct atatatatgg atggctgtca
caatcagaaa cgtgatcgaa aaaagacaaa 900cagtgtttgc ataaaaagac
tatttcgttt cattgacaat ttgtgtttat ttgtaaagaa 960aagtggcaaa
gtggaatttg agttcctgca agtaagaaag atgaaataaa agacttgagt
1020gtgtgttttt ttcttttatc tgaaagctgc aatgaaatat tcctaccaag
cccgtttgat 1080tattaattgg ggtttggttt tcttgatgcg aactaattgg
ttatataaga aactatacaa 1140tccatgttaa ttcaaaaatt ttgatttctc
ttgtaggaat atgatttact atatgagact 1200ttcttttcgc caataatagt
aaatccaaag atatttgacc ggaccaaaac acattgatct 1260attttttagt
ttatttaatc cagtttctct gagataattc attaaggaaa acttagtatt
1320aacccatcct aagattaaat aggagccaaa ctcacatttc aaatattaaa
taacataaaa 1380tggatttaaa aaatctatac gtcaaatttt atttatgaca
tttcttattt aaatttatat 1440ttaatgaaat acagctaaga caaaccaaaa
aaaaaatact ttctaagtgg tccaaaacat 1500caattccgtt caatattatt
aggtagaatc gtacgaccaa aaaaggtagg ttaatacgaa 1560attacaaaca
tatctatata catagtatat attattacct attatgagga atcaaaatgc
1620atcaaatatg gatttaagga atccataaaa gaataaattc tacggaaaaa
aaaaaaagaa 1680taaattcttt taagttttta atttgttttt tatttggtag
ttctccattt tgttttattt 1740cgtttggatt tattgtgtcc aaatactttg
taaaccaccg ttgtaattct taaacggggt 1800tttcacttct tttttatatt
cagacataaa gcatcggctg gtttaatcaa tcaatagatt 1860ttatttttct
tctcaattat tagtaggttt gatgtgaact ttacaaaaaa aacaaaaaca
1920aatcaatgca gagaaaagaa accacgtggg ctagtcccac cttgtttcat
ttccaccaca 1980ggttcgatct tcgttaccgt ctccaatagg aaaataaacg
tgaccacaaa aaaaaaacaa 2040aaaaaaagtc tatatattgc ttctctcaag
tctctgagtg tcatgaacca aagtaaaaaa 2100caaagactcg agtggatccc
cgggccgcca tggccgcggg atggatccga tgagcgcgtc 2160caggttcata
aagtgcgtca cggtcgggga cggcgccgtc ggcaagacct gcatgctcat
2220ctcctacacc tccaacacct tccccaccga ctatgttccg acagtgtttg
ataacttcag 2280tgccaacgtt gtggttgatg gtaatactgt caacctcggc
ctctgggaca ctgcaggtca 2340agaggattac aacagactga gaccactgag
ctatcgtgga gctgatgttt ttcttctggc 2400tttctcactg atcagtaagg
ccagctatga gaatgtttcg aagaagtgga tacctgaact 2460gaagcattat
gcacctggtg tgccaattat tctcgtaggg acaaagcttg atcttcgaga
2520cgacaagcag ttctttgtgg accatcctgg tgctgtccct atcactactg
ctcagggaga 2580ggagctaaga aagcaaatag gcgctccata ctacatcgaa
tgcagctcga agacccaact 2640aaacgtgaag ggcgtcttcg atgcggcgat
aaaggttgtg ctgcagccgc ctaaggcgaa 2700gaagaagaaa aaggtgcaga
ggggggcgtg ctccattttg tgaaattcac tggccgtcgt 2760tttacaacga
ctcagagctt gacaggaggc ccgatctagt aacatagatg acaccgcgcg
2820cgataattta tcctagtttg cgcgctatat tttgttttct atcgcgtatt
aaatgtataa 2880ttgcgggact ctaatcataa aaacccatct cataaataac
gtcatgcatt acatgttaat 2940tattacatgc ttaacgtaat tcaacagaaa
ttatatgata atcatcgcaa gaccggcaac 3000aggattcaat cttaagaaac
tttattgcca aatgtttgaa cgatcgggga tcatccgggt 3060ctgtggcggg
aactccacga aaatatccga acgcagcaag atctagagct tgggtcccgc
3120tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg
gagcggcgat 3180accgtaaagc acgaggaagc ggtcagccca ttcgccgcca
agctcttcag caatatcacg 3240ggtagccaac gctatgtcct gatagcggtc
cgccacaccc agccggccac agtcgatgaa 3300tccagaaaag cggccatttt
ccaccatgat attcggcaag caggcatcgc catgggtcac 3360gacgagatcc
tcgccgtcgg gcatgcgcgc cttgagcctg gcgaacagtt cggctggcgc
3420gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt
ccatccgagt 3480acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat
gggcaggtag ccggatcaag 3540cgtatgcagc cgccgcattg catcagccat
gatggatact ttctcggcag gagcaaggtg 3600agatgacagg agatcctgcc
ccggcacttc gcccaatagc agccagtccc ttcccgcttc 3660agtgacaacg
tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg
3720cgctgcctcg tcctgcagtt cattcagggc accggacagg tcggtcttga
caaaaagaac 3780cgggcgcccc tgcgctgaca gccggaacac ggcggcatca
gagcagccga ttgtctgttg 3840tgcccagtca tagccgaata gcctctccac
ccaagcggcc ggagaacctg cgtgcaatcc 3900atcttgttca atcatgcgaa
acgatccaga tccggtgcag attatttgga ttgagagtga 3960atatgagact
ctaattggat accgagggga atttatggaa cgtcagtgga gcatttttga
4020caagaaatat ttgctagctg atagtgacct taggcgactt ttgaacgcgc
aataatggtt 4080tctgacgtat gtgcttagct cattaaactc cagaaacccg
cggctgagtg gctccttcaa 4140cgttgcggtt ctgtcagttc caaacgtaaa
acggcttgtc ccgcgtcatc ggcgggggtc 4200ataacgtgac tcccttaatt
ctccgctcat gatcagattg tcgtttcccg ccttcagttt 4260aaactatcag
tgtttgacag gatcctgctt ggtaataatt gtcattagat tgtttttatg
4320catagatgca ctcgaaatca gccaatttta gacaagtatc aaacggatgt
taattcagta 4380cattaaagac gtccgcaatg tgttattaag ttgtctaagc
gtcaatttgt ttacaccaca 4440atatatcctg ccaccagcca gccaacagct
ccccgaccgg cagctcggca caaaatcacc 4500acgcgttacc accacgccgg
ccggccgcat ggtgttgacc gtgttcgccg gcattgccga 4560gttcgagcgt
tccctaatca tcgaccgcac ccggagcggg cgcgaggccg ccaaggcccg
4620aggcgtgaag tttggccccc gccctaccct caccccggca cagatcgcgc
acgcccgcga 4680gctgatcgac caggaaggcc gcaccgtgaa agaggcggct
gcactgcttg gcgtgcatcg 4740ctcgaccctg taccgcgcac ttgagcgcag
cgaggaagtg acgcccaccg aggccaggcg 4800gcgcggtgcc ttccgtgagg
acgcattgac cgaggccgac gccctggcgg ccgccgagaa 4860tgaacgccaa
gaggaacaag catgaaaccg caccaggacg gccaggacga accgtttttc
4920attaccgaag agatcgaggc ggagatgatc gcggccgggt acgtgttcga
gccgcccgcg 4980cacgtctcaa ccgtgcggct gcatgaaatc ctggccggtt
tgtctgatgc caagctggcg 5040gcctggccgg ccagcttggc cgctgaagaa
accgagcgcc gccgtctaaa aaggtgatgt 5100gtatttgagt aaaacagctt
gcgtcatgcg gtcgctgcgt atatgatgcg atgagtaaat 5160aaacaaatac
gcaaggggaa cgcatgaagg ttatcgctgt acttaaccag aaaggcgggt
5220caggcaagac gaccatcgca acccatctag cccgcgccct gcaactcgcc
ggggccgatg 5280ttctgttagt cgattccgat ccccagggca gtgcccgcga
ttgggcggcc gtgcgggaag 5340atcaaccgct aaccgttgtc ggcatcgacc
gcccgacgat tgaccgcgac gtgaaggcca 5400tcggccggcg
cgacttcgta gtgatcgacg gagcgcccca ggcggcggac ttggctgtgt
5460ccgcgatcaa ggcagccgac ttcgtgctga ttccggtgca gccaagccct
tacgacatat 5520gggccaccgc cgacctggtg gagctggtta agcagcgcat
tgaggtcacg gatggaaggc 5580tacaagcggc ctttgtcgtg tcgcgggcga
tcaaaggcac gcgcatcggc ggtgaggttg 5640ccgaggcgct ggccgggtac
gagctgccca ttcttgagtc ccgtatcacg cagcgcgtga 5700gctacccagg
cactgccgcc gccggcacaa ccgttcttga atcagaaccc gagggcgacg
5760ctgcccgcga ggtccaggcg ctggccgctg aaattaaatc aaaactcatt
tgagttaatg 5820aggtaaagag aaaatgagca aaagcacaaa cacgctaagt
gccggccgtc cgagcgcacg 5880cagcagcaag gctgcaacgt tggccagcct
ggcagacacg ccagccatga agcgggtcaa 5940ctttcagttg ccggcggagg
atcacaccaa gctgaagatg tacgcggtac gccaaggcaa 6000gaccattacc
gagctgctat ctgaatacat cgcgcagcta ccagagtaaa tgagcaaatg
6060aataaatgag tagatgaatt ttagcggcta aaggaggcgg catggaaaat
caagaacaac 6120caggcaccga cgccgtggaa tgccccatgt gtggaggaac
gggcggttgg ccaggcgtaa 6180gcggctgggt tgtctgccgg ccctgcaatg
gcactggaac ccccaagccc gaggaatcgg 6240cgtgagcggt cgcaaaccat
ccggcccggt acaaatcggc gcggcgctgg gtgatgacct 6300ggtggagaag
ttgaaggccg cgcaggccgc ccagcggcaa cgcatcgagg cagaagcacg
6360ccccggtgaa tcgtggcaag cggccgctga tcgaatccgc aaagaatccc
ggcaaccgcc 6420ggcagccggt gcgccgtcga ttaggaagcc gcccaagggc
gacgagcaac cagatttttt 6480cgttccgatg ctctatgacg tgggcacccg
cgatagtcgc agcatcatgg acgtggccgt 6540tttccgtctg tcgaagcgtg
accgacgagc tggcgaggtg atccgctacg agcttccaga 6600cgggcacgta
gaggtttccg cagggccggc cggcatggcc agtgtgtggg attacgacct
6660ggtactgatg gcggtttccc atctaaccga atccatgaac cgataccggg
aagggaaggg 6720agacaagccc ggccgcgtgt tccgtccaca cgttgcggac
gtactcaagt tctgccggcg 6780agccgatggc ggaaagcaga aagacgacct
ggtagaaacc tgcattcggt taaacaccac 6840gcacgttgcc atgcagcgta
cgaagaaggc caagaacggc cgcctggtga cggtatccga 6900gggtgaagcc
ttgattagcc gctacaagat cgtaaagagc gaaaccgggc ggccggagta
6960catcgagatc gagctagctg attggatgta ccgcgagatc acagaaggca
agaacccgga 7020cgtgctgacg gttcaccccg attacttttt gatcgatccc
ggcatcggcc gttttctcta 7080ccgcctggca cgccgcgccg caggcaaggc
agaagccaga tggttgttca agacgatcta 7140cgaacgcagt ggcagcgccg
gagagttcaa gaagttctgt ttcaccgtgc gcaagctgat 7200cgggtcaaat
gacctgccgg agtacgattt gaaggaggag gcggggcagg ctggcccgat
7260cctagtcatg cgctaccgca acctgatcga gggcgaagca tccgccggtt
cctaatgtac 7320ggagcagatg ctagggcaaa ttgccctagc aggggaaaaa
ggtcgaaaag gtctctttcc 7380tgtggatagc acgtacattg ggaacccaaa
gccgtacatt gggaaccgga acccgtacat 7440tgggaaccca aagccgtaca
ttgggaaccg gtcacacatg taagtgactg atataaaaga 7500gaaaaaaggc
gatttttccg cctaaaactc tttaaaactt attaaaactc ttaaaacccg
7560cctggcctgt gcataactgt ctggccagcg cacagccgaa gagctgcaaa
aagcgcctac 7620ccttcggtcg ctgcgctccc tacgccccgc cgcttcgcgt
cggcctatcg cggccgctgg 7680ccgctcaaaa atggctggcc tacggccagg
caatctacca gggcgcggac aagccgcgcc 7740gtcgccactc gaccgccggc
gcccacatca aggcaccctg cctcgcgcgt ttcggtgatg 7800acggtgaaaa
cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
7860atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
tgtcggggcg 7920cagccatgac ccagtcacgt agcgatagcg gagtgtatac
tggcttaact atgcggcatc 7980agagcagatt gtactgagag tgcaccatat
gcggtgtgaa ataccgcaca gatgcgtaag 8040gagaaaatac cgcatcaggc
gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 8100cgttcggctg
cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
8160atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
ccaggaaccg 8220taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
ccccctgacg agcatcacaa 8280aaatcgacgc tcaagtcaga ggtggcgaaa
cccgacagga ctataaagat accaggcgtt 8340tccccctgga agctccctcg
tgcgctctcc tgttccgacc ctgccgctta ccggatacct 8400gtccgccttt
ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
8460cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
ccgttcagcc 8520cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
aacccggtaa gacacgactt 8580atcgccactg gcagcagcca ctggtaacag
gattagcaga gcgaggtatg taggcggtgc 8640tacagagttc ttgaagtggt
ggcctaacta cggctacact agaaggacag tatttggtat 8700ctgcgctctg
ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
8760acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
cgcgcagaaa 8820aaaaggatct caagaagatc ctttgatctt ttctacgggg
tctgacgctc agtggaacga 8880aaactcacgt taagggattt tggtcatgca
tgatatatct cccaatttgt gtagggctta 8940ttatgcacgc ttaaaaataa
taaaagcaga cttgacctga tagtttggct gtgagcaatt 9000atgtgcttag
tgcatctaac gcttgagtta agccgcgccg cgaagcggcg tcggcttgaa
9060cgaatttcta gctagacatt atttgccgac taccttggtg atctcgcctt
tcacgtagtg 9120gacaaattct tccaactgat ctgcgcgcga ggccaagcga
tcttcttctt gtccaagata 9180agcctgtcta gcttcaagta tgacgggctg
atactgggcc ggcaggcgct ccattgccca 9240gtcggcagcg acatccttcg
gcgcgatttt gccggttact gcgctgtacc aaatgcggga 9300caacgtaagc
actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt
9360taaggtttca tttagcgcct caaatagatc ctgttcagga accggatcaa
agagttcctc 9420cgccgctgga cctaccaagg caacgctatg ttctcttgct
tttgtcagca agatagccag 9480atcaatgtcg atcgtggctg gctcgaagat
acctgcaaga atgtcattgc gctgccattc 9540tccaaattgc agttcgcgct
tagctggata acgccacgga atgatgtcgt cgtgcacaac 9600aatggtgact
tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa
9660aaggtcgttg atcaaagctc gccgcgttgt ttcatcaagc cttacggtca
ccgtaaccag 9720caaatcaata tcactgtgtg gcttcaggcc gccatccact
gcggagccgt acaaatgtac 9780ggccagcaac gtcggttcga gatggcgctc
gatgacgcca actacctctg atagttgagt 9840cgatacttcg gcgatcaccg
cttcccccat gatgtttaac tttgttttag ggcgactgcc 9900ctgctgcgta
acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc
9960ttgctgcttg gatgcccgag gcatagactg taccccaaaa aaacagtcat
aacaagccat 10020gaaaaccgcc actgcg 100367610036DNAArtificial
sequenceDescription of the artificial sequence transgenic
expression vector pSUN3NIT_HvRacB_as for expression of barley RacB
anti sense RNA fragment 76ttccatggac atacaaatgg acgaacggat
aaaccttttc acgccctttt aaatatccga 60ttattctaat aaacgctctt ttctcttagg
tttacccgcc aatatatcct gtcaaacact 120gatagtttaa actgaaggcg
ggaaacgaca atcagatcta gtaggaaaca gctatgacca 180tgattacgcc
aagcttgcat gcctgcaggt cgactctaga ggatccccca tcaagatctt
240ggtgatgtag caagagctaa gttgtacttc gatycggttg gacattactc
gagaccagat 300gttttacact tgaccgtaaa tgagcacccg aagaaaccgg
taacattcat ttcgaaggta 360gagaaagcgg aagatgactc aaacaagtaa
tcggttgtga ttcgtcagtt catgtcactc 420ctatgaagga gtcaagttca
aaatgttatg ttgagtttca aacttttatg ctaaactttt 480tttcttaatt
ttcgttaata atggaagaga accaattctc ttgtatctaa agattatcca
540tctatcatcc aatttgagtg ttcaattctg gatgttgtgt taccctacat
tctacaacca 600tgtagccaat tattatgaat ctggctttga tttcagttgt
gttcttttct ttttttcttt 660gcatatttgc atttagaatg tttaataatt
aagttactgt atttccacat acattagttc 720caagaatata catatattaa
tttatttttc ttaaaaatgt tttggaatga ctaatattga 780caacgaaaat
agaagctatg ctaaaccatt acgtatatgt gacttcacat gttgttgttt
840tacattccct atatatatgg atggctgtca caatcagaaa cgtgatcgaa
aaaagacaaa 900cagtgtttgc ataaaaagac tatttcgttt cattgacaat
ttgtgtttat ttgtaaagaa 960aagtggcaaa gtggaatttg agttcctgca
agtaagaaag atgaaataaa agacttgagt 1020gtgtgttttt ttcttttatc
tgaaagctgc aatgaaatat tcctaccaag cccgtttgat 1080tattaattgg
ggtttggttt tcttgatgcg aactaattgg ttatataaga aactatacaa
1140tccatgttaa ttcaaaaatt ttgatttctc ttgtaggaat atgatttact
atatgagact 1200ttcttttcgc caataatagt aaatccaaag atatttgacc
ggaccaaaac acattgatct 1260attttttagt ttatttaatc cagtttctct
gagataattc attaaggaaa acttagtatt 1320aacccatcct aagattaaat
aggagccaaa ctcacatttc aaatattaaa taacataaaa 1380tggatttaaa
aaatctatac gtcaaatttt atttatgaca tttcttattt aaatttatat
1440ttaatgaaat acagctaaga caaaccaaaa aaaaaatact ttctaagtgg
tccaaaacat 1500caattccgtt caatattatt aggtagaatc gtacgaccaa
aaaaggtagg ttaatacgaa 1560attacaaaca tatctatata catagtatat
attattacct attatgagga atcaaaatgc 1620atcaaatatg gatttaagga
atccataaaa gaataaattc tacggaaaaa aaaaaaagaa 1680taaattcttt
taagttttta atttgttttt tatttggtag ttctccattt tgttttattt
1740cgtttggatt tattgtgtcc aaatactttg taaaccaccg ttgtaattct
taaacggggt 1800tttcacttct tttttatatt cagacataaa gcatcggctg
gtttaatcaa tcaatagatt 1860ttatttttct tctcaattat tagtaggttt
gatgtgaact ttacaaaaaa aacaaaaaca 1920aatcaatgca gagaaaagaa
accacgtggg ctagtcccac cttgtttcat ttccaccaca 1980ggttcgatct
tcgttaccgt ctccaatagg aaaataaacg tgaccacaaa aaaaaaacaa
2040aaaaaaagtc tatatattgc ttctctcaag tctctgagtg tcatgaacca
aagtaaaaaa 2100caaagactcg agtggatccc cggtcacaaa atggagcacg
cccccctctg cacctttttc 2160ttcttcttcg ccttaggcgg ctgcagcaca
acctttatcg ccgcatcgaa gacgcccttc 2220acgtttagtt gggtcttcga
gctgcattcg atgtagtatg gagcgcctat ttgctttctt 2280agctcctctc
cctgagcagt agtgataggg acagcaccag gatggtccac aaagaactgc
2340ttgtcgtctc gaagatcaag ctttgtccct acgagaataa ttggcacacc
aggtgcataa 2400tgcttcagtt caggtatcca cttcttcgaa acattctcat
agctggcctt actgatcagt 2460gagaaagcca gaagaaaaac atcagctcca
cgatagctca gtggtctcag tctgttgtaa 2520tcctcttgac ctgcagtgtc
ccagaggccg aggttgacag tattaccatc aaccacaacg 2580ttggcactga
agttatcaaa cactgtcgga acatagtcgg tggggaaggt gttggaggtg
2640taggagatga gcatgcaggt cttgccgacg gcgccgtccc cgaccgtgac
gcactttatg 2700aacctggacg cgctcatcgg atccatcccg cggccatggc
ggcaattcac tggccgtcgt 2760tttacaacga ctcagagctt gacaggaggc
ccgatctagt aacatagatg acaccgcgcg 2820cgataattta tcctagtttg
cgcgctatat tttgttttct atcgcgtatt aaatgtataa 2880ttgcgggact
ctaatcataa aaacccatct cataaataac gtcatgcatt acatgttaat
2940tattacatgc ttaacgtaat tcaacagaaa ttatatgata atcatcgcaa
gaccggcaac 3000aggattcaat cttaagaaac tttattgcca aatgtttgaa
cgatcgggga tcatccgggt 3060ctgtggcggg aactccacga aaatatccga
acgcagcaag atctagagct tgggtcccgc 3120tcagaagaac tcgtcaagaa
ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat 3180accgtaaagc
acgaggaagc ggtcagccca ttcgccgcca agctcttcag caatatcacg
3240ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac
agtcgatgaa 3300tccagaaaag cggccatttt ccaccatgat attcggcaag
caggcatcgc catgggtcac 3360gacgagatcc tcgccgtcgg gcatgcgcgc
cttgagcctg gcgaacagtt cggctggcgc 3420gagcccctga tgctcttcgt
ccagatcatc ctgatcgaca agaccggctt ccatccgagt 3480acgtgctcgc
tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag
3540cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag
gagcaaggtg 3600agatgacagg agatcctgcc ccggcacttc gcccaatagc
agccagtccc ttcccgcttc 3660agtgacaacg tcgagcacag ctgcgcaagg
aacgcccgtc gtggccagcc acgatagccg 3720cgctgcctcg tcctgcagtt
cattcagggc accggacagg tcggtcttga caaaaagaac 3780cgggcgcccc
tgcgctgaca gccggaacac ggcggcatca gagcagccga ttgtctgttg
3840tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg
cgtgcaatcc 3900atcttgttca atcatgcgaa acgatccaga tccggtgcag
attatttgga ttgagagtga 3960atatgagact ctaattggat accgagggga
atttatggaa cgtcagtgga gcatttttga 4020caagaaatat ttgctagctg
atagtgacct taggcgactt ttgaacgcgc aataatggtt 4080tctgacgtat
gtgcttagct cattaaactc cagaaacccg cggctgagtg gctccttcaa
4140cgttgcggtt ctgtcagttc caaacgtaaa acggcttgtc ccgcgtcatc
ggcgggggtc 4200ataacgtgac tcccttaatt ctccgctcat gatcagattg
tcgtttcccg ccttcagttt 4260aaactatcag tgtttgacag gatcctgctt
ggtaataatt gtcattagat tgtttttatg 4320catagatgca ctcgaaatca
gccaatttta gacaagtatc aaacggatgt taattcagta 4380cattaaagac
gtccgcaatg tgttattaag ttgtctaagc gtcaatttgt ttacaccaca
4440atatatcctg ccaccagcca gccaacagct ccccgaccgg cagctcggca
caaaatcacc 4500acgcgttacc accacgccgg ccggccgcat ggtgttgacc
gtgttcgccg gcattgccga 4560gttcgagcgt tccctaatca tcgaccgcac
ccggagcggg cgcgaggccg ccaaggcccg 4620aggcgtgaag tttggccccc
gccctaccct caccccggca cagatcgcgc acgcccgcga 4680gctgatcgac
caggaaggcc gcaccgtgaa agaggcggct gcactgcttg gcgtgcatcg
4740ctcgaccctg taccgcgcac ttgagcgcag cgaggaagtg acgcccaccg
aggccaggcg 4800gcgcggtgcc ttccgtgagg acgcattgac cgaggccgac
gccctggcgg ccgccgagaa 4860tgaacgccaa gaggaacaag catgaaaccg
caccaggacg gccaggacga accgtttttc 4920attaccgaag agatcgaggc
ggagatgatc gcggccgggt acgtgttcga gccgcccgcg 4980cacgtctcaa
ccgtgcggct gcatgaaatc ctggccggtt tgtctgatgc caagctggcg
5040gcctggccgg ccagcttggc cgctgaagaa accgagcgcc gccgtctaaa
aaggtgatgt 5100gtatttgagt aaaacagctt gcgtcatgcg gtcgctgcgt
atatgatgcg atgagtaaat 5160aaacaaatac gcaaggggaa cgcatgaagg
ttatcgctgt acttaaccag aaaggcgggt 5220caggcaagac gaccatcgca
acccatctag cccgcgccct gcaactcgcc ggggccgatg 5280ttctgttagt
cgattccgat ccccagggca gtgcccgcga ttgggcggcc gtgcgggaag
5340atcaaccgct aaccgttgtc ggcatcgacc gcccgacgat tgaccgcgac
gtgaaggcca 5400tcggccggcg cgacttcgta gtgatcgacg gagcgcccca
ggcggcggac ttggctgtgt 5460ccgcgatcaa ggcagccgac ttcgtgctga
ttccggtgca gccaagccct tacgacatat 5520gggccaccgc cgacctggtg
gagctggtta agcagcgcat tgaggtcacg gatggaaggc 5580tacaagcggc
ctttgtcgtg tcgcgggcga tcaaaggcac gcgcatcggc ggtgaggttg
5640ccgaggcgct ggccgggtac gagctgccca ttcttgagtc ccgtatcacg
cagcgcgtga 5700gctacccagg cactgccgcc gccggcacaa ccgttcttga
atcagaaccc gagggcgacg 5760ctgcccgcga ggtccaggcg ctggccgctg
aaattaaatc aaaactcatt tgagttaatg 5820aggtaaagag aaaatgagca
aaagcacaaa cacgctaagt gccggccgtc cgagcgcacg 5880cagcagcaag
gctgcaacgt tggccagcct ggcagacacg ccagccatga agcgggtcaa
5940ctttcagttg ccggcggagg atcacaccaa gctgaagatg tacgcggtac
gccaaggcaa 6000gaccattacc gagctgctat ctgaatacat cgcgcagcta
ccagagtaaa tgagcaaatg 6060aataaatgag tagatgaatt ttagcggcta
aaggaggcgg catggaaaat caagaacaac 6120caggcaccga cgccgtggaa
tgccccatgt gtggaggaac gggcggttgg ccaggcgtaa 6180gcggctgggt
tgtctgccgg ccctgcaatg gcactggaac ccccaagccc gaggaatcgg
6240cgtgagcggt cgcaaaccat ccggcccggt acaaatcggc gcggcgctgg
gtgatgacct 6300ggtggagaag ttgaaggccg cgcaggccgc ccagcggcaa
cgcatcgagg cagaagcacg 6360ccccggtgaa tcgtggcaag cggccgctga
tcgaatccgc aaagaatccc ggcaaccgcc 6420ggcagccggt gcgccgtcga
ttaggaagcc gcccaagggc gacgagcaac cagatttttt 6480cgttccgatg
ctctatgacg tgggcacccg cgatagtcgc agcatcatgg acgtggccgt
6540tttccgtctg tcgaagcgtg accgacgagc tggcgaggtg atccgctacg
agcttccaga 6600cgggcacgta gaggtttccg cagggccggc cggcatggcc
agtgtgtggg attacgacct 6660ggtactgatg gcggtttccc atctaaccga
atccatgaac cgataccggg aagggaaggg 6720agacaagccc ggccgcgtgt
tccgtccaca cgttgcggac gtactcaagt tctgccggcg 6780agccgatggc
ggaaagcaga aagacgacct ggtagaaacc tgcattcggt taaacaccac
6840gcacgttgcc atgcagcgta cgaagaaggc caagaacggc cgcctggtga
cggtatccga 6900gggtgaagcc ttgattagcc gctacaagat cgtaaagagc
gaaaccgggc ggccggagta 6960catcgagatc gagctagctg attggatgta
ccgcgagatc acagaaggca agaacccgga 7020cgtgctgacg gttcaccccg
attacttttt gatcgatccc ggcatcggcc gttttctcta 7080ccgcctggca
cgccgcgccg caggcaaggc agaagccaga tggttgttca agacgatcta
7140cgaacgcagt ggcagcgccg gagagttcaa gaagttctgt ttcaccgtgc
gcaagctgat 7200cgggtcaaat gacctgccgg agtacgattt gaaggaggag
gcggggcagg ctggcccgat 7260cctagtcatg cgctaccgca acctgatcga
gggcgaagca tccgccggtt cctaatgtac 7320ggagcagatg ctagggcaaa
ttgccctagc aggggaaaaa ggtcgaaaag gtctctttcc 7380tgtggatagc
acgtacattg ggaacccaaa gccgtacatt gggaaccgga acccgtacat
7440tgggaaccca aagccgtaca ttgggaaccg gtcacacatg taagtgactg
atataaaaga 7500gaaaaaaggc gatttttccg cctaaaactc tttaaaactt
attaaaactc ttaaaacccg 7560cctggcctgt gcataactgt ctggccagcg
cacagccgaa gagctgcaaa aagcgcctac 7620ccttcggtcg ctgcgctccc
tacgccccgc cgcttcgcgt cggcctatcg cggccgctgg 7680ccgctcaaaa
atggctggcc tacggccagg caatctacca gggcgcggac aagccgcgcc
7740gtcgccactc gaccgccggc gcccacatca aggcaccctg cctcgcgcgt
ttcggtgatg 7800acggtgaaaa cctctgacac atgcagctcc cggagacggt
cacagcttgt ctgtaagcgg 7860atgccgggag cagacaagcc cgtcagggcg
cgtcagcggg tgttggcggg tgtcggggcg 7920cagccatgac ccagtcacgt
agcgatagcg gagtgtatac tggcttaact atgcggcatc 7980agagcagatt
gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag
8040gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
tgcgctcggt 8100cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
gtaatacggt tatccacaga 8160atcaggggat aacgcaggaa agaacatgtg
agcaaaaggc cagcaaaagg ccaggaaccg 8220taaaaaggcc gcgttgctgg
cgtttttcca taggctccgc ccccctgacg agcatcacaa 8280aaatcgacgc
tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
8340tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
ccggatacct 8400gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
agctcacgct gtaggtatct 8460cagttcggtg taggtcgttc gctccaagct
gggctgtgtg cacgaacccc ccgttcagcc 8520cgaccgctgc gccttatccg
gtaactatcg tcttgagtcc aacccggtaa gacacgactt 8580atcgccactg
gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
8640tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
tatttggtat 8700ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
ggtagctctt gatccggcaa 8760acaaaccacc gctggtagcg gtggtttttt
tgtttgcaag cagcagatta cgcgcagaaa 8820aaaaggatct caagaagatc
ctttgatctt ttctacgggg tctgacgctc agtggaacga 8880aaactcacgt
taagggattt tggtcatgca tgatatatct cccaatttgt gtagggctta
8940ttatgcacgc ttaaaaataa taaaagcaga cttgacctga tagtttggct
gtgagcaatt 9000atgtgcttag tgcatctaac gcttgagtta agccgcgccg
cgaagcggcg tcggcttgaa 9060cgaatttcta gctagacatt atttgccgac
taccttggtg atctcgcctt tcacgtagtg 9120gacaaattct tccaactgat
ctgcgcgcga ggccaagcga tcttcttctt gtccaagata 9180agcctgtcta
gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca
9240gtcggcagcg acatccttcg gcgcgatttt gccggttact gcgctgtacc
aaatgcggga 9300caacgtaagc actacatttc gctcatcgcc agcccagtcg
ggcggcgagt tccatagcgt 9360taaggtttca tttagcgcct caaatagatc
ctgttcagga accggatcaa agagttcctc 9420cgccgctgga cctaccaagg
caacgctatg ttctcttgct tttgtcagca agatagccag 9480atcaatgtcg
atcgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc
9540tccaaattgc agttcgcgct tagctggata acgccacgga atgatgtcgt
cgtgcacaac 9600aatggtgact tctacagcgc ggagaatctc gctctctcca
ggggaagccg aagtttccaa 9660aaggtcgttg atcaaagctc gccgcgttgt
ttcatcaagc cttacggtca ccgtaaccag 9720caaatcaata tcactgtgtg
gcttcaggcc gccatccact gcggagccgt acaaatgtac 9780ggccagcaac
gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt
9840cgatacttcg gcgatcaccg cttcccccat gatgtttaac tttgttttag
ggcgactgcc 9900ctgctgcgta acatcgttgc tgctccataa catcaaacat
cgacccacgg cgtaacgcgc 9960ttgctgcttg gatgcccgag gcatagactg
taccccaaaa aaacagtcat aacaagccat 10020gaaaaccgcc actgcg 10036
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