U.S. patent application number 12/573869 was filed with the patent office on 2010-09-30 for hypersensitive response induced resistance in plants by seed treatment.
Invention is credited to Steven V. Beer, Dewen Qiu, Zhong-Min Wei.
Application Number | 20100242358 12/573869 |
Document ID | / |
Family ID | 21869247 |
Filed Date | 2010-09-30 |
United States Patent
Application |
20100242358 |
Kind Code |
A1 |
Qiu; Dewen ; et al. |
September 30, 2010 |
HYPERSENSITIVE RESPONSE INDUCED RESISTANCE IN PLANTS BY SEED
TREATMENT
Abstract
The present invention relates to a method of imparting pathogen
resistance to plants. This involves applying a hypersensitive
response elicitor polypeptide or protein in a non-infectious form
to a plant seed under conditions where the polypeptide or protein
contacts cells of the plant seed. The present invention is also
directed to a pathogen resistance imparting plant seed.
Alternatively, transgenic plant seeds containing a DNA molecule
encoding a hypersensitive response elicitor polypeptide or protein
can be planted in soil and a plant can be propagated from the
planted seed under conditions effective to impart pathogen
resistance to the plant.
Inventors: |
Qiu; Dewen; (Seattle,
WA) ; Wei; Zhong-Min; (Kirkland, WA) ; Beer;
Steven V.; (Ithaca, NY) |
Correspondence
Address: |
NIXON PEABODY LLP - PATENT GROUP
1100 CLINTON SQUARE
ROCHESTER
NY
14604
US
|
Family ID: |
21869247 |
Appl. No.: |
12/573869 |
Filed: |
October 5, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09766348 |
Jan 19, 2001 |
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12573869 |
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08984207 |
Dec 3, 1997 |
6235974 |
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09766348 |
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60033230 |
Dec 5, 1996 |
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Current U.S.
Class: |
47/58.1SE ;
111/200 |
Current CPC
Class: |
A01H 3/02 20130101; A01N
63/10 20200101; A01N 63/30 20200101; C07K 14/27 20130101; C12N
15/8279 20130101 |
Class at
Publication: |
47/58.1SE ;
111/200 |
International
Class: |
A01C 1/08 20060101
A01C001/08; A01C 7/00 20060101 A01C007/00; A01G 1/00 20060101
A01G001/00 |
Goverment Interests
[0002] This invention was made with support from the U.S.
Government under USDA NRI Competitive Research Grant No.
91-37303-6430.
Claims
1. A method of imparting pathogen resistance to plants, the method
comprising: providing a transgenic plant seed transformed with a
transgene comprising a DNA molecule encoding a hypersensitive
response elicitor polypeptide or protein comprising an amino acid
sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7
and a promoter that is not pathogen-inducible, the promoter being
operatively coupled to the DNA molecule encoding the hypersensitive
response elicitor polypeptide or protein; planting the transgenic
plant seed in soil; and propagating a plant from the planted seed,
whereby expression of the hypersensitive response elicitor
polypeptide or protein by the plant imparts systemic pathogen
resistance to the plant.
Description
[0001] This is a continuation of U.S. patent application Ser. No.
09/766,348 filed Jan. 19, 2001, which is a division of U.S. patent
application Ser. No. 08/984,207 filed Dec. 3, 1997, now U.S. Pat.
No. 6,235,974, which claims the benefit of U.S. Provisional Patent
Application Ser. No. 60/033,230 filed Dec. 5, 1996.
FIELD OF THE INVENTION
[0003] The present invention relates to imparting hypersensitive
response induced resistance to plants by treatment of seeds.
BACKGROUND OF THE INVENTION
[0004] Living organisms have evolved a complex array of biochemical
pathways that enable them to recognize and respond to signals from
the environment. These pathways include receptor organs, hormones,
second messengers, and enzymatic modifications. At present, little
is known about the signal transduction pathways that are activated
during a plant's response to attack by a pathogen, although this
knowledge is central to an understanding of disease susceptibility
and resistance. A common form of plant resistance is the
restriction of pathogen proliferation to a small zone surrounding
the site of infection. In many cases, this restriction is
accompanied by localized death (i.e., necrosis) of host tissues.
Together, pathogen restriction and local tissue necrosis
characterize the hypersensitive response. In addition to local
defense responses, many plants respond to infection by activating
defenses in uninfected parts of the plant. As a result, the entire
plant is more resistant to a secondary infection. This systemic
acquired resistance can persist for several weeks or more (R. E. F.
Matthews, Plant Virology (Academic Press, New York, ed. 2, 1981))
and often confers cross-resistance to unrelated pathogens (J. Kuc,
in Innovative Approaches to Plant Disease Control, I. Chet, Ed.
(Wiley, New York, 1987), pp. 255-274, which is hereby incorporated
by reference). See also Kessman, et al., "Induction of Systemic
Acquired Disease Resistance in Plants By Chemicals," Ann. Rev.
Phytopathol. 32:439-59 (1994), Ryals, et al., "Systemic Acquired
Resistance," The Plant Cell 8:1809-19 (October 1996), and
Neuenschwander, et al., "Systemic Acquired Resistance,"
Plant-Microbe Interactions vol. 1, G. Stacey, et al. ed. pp. 81-106
(1996), which are hereby incorporated by reference.
[0005] Expression of systemic acquired resistance is associated
with the failure of normally virulent pathogens to ingress the
immunized tissue (Kuc, J., "Induced Immunity to Plant Disease,"
Bioscience, 32:854-856 (1982), which is hereby incorporated by
reference). Establishment of systemic acquired resistance is
correlated with systemic increases in cell wall hydroxyproline
levels and peroxidase activity (Smith, J. A., et al., "Comparative
Study of Acidic Peroxidases Associated with Induced Resistance in
Cucumber, Muskmelon and Watermelon," Physiol. Mol. Plant. Pathol.
14:329-338 (1988), which is hereby incorporated by reference) and
with the expression of a set of nine families of so-called systemic
acquired resistance gene (Ward, E. R., et al., "Coordinate Gene
Activity in Response to Agents that Induce Systemic Acquired
Resistance," Plant Cell 3:49-59 (1991), which is hereby
incorporated by reference). Five of these defense gene families
encode pathogenesis-related proteins whose physiological functions
have not been established. However, some of these proteins have
antifungal activity in vitro (Bol, J. F., et al., "Plant
Pathogenesis-Related Proteins Induced by Virus Infection," Ann.
Rev. Phytopathol. 28:113-38 (1990), which is hereby incorporated by
reference) and the constitutive expression of a bean chitinase gene
in transgenic tobacco protects against infection by the fungus
Rhizoctonia solani (Broglie, K., et al., "Transgenic Plants with
Enhanced Resistance to the Fungal Pathogen Rhizoctonia Solani,"
Science 254:1194-1197 (1991), which is hereby incorporated by
reference), suggesting that these systemic acquired resistance
proteins may contribute to the immunized state (Uknes, S., et al.,
"Acquired Resistance in Arabidopsis," Plant Cell 4:645-656 (1992),
which is hereby incorporated by reference).
[0006] Salicylic acid appears to play a signal function in the
induction of systemic acquired resistance since endogenous levels
increase after immunization (Malamy, J., et al., "Salicylic Acid: A
Likely Endogenous Signal in the Resistance Response of Tobacco to
Viral Infection," Science 250:1002-1004 (1990), which is hereby
incorporated by reference) and exogenous salicylate induces
systemic acquired resistance genes (Yalpani, N., et al., "Salicylic
Acid is a Systemic Signal and an Inducer of Pathogenesis-Related
Proteins in Virus-Infected Tobacco," Plant Cell 3:809-818 (1991),
which is hereby incorporated by reference), and acquired resistance
(Uknes, S., et al., "Acquired Resistance in Arabidopsis," Plant
Cell 4:645-656 (1992), which is hereby incorporated by reference).
Moreover, transgenic tobacco plants in which salicylate is
destroyed by the action of a bacterial transgene encoding
salicylate hydroxylase do not exhibit systemic acquired resistance
(Gaffney, T., et al., "Requirement of Salicylic Acid for the
Induction of Systemic Acquired Resistance," Science 261:754-56
(1993), which is hereby incorporated by reference). However, this
effect may reflect inhibition of a local rather than a systemic
signal function, and detailed kinetic analysis of signal
transmission in cucumber suggests that salicylate may not be
essential for long-distance signaling (Rasmussen, J. B., et al.,
"Systemic Induction of Salicylic Acid Accumulation in Cucumber
after Inoculation with Pseudomonas Syringae pv. Syringae," Plant
Physiol. 97:1342-1347) (1991), which is hereby incorporated by
reference).
[0007] Immunization using biotic agents has been extensively
studied. Green beans were systemically immunized against disease
caused by cultivar-pathogenic races of Colletotrichum
lindemuthianum by prior infection with either
cultivar-nonpathogenic races (Rahe, J. E., "Induced Resistance in
Phaseolus Vulgaris to Bean Anthracnose," Phytopathology 59:1641-5
(1969); Elliston, J., et al., "Induced Resistance to Anthracnose at
a Distance from the Site of the Inducing Interaction,"
Phytopathology 61:1110-12 (1971); Skipp, R., et al., "Studies on
Cross Protection in the Anthracnose Disease of Bean," Physiological
Plant Pathology 3:299-313 (1973), which are hereby incorporated by
reference), cultivar-pathogenic races attenuated by heat in host
tissue prior to symptom appearance (Rahe, J. E., et al., "Metabolic
Nature of the Infection-Limiting Effect of Heat on Bean
Anthracnose," Phytopathology 60:1005-9 (1970), which is hereby
incorporated by reference) or nonpathogens of bean. The anthracnose
pathogen of cucumber, Colletotrichum lagenarium, was equally
effective as non-pathogenic races as an inducer of systemic
protection against all races of bean anthracnose. Protection was
induced by C. lagenarium in cultivars resistant to one or more
races of C. lindemuthianum as well as in cultivars susceptible to
all reported races of the fungus and which accordingly had been
referred to as `lacking genetic resistance` to the pathogen
(Elliston, J., et al., "Protection of Bean Against Anthracnose by
Colletotrichum Species Nonpathogenic on Bean," Phytopathologische
Zeitschrift 86:117-26 (1976); Elliston, J., et al., "A Comparative
Study on the Development of Compatible, Incompatible and Induced
Incompatible Interactions Between Collectotrichum Species and
Phaseolus Vulgaris," Phytopathologische Zeitschrift 87:289-303
(1976), which are hereby incorporated by reference). These results
suggest that the same mechanisms may be induced in cultivars
reported as `possessing` or `lacking` resistance genes (Elliston,
J., et al., "Relation of Phytoalexin Accumulation to Local and
Systemic Protection of Bean Against Anthracnose,"
Phytopathologische Zeitschrift 88:114-30 (1977), which is hereby
incorporated by reference). It also is apparent that cultivars
susceptible to all races of C. lindemuthianum do not lack genes for
induction of resistance mechanisms against the pathogen.
[0008] Kuc, J., et al., "Protection of Cucumber Against
Collectotrichum Lagenarium by Colletotrichum Lagenarium,"
Physiological Plant Pathology 7:195-9 (1975), which is hereby
incorporated by reference), showed that cucumber plants could be
systemically protected against disease caused by Colletotrichum
lagenarium by prior inoculation of the cotyledons or the first true
leaf with the same fungus. Subsequently, cucumbers have been
systemically protected against fungal, bacterial, and viral
diseases by prior localized infection with either fungi, bacteria,
or viruses (Hammerschmidt, R., et al., "Protection of Cucumbers
Against Colletotrichum Lagenarium and Cladosporium Cucumerinum,"
Phytopathology 66:790-3 (1976); Jenns, A. E., et al., "Localized
Infection with Tobacco Necrosis Virus Protects Cucumber Against
Colletotrichum Lagenarium," Physiological Plant Pathology 11:207-12
(1977); Caruso, F. L., et al. "Induced Resistance of Cucumber to
Anthracnose and Angular Leaf Spot by Pseudomonas Lachrymans and
Colletotrichum Lagenarium," Physiological Plant Pathology
14:191-201 (1979); Staub, T., et al., "Systemic Protection of
Cucumber Plants Against Disease Caused by Cladosporium Cucumerinum
and Colletotrichum Lagenarium by Prior Localized Infection with
Either Fungus," Physiological Plant Pathology, 17:389-93 (1980);
Bergstrom, G. C., et al., "Effects of Local Infection of Cucumber
by Colletotrichum Lagenarium, Pseudomonas Lachrymans or Tobacco
Necrosis Virus on Systemic Resistance to Cucumber Mosaic Virus,"
Phytopathology 72:922-6 (1982); Gessler, C., et al., "Induction of
Resistance to Fusarium Wilt in Cucumber by Root and Foliar
Pathogens," Phytopathology 72:1439-41 (1982); Basham, B., et al.,
"Tobacco Necrosis Virus Induces Systemic Resistance in Cucumbers
Against Sphaerotheca Fuliginea," Physiological Plant Pathology
23:137-44 (1983), which are hereby incorporated by reference).
Non-specific protection induced by infection with C. lagenarium or
tobacco necrosis virus was effective against at least 13 pathogens,
including obligatory and facultative parasitic fungi, local lesion
and systemic viruses, wilt fungi, and bacteria. Similarly,
protection was induced by and was also effective against root
pathogens. Other curcurbits, including watermelon and muskmelon
have been systemically protected against C. lagenarium (Caruso, F.
L., et al., "Protection of Watermelon and Muskmelon Against
Colletotrichum Lagenarium by Colletotrichum Lagenarium,"
Phytopathology 67:1285-9 (1977), which is hereby incorporated by
reference).
[0009] Systemic protection in tobacco has also been induced against
a wide variety of diseases (Kuc, J., et al., "Immunization for
Disease Resistance in Tobacco," Recent Advances in Tobacco Science
9:179-213 (1983), which is hereby incorporated by reference).
Necrotic lesions caused by tobacco mosaic virus enhanced resistance
in the upper leaves to disease caused by the virus (Ross, A. F., et
al., "Systemic Acquired Resistance Induced by Localized Virus
Infections in Plants," Virology 14:340-58 (1961); Ross, A. F., et
al., "Systemic Effects of Local Lesion Formation," In: Viruses of
Plants pp. 127-50 (1966), which are hereby incorporated by
reference). Phytophthora parasitica var. nicotianae, P. tabacina
and Pseudomonas tabaci and reduced reproduction of the aphid Myzus
persicae (McIntyre, J. L., et al., "Induction of Localized and
Systemic Protection Against Phytophthora Parasitica var. nicotianae
by Tobacco Mosaic Virus Infection of Tobacco Hypersensitive to the
Virus," Physiological Plant Pathology 15:321-30 (1979); McIntyre,
J. L., et al., "Effects of Localized Infections of Nicotiana
Tabacum by Tobacco Mosaic Virus on Systemic Resistance Against
Diverse Pathogens and an Insect," Phytopathology 71:297-301 (1981),
which are hereby incorporated by reference). Infiltration of
heat-killed Pseudomonas tabacin (Lovrekovich, L., et al., "Induced
Reaction Against Wildfire Disease in Tobacco Leaves Treated with
Heat-Killed Bacteria," Nature 205:823-4 (1965), which is hereby
incorporated by reference), and Pseudomonas solanacearum (Sequeira,
L, et al., "Interaction of Bacteria and Host Cell Walls: Its
Relation to Mechanisms of Induced Resistance," Physiological Plant
Pathology 10:43-50 (1977), which is hereby incorporated by
reference), into tobacco leaves induced resistance against the same
bacteria used for infiltration. Tobacco plants were also protected
by the nematode Pratylenchus penetrans against P. parasitica var.
nicotiana (McIntyre, J. L., et al. "Protection of Tobacco Against
Phytophthora Parasitica Var. Nicotianae by Cultivar-Nonpathogenic
Races, Cell-Free Sonicates and Pratylenchus Penetrans,"
Phytopathology 68:235-9 (1978), which is hereby incorporated by
reference).
[0010] Cruikshank, I. A. M., et al., "The Effect of Stem
Infestation of Tobacco with Peronospora Tabacina Adam on Foliage
Reaction to Blue Mould," Journal of the Australian Institute of
Agricultural Science 26:369-72 (1960), which is hereby incorporated
by reference, were the first to report immunization of tobacco
foliage against blue mould (i.e., P. tabacina) by stem injection
with the fungus, which also resulted in dwarfing and premature
senescence. It was recently discovered that injection external to
the xylem not only alleviated stunting but also promoted growth and
development. Immunized tobacco plants, in both glasshouse and field
experiments, were approximately 40% taller, had a 40% increase in
dry weight, a 30% increase in fresh weight, and 4-6 more leaves
than control plants (Tuzun, S., et al., "The Effect of Stem
Injections with Peronospora Tabacina and Metalaxyl Treatment on
Growth of Tobacco and Protection Against Blue Mould in the Field,"
Phytopathology 74:804 (1984), which is hereby incorporated by
reference). These plants flowered approximately 2-3 weeks earlier
than control plants (Tuzun, S., et al., "Movement of a Factor in
Tobacco Infected with Peronospora Tabacina Adam which Systemically
Protects Against Blue Mould," Physiological Plant Pathology
26:321-30 (1985), which is hereby incorporated by reference).
[0011] Systemic protection does not confer absolute immunity
against infection, but reduces the severity of the disease and
delays symptom development. Lesion number, lesion size, and extent
of sporulation of fungal pathogens are all decreased. The diseased
area may be reduced by more than 90%.
[0012] When cucumbers were given a `booster` inoculation 3-6 weeks
after the initial inoculation, immunization induced by C.
lagenarium lasted through flowering and fruiting (Kuc, J., et al.,
"Aspects of the Protection of Cucumber Against Colletotrichum
Lagenarium by Colletotrichum Lagenarium," Phytopathology 67:533-6
(1977), which is hereby incorporated by reference). Protection
could not be induced once plants had set fruit. Tobacco plants were
immunized for the growing season by stem injection with sporangia
of P. tabacina. However, to prevent systemic blue mould
development, this technique was only effective when the plants were
above 20 cm in height.
[0013] Removal of the inducer leaf from immunized cucumber plants
did not reduce the level of immunization of pre-existing expanded
leaves. However, leaves which subsequently emerged from the apical
bud were progressively less protected than their predecessors
(Dean, R. A., et al., "Induced Systemic Protection in Cucumber:
Time of Production and Movement of the `Signal`," Phytopathology
76:966-70 (1986), which is hereby incorporated by reference).
Similar results were reported by Ross, A. F., "Systemic Effects of
Local Lesion Formation," In: Viruses of Plants pp. 127-50 (1966),
which is hereby incorporated by reference, with tobacco (local
lesion host) immunized against tobacco mosaic virus by prior
infection with tobacco mosaic virus. In contrast, new leaves which
emerged from scions excised from tobacco plants immunized by
stem-injection with P. tabacina were highly protected (Tuzun, S.,
et al., "Transfer of Induced Resistance in Tobacco to Blue Mould
(Peronospora tabacina Adam.) Via Callus," Phytopathology 75:1304
(1985), which is hereby incorporated by reference). Plants
regenerated via tissue culture from leaves of immunized plants
showed a significant reduction in blue mould compared to plants
regenerated from leaves of non-immunized parents. Young regenerants
only showed reduced sporulation. As plants aged, both lesion
development and sporulation were reduced. Other investigators,
however, did not reach the same conclusion, although a significant
reduction in sporulation in one experiment was reported (Lucas, J.
A., et al., "Nontransmissibility to Regenerants from Protected
Tobacco Explants of Induced Resistance to Peronospora Hyoscyami,"
Phytopathology 75:1222-5 (1985), which is hereby incorporated by
reference).
[0014] Protection of cucumber and watermelon is effective in the
glasshouse and in the field (Caruso, F. L., et al., "Field
Protection of Cucumber Against Colletotrichum Lagenarium by C.
Lagenarium," Phytopathology 67:1290-2 (1977), which is hereby
incorporated by reference). In one trial, the total lesion area of
C. lagenarium on protected cucumber was less than 2% of the lesion
areas on unprotected control plants. Similarly, only 1 of 66
protected, challenged plants died, whereas 47 of 69 unprotected,
challenged watermelons died. In extensive field trials in Kentucky
and Puerto Rico, stem injection of tobacco with sporangia of P.
tabacina was at least as effective in controlling blue mould as the
best fungicide, metalaxyl. Plants were protected, leading to a
yield increase of 10-25% in cured tobacco.
[0015] Induced resistance against bacteria and viruses appears to
be expressed as suppression of disease symptoms or pathogen
multiplication or both (Caruso, F. L., et al., "Induced Resistance
of Cucumber to Anthracnose and Angular Leaf Spot by Pseudomonas
Lachrymans and Colletotrichum Lagenarium," Physiological Plant
Pathology 14:191-201 (1979); Doss, M., et al., "Systemic Acquired
Resistance of Cucumber to Pseudomonas Lachrymans as Expressed in
Suppression of Symptoms, but not in Multiplication of Bacteria,"
Acta Phytopathologia Academiae Scientiarum Hungaricae 16:(3-4),
269-72 (1981); Jenns, A. E., et al., "Non-Specific Resistance to
Pathogens Induced Systemically by Local Infection of Cucumber with
Tobacco Necrosis Virus, Colletotrichum Lagenarium or Pseudomonas
Lachrymans," Phytopathologia Mediterranea 18:129-34 (1979), which
are hereby incorporated by reference).
[0016] As described above, research concerning systemic acquired
resistance involves infecting plants with infectious pathogens.
Although studies in this area are useful in understanding how
systemic acquired resistance works, eliciting such resistance with
infectious agents is not commercially useful, because such
plant-pathogen contact can weaken or kill plants. The present
invention is directed to overcoming this deficiency.
SUMMARY OF THE INVENTION
[0017] The present invention relates to a method of producing plant
seeds which impart pathogen resistance to plants grown from the
seeds. This method involves applying a hypersensitive response
elicitor polypeptide or protein in a non-infectious form to plant
seeds under conditions where the polypeptide or protein contacts
cells of the plant seeds.
[0018] As an alternative to applying a hypersensitive response
elicitor polypeptide or protein to plant seeds in order to impart
pathogen resistance to plants grown from the seeds, transgenic
seeds can be utilized. This involves providing a transgenic plant
seed transformed with a DNA molecule encoding a hypersensitive
response elicitor polypeptide or protein and planting that seed in
soil. A plant is then propagated from the planted seed under
conditions effective to impart pathogen resistance to the
plant.
[0019] Another aspect of the present invention relates to a
pathogen-resistance imparting plant seed to which a non-infectious
hypersensitive response elicitor polypeptide or protein has been
applied.
[0020] The present invention has the potential to: treat plant
diseases which were previously untreatable; treat diseases
systemically that one would not want to treat separately due to
cost; and avoid the use of agents that have an unpredictable effect
on the environment and even the plants. The present invention can
impart resistance without using agents which are harmful to the
environment or pathogenic to the plant seeds being treated or to
plants situated near the location that treated seeds are planted.
Since the present invention involves use of a natural product that
is fully and rapidly biodegradable, the environment would not be
contaminated.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The present invention relates to a method of producing plant
seeds which impart pathogen resistance to plants grown from the
seeds. This method involves applying a hypersensitive response
elicitor polypeptide or protein in a non-infectious form to a plant
seed under conditions effective to impart disease resistance to a
plant grown from the seed.
[0022] As an alternative to applying a hypersensitive response
elicitor polypeptide or protein to plant seeds in order to impart
pathogen resistance to plants grown from the seeds, transgenic
seeds can be utilized. This involves providing a transgenic plant
seed transformed with a DNA molecule encoding a hypersensitive
response elicitor polypeptide or protein and planting that seed in
soil. A plant is then propagated from the planted seed under
conditions effective to impart pathogen resistance to the
plant.
[0023] Another aspect of the present invention relates to a
pathogen-resistance imparting plant seed to which a non-infectious
hypersensitive response elicitor polypeptide or protein has been
applied.
[0024] The hypersensitive response elicitor polypeptide or protein
utilized in the present invention can correspond to hypersensitive
response elicitor polypeptides or proteins derived from a wide
variety of fungal and bacterial pathogens. Such polypeptides or
proteins are able to elicit local necrosis in plant tissue
contacted by the elicitor.
[0025] Examples of suitable bacterial sources of polypeptide or
protein elicitors include Erwinia, Pseudomonas, and Xanthamonas
species (e.g., the following bacteria: Erwinia amylovora, Erwinia
chrysanthemi, Erwinia stewartii, Erwinia carotovora, Pseudomonas
syringae, Pseudomonas solancearum, Xanthomonas campestris, or
mixtures thereof).
[0026] An example of a fungal source of a hypersensitive response
elicitor protein or polypeptide is Phytophthora. Suitable species
of such fungal pathogens include Phytophthora parasitica,
Phytophthora cryptogea, Phytophthora cinnamomi, Phytophthora
capsici, Phytophthora megasperma, and Phytophthora
citrophthora.
[0027] The embodiment of the present invention where the
hypersensitive response elicitor polypeptide or protein is applied
to the plant seed can be carried out in a number of ways,
including: 1) application of an isolated elicitor polypeptide or
protein; 2) application of bacteria which do not cause disease and
are transformed with genes encoding a hypersensitive response
elicitor polypeptide or protein; and 3) application of bacteria
which cause disease in some plant species (but not in those to
which they are applied) and naturally contain a gene encoding the
hypersensitive response elicitor polypeptide or protein. In
addition, seeds in accordance with the present invention can be
recovered from plants which have been treated with a hypersensitive
response elicitor protein or polypeptide in accordance with the
present invention.
[0028] In one embodiment of the present invention, the
hypersensitive response elicitor polypeptides or proteins to be
applied can be isolated from their corresponding organisms and
applied to plants. Such isolation procedures are well known, as
described in Arlat, M., F. Van Gijsegem, J. C. Huet, J. C.
Pemollet, and C. A. Boucher, "PopA1, a Protein which Induces a
Hypersensitive-like Response in Specific Petunia Genotypes is
Secreted via the Hrp Pathway of Pseudomonas solanacearum," EMBO J.
13:543-553 (1994); He, S. Y., H. C. Huang, and A. Collmer,
"Pseudomonas syringae pv. syringae Harpin.sub.Pss: a Protein that
is Secreted via the Hrp Pathway and Elicits the Hypersensitive
Response in Plants," Cell 73:1255-1266 (1993); and Wei, Z.-M., R.
J. Laby, C. H. Zumoff, D. W. Bauer, S.-Y. He, A. Collmer, and S. V.
Beer, "Harpin Elicitor of the Hypersensitive Response Produced by
the Plant Pathogen Erwinia amylovora, Science 257:85-88 (1992),
which are hereby incorporated by reference. See also pending U.S.
patent application Ser. Nos. 08/200,024 and 08/062,024, which are
hereby incorporated by reference. Preferably, however, the isolated
hypersensitive response elicitor polypeptides or proteins of the
present invention are produced recombinantly and purified as
described below.
[0029] In other embodiments of the present invention, the
hypersensitive response elicitor polypeptide or protein of the
present invention can be applied to plant seeds by applying
bacteria containing genes encoding the hypersensitive response
elicitor polypeptide or protein. Such bacteria must be capable of
secreting or exporting the polypeptide or protein so that the
elicitor can contact plant seed cells. In these embodiments, the
hypersensitive response elicitor polypeptide or protein is produced
by the bacteria after application to the seeds or just prior to
introduction of the bacteria to the seeds.
[0030] In one embodiment of the bacterial application mode of the
present invention, the bacteria to be applied do not cause the
disease and have been transformed (e.g., recombinantly) with genes
encoding a hypersensitive response elicitor polypeptide or protein.
For example, E. coli, which do not elicit a hypersensitive response
in plants, can be transformed with genes encoding a hypersensitive
response elicitor polypeptide and other related proteins required
for production and secretion of the elicitor which is then applied
to plant seeds. Expression of this polypeptide or protein can then
be caused to occur. Bacterial species (other than E. coli) can also
be used in this embodiment of the present invention.
[0031] In another embodiment of the bacterial application mode of
the present invention, the bacteria do cause disease and naturally
contain a gene encoding a hypersensitive response elicitor
polypeptide or protein. Examples of such bacteria are noted above.
However, in this embodiment these bacteria are applied to plant
seeds for plants which are not susceptible to the disease carried
by the bacteria. For example, Erwinia amylovora causes disease in
apple or pear but not in tomato. However, such bacteria will elicit
a hypersensitive response in tomato. Accordingly, in accordance
with this embodiment of the present invention, Erwinia amylovora
can be applied to tomato seeds to impart pathogen resistance
without causing disease in plants of that species.
[0032] The hypersensitive response elicitor polypeptide or protein
from Erwinia chrysanthemi has an amino acid sequence corresponding
to SEQ. ID. No. 1 as follows:
TABLE-US-00001 Met Gln Ile Thr Ile Lys Ala His Ile Gly Gly Asp Leu
Gly Val Ser 1 5 10 15 Gly Leu Gly Ala Gln Gly Leu Lys Gly Leu Asn
Ser Ala Ala Ser Ser 20 25 30 Leu Gly Ser Ser Val Asp Lys Leu Ser
Ser Thr Ile Asp Lys Leu Thr 35 40 45 Ser Ala Leu Thr Ser Met Met
Phe Gly Gly Ala Leu Ala Gln Gly Leu 50 55 60 Gly Ala Ser Ser Lys
Gly Leu Gly Met Ser Asn Gln Leu Gly Gln Ser 65 70 75 80 Phe Gly Asn
Gly Ala Gln Gly Ala Ser Asn Leu Leu Ser Val Pro Lys 85 90 95 Ser
Gly Gly Asp Ala Leu Ser Lys Met Phe Asp Lys Ala Leu Asp Asp 100 105
110 Leu Leu Gly His Asp Thr Val Thr Lys Leu Thr Asn Gln Ser Asn Gln
115 120 125 Leu Ala Asn Ser Met Leu Asn Ala Ser Gln Met Thr Gln Gly
Asn Met 130 135 140 Asn Ala Phe Gly Ser Gly Val Asn Asn Ala Leu Ser
Ser Ile Leu Gly 145 150 155 160 Asn Gly Leu Gly Gln Ser Met Ser Gly
Phe Ser Gln Pro Ser Leu Gly 165 170 175 Ala Gly Gly Leu Gln Gly Leu
Ser Gly Ala Gly Ala Phe Asn Gln Leu 180 185 190 Gly Asn Ala Ile Gly
Met Gly Val Gly Gln Asn Ala Ala Leu Ser Ala 195 200 205 Leu Ser Asn
Val Ser Thr His Val Asp Gly Asn Asn Arg His Phe Val 210 215 220 Asp
Lys Glu Asp Arg Gly Met Ala Lys Glu Ile Gly Gln Phe Met Asp 225 230
235 240 Gln Tyr Pro Glu Ile Phe Gly Lys Pro Glu Tyr Gln Lys Asp Gly
Trp 245 250 255 Ser Ser Pro Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala
Leu Ser Lys 260 265 270 Pro Asp Asp Asp Gly Met Thr Gly Ala Ser Met
Asp Lys Phe Arg Gln 275 280 285 Ala Met Gly Met Ile Lys Ser Ala Val
Ala Gly Asp Thr Gly Asn Thr 290 295 300 Asn Leu Asn Leu Arg Gly Ala
Gly Gly Ala Ser Leu Gly Ile Asp Ala 305 310 315 320 Ala Val Val Gly
Asp Lys Ile Ala Asn Met Ser Leu Gly Lys Leu Ala 325 330 335 Asn
Ala
This hypersensitive response elicitor polypeptide or protein has a
molecular weight of 34 kDa, is heat stable, has a glycine content
of greater than 16%, and contains substantially no cysteine. The
Erwinia chrysanthemi hypersensitive response elicitor polypeptide
or protein is encoded by a DNA molecule having a nucleotide
sequence corresponding to SEQ. ID. No. 2 as follows:
TABLE-US-00002 CGATTTTACC CGGGTGAACG TGCTATGACC GACAGCATCA
CGGTATTCGA CACCGTTACG 60 GCGTTTATGG CCGCGATGAA CCGGCATCAG
GCGGCGCGCT GGTCGCCGCA ATCCGGCGTC 120 GATCTGGTAT TTCAGTTTGG
GGACACCGGG CGTGAACTCA TGATGCAGAT TCAGCCGGGG 180 CAGCAATATC
CCGGCATGTT GCGCACGCTG CTCGCTCGTC GTTATCAGCA GGCGGCAGAG 240
TGCGATGGCT GCCATCTGTG CCTGAACGGC AGCGATGTAT TGATCCTCTG GTGGCCGCTG
300 CCGTCGGATC CCGGCAGTTA TCCGCAGGTG ATCGAACGTT TGTTTGAACT
GGCGGGAATG 360 ACGTTGCCGT CGCTATCCAT AGCACCGACG GCGCGTCCGC
AGACAGGGAA CGGACGCGCC 420 CGATCATTAA GATAAAGGCG GCTTTTTTTA
TTGCAAAACG GTAACGGTGA GGAACCGTTT 480 CACCGTCGGC GTCACTCAGT
AACAAGTATC CATCATGATG CCTACATCGG GATCGGCGTG 540 GGCATCCGTT
GCAGATACTT TTGCGAACAC CTGACATGAA TGAGGAAACG AAATTATGCA 600
AATTACGATC AAAGCGCACA TCGGCGGTGA TTTGGGCGTC TCCGGTCTGG GGCTGGGTGC
660 TCAGGGACTG AAAGGACTGA ATTCCGCGGC TTCATCGCTG GGTTCCAGCG
TGGATAAACT 720 GAGCAGCACC ATCGATAAGT TGACCTCCGC GCTGACTTCG
ATGATGTTTG GCGGCGCGCT 780 GGCGCAGGGG CTGGGCGCCA GCTCGAAGGG
GCTGGGGATG AGCAATCAAC TGGGCCAGTC 840 TTTCGGCAAT GGCGCGCAGG
GTGCGAGCAA CCTGCTATCC GTACCGAAAT CCGGCGGCGA 900 TGCGTTGTCA
AAAATGTTTG ATAAAGCGCT GGACGATCTG CTGGGTCATG ACACCGTGAC 960
CAAGCTGACT AACCAGAGCA ACCAACTGGC TAATTCAATG CTGAACGCCA GCCAGATGAC
1020 CCAGGGTAAT ATGAATGCGT TCGGCAGCGG TGTGAACAAC GCACTGTCGT
CCATTCTCGG 1080 CAACGGTCTC GGCCAGTCGA TGAGTGGCTT CTCTCAGCCT
TCTCTGGGGG CAGGCGGCTT 1140 GCAGGGCCTG AGCGGCGCGG GTGCATTCAA
CCAGTTGGGT AATGCCATCG GCATGGGCGT 1200 GGGGCAGAAT GCTGCGCTGA
GTGCGTTGAG TAACGTCAGC ACCCACGTAG ACGGTAACAA 1260 CCGCCACTTT
GTAGATAAAG AAGATCGCGG CATGGCGAAA GAGATCGGCC AGTTTATGGA 1320
TCAGTATCCG GAAATATTCG GTAAACCGGA ATACCAGAAA GATGGCTGGA GTTCGCCGAA
1380 GACGGACGAC AAATCCTGGG CTAAAGCGCT GAGTAAACCG GATGATGACG
GTATGACCGG 1440 CGCCAGCATG GACAAATTCC GTCAGGCGAT GGGTATGATC
AAAAGCGCGG TGGCGGGTGA 1500 TACCGGCAAT ACCAACCTGA ACCTGCGTGG
CGCGGGCGGT GCATCGCTGG GTATCGATGC 1560 GGCTGGGTGC GGCGATAAAA
TAGCCAACAT GTCGCTGGGT AAGCTGGCCA ACGCCTGATA 1620 ATCTGTGCTG
GCCTGATAAA GCGGAAACGA AAAAAGAGAC GGGGAAGCCT GTCTCTTTTC 1680
TTATTATGCG GTTTATGCGG TTACCTGGAC CGGTTAATCA TCGTCATCGA TCTGGTACAA
1740 ACGCACATTT TCCCGTTCAT TCGCGTCGTT ACGCGCCACA ATCGCGATGG
CATCTTCCTC 1800 GTCGCTCAGA TTGCGCGGCT GATGGGGAAC GCCGGGTGGA
ATATAGAGAA ACTCGCCGGC 1860 CAGATGGAGA CACGTCTGCG ATAAATCTGT
GCCGTAACGT GTTTCTATCC GCCCCTTTAG 1920 CAGATAGATT GCGGTTTCGT
AATCAACATG GTAATGCGGT TCCGCCTGTG CGCCGGCCGG 1980 GATCACCACA
ATATTCATAG AAAGCTGTCT TGCACCTACC GTATCGCGGG AGATACCGAC 2040
AAAATAGGGC AGTTTTTGCG TGGTATCCGT GGGGTGTTCC GGCCTGACAA TCTTGAGTTG
2100 GTTCGTCATC ATCTTTCTCC ATCTGGGCGA CCTGATCGGT T 2141
[0033] The hypersensitive response elicitor polypeptide or protein
derived from Erwinia amylovora has an amino acid sequence
corresponding to SEQ. ID. No. 3 as follows:
TABLE-US-00003 Met Ser Leu Asn Thr Ser Gly Leu Gly Ala Ser Thr Met
Gln Ile Ser 1 5 10 15 Ile Gly Gly Ala Gly Gly Asn Asn Gly Leu Leu
Gly Thr Ser Arg Gln 20 25 30 Asn Ala Gly Leu Gly Gly Asn Ser Ala
Leu Gly Leu Gly Gly Gly Asn 35 40 45 Gln Asn Asp Thr Val Asn Gln
Leu Ala Gly Leu Leu Thr Gly Met Met 50 55 60 Met Met Met Ser Met
Met Gly Gly Gly Gly Leu Met Gly Gly Gly Leu 65 70 75 80 Gly Gly Gly
Leu Gly Asn Gly Leu Gly Gly Ser Gly Gly Leu Gly Glu 85 90 95 Gly
Leu Ser Asn Ala Leu Asn Asp Met Leu Gly Gly Ser Leu Asn Thr 100 105
110 Leu Gly Ser Lys Gly Gly Asn Asn Thr Thr Ser Thr Thr Asn Ser Pro
115 120 125 Leu Asp Gln Ala Leu Gly Ile Asn Ser Thr Ser Gln Asn Asp
Asp Ser 130 135 140 Thr Ser Gly Thr Asp Ser Thr Ser Asp Ser Ser Asp
Pro Met Gln Gln 145 150 155 160 Leu Leu Lys Met Phe Ser Glu Ile Met
Gln Ser Leu Phe Gly Asp Gly 165 170 175 Gln Asp Gly Thr Gln Gly Ser
Ser Ser Gly Gly Lys Gln Pro Thr Glu 180 185 190 Gly Glu Gln Asn Ala
Tyr Lys Lys Gly Val Thr Asp Ala Leu Ser Gly 195 200 205 Leu Met Gly
Asn Gly Leu Ser Gln Leu Leu Gly Asn Gly Gly Leu Gly 210 215 220 Gly
Gly Gln Gly Gly Asn Ala Gly Thr Gly Leu Asp Gly Ser Ser Leu 225 230
235 240 Gly Gly Lys Gly Leu Gln Asn Leu Ser Gly Pro Val Asp Tyr Gln
Gln 245 250 255 Leu Gly Asn Ala Val Gly Thr Gly Ile Gly Met Lys Ala
Gly Ile Gln 260 265 270 Ala Leu Asn Asp Ile Gly Thr His Arg His Ser
Ser Thr Arg Ser Phe 275 280 285 Val Asn Lys Gly Asp Arg Ala Met Ala
Lys Glu Ile Gly Gln Phe Met 290 295 300 Asp Gln Tyr Pro Glu Val Phe
Gly Lys Pro Gln Tyr Gln Lys Gly Pro 305 310 315 320 Gly Gln Glu Val
Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser 325 330 335 Lys Pro
Asp Asp Asp Gly Met Thr Pro Ala Ser Met Glu Gln Phe Asn 340 345 350
Lys Ala Lys Gly Met Ile Lys Arg Pro Met Ala Gly Asp Thr Gly Asn 355
360 365 Gly Asn Leu Gln Ala Arg Gly Ala Gly Gly Ser Ser Leu Gly Ile
Asp 370 375 380 Ala Met Met Ala Gly Asp Ala Ile Asn Asn Met Ala Leu
Gly Lys Leu 385 390 395 400 Gly Ala Ala
This hypersensitive response elicitor polypeptide or protein has a
molecular weight of about 39 kDa, it has a pI of approximately 4.3,
and is heat stable at 100.degree. C. for at least 10 minutes. This
hypersensitive response elicitor polypeptide or protein has
substantially no cysteine. The hypersensitive response elicitor
polypeptide or protein derived from Erwinia amylovora is more fully
described in Wei, Z.-M., R. J. Laby, C. H. Zumoff, D. W. Bauer,
S.-Y. He, A. Collmer, and S. V. Beer, "Harpin, Elicitor of the
Hypersensitive Response Produced by the Plant Pathogen Erwinia
amylovora," Science 257:85-88 (1992), which is hereby incorporated
by reference. The DNA molecule encoding this polypeptide or protein
has a nucleotide sequence corresponding to SEQ. ID. No. 4 as
follows:
TABLE-US-00004 AAGCTTCGGC ATGGCACGTT TGACCGTTGG GTCGGCAGGG
TACGTTTGAA TTATTCATAA 60 GAGGAATACG TTATGAGTCT GAATACAAGT
GGGCTGGGAG CGTCAACGAT GCAAATTTCT 120 ATCGGCGGTG CGGGCGGAAA
TAACGGGTTG CTGGGTACCA GTCGCCAGAA TGCTGGGTTG 180 GGTGGCAATT
CTGCACTGGG GCTGGGCGGC GGTAATCAAA ATGATACCGT CAATCAGCTG 240
GCTGGCTTAC TCACCGGCAT GATGATGATG ATGAGCATGA TGGGCGGTGG TGGGCTGATG
300 GGCGGTGGCT TAGGCGGTGG CTTAGGTAAT GGCTTGGGTG GCTCAGGTGG
CCTGGGCGAA 360 GGACTGTCGA ACGCGCTGAA CGATATGTTA GGCGGTTCGC
TGAACACGCT GGGCTCGAAA 420 GGCGGCAACA ATACCACTTC AACAACAAAT
TCCCCGCTGG ACCAGGCGCT GGGTATTAAC 480 TCAACGTCCC AAAACGACGA
TTCCACCTCC GGCACAGATT CCACCTCAGA CTCCAGCGAC 540 CCGATGCAGC
AGCTGCTGAA GATGTTCAGC GAGATAATGC AAAGCCTGTT TGGTGATGGG 600
CAAGATGGCA CCCAGGGCAG TTCCTCTGGG GGCAAGCAGC CGACCGAAGG CGAGCAGAAC
660 GCCTATAAAA AAGGAGTCAC TGATGCGCTG TCGGGCCTGA TGGGTAATGG
TCTGAGCCAG 720 CTCCTTGGCA ACGGGGGACT GGGAGGTGGT CAGGGCGGTA
ATGCTGGCAC GGGTCTTGAC 780 GGTTCGTCGC TGGGCGGCAA AGGGCTGCAA
AACCTGAGCG GGCCGGTGGA CTACCAGCAG 840 TTAGGTAACG CCGTGGGTAC
CGGTATCGGT ATGAAAGCGG GCATTCAGGC GCTGAATGAT 900 ATCGGTACGC
ACAGGCACAG TTCAACCCGT TCTTTCGTCA ATAAAGGCGA TCGGGCGATG 960
GCGAAGGAAA TCGGTCAGTT CATGGACCAG TATCCTGAGG TGTTTGGCAA GCCGCAGTAC
1020 CAGAAAGGCC CGGGTCAGGA GGTGAAAACC GATGACAAAT CATGGGCAAA
AGCACTGAGC 1080 AAGCCAGATG ACGACGGAAT GACACCAGCC AGTATGGAGC
AGTTCAACAA AGCCAAGGGC 1140 ATGATCAAAA GGCCCATGGC GGGTGATACC
GGCAACGGCA ACCTGCAGGC ACGCGGTGCC 1200 GGTGGTTCTT CGCTGGGTAT
TGATGCCATG ATGGCCGGTG ATGCCATTAA CAATATGGCA 1260 CTTGGCAAGC
TGGGCGCGGC TTAAGCTT 1288
[0034] The hypersensitive response elicitor polypeptide or protein
derived from Pseudomonas syringae has an amino acid sequence
corresponding to SEQ. ID. No. 5 as follows:
TABLE-US-00005 Met Gln Ser Leu Ser Leu Asn Ser Ser Ser Leu Gln Thr
Pro Ala Met 1 5 10 15 Ala Leu Val Leu Val Arg Pro Glu Ala Glu Thr
Thr Gly Ser Thr Ser 20 25 30 Ser Lys Ala Leu Gln Glu Val Val Val
Lys Leu Ala Glu Glu Leu Met 35 40 45 Arg Asn Gly Gln Leu Asp Asp
Ser Ser Pro Leu Gly Lys Leu Leu Ala 50 55 60 Lys Ser Met Ala Ala
Asp Gly Lys Ala Gly Gly Gly Ile Glu Asp Val 65 70 75 80 Ile Ala Ala
Leu Asp Lys Leu Ile His Glu Lys Leu Gly Asp Asn Phe 85 90 95 Gly
Ala Ser Ala Asp Ser Ala Ser Gly Thr Gly Gln Gln Asp Leu Met 100 105
110 Thr Gln Val Leu Asn Gly Leu Ala Lys Ser Met Leu Asp Asp Leu Leu
115 120 125 Thr Lys Gln Asp Gly Gly Thr Ser Phe Ser Glu Asp Asp Met
Pro Met 130 135 140 Leu Asn Lys Ile Ala Gln Phe Met Asp Asp Asn Pro
Ala Gln Phe Pro 145 150 155 160 Lys Pro Asp Ser Gly Ser Trp Val Asn
Glu Leu Lys Glu Asp Asn Phe 165 170 175 Leu Asp Gly Asp Glu Thr Ala
Ala Phe Arg Ser Ala Leu Asp Ile Ile 180 185 190 Gly Gln Gln Leu Gly
Asn Gln Gln Ser Asp Ala Gly Ser Leu Ala Gly 195 200 205 Thr Gly Gly
Gly Leu Gly Thr Pro Ser Ser Phe Ser Asn Asn Ser Ser 210 215 220 Val
Met Gly Asp Pro Leu Ile Asp Ala Asn Thr Gly Pro Gly Asp Ser 225 230
235 240 Gly Asn Thr Arg Gly Glu Ala Gly Gln Leu Ile Gly Glu Leu Ile
Asp 245 250 255 Arg Gly Leu Gln Ser Val Leu Ala Gly Gly Gly Leu Gly
Thr Pro Val 260 265 270 Asn Thr Pro Gln Thr Gly Thr Ser Ala Asn Gly
Gly Gln Ser Ala Gln 275 280 285 Asp Leu Asp Gln Leu Leu Gly Gly Leu
Leu Leu Lys Gly Leu Glu Ala 290 295 300 Thr Leu Lys Asp Ala Gly Gln
Thr Gly Thr Asp Val Gln Ser Ser Ala 305 310 315 320 Ala Gln Ile Ala
Thr Leu Leu Val Ser Thr Leu Leu Gln Gly Thr Arg 325 330 335 Asn Gln
Ala Ala Ala 340
This hypersensitive response elicitor polypeptide or protein has a
molecular weight of 34-35 kDa. It is rich in glycine (about 13.5%)
and lacks cysteine and tyrosine. Further information about the
hypersensitive response elicitor derived from Pseudomonas syringae
is found in He, S. Y., H. C. Huang, and A. Collmer, "Pseudomonas
syringae pv. syringae Harpin.sub.Pss: a Protein that is Secreted
via the Hrp Pathway and Elicits the Hypersensitive Response in
Plants," Cell 73:1255-1266 (1993), which is hereby incorporated by
reference. The DNA molecule encoding the hypersensitive response
elicitor from Pseudomonas syringae has a nucleotide sequence
corresponding to SEQ. ID. No. 6 as follows:
TABLE-US-00006 ATGCAGAGTC TCAGTCTTAA CAGCAGCTCG CTGCAAACCC
CGGCAATGGC CCTTGTCCTG 60 GTACGTCCTG AAGCCGAGAC GACTGGCAGT
ACGTCGAGCA AGGCGCTTCA GGAAGTTGTC 120 GTGAAGCTGG CCGAGGAACT
GATGCGCAAT GGTCAACTCG ACGACAGCTC GCCATTGGGA 180 AAACTGTTGG
CCAAGTCGAT GGCCGCAGAT GGCAAGGCGG GCGGCGGTAT TGAGGATGTC 240
ATCGCTGCGC TGGACAAGCT GATCCATGAA AAGCTCGGTG ACAACTTCGG CGCGTCTGCG
300 GACAGCGCCT CGGGTACCGG ACAGCAGGAC CTGATGACTC AGGTGCTCAA
TGGCCTGGCC 360 AAGTCGATGC TCGATGATCT TCTGACCAAG CAGGATGGCG
GGACAAGCTT CTCCGAAGAC 420 GATATGCCGA TGCTGAACAA GATCGCGCAG
TTCATGGATG ACAATCCCGC ACAGTTTCCC 480 AAGCCGGACT CGGGCTCCTG
GGTGAACGAA CTCAAGGAAG ACAACTTCCT TGATGGCGAC 540 GAAACGGCTG
CGTTCCGTTC GGCACTCGAC ATCATTGGCC AGCAACTGGG TAATCAGCAG 600
AGTGACGCTG GCAGTCTGGC AGGGACGGGT GGAGGTCTGG GCACTCCGAG CAGTTTTTCC
660 AACAACTCGT CCGTGATGGG TGATCCGCTG ATCGACGCCA ATACCGGTCC
CGGTGACAGC 720 GGCAATACCC GTGGTGAAGC GGGGCAACTG ATCGGCGAGC
TTATCGACCG TGGCCTGCAA 780 TCGGTATTGG CCGGTGGTGG ACTGGGCACA
CCCGTAAACA CCCCGCAGAC CGGTACGTCG 840 GCGAATGGCG GACAGTCCGC
TCAGGATCTT GATCAGTTGC TGGGCGGCTT GCTGCTCAAG 900 GGCCTGGAGG
CAACGCTCAA GGATGCCGGG CAAACAGGCA CCGACGTGCA GTCGAGCGCT 960
GCGCAAATCG CCACCTTGCT GGTCAGTACG CTGCTGCAAG GCACCCGCAA TCAGGCTGCA
1020 GCCTGA 1026
[0035] The hypersensitive response elicitor polypeptide or protein
derived from Pseudomonas solanacearum has an amino acid sequence
corresponding to SEQ. ID. No. 7 as follows:
TABLE-US-00007 Met Ser Val Gly Asn Ile Gln Ser Pro Ser Asn Leu Pro
Gly Leu Gln 1 5 10 15 Asn Leu Asn Leu Asn Thr Asn Thr Asn Ser Gln
Gln Ser Gly Gln Ser 20 25 30 Val Gln Asp Leu Ile Lys Gln Val Glu
Lys Asp Ile Leu Asn Ile Ile 35 40 45 Ala Ala Leu Val Gln Lys Ala
Ala Gln Ser Ala Gly Gly Asn Thr Gly 50 55 60 Asn Thr Gly Asn Ala
Pro Ala Lys Asp Gly Asn Ala Asn Ala Gly Ala 65 70 75 80 Asn Asp Pro
Ser Lys Asn Asp Pro Ser Lys Ser Gln Ala Pro Gln Ser 85 90 95 Ala
Asn Lys Thr Gly Asn Val Asp Asp Ala Asn Asn Gln Asp Pro Met 100 105
110 Gln Ala Leu Met Gln Leu Leu Glu Asp Leu Val Lys Leu Leu Lys Ala
115 120 125 Ala Leu His Met Gln Gln Pro Gly Gly Asn Asp Lys Gly Asn
Gly Val 130 135 140 Gly Gly Ala Asn Gly Ala Lys Gly Ala Gly Gly Gln
Gly Gly Leu Ala 145 150 155 160 Glu Ala Leu Gln Glu Ile Glu Gln Ile
Leu Ala Gln Leu Gly Gly Gly 165 170 175 Gly Ala Gly Ala Gly Gly Ala
Gly Gly Gly Val Gly Gly Ala Gly Gly 180 185 190 Ala Asp Gly Gly Ser
Gly Ala Gly Gly Ala Gly Gly Ala Asn Gly Ala 195 200 205 Asp Gly Gly
Asn Gly Val Asn Gly Asn Gln Ala Asn Gly Pro Gln Asn 210 215 220 Ala
Gly Asp Val Asn Gly Ala Asn Gly Ala Asp Asp Gly Ser Glu Asp 225 230
235 240 Gln Gly Gly Leu Thr Gly Val Leu Gln Lys Leu Met Lys Ile Leu
Asn 245 250 255 Ala Leu Val Gln Met Met Gln Gln Gly Gly Leu Gly Gly
Gly Asn Gln 260 265 270 Ala Gln Gly Gly Ser Lys Gly Ala Gly Asn Ala
Ser Pro Ala Ser Gly 275 280 285 Ala Asn Pro Gly Ala Asn Gln Pro Gly
Ser Ala Asp Asp Gln Ser Ser 290 295 300 Gly Gln Asn Asn Leu Gln Ser
Gln Ile Met Asp Val Val Lys Glu Val 305 310 315 320 Val Gln Ile Leu
Gln Gln Met Leu Ala Ala Gln Asn Gly Gly Ser Gln 325 330 335 Gln Ser
Thr Ser Thr Gln Pro Met 340
It is encoded by a DNA molecule having a nucleotide sequence
corresponding SEQ. ID. No. 8 as follows:
TABLE-US-00008 ATGTCAGTCG GAAACATCCA GAGCCCGTCG AACCTCCCGG
GTCTGCAGAA CCTGAACCTC 60 AACACCAACA CCAACAGCCA GCAATCGGGC
CAGTCCGTGC AAGACCTGAT CAAGCAGGTC 120 GAGAAGGACA TCCTCAACAT
CATCGCAGCC CTCGTGCAGA AGGCCGCACA GTCGGCGGGC 180 GGCAACACCG
GTAACACCGG CAACGCGCCG GCGAAGGACG GCAATGCCAA CGCGGGCGCC 240
AACGACCCGA GCAAGAACGA CCCGAGCAAG AGCCAGGCTC CGCAGTCGGC CAACAAGACC
300 GGCAACGTCG ACGACGCCAA CAACCAGGAT CCGATGCAAG CGCTGATGCA
GCTGCTGGAA 360 GACCTGGTGA AGCTGCTGAA GGCGGCCCTG CACATGCAGC
AGCCCGGCGG CAATGACAAG 420 GGCAACGGCG TGGGCGGTGC CAACGGCGCC
AAGGGTGCCG GCGGCCAGGG CGGCCTGGCC 480 GAAGCGCTGC AGGAGATCGA
GCAGATCCTC GCCCAGCTCG GCGGCGGCGG TGCTGGCGCC 540 GGCGGCGCGG
GTGGCGGTGT CGGCGGTGCT GGTGGCGCGG ATGGCGGCTC CGGTGCGGGT 600
GGCGCAGGCG GTGCGAACGG CGCCGACGGC GGCAATGGCG TGAACGGCAA CCAGGCGAAC
660 GGCCCGCAGA ACGCAGGCGA TGTCAACGGT GCCAACGGCG CGGATGACGG
CAGCGAAGAC 720 CAGGGCGGCC TCACCGGCGT GCTGCAAAAG CTGATGAAGA
TCCTGAACGC GCTGGTGCAG 780 ATGATGCAGC AAGGCGGCCT CGGCGGCGGC
AACCAGGCGC AGGGCGGCTC GAAGGGTGCC 840 GGCAACGCCT CGCCGGCTTC
CGGCGCGAAC CCGGGCGCGA ACCAGCCCGG TTCGGCGGAT 900 GATCAATCGT
CCGGCCAGAA CAATCTGCAA TCCCAGATCA TGGATGTGGT GAAGGAGGTC 960
GTCCAGATCC TGCAGCAGAT GCTGGCGGCG CAGAACGGCG GCAGCCAGCA GTCCACCTCG
1020 ACGCAGCCGA TGTAA 1035
Further information regarding the hypersensitive response elicitor
polypeptide or protein derived from Pseudomonas solanacearum is set
forth in Arlat, M., F. Van Gijsegem, J. C. Huet, J. C. Pemollet,
and C. A. Boucher, "PopA1, a Protein which Induces a
Hypersensitive-like Response in Specific Petunia Genotypes, is
Secreted via the Hrp Pathway of Pseudomonas solanacearum," EMBO J.
13:543-533 (1994), which is hereby incorporated by reference.
[0036] The hypersensitive response elicitor polypeptide or protein
from Xanthomonas campestris pv. glycines has an amino acid sequence
corresponding to SEQ. ID. No. 9 as follows:
TABLE-US-00009 Thr Leu Ile Glu Leu Met Ile Val Val Ala Ile Ile Ala
Ile Leu Ala 1 5 10 15 Ala Ile Ala Leu Pro Ala Tyr Gln Asp Tyr 20
25
This sequence is an amino terminal sequence having 26 residues only
from the hypersensitive response elicitor polypeptide or protein of
Xanthomonas campestris pv. glycines. It matches with fimbrial
subunit proteins determined in other Xanthomonas campestris
pathovars.
[0037] The hypersensitive response elicitor polypeptide or protein
from Xanthomonas campestris pelargonii is heat stable, protease
sensitive, and has a molecular weight of 20 kDa. It includes an
amino acid sequence corresponding to SEQ. ID. No. 10 as
follows:
TABLE-US-00010 Ser Ser Gln Gln Ser Pro Ser Ala Gly Ser Glu Gln Gln
Leu Asp Gln 1 5 10 15 Leu Leu Ala Met 20
[0038] Isolation of Erwinia carotovora hypersensitive response
elicitor protein or polypeptide is described in Cai, et al., "The
RsmA.sup.- Mutants of Erwinia carotovora subsp. carotova Strain
Ecc71 Overexpress hrpN.sub.Ecc and Elicit a Hypersensitive
Reaction-Like Response in Tobacco Leaves," MPMI, 9(7):565-73
(1996), which is hereby incorporated by reference. The
hypersensitive response elicitor protein or polypeptide for Erwinia
stewartii is disclosed in Ahmad, et al., "Harpin is Not Necessary
for the Pathogenicity of Erwinia stewartii on Maize," 8th Int'l.
Cong. Molec. Plant-Microbe Interact, Jul. 14-19, 1996 and Ahmad, et
al., "Harpin is Not Necessary for the Pathogenicity of Erwinia
stewartii on Maize," Ann. Mtg. Am. Phytopath. Soc., Jul. 27-31,
1996, which are hereby incorporated by reference.
[0039] Hypersensitive response elicitor proteins or polypeptides
from Phytophora parasitica, Phytophora cryptogea, Phytophora
cinnamoni, Phytophora capsici, Phytophora megasperma, and
Phytophora citrophthora are described in Kamoun, et al.,
"Extracellular Protein Elicitors from Phytophora: Host-Specificity
and Induction of Resistance to Bacterial and Fungal
Phytopathogens," Molec. Plant-Microbe Interact., 6(1):15-25 (1993),
Ricci, et al., "Structure and Activity of Proteins from Pathogenic
Fungi Phytophora Eliciting Necrosis and Acquired Resistance in
Tobacco," Eur. J. Biochem., 183:555-63 (1989), Ricci, et al.,
"Differential Production of Parasiticein, an Elicitor of Necrosis
and Resistance in Tobacco by Isolates of Phytophora paraticica,"
Plant Path., 41:298-307 (1992), Baillieul, et al., "A New Elicitor
of the Hypersensitive Response in Tobacco: A Fungal Glycoprotein
Elicits Cell Death, Expression of Defense Genes, Production of
Salicylic Acid, and Induction of Systemic Acquired Resistance,"
Plant J., 8(4):551-60 (1995), and Bonnet, et al., "Acquired
Resistance Triggered by Elicitins in Tobacco and Other Plants,"
Eur. J. Plant Path., 102:181-92 (1996), which are hereby
incorporated by reference.
[0040] The above elicitors are exemplary. Other elicitors can be
identified by growing fungi or bacteria that elicit a
hypersensitive response under which genes encoding an elicitor are
expressed. Cell-free preparations from culture supernatants can be
tested for elicitor activity (i.e. local necrosis) by using them to
infiltrate appropriate plant tissues.
[0041] It is also possible to use fragments of the above
hypersensitive response elicitor polypeptides or proteins as well
as fragments of full length elicitors from other pathogens, in the
method of the present invention.
[0042] Suitable fragments can be produced by several means. In the
first, subclones of the gene encoding a known elicitor protein are
produced by conventional molecular genetic manipulation by
subcloning gene fragments. The subclones then are expressed in
vitro or in vivo in bacterial cells to yield a smaller protein or a
peptide that can be tested for elicitor activity according to the
procedure described below.
[0043] As an alternative, fragments of an elicitor protein can be
produced by digestion of a full-length elicitor protein with
proteolytic enzymes like chymotrypsin or Staphylococcus proteinase
A, or trypsin. Different proteolytic enzymes are likely to cleave
elicitor proteins at different sites based on the amino acid
sequence of the elicitor protein. Some of the fragments that result
from proteolysis may be active elicitors of resistance.
[0044] In another approach, based on knowledge of the primary
structure of the protein, fragments of the elicitor protein gene
may be synthesized by using the PCR technique together with
specific sets of primers chosen to represent particular portions of
the protein. These then would be cloned into an appropriate vector
for increased expression of a truncated peptide or protein.
[0045] An example of a suitable fragment is the popA1 fragment of
the hypersensitive response elicitor polypeptide or protein from
Pseudomonas solanacearum. See Arlat, M., F. Van Gijsegem, J. C.
Huet, J. C. Pemollet, and C. A. Boucher, "PopA1, a Protein Which
Induces a Hypersensitive-like Response in Specific Petunia
Genotypes is Secreted via the Hrp Pathway of Pseudomonas
solanacearum," EMBO J. 13:543-53 (1994), which is hereby
incorporated by reference. As to Erwinia amylovora, a suitable
fragment can be, for example, either or both the polypeptide
extending between and including amino acids 1 and 98 of SEQ. ID.
NO. 3 and the polypeptide extending between and including amino
acids 137 and 204 of SEQ. ID. No. 3.
[0046] Variants may be made by, for example, the deletion or
addition of amino acids that have minimal influence on the
properties, secondary structure and hydropathic nature of the
polypeptide. For example, a polypeptide may be conjugated to a
signal (or leader) sequence at the N-terminal end of the protein
which co-translationally or post-translationally directs transfer
of the protein. The polypeptide may also be conjugated to a linker
or other sequence for ease of synthesis, purification or
identification of the polypeptide.
[0047] The protein or polypeptide of the present invention is
preferably produced in purified form (preferably at least about
60%, more preferably 80%, pure) by conventional techniques.
Typically, the protein or polypeptide of the present invention is
produced but not secreted into the growth medium of recombinant E.
coli. Alternatively, the protein or polypeptide of the present
invention is secreted into the growth medium. In the case of
unsecreted protein, to isolate the protein, the E. coli host cell
carrying a recombinant plasmid is propagated, homogenized, and the
homogenate is centrifuged to remove bacterial debris. The
supernatant is then subjected to heat treatment and the
hypersensitive response elicitor protein is separated by
centrifugation. The supernatant fraction containing the polypeptide
or protein of the present invention is subjected to gel filtration
in an appropriately sized dextran or polyacrylamide column to
separate the proteins. If necessary, the protein fraction may be
further purified by ion exchange or HPLC.
[0048] Alternatively, the hypersensitive response elicitor protein
can be prepared by chemical synthesis using conventional
techniques.
[0049] The DNA molecule encoding the hypersensitive response
elicitor polypeptide or protein can be incorporated in cells using
conventional recombinant DNA technology. Generally, this involves
inserting the DNA molecule into an expression system to which the
DNA molecule is heterologous (i.e. not normally present). The
heterologous DNA molecule is inserted into the expression system or
vector in proper sense orientation and correct reading frame. The
vector contains the necessary elements for the transcription and
translation of the inserted protein-coding sequences.
[0050] U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby
incorporated by reference, describes the production of expression
systems in the form of recombinant plasmids using restriction
enzyme cleavage and ligation with DNA ligase. These recombinant
plasmids are then introduced by means of transformation and
replicated in unicellular cultures including procaryotic organisms
and eucaryotic cells grown in tissue culture.
[0051] Recombinant genes may also be introduced into viruses, such
as vaccina virus. Recombinant viruses can be generated by
transection of plasmids into cells infected with virus.
[0052] Suitable vectors include, but are not limited to, the
following viral vectors such as lambda vector system gt11, gt
WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325,
pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290,
pKC37, pKC101, SV 40, pBluescript II SK +/- or KS +/- (see
"Stratagene Cloning Systems" Catalog (1993) from Stratagene, La
Jolla, Calif., which is hereby incorporated by reference), pQE,
pIH821, pGEX, pET series (see F. W. Studier et. al., "Use of T7 RNA
Polymerase to Direct Expression of Cloned Genes," Gene Expression
Technology vol. 185 (1990), which is hereby incorporated by
reference), and any derivatives thereof. Recombinant molecules can
be introduced into cells via transformation, particularly
transduction, conjugation, mobilization, or electroporation. The
DNA sequences are cloned into the vector using standard cloning
procedures in the art, as described by Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs
Harbor, N.Y. (1989), which is hereby incorporated by reference.
[0053] A variety of host-vector systems may be utilized to express
the protein-encoding sequence(s). Primarily, the vector system must
be compatible with the host cell used. Host-vector systems include
but are not limited to the following: bacteria transformed with
bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such
as yeast containing yeast vectors; mammalian cell systems infected
with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell
systems infected with virus (e.g., baculovirus); and plant cells
infected by bacteria. The expression elements of these vectors vary
in their strength and specificities. Depending upon the host-vector
system utilized, any one of a number of suitable transcription and
translation elements can be used.
[0054] Different genetic signals and processing events control many
levels of gene expression (e.g., DNA transcription and messenger
RNA (mRNA) translation).
[0055] Transcription of DNA is dependent upon the presence of a
promotor which is a DNA sequence that directs the binding of RNA
polymerase and thereby promotes mRNA synthesis. The DNA sequences
of eucaryotic promotors differ from those of procaryotic promotors.
Furthermore, eucaryotic promotors and accompanying genetic signals
may not be recognized in or may not function in a procaryotic
system, and, further, procaryotic promotors are not recognized and
do not function in eucaryotic cells.
[0056] Similarly, translation of mRNA in procaryotes depends upon
the presence of the proper procaryotic signals which differ from
those of eucaryotes. Efficient translation of mRNA in procaryotes
requires a ribosome binding site called the Shine-Dalgarno ("SD")
sequence on the mRNA. This sequence is a short nucleotide sequence
of mRNA that is located before the start codon, usually AUG, which
encodes the amino-terminal methionine of the protein. The SD
sequences are complementary to the 3'-end of the 16S rRNA
(ribosomal RNA) and probably promote binding of mRNA to ribosomes
by duplexing with the rRNA to allow correct positioning of the
ribosome. For a review on maximizing gene expression, see Roberts
and Lauer, Methods in Enzymology, 68:473 (1979), which is hereby
incorporated by reference.
[0057] Promotors vary in their "strength" (i.e. their ability to
promote transcription). For the purposes of expressing a cloned
gene, it is desirable to use strong promotors in order to obtain a
high level of transcription and, hence, expression of the gene.
Depending upon the host cell system utilized, any one of a number
of suitable promotors may be used. For instance, when cloning in E.
coli, its bacteriophages, or plasmids, promotors such as the T7
phage promoter, lac promotor, trp promotor, recA promotor,
ribosomal RNA promotor, the P.sub.R and P.sub.L promotors of
coliphage lambda and others, including but not limited, to lacUV 5,
ompF, bla, 1 pp, and the like, may be used to direct high levels of
transcription of adjacent DNA segments. Additionally, a hybrid
trp-lacUV 5 (tac) promotor or other E. coli promotors produced by
recombinant DNA or other synthetic DNA techniques may be used to
provide for transcription of the inserted gene.
[0058] Bacterial host cell strains and expression vectors may be
chosen which inhibit the action of the promotor unless specifically
induced. In certain operations, the addition of specific inducers
is necessary for efficient transcription of the inserted DNA. For
example, the lac operon is induced by the addition of lactose or
IPTG (isopropylthio-beta-D-galactoside). A variety of other
operons, such as trp, pro, etc., are under different controls.
[0059] Specific initiation signals are also required for efficient
gene transcription and translation in procaryotic cells. These
transcription and translation initiation signals may vary in
"strength" as measured by the quantity of gene specific messenger
RNA and protein synthesized, respectively. The DNA expression
vector, which contains a promotor, may also contain any combination
of various "strong" transcription and/or translation initiation
signals. For instance, efficient translation in E. coli requires a
Shine-Dalgarno (SD) sequence about 7-9 bases 5' to the initiation
codon (ATG) to provide a ribosome binding site. Thus, any SD-ATG
combination that can be utilized by host cell ribosomes may be
employed. Such combinations include but are not limited to the
SD-ATG combination from the cro gene or the N gene of coliphage
lambda, or from the E. coli tryptophan E, D, C, B or A genes.
Additionally, any SD-ATG combination produced by recombinant DNA or
other techniques involving incorporation of synthetic nucleotides
may be used.
[0060] Once the isolated DNA molecule encoding the hypersensitive
response elicitor polypeptide or protein has been cloned into an
expression system, it is ready to be incorporated into a host cell.
Such incorporation can be carried out by the various forms of
transformation noted above, depending upon the vector/host cell
system. Suitable host cells include, but are not limited to,
bacteria, virus, yeast, mammalian cells, insect, plant, and the
like.
[0061] The method of the present invention can be utilized to treat
seeds for a wide variety of plants to impart pathogen resistance to
the plants. Suitable seeds are for plants which are dicots and
monocots. More particularly, useful crop plants can include: rice,
wheat, barley, rye, oats, cotton, sunflower, canola, peanut, corn,
potato, sweet potato, bean, pea, chicory, lettuce, endive, cabbage,
cauliflower, broccoli, turnip, radish, spinach, onion, garlic,
eggplant, pepper, celery, carrot, squash, pumpkin, zucchini,
cucumber, apple, pear, melon, strawberry, grape, raspberry,
pineapple, soybean, tobacco, tomato, sorghum, and sugarcane.
Examples of suitable ornamental plants are: rose, Saintpaulia,
petunia, Pelargonium, poinsettia, chrysanthemum, carnation, and
zinnia.
[0062] The method of imparting pathogen resistance to plants in
accordance with the present invention is useful in imparting
resistance to a wide variety of pathogens including viruses,
bacteria, and fungi.
[0063] Resistance, inter alia, to the following viruses can be
achieved by the method of the present invention: Tobacco mosaic
virus, cucumber mosaic virus, potato x virus, potato y virus, and
tomato mosaic virus.
[0064] Resistance, inter alia, to the following bacteria can also
be imparted to plants in accordance with the present invention:
Pseudomonas solancearum, Pseudomonas syringae pv. tabaci, and
Xanthamonas campestris pv. pelargonii.
[0065] Plants can be made resistant, inter alia, to the following
fungi by use of the method of the present invention: Fusarium
oxysporum and Phytophthora infestans.
[0066] The embodiment of the present invention involving applying
the hypersensitive response elicitor polypeptide or protein to all
or part of the plant seeds being treated can be carried out through
a variety of procedures. Suitable application methods include high
or low pressure spraying, injection, coating, dusting, and
immersion. Other suitable application procedures can be envisioned
by those skilled in the art. Once treated with the hypersensitive
response elicitor of the present invention, the seeds can be
planted and cultivated using conventional procedures to produce
plants. After plants have been propagated from seeds treated in
accordance with the present invention, the plants may be treated
with one or more applications of the hypersensitive response
elicitor protein or polypeptide to enhance hypersensitive response
induced resistance in the plants. See U.S. patent application Ser.
No. 08/475,775, which is hereby incorporated by reference. Such
propagated plants, which are resistant to disease, may, in turn, be
useful in producing seeds or propagules (e.g. cuttings) that
produce resistant plants.
[0067] The hypersensitive response elicitor polypeptide or protein
can be applied to plant seeds in accordance with the present
invention alone or in a mixture with other materials.
[0068] A composition suitable for treating plant seeds in
accordance with the present invention contains a hypersensitive
response elicitor polypeptide or protein in a carrier. Suitable
carriers include water, aqueous solutions, slurries, or dry
powders. In this embodiment, the composition contains greater than
0.5 nM hypersensitive response elicitor polypeptide or protein.
[0069] Although not required, this composition may contain
additional additives including fertilizer, insecticide, fungicide,
nematicide, herbicide, and mixtures thereof. Suitable fertilizers
include (NH.sub.4).sub.2NO.sub.3. An example of a suitable
insecticide is Malathion. Useful fungicides include Captan.
[0070] Other suitable additives include buffering agents, wetting
agents, coating agents, and abrading agents. These materials can be
used to facilitate the process of the present invention. In
addition, the hypersensitive response elicitor polypeptide or
protein can be applied to plant seeds with other conventional seed
formulation and treatment materials, including clays and
polysaccharides.
[0071] In the alternative embodiment of the present invention
involving the use of transgenic seeds, a hypersensitive response
elicitor polypeptide or protein need not be applied topically to
the seeds. Instead, transgenic plants transformed with a DNA
molecule encoding a hypersensitive response elicitor polypeptide or
protein are produced according to procedures well known in the art,
such as biolistics or Agrobacterium mediated transformation.
Examples of suitable hypersensitive response elicitor polypeptides
or proteins and the nucleic acid sequences for their encoding DNA
are disclosed supra. As is conventional in the art, such transgenic
plants would contain suitable vectors with various promoters
including pathogen-induced promoters, and other components needed
for transformation, transcription, and, possibly, translation. Such
transgenic plants themselves could be grown under conditions
effective to be imparted with pathogen resistance. In any event,
once transgenic plants of this type are produced, transgenic seeds
are recovered. These seeds can then be planted in the soil and
cultivated using conventional procedures to produce plants. The
plants are propagated from the planted transgenic seeds under
conditions effective to impart pathogen resistance to the
plants.
[0072] When transgenic plant seeds are used in accordance with the
present invention, they additionally can be treated with the same
materials (noted above) as are used to treat the seeds to which a
hypersensitive response elicitor polypeptide or protein is applied.
These other materials, including hypersensitive response elicitors,
can be applied to the transgenic plant seeds by high or low
pressure spraying, injection, coating, dusting, and immersion.
Similarly, transgenic plants additionally may be treated with one
or more applications of the hypersensitive response elicitor to
enhance hypersensitive response induced resistance in the plants.
Such plants may also be treated with conventional plant treatment
agents (e.g., insecticides, fertilizers, etc.). The transgenic
plants of the present invention are useful in producing seeds or
propagules (e.g. cuttings) from which disease resistant plants
grow.
EXAMPLES
Example 1
Effect of Treating Seeds with Hypersensitive Response Elicitor
Protein
[0073] Marglobe tomato seeds were submerged in hypersensitive
response elicitor protein (ca. 26 .mu.m/ml) from Erwinia amylovora
solution or buffer in beakers on day 0 for 24 hours at 28.degree.
C. in a growth chamber. After soaking seeds in hypersensitive
response elicitor protein from Erwinia amylovora or buffer, they
were sown in germination pots with artificial soil on day 1.
Seedlings were transplanted to individual pots at the two-true-leaf
stage on day 12. After transplanting, some plants that arose from
treated seed also were sprayed with hypersensitive response
elicitor protein (ca. 13 .mu.m/ml) from Erwinia amylovora
(Treatments 3 and 4).
[0074] Tomato treated as noted in the preceding paragraph were
inoculated with Burkholderia (Pseudomonas) solanacearum K60 strain
(See Kelman, "The Relationship of Pathogenicity in Pseudomonas
solanacearum to Colony Appearance on a Tetrazolium Medium,"
Phytopathology 44:693-95 (1954)) on day 23 by making vertical cuts
through the roots and potting medium of tomato plants (on a tangent
2 cm from the stem and 2 times/pot) and putting 10 ml
(5.times.10.sup.8 cfu/ml) suspension into the soil.
[0075] The above procedure involved use of 10 seeds treated with
hypersensitive response elicitor protein from Erwinia amylovora per
treatment.
[0076] Treatments: [0077] 1. Seeds soaked in hypersensitive
response elicitor protein from Erwinia amylovora (ca. 26
.mu.mg/ml). [0078] 2. Seeds soaked in buffer (5 mM KPO.sub.4, pH
6.8). [0079] 3. Seeds soaked in hypersensitive response elicitor
protein from Erwinia amylovora (ca. 26 .mu.mg/ml) and seedlings
sprayed with hypersensitive response elicitor protein from Erwinia
amylovora (ca. 13 .mu.m/ml) at transplanting. [0080] 4. Seeds
soaked in buffer and seedlings sprayed with hypersensitive response
elicitor protein from Erwinia amylovora (ca. 13 .mu.m/ml) at
transplanting.
[0081] The results of these treatments are set forth in Tables
1-4.
TABLE-US-00011 TABLE 1 Infection Data - 28 Days After Seed
Treatment and 5 Days After Inoculation Number of Plants of Given
Disease Rating* Treatm. Plants 0 1 2 3 4 5 1 10 10 0 0 0 0 0 2 10 9
1 0 0 0 0 3 10 9 1 0 0 0 0 4 10 10 0 0 0 0 0 *Disease Scale: Grade
O: No symptoms Grade 1: One leaf partially wilted. Grade 2: 2-3
leaves wilted. Grade 3: All except the top 2-3 leaves wilted. Grade
4: All leaves wilted. Grade 5: Plant Dead
TABLE-US-00012 TABLE 2 Infection Data - 31 Days After Seed
Treatment and 8 Days After Inoculation Number of Plants of Given
Disease Rating* Treatm. Plants 0 1 2 3 4 5 1 10 6 4 0 0 0 0 2 10 4
3 2 1 0 0 3 10 8 2 0 0 0 0 4 10 7 2 1 0 0 0
TABLE-US-00013 TABLE 3 Infection Data - 35 Days After Seed
Treatment and 12 Days After Inoculation Number of Plants of Given
Disease Rating* Treatm. Plants 0 1 2 3 4 5 1 10 5 3 0 1 1 0 2 10 1
3 3 2 1 0 3 10 4 3 3 0 0 0 4 10 3 3 3 1 0 0
TABLE-US-00014 TABLE 4 Disease Indices of Seed Treatment With
Hypersensitive Response Elicitor Protein Treatment Inoculation
Disease Index (%)* Day 0 Day 14 Day 23 Day 28 Day 31 Day 35 1.
Hypersensitive Inoculate 0 8 20 response elicitor protein seed soak
2. Buffer seed soak Inoculate 2 20 38 3. Hypersensitive Spray
Inoculate 2 4 18 response elicitor Hypersensitive protein seed soak
response elicitor protein 4. Buffer seed soak Spray Inoculate 0 8
24 Hypersensitive response elicitor protein *The Disease Index was
determined using the procedure set forth in N. N. Winstead, et al.,
"Inoculation Techniques for Evaluating Resistance to Pseudomonas
Solanacearum, " Phytopathology 42: 628-34 (1952), particularly at
page 629.
[0082] The above data shows that the hypersensitive response
elicitor protein was more effective than buffer as a seed treatment
in reducing disease index and was as effective as spraying leaves
of young plants with hypersensitive response elicitor protein.
Example 2
Effect of Treating Tomato Seeds with Hypersensitive Response
Elicitor Protein from pCPP2139 Versus pCPP50 Vector on Southern
Bacteria Wilt of Tomato
[0083] Marglobe tomato seeds were submerged in hypersensitive
response elicitor protein from pCPP2139 or in pCPP50 vector
solution (1:50, 1:100 and 1:200) in beakers on day 0 for 24 hours
at 28.degree. C. in a growth chamber. After soaking seeds in
hypersensitive response elicitor protein or vector, they were sown
in germination pots with artificial soil on day 0. Ten uniform
appearing plants were chosen randomly from each of the following
treatments:
TABLE-US-00015 Treatment Strain Dilution Harpin Content 1.
DH5.alpha.(pCPP2139) 1:50 8 .mu.g/ml 2. DH5.alpha.(pCCP50) 1:50 0
3. DH5.alpha.(pCPP2139) 1:100 4 .mu.g/ml 4. DH5.alpha.(pCPP50)
1:100 0 5. DH5.alpha.(pCPP2139) 1:200 2 .mu.g/ml 6.
DH5.alpha.(pCPP50) 1:200 0
The resulting seedlings were inoculated with Pseudomonas
solanacearum K60 by dipping the roots of tomato seedling plants for
about 30 seconds in a 40 ml (1.times.10.sup.8 cfu/ml) suspension.
The seedlings were then transplanted into the pots with artificial
soil on day 12.
[0084] The results of these treatments are set forth in Tables
5-8.
TABLE-US-00016 TABLE 5 16 Days After Seed Treatment and 3 Days
After Inoculation Number of Plants of Given Disease Rating* Treatm.
Plants 0 1 2 3 4 5 1 10 7 3 0 0 0 0 2 10 5 5 0 0 0 0 3 10 6 4 0 0 0
0 4 10 6 4 0 0 0 0 5 10 7 4 0 0 0 0 6 10 4 6 0 0 0 0
TABLE-US-00017 TABLE 6 19 Days After Seed Treatment and 6 Days
After Inoculation Number of Plants of Given Disease Rating* Treatm.
Plants 0 1 2 3 4 5 1 10 6 0 0 0 0 0 2 10 2 0 2 2 1 3 3 10 2 0 2 0 2
4 4 10 3 1 2 0 2 2 5 10 2 1 0 2 2 3 6 10 1 0 1 1 3 4
TABLE-US-00018 TABLE 7 21 Days After Seed Treatment and 8 Days
After Inoculation Number of Plants of Given Disease Rating* Treatm.
Plants 0 1 2 3 4 5 1 10 6 0 0 0 1 3 2 10 2 0 0 1 3 4 3 10 2 0 0 2 2
3 4 10 3 0 0 2 2 3 5 10 2 0 0 0 4 4 6 10 1 0 1 2 1 5
TABLE-US-00019 TABLE 8 Disease Indices of Seed Treatment With
Hypersensitive Response Elicitor and Vector Treatment Disease Index
(%) Day 0 Day 12 Day 15 Day 18 Day 20 Hypersensitive inoculate 6.0
32.0 38.0 response elicitor protein seed dip (1:50) Vector seed dip
(1:50) inoculate 10.0 58.0 70.0 Hypersensitive inoculate 8.0 64.0
68.0 response elicitor protein seed dip (1:100) Vector seed dip
inoculate 8.0 46.0 58.0 (1:100) Hypersensitive inoculate 6.0 60.00
72.0 response elicitor protein seed dip (1:200) Vector seed dip
inoculate 12.0 74.0 74.0 (1:200)
[0085] The above data shows that the hypersensitive response
elicitor protein is much more effective than the vector solution in
preventing Tomato Southern Bacteria Wilt.
Example 3
Effect of Treating Tomato Seeds with Hypersensitive Response
Elicitor Protein from pCPP2139 Versus pCPP50 Vector on Tomato
Southern Bacteria Wilt
[0086] Marglobe tomato seeds were submerged in hypersensitive
response elicitor protein from pCPP2139 or in pCPP50 vector
solution (1:50, 1:100 and 1:200) in beakers on day 0 for 24 hours
at 28.degree. C. in a growth chamber. After soaking seeds in the
hypersensitive response elicitor protein or vector, the seeds were
sown in germination pots with artificial soil on day 1. Ten uniform
appearing plants were chosen randomly from each of the following
treatments:
TABLE-US-00020 Hypersensitive Response Elicitor Treatment Strain
Dilution Content 1. DH5.alpha.(pCPP2139) 1:50 8 .mu.g/ml 2.
DH5.alpha.(pCCP50) 1:50 0 3. DH5.alpha.(pCPP2139) 1:100 4 .mu.g/ml
4. DH5.alpha.(pCPP50) 1:100 0 5. DH5.alpha.(pCPP2139) 1:200 2
.mu.g/ml 6. DH5.alpha.(pCPP50) 1:200 0
The resulting seedlings were inoculated with Pseudomonas
solanacearum K60 by dipping the roots of tomato seedling plants for
about 30 seconds in a 40 ml (1.times.10.sup.6 cfu/ml) suspension.
The seedlings were then transplanted into the pots with artificial
soil on day 12.
[0087] The results of these treatments are set forth in Tables
9-12.
TABLE-US-00021 TABLE 9 16 Days After Seed Treatment and 3 Days
After Inoculation Number of Plants of Given Diease Rating* Treatm.
Plants 0 1 2 3 4 5 1 10 8 2 0 0 0 0 2 10 7 3 0 0 0 0 3 10 7 3 0 0 0
0 4 10 7 3 0 0 0 0 5 10 8 2 0 0 0 0 6 10 7 3 0 0 0 0
TABLE-US-00022 TABLE 10 19 Days After Seed Treatment and 6 Days
After Inoculation Number of Plants of Given Disease Rating* Treatm.
Plants 0 1 2 3 4 5 1 10 5 0 0 1 2 2 2 10 1 0 1 2 3 3 3 10 4 1 0 0 2
3 4 10 2 0 2 1 2 3 5 10 1 0 1 1 4 3 6 10 1 0 0 2 4 3
TABLE-US-00023 TABLE 11 21 Days After Hypersensitive Response
Elicitor Protein Seed Treatment and 8 Days After Inoculation Number
of Plants of Driven Disease Rating* Treatm. Plants 0 1 2 3 4 5 1 10
5 0 0 0 2 3 2 10 2 0 2 0 2 4 3 10 5 0 0 0 2 3 4 10 2 0 2 0 2 4 5 10
1 0 1 0 2 6 6 10 1 0 0 0 2 7
TABLE-US-00024 TABLE 12 Disease Indices of Seed Treatment With
Hypersensitive Response Elicitor Protein and Vector Day 1 Day 13
Day 16 Day 19 Day 21 Hypersensitive inoculate 4.0 42.0 46.0
response elicitor protein seed dip (1:50) Vector seed dip (1:50)
inoculate 6.0 70.0 64.0 Hypersensitive inoculate 6.0 48.0 46.0
response elicitor protein seed dip (1:100) Vector seed dip
inoculate 6.0 60.0 64.0 (1:100) Hypersensitive inoculate 4.0 72.0
80.0 response elicitor protein seed dip (1:200) Vector seed dip
inoculate 6.0 74.0 86.0 (1:200)
[0088] The above data shows that the hypersensitive response
elicitor protein is much more effective in preventing Tomato
Southern Bacteria Wilt.
Example 4
Effect of Treating Tomato Seeds With Hypersensitive Response
Elicitor Protein from pCPP2139 Versus pCPP50 Vector on Southern
Bacteria Wilt of Tomato
[0089] Marglobe tomato seeds were submerged in hypersensitive
response elicitor protein from pCPP2139 or in pCPP50 vector
solution (1:25, 1:50 and 1:100) in beakers on day 0 for 24 hours at
28.degree. C. in a growth chamber. After soaking seeds in
hypersensitive response elicitor protein or vector, they were sown
in germination pots with artificial soil on day 1. Ten uniform
appearing plants were chosen randomly from each of the following
treatments:
TABLE-US-00025 Treatment Strain Dilution Harpin Content 1.
DH5.alpha.(pCPP2139) 1:25 16 .mu.g/ml 2. DH5.alpha.(pCCP50) 1:25 0
3. DH5.alpha.(pCPP2139) 1:50 8 .mu.g/ml 4. DH5.alpha.(pCPP50) 1:50
0 5. DH5.alpha.(pCPP2139) 1:100 2 .mu.g/ml 6. DH5.alpha.(pCPP50)
1:100 0
The resulting seedlings were inoculated with Pseudomonas
solanacearum K60 by dipping the roots of tomato seedling plants for
about 30 seconds in a 40 ml (1.times.10.sup.8 cfu/ml) suspension.
The seedlings were then transplanted into the pots with artificial
soil on day 14.
[0090] The results of these treatments are set forth in Tables
13-16.
TABLE-US-00026 TABLE 13 19 Days After Seed Treatment and 4 Days
After Inoculation Number of Plants of Given Disease Rating* Treatm.
Plants 0 1 2 3 4 5 1 10 8 2 0 0 0 0 2 10 5 2 2 1 0 0 3 10 9 1 0 0 0
0 4 10 5 2 1 2 0 0 5 10 5 3 1 1 0 0 6 10 6 1 2 1 0 0
TABLE-US-00027 TABLE 14 21 Days After Seed Treatments and 6 Days
After Inoculation Number of Plants of Given Disease Rating* Treatm.
Plants 0 1 2 3 4 5 1 10 6 3 0 0 1 0 2 10 3 2 1 0 0 0 3 10 6 3 1 0 0
0 4 10 3 2 1 2 2 0 5 10 5 1 2 2 0 0 6 10 3 1 3 2 1 0
TABLE-US-00028 TABLE 15 23 Days After Seed Treatment and 8 Days
After Inoculation Number of Plants of Given Disease Rating* Treatm.
Plants 0 1 2 3 4 5 1 10 7 2 0 0 0 1 2 10 2 2 2 3 0 1 3 10 7 2 0 1 0
0 4 10 2 1 2 3 0 2 5 10 3 1 2 3 0 1 6 10 2 2 2 3 0 1
TABLE-US-00029 TABLE 16 Disease Indices of Seed Treatment With
Hypersensitive Elicitor Protein and Vector Treatment Disease Index
(%) Day 1 Day 15 Day 19 Day 21 Day 23 Hypersensitive inoculate 4.0
14.0 14.0 response elicitor protein seed dip (1:25) Vector seed dip
(1:25) inoculate 18.0 28.0 40.0 Hypersensitive inoculate 2.0 10.0
10.0 response elicitor protein seed dip (1:50) Vector seed dip
(1:50) inoculate 20.0 36.0 48.0 Hypersensitive inoculate 16.0 22.0
38.0 response elicitor protein seed dip (1:100) Vector seed dip
inoculate 16.0 34.0 40.0 (1:100)
[0091] The above data shows that the hypersensitive response
elicitor protein is much more effective than the vector solution in
preventing Tomato Southern Bacteria Wilt. A hypersensitive response
protein concentration of 1:50 is particularly effective in disease
control.
Example 5
Effect of Treating Tomato Seeds With Hypersensitive Response
Elicitor Protein From pCPP2139 versus pCPP50 Vector on Southern
Bacteria Wilt of Tomato
[0092] Marglobe tomato seeds were submerged in hypersensitive
response elicitor protein from pCPP2139 or pCPP50 vector solution
(1:25, 1:50 and 1:100) in beakers on day 0 for 24 hours at
28.degree. C. in a growth chamber. After soaking seeds in
hypersensitive response elicitor protein or vector, they were sown
in germination pots with artificial soil on day 1. Ten uniform
appearing plants were chosen randomly from each of the following
treatments:
TABLE-US-00030 Treatment Strain Dilution Harpin Content 1.
DH5.alpha.(pCPP2139) 1:25 16 .mu.g/ml 2. DH5.alpha.(pCCP50) 1:25 0
3. DH5.alpha.(pCPP2139) 1:50 8 .mu.g/ml 4. DH5.alpha.(pCPP50) 1:50
0 5. DH5.alpha.(pCPP2139) 1:100 4 .mu.g/ml 6. DH5.alpha.(pCPP50)
1:100 0
The resulting seedlings were inoculated with Pseudomonas
solanacearum K60 by dipping the roots of tomato seedling plants for
about 30 seconds in a 40 ml (1.times.10.sup.6 cfu/ml) suspension.
The seedlings were then transplanted into the pots with artificial
soil on day 14.
[0093] The results of these treatments are set forth in Tables
17-20.
TABLE-US-00031 TABLE 17 19 Days After Seed Treatment and 4 Days
After Inoculation Number of Plants of Given Disease Rating* Treatm.
Plants 0 1 2 3 4 5 1 10 8 2 0 0 0 0 2 10 6 3 1 0 0 0 3 10 9 1 0 0 0
0 4 10 6 4 0 0 0 0 5 10 6 2 1 1 0 0 6 10 6 4 0 0 0 0
TABLE-US-00032 TABLE 18 21 Days After Seed Treatment and 6 Days
After Inoculation Number of Plants of Given Disease Rating* Treatm.
Plants 0 1 2 3 4 5 1 10 7 1 1 1 0 0 2 10 3 3 2 2 0 0 3 10 8 2 0 0 0
0 4 10 3 3 2 2 0 0 5 10 6 1 1 2 0 0 6 10 3 2 3 1 1 0
TABLE-US-00033 TABLE 19 23 Days After Seed Treatment and 8 Days
After Inoculation Number of Plants of Given Disease Rating* Treatm.
Plants 0 1 2 3 4 5 1 10 7 0 2 1 0 0 2 10 3 1 2 3 0 1 3 10 8 1 0 1 0
0 4 10 3 3 1 2 0 1 5 10 3 3 0 2 1 1 6 10 3 2 0 3 0 2
TABLE-US-00034 TABLE 20 Disease Indices of Seed Treatment With
Hypersensitive Response Elicitor Protein and Vector Treatment
Disease Index (%) Day 0 Day 15 Day 19 Day 21 Day 23 Hypersensitive
inoculate 4.0 12.0 14.0 response elicitor protein seed dip (1:25)
Vector seed dip (1:25) inoculate 10.0 26.0 38.0 Hypersensitive
inoculate 2.0 4.0 8.0 response elicitor protein seed dip (1:50)
Vector seed dip (1:50) inoculate 8.0 26.0 32.0 Hypersensitive
inoculate 14.0 18.0 36.0 response elicitor protein seed dip (1:100)
Vector seed dip inoculate 8.0 30.0 42.0 (1:100)
[0094] The above data shows that the hypersensitive response
elicitor protein is much more effective than the vector solution in
preventing Tomato Southern Bacteria Wilt. A hypersensitive response
elicitor protein concentration of 1:50 is more effective in disease
control.
Example 6
Treating Rice Seeds with Hypersensitive Response Elicitor Protein
to Reduce Rice Stem Rot
[0095] Rice seeds (variety, M-202) were submerged in two gallons of
hypersensitive response elicitor protein solution at a
concentration of 20 .mu.g for 24 hours at room temperature. Rice
seeds submerged in the same solution without hypersensitive
response elicitor protein were used as a control. After soaking,
the seeds were sown in a rice field by air plane spray. There were
four replicates for both hypersensitive response elicitor protein
and control treatment. The lot size of each replicate is 150
Ft.sup.2. The design of each plot was completely randomized, and
each plot had substantial level contamination of Sclerotium oryzae.
Three months after sowing, stem rot was evaluated according to the
following rating scale: Scale 1=no disease, 2=disease present on
the exterior of the leaf sheath, 3=disease penetrates leaf sheath
completely but is not present on culm, 4=disease is present on culm
exterior but does not penetrate to interior of culm, and 5=disease
penetrates to interior of culm. 40 plants from each replicate were
sampled and assessed for the disease incidence and severity. From
Table 21, it is apparent that treating seeds with hypersensitive
response elicitor reduced both disease incidence and severity. More
particularly, regarding incidence, 67% of the plants were infected
by stem rot for the control treatment, however, only 40% plants
were infected for the hypersensitive response elicitor protein
treatment. As to severity, the disease index* for the
hypersensitive response elicitor protein treatment was 34% and 60%
for the control. Accordingly, treating rice seed with
hypersensitive response elicitor protein resulted in a significant
reduction of stem rot disease. The hypersensitive response elicitor
protein-induced resistance in rice can last a season long. In
addition to disease resistance, it was also observed that
hypersensitive response elicitor protein-treated rice had little or
no damage by army worm (Spodoptera praefica). In addition, the
treated plants were larger and had deeper green color than the
control plants.
TABLE-US-00035 TABLE 21 Incidence and Severity of Stem Rot
(Schlerotium oryzae) on Rice, M-202 % plants given disease rating
Disease index (%) Treatment 1 2 3 4 5 (severity) Harpin 20 .mu.g/ml
60 5 8 18 10 34 Control 33 5 18 28 18 60 * Disease Index ( % ) for
the hairpin treatment = 1 .times. 60 + 2 .times. 5 + 3 .times. 8 +
4 .times. 18 + 5 .times. 10 5 .times. 100 .times. 100 / 100
##EQU00001## * Disease Index ( % ) for the control treatment = 1
.times. 33 + 2 .times. 5 + 3 .times. 18 + 4 .times. 28 + 5 .times.
18 5 .times. 100 .times. 100 / 100 .times. 100 / 100
##EQU00002##
Example 7
Effect of Treating Onion Seed with Hypersensitive Response Elicitor
Protein on the Development of Onion Smut Disease (Urocystis
cepulae) and on Seedling Emergence
[0096] Onion seed, variety Pennant, (Seed Lot #64387), obtained
from the Crookham Co., Caldwell, Id. 83606, treated with
hypersensitive response elicitor protein or a control was planted
in a natural organic or "muck" soil. Some of the seedlings that
grew from the sown seed were healthy, some had lesions
characteristic of the Onion Smut disease, and some of the sown seed
did not produce seedlings that emerged from the soil. Thus, the
effect of treating onion seed with various concentrations of
hypersensitive response elicitor protein was determined.
[0097] Naturally infested muck soil was obtained from a field in
Oswego County, N.Y., where onions had been grown for several years
and where the Onion Smut disease commonly had been problematic.
Buckets of muck (5-gallon plastic) were stored at 4.degree. C.
until used. The soil was mixed, sieved, and put in plastic flats 10
inches wide, 20 inches long, and 2 inches deep for use in the tests
described. Based on preliminary experiments, the soil contained
many propagules of the Onion Smut fungus, Urocystis cepulae, such
that when onion seed was sown in the soil, smut lesions developed
on many of the seedlings that emerged from the soil. In addition,
the soil harbored other microorganisms, including those that cause
the "damping-off" disease. Among the several fungi that cause
damping off are Pythium, Fusarium, and Rhizoctonia species.
[0098] The hypersensitive response elicitor protein encoded by the
hrpN gene of Erwinia amylovora was used to treat seeds. It was
produced by fermentation of the cloned gene in a high-expression
vector in E. coli. Analysis of the cell-free elicitor preparation
by high-pressure liquid chromatography indicated its hypersensitive
response elicitor protein content and on that basis appropriate
dilutions were prepared in water. Seeds were soaked in a beaker
containing hypersensitive response elicitor protein concentrations
of 0, 5, 25, and 50 .mu.gm/ml of hypersensitive response elicitor
protein for 24 hours. They were removed, dried briefly on paper
towels, and sown in the muck soil. Treated seed was arranged by
row, 15 seeds in each row for each treatment; each flat contained
two replicates, and there were six replicates. Thus, a total of 90
seeds were treated with each concentration of hypersensitive
response elicitor protein. The flats containing the seeds were held
in a controlled environment chamber operating at 60.degree. F.
(15.6.degree. C.), with a 14-hour day/10-hour night. Observations
were made on seedling emergence symptoms (smut lesions). The data
were recorded 23 days after sowing.
[0099] The effect of soaking onion seed in different concentrations
of hypersensitive response elicitor protein on emergence of onion
seedlings and on the incidence of onion smut is shown in Table 22.
Only slight differences in emergence were noted, suggesting that
there is no significant effect of treating with hypersensitive
response elicitor protein at the concentrations used. Among the
seedlings that emerged, substantially more of the seeds that
received no hypersensitive response elicitor protein exhibited
symptoms of Onion Smut than seedlings that grew from seed that had
been treated with hypersensitive response elicitor protein.
Treating seed with 25 .mu.gm/ml of hypersensitive response elicitor
protein was the most effective concentration tested in reducing
Onion Smut. Thus, this example demonstrates that treating onion
seed with hypersensitive response elicitor protein reduces the
Onion Smut disease.
TABLE-US-00036 TABLE 22 Effect of Treating Onion Seed With
Hypersensitive Response Elicitor Protein (i.e. Harpin) on the
Development of Onion Smut Disease (Urocystis cepulae). Mean
Treatment Seedlings Mean Emerged harpin Emerged Percent Percent
Percent (.mu.g/ml) (of 15) Emerged Healthy with Smut 0 5.00 33.3
20.0 80.0 5 3.67 24.4 40.9 59.1 25 .sup. 4.33.sup.1 28.8 50.0 46.2
50 4.17 27.7 44.0 56.0 .sup.1One seedling emerged then died.
Example 8
Effect of Treating Tomato Seed with Hypersensitive Response
Elicitor Protein on the Development of Bacterial Speck of Tomato
(Pseudomonas syringae pv. tomato)
[0100] Tomato seed, variety New Yorker (Seed lot #2273-2B),
obtained from Harris Seeds, Rochester, N.Y., were treated with four
concentrations of hypersensitive response elicitor protein
(including a no-elicitor protein, water-treated control) and
planted in peatlite soil mix. After 12 days and when the seedlings
were in the second true-leaf stage, they were inoculated with the
Bacterial Speck pathogen. Ten days later, the treated and
inoculated plants were evaluated for extent of infection. Thus, the
effect of treating tomato seed with various concentrations of
hypersensitive response elicitor protein on resistance to
Pseudomonas syringae pv. tomato was determined.
[0101] The hypersensitive response elicitor protein encoded by the
hrpN gene of Erwinia amylovora was used to treat seeds. It was
produced by fermentation of the cloned gene in a high-expression
vector in E. coli. Analysis of the cell-free elicitor preparation
by high-pressure liquid chromatography indicated its hypersensitive
response elicitor protein content and, on that basis, appropriate
dilutions were prepared in water. Seeds were soaked in a beaker
containing hypersensitive response elicitor protein concentrations
of 0, 5, 10, and 20 nm/ml of hypersensitive response elicitor
protein for 24 hours. They were removed, dried briefly on paper
towels, and sown. The soil was a mixture of peat and Pearlite.TM.
in plastic flats 10 inches wide, 20 inches long, and 2 inches deep.
Treated seed was arranged by row, 6 seeds in each row for each
treatment; each flat contained two replicates, and there were four
replicates and thus a total of 24 seeds that were treated with each
concentration of hypersensitive response elicitor protein. The
flats containing the seeds were held in a controlled environment
chamber operating at 75.degree. F. (25.degree. C.), with a 14-hour
day/10-hour night.
[0102] When twelve-days old, the tomato seedlings were inoculated
with 10.sup.8 colony forming units/ml of the pathogen, applied as a
foliar spray. The flats containing the seedlings were covered with
a plastic dome for 48 hours after inoculation to maintain high
humidity. Observations were made on symptom severity using a rating
scale of 0-5. The rating was based on the number of lesions that
developed on the leaflets and the cotyledons and on the relative
damage caused to the plant parts by necrosis that accompanied the
lesions. The cotyledons and (true) leaflets were separately rated
for disease severity 11 days after inoculation
[0103] The effect of soaking tomato seed in different
concentrations of hypersensitive response elicitor protein (i.e.
harpin) on the development of Bacterial Speck on leaflets and
cotyledons of tomato is shown in Table 23. The seedlings that grew
from seed treated with the highest amount of hypersensitive
response elicitor protein tested (20 .mu.gm/ml) had fewer diseased
leaflets and cotyledons than the treatments. The water-treated
control seedlings did not differ substantially from the plants
treated with the two lower concentrations of hypersensitive
response elicitor protein. Considering the disease ratings, the
results were similar. Only plants treated with the highest
concentration of hypersensitive response elicitor protein had
disease ratings that were less than those of the other treatments.
This example demonstrates that treatment of tomato seed with
hypersensitive response elicitor protein reduces the incidence and
severity of Bacterial Speck of tomato.
TABLE-US-00037 TABLE 23 Effect of Treating Tomato Seed With
Hypersensitive Response Elicitor Protein (i.e. Harpin) on the
Subsequent Development of Bacterial Speck Disease (Pseudomonas
syringae pv. tomato) on Tomato Cotyledons and Tomato Leaflets
Cotyledons Leaflets Treatment Mean Percent Disease Percent Disease
Harpin (.mu.g/ml) Diseased Diseased Rating Mean Diseased Diseased
Rating 0 6.0/9.0 66.6 0.8 25.8/68.8 37.5 0.5 5 5.3/7.3 72.4 0.8
22.5/68.0 37.5 0.5 10 5.8/8.0 72.3 0.8 25.5/66.0 38.6 0.5 20
5.3/8.5 61.8 0.6 23.8/73.5 32.3 0.4
[0104] Although the invention has been described in detail for the
purpose of illustration, it is understood that such detail is
solely for that purpose, and variations can be made therein by
those skilled in the art without departing from the spirit and
scope of the invention which is defined by the following claims.
Sequence CWU 1
1
101338PRTErwinia chrysanthemi 1Met Gln Ile Thr Ile Lys Ala His Ile
Gly Gly Asp Leu Gly Val Ser1 5 10 15Gly Leu Gly Ala Gln Gly Leu Lys
Gly Leu Asn Ser Ala Ala Ser Ser 20 25 30Leu Gly Ser Ser Val Asp Lys
Leu Ser Ser Thr Ile Asp Lys Leu Thr 35 40 45Ser Ala Leu Thr Ser Met
Met Phe Gly Gly Ala Leu Ala Gln Gly Leu 50 55 60Gly Ala Ser Ser Lys
Gly Leu Gly Met Ser Asn Gln Leu Gly Gln Ser65 70 75 80Phe Gly Asn
Gly Ala Gln Gly Ala Ser Asn Leu Leu Ser Val Pro Lys 85 90 95Ser Gly
Gly Asp Ala Leu Ser Lys Met Phe Asp Lys Ala Leu Asp Asp 100 105
110Leu Leu Gly His Asp Thr Val Thr Lys Leu Thr Asn Gln Ser Asn Gln
115 120 125Leu Ala Asn Ser Met Leu Asn Ala Ser Gln Met Thr Gln Gly
Asn Met 130 135 140Asn Ala Phe Gly Ser Gly Val Asn Asn Ala Leu Ser
Ser Ile Leu Gly145 150 155 160Asn Gly Leu Gly Gln Ser Met Ser Gly
Phe Ser Gln Pro Ser Leu Gly 165 170 175Ala Gly Gly Leu Gln Gly Leu
Ser Gly Ala Gly Ala Phe Asn Gln Leu 180 185 190Gly Asn Ala Ile Gly
Met Gly Val Gly Gln Asn Ala Ala Leu Ser Ala 195 200 205Leu Ser Asn
Val Ser Thr His Val Asp Gly Asn Asn Arg His Phe Val 210 215 220Asp
Lys Glu Asp Arg Gly Met Ala Lys Glu Ile Gly Gln Phe Met Asp225 230
235 240Gln Tyr Pro Glu Ile Phe Gly Lys Pro Glu Tyr Gln Lys Asp Gly
Trp 245 250 255Ser Ser Pro Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala
Leu Ser Lys 260 265 270Pro Asp Asp Asp Gly Met Thr Gly Ala Ser Met
Asp Lys Phe Arg Gln 275 280 285Ala Met Gly Met Ile Lys Ser Ala Val
Ala Gly Asp Thr Gly Asn Thr 290 295 300Asn Leu Asn Leu Arg Gly Ala
Gly Gly Ala Ser Leu Gly Ile Asp Ala305 310 315 320Ala Val Val Gly
Asp Lys Ile Ala Asn Met Ser Leu Gly Lys Leu Ala 325 330 335Asn
Ala22141DNAErwinia chrysanthemi 2cgattttacc cgggtgaacg tgctatgacc
gacagcatca cggtattcga caccgttacg 60gcgtttatgg ccgcgatgaa ccggcatcag
gcggcgcgct ggtcgccgca atccggcgtc 120gatctggtat ttcagtttgg
ggacaccggg cgtgaactca tgatgcagat tcagccgggg 180cagcaatatc
ccggcatgtt gcgcacgctg ctcgctcgtc gttatcagca ggcggcagag
240tgcgatggct gccatctgtg cctgaacggc agcgatgtat tgatcctctg
gtggccgctg 300ccgtcggatc ccggcagtta tccgcaggtg atcgaacgtt
tgtttgaact ggcgggaatg 360acgttgccgt cgctatccat agcaccgacg
gcgcgtccgc agacagggaa cggacgcgcc 420cgatcattaa gataaaggcg
gcttttttta ttgcaaaacg gtaacggtga ggaaccgttt 480caccgtcggc
gtcactcagt aacaagtatc catcatgatg cctacatcgg gatcggcgtg
540ggcatccgtt gcagatactt ttgcgaacac ctgacatgaa tgaggaaacg
aaattatgca 600aattacgatc aaagcgcaca tcggcggtga tttgggcgtc
tccggtctgg ggctgggtgc 660tcagggactg aaaggactga attccgcggc
ttcatcgctg ggttccagcg tggataaact 720gagcagcacc atcgataagt
tgacctccgc gctgacttcg atgatgtttg gcggcgcgct 780ggcgcagggg
ctgggcgcca gctcgaaggg gctggggatg agcaatcaac tgggccagtc
840tttcggcaat ggcgcgcagg gtgcgagcaa cctgctatcc gtaccgaaat
ccggcggcga 900tgcgttgtca aaaatgtttg ataaagcgct ggacgatctg
ctgggtcatg acaccgtgac 960caagctgact aaccagagca accaactggc
taattcaatg ctgaacgcca gccagatgac 1020ccagggtaat atgaatgcgt
tcggcagcgg tgtgaacaac gcactgtcgt ccattctcgg 1080caacggtctc
ggccagtcga tgagtggctt ctctcagcct tctctggggg caggcggctt
1140gcagggcctg agcggcgcgg gtgcattcaa ccagttgggt aatgccatcg
gcatgggcgt 1200ggggcagaat gctgcgctga gtgcgttgag taacgtcagc
acccacgtag acggtaacaa 1260ccgccacttt gtagataaag aagatcgcgg
catggcgaaa gagatcggcc agtttatgga 1320tcagtatccg gaaatattcg
gtaaaccgga ataccagaaa gatggctgga gttcgccgaa 1380gacggacgac
aaatcctggg ctaaagcgct gagtaaaccg gatgatgacg gtatgaccgg
1440cgccagcatg gacaaattcc gtcaggcgat gggtatgatc aaaagcgcgg
tggcgggtga 1500taccggcaat accaacctga acctgcgtgg cgcgggcggt
gcatcgctgg gtatcgatgc 1560ggctgtcgtc ggcgataaaa tagccaacat
gtcgctgggt aagctggcca acgcctgata 1620atctgtgctg gcctgataaa
gcggaaacga aaaaagagac ggggaagcct gtctcttttc 1680ttattatgcg
gtttatgcgg ttacctggac cggttaatca tcgtcatcga tctggtacaa
1740acgcacattt tcccgttcat tcgcgtcgtt acgcgccaca atcgcgatgg
catcttcctc 1800gtcgctcaga ttgcgcggct gatggggaac gccgggtgga
atatagagaa actcgccggc 1860cagatggaga cacgtctgcg ataaatctgt
gccgtaacgt gtttctatcc gcccctttag 1920cagatagatt gcggtttcgt
aatcaacatg gtaatgcggt tccgcctgtg cgccggccgg 1980gatcaccaca
atattcatag aaagctgtct tgcacctacc gtatcgcggg agataccgac
2040aaaatagggc agtttttgcg tggtatccgt ggggtgttcc ggcctgacaa
tcttgagttg 2100gttcgtcatc atctttctcc atctgggcga cctgatcggt t
21413403PRTErwinia amylovora 3Met Ser Leu Asn Thr Ser Gly Leu Gly
Ala Ser Thr Met Gln Ile Ser1 5 10 15Ile Gly Gly Ala Gly Gly Asn Asn
Gly Leu Leu Gly Thr Ser Arg Gln 20 25 30Asn Ala Gly Leu Gly Gly Asn
Ser Ala Leu Gly Leu Gly Gly Gly Asn 35 40 45Gln Asn Asp Thr Val Asn
Gln Leu Ala Gly Leu Leu Thr Gly Met Met 50 55 60Met Met Met Ser Met
Met Gly Gly Gly Gly Leu Met Gly Gly Gly Leu65 70 75 80Gly Gly Gly
Leu Gly Asn Gly Leu Gly Gly Ser Gly Gly Leu Gly Glu 85 90 95Gly Leu
Ser Asn Ala Leu Asn Asp Met Leu Gly Gly Ser Leu Asn Thr 100 105
110Leu Gly Ser Lys Gly Gly Asn Asn Thr Thr Ser Thr Thr Asn Ser Pro
115 120 125Leu Asp Gln Ala Leu Gly Ile Asn Ser Thr Ser Gln Asn Asp
Asp Ser 130 135 140Thr Ser Gly Thr Asp Ser Thr Ser Asp Ser Ser Asp
Pro Met Gln Gln145 150 155 160Leu Leu Lys Met Phe Ser Glu Ile Met
Gln Ser Leu Phe Gly Asp Gly 165 170 175Gln Asp Gly Thr Gln Gly Ser
Ser Ser Gly Gly Lys Gln Pro Thr Glu 180 185 190Gly Glu Gln Asn Ala
Tyr Lys Lys Gly Val Thr Asp Ala Leu Ser Gly 195 200 205Leu Met Gly
Asn Gly Leu Ser Gln Leu Leu Gly Asn Gly Gly Leu Gly 210 215 220Gly
Gly Gln Gly Gly Asn Ala Gly Thr Gly Leu Asp Gly Ser Ser Leu225 230
235 240Gly Gly Lys Gly Leu Gln Asn Leu Ser Gly Pro Val Asp Tyr Gln
Gln 245 250 255Leu Gly Asn Ala Val Gly Thr Gly Ile Gly Met Lys Ala
Gly Ile Gln 260 265 270Ala Leu Asn Asp Ile Gly Thr His Arg His Ser
Ser Thr Arg Ser Phe 275 280 285Val Asn Lys Gly Asp Arg Ala Met Ala
Lys Glu Ile Gly Gln Phe Met 290 295 300Asp Gln Tyr Pro Glu Val Phe
Gly Lys Pro Gln Tyr Gln Lys Gly Pro305 310 315 320Gly Gln Glu Val
Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser 325 330 335Lys Pro
Asp Asp Asp Gly Met Thr Pro Ala Ser Met Glu Gln Phe Asn 340 345
350Lys Ala Lys Gly Met Ile Lys Arg Pro Met Ala Gly Asp Thr Gly Asn
355 360 365Gly Asn Leu Gln Ala Arg Gly Ala Gly Gly Ser Ser Leu Gly
Ile Asp 370 375 380Ala Met Met Ala Gly Asp Ala Ile Asn Asn Met Ala
Leu Gly Lys Leu385 390 395 400Gly Ala Ala41288DNAErwinia amylovora
4aagcttcggc atggcacgtt tgaccgttgg gtcggcaggg tacgtttgaa ttattcataa
60gaggaatacg ttatgagtct gaatacaagt gggctgggag cgtcaacgat gcaaatttct
120atcggcggtg cgggcggaaa taacgggttg ctgggtacca gtcgccagaa
tgctgggttg 180ggtggcaatt ctgcactggg gctgggcggc ggtaatcaaa
atgataccgt caatcagctg 240gctggcttac tcaccggcat gatgatgatg
atgagcatga tgggcggtgg tgggctgatg 300ggcggtggct taggcggtgg
cttaggtaat ggcttgggtg gctcaggtgg cctgggcgaa 360ggactgtcga
acgcgctgaa cgatatgtta ggcggttcgc tgaacacgct gggctcgaaa
420ggcggcaaca ataccacttc aacaacaaat tccccgctgg accaggcgct
gggtattaac 480tcaacgtccc aaaacgacga ttccacctcc ggcacagatt
ccacctcaga ctccagcgac 540ccgatgcagc agctgctgaa gatgttcagc
gagataatgc aaagcctgtt tggtgatggg 600caagatggca cccagggcag
ttcctctggg ggcaagcagc cgaccgaagg cgagcagaac 660gcctataaaa
aaggagtcac tgatgcgctg tcgggcctga tgggtaatgg tctgagccag
720ctccttggca acgggggact gggaggtggt cagggcggta atgctggcac
gggtcttgac 780ggttcgtcgc tgggcggcaa agggctgcaa aacctgagcg
ggccggtgga ctaccagcag 840ttaggtaacg ccgtgggtac cggtatcggt
atgaaagcgg gcattcaggc gctgaatgat 900atcggtacgc acaggcacag
ttcaacccgt tctttcgtca ataaaggcga tcgggcgatg 960gcgaaggaaa
tcggtcagtt catggaccag tatcctgagg tgtttggcaa gccgcagtac
1020cagaaaggcc cgggtcagga ggtgaaaacc gatgacaaat catgggcaaa
agcactgagc 1080aagccagatg acgacggaat gacaccagcc agtatggagc
agttcaacaa agccaagggc 1140atgatcaaaa ggcccatggc gggtgatacc
ggcaacggca acctgcaggc acgcggtgcc 1200ggtggttctt cgctgggtat
tgatgccatg atggccggtg atgccattaa caatatggca 1260cttggcaagc
tgggcgcggc ttaagctt 12885341PRTPseudomonas syringae 5Met Gln Ser
Leu Ser Leu Asn Ser Ser Ser Leu Gln Thr Pro Ala Met1 5 10 15Ala Leu
Val Leu Val Arg Pro Glu Ala Glu Thr Thr Gly Ser Thr Ser 20 25 30Ser
Lys Ala Leu Gln Glu Val Val Val Lys Leu Ala Glu Glu Leu Met 35 40
45Arg Asn Gly Gln Leu Asp Asp Ser Ser Pro Leu Gly Lys Leu Leu Ala
50 55 60Lys Ser Met Ala Ala Asp Gly Lys Ala Gly Gly Gly Ile Glu Asp
Val65 70 75 80Ile Ala Ala Leu Asp Lys Leu Ile His Glu Lys Leu Gly
Asp Asn Phe 85 90 95Gly Ala Ser Ala Asp Ser Ala Ser Gly Thr Gly Gln
Gln Asp Leu Met 100 105 110Thr Gln Val Leu Asn Gly Leu Ala Lys Ser
Met Leu Asp Asp Leu Leu 115 120 125Thr Lys Gln Asp Gly Gly Thr Ser
Phe Ser Glu Asp Asp Met Pro Met 130 135 140Leu Asn Lys Ile Ala Gln
Phe Met Asp Asp Asn Pro Ala Gln Phe Pro145 150 155 160Lys Pro Asp
Ser Gly Ser Trp Val Asn Glu Leu Lys Glu Asp Asn Phe 165 170 175Leu
Asp Gly Asp Glu Thr Ala Ala Phe Arg Ser Ala Leu Asp Ile Ile 180 185
190Gly Gln Gln Leu Gly Asn Gln Gln Ser Asp Ala Gly Ser Leu Ala Gly
195 200 205Thr Gly Gly Gly Leu Gly Thr Pro Ser Ser Phe Ser Asn Asn
Ser Ser 210 215 220Val Met Gly Asp Pro Leu Ile Asp Ala Asn Thr Gly
Pro Gly Asp Ser225 230 235 240Gly Asn Thr Arg Gly Glu Ala Gly Gln
Leu Ile Gly Glu Leu Ile Asp 245 250 255Arg Gly Leu Gln Ser Val Leu
Ala Gly Gly Gly Leu Gly Thr Pro Val 260 265 270Asn Thr Pro Gln Thr
Gly Thr Ser Ala Asn Gly Gly Gln Ser Ala Gln 275 280 285Asp Leu Asp
Gln Leu Leu Gly Gly Leu Leu Leu Lys Gly Leu Glu Ala 290 295 300Thr
Leu Lys Asp Ala Gly Gln Thr Gly Thr Asp Val Gln Ser Ser Ala305 310
315 320Ala Gln Ile Ala Thr Leu Leu Val Ser Thr Leu Leu Gln Gly Thr
Arg 325 330 335Asn Gln Ala Ala Ala 34061026DNAPseudomonas syringae
6atgcagagtc tcagtcttaa cagcagctcg ctgcaaaccc cggcaatggc ccttgtcctg
60gtacgtcctg aagccgagac gactggcagt acgtcgagca aggcgcttca ggaagttgtc
120gtgaagctgg ccgaggaact gatgcgcaat ggtcaactcg acgacagctc
gccattggga 180aaactgttgg ccaagtcgat ggccgcagat ggcaaggcgg
gcggcggtat tgaggatgtc 240atcgctgcgc tggacaagct gatccatgaa
aagctcggtg acaacttcgg cgcgtctgcg 300gacagcgcct cgggtaccgg
acagcaggac ctgatgactc aggtgctcaa tggcctggcc 360aagtcgatgc
tcgatgatct tctgaccaag caggatggcg ggacaagctt ctccgaagac
420gatatgccga tgctgaacaa gatcgcgcag ttcatggatg acaatcccgc
acagtttccc 480aagccggact cgggctcctg ggtgaacgaa ctcaaggaag
acaacttcct tgatggcgac 540gaaacggctg cgttccgttc ggcactcgac
atcattggcc agcaactggg taatcagcag 600agtgacgctg gcagtctggc
agggacgggt ggaggtctgg gcactccgag cagtttttcc 660aacaactcgt
ccgtgatggg tgatccgctg atcgacgcca ataccggtcc cggtgacagc
720ggcaataccc gtggtgaagc ggggcaactg atcggcgagc ttatcgaccg
tggcctgcaa 780tcggtattgg ccggtggtgg actgggcaca cccgtaaaca
ccccgcagac cggtacgtcg 840gcgaatggcg gacagtccgc tcaggatctt
gatcagttgc tgggcggctt gctgctcaag 900ggcctggagg caacgctcaa
ggatgccggg caaacaggca ccgacgtgca gtcgagcgct 960gcgcaaatcg
ccaccttgct ggtcagtacg ctgctgcaag gcacccgcaa tcaggctgca 1020gcctga
10267344PRTPseudomonas solanacearum 7Met Ser Val Gly Asn Ile Gln
Ser Pro Ser Asn Leu Pro Gly Leu Gln1 5 10 15Asn Leu Asn Leu Asn Thr
Asn Thr Asn Ser Gln Gln Ser Gly Gln Ser 20 25 30Val Gln Asp Leu Ile
Lys Gln Val Glu Lys Asp Ile Leu Asn Ile Ile 35 40 45Ala Ala Leu Val
Gln Lys Ala Ala Gln Ser Ala Gly Gly Asn Thr Gly 50 55 60Asn Thr Gly
Asn Ala Pro Ala Lys Asp Gly Asn Ala Asn Ala Gly Ala65 70 75 80Asn
Asp Pro Ser Lys Asn Asp Pro Ser Lys Ser Gln Ala Pro Gln Ser 85 90
95Ala Asn Lys Thr Gly Asn Val Asp Asp Ala Asn Asn Gln Asp Pro Met
100 105 110Gln Ala Leu Met Gln Leu Leu Glu Asp Leu Val Lys Leu Leu
Lys Ala 115 120 125Ala Leu His Met Gln Gln Pro Gly Gly Asn Asp Lys
Gly Asn Gly Val 130 135 140Gly Gly Ala Asn Gly Ala Lys Gly Ala Gly
Gly Gln Gly Gly Leu Ala145 150 155 160Glu Ala Leu Gln Glu Ile Glu
Gln Ile Leu Ala Gln Leu Gly Gly Gly 165 170 175Gly Ala Gly Ala Gly
Gly Ala Gly Gly Gly Val Gly Gly Ala Gly Gly 180 185 190Ala Asp Gly
Gly Ser Gly Ala Gly Gly Ala Gly Gly Ala Asn Gly Ala 195 200 205Asp
Gly Gly Asn Gly Val Asn Gly Asn Gln Ala Asn Gly Pro Gln Asn 210 215
220Ala Gly Asp Val Asn Gly Ala Asn Gly Ala Asp Asp Gly Ser Glu
Asp225 230 235 240Gln Gly Gly Leu Thr Gly Val Leu Gln Lys Leu Met
Lys Ile Leu Asn 245 250 255Ala Leu Val Gln Met Met Gln Gln Gly Gly
Leu Gly Gly Gly Asn Gln 260 265 270Ala Gln Gly Gly Ser Lys Gly Ala
Gly Asn Ala Ser Pro Ala Ser Gly 275 280 285Ala Asn Pro Gly Ala Asn
Gln Pro Gly Ser Ala Asp Asp Gln Ser Ser 290 295 300Gly Gln Asn Asn
Leu Gln Ser Gln Ile Met Asp Val Val Lys Glu Val305 310 315 320Val
Gln Ile Leu Gln Gln Met Leu Ala Ala Gln Asn Gly Gly Ser Gln 325 330
335Gln Ser Thr Ser Thr Gln Pro Met 34081035DNAPseudomonas
solanacearum 8atgtcagtcg gaaacatcca gagcccgtcg aacctcccgg
gtctgcagaa cctgaacctc 60aacaccaaca ccaacagcca gcaatcgggc cagtccgtgc
aagacctgat caagcaggtc 120gagaaggaca tcctcaacat catcgcagcc
ctcgtgcaga aggccgcaca gtcggcgggc 180ggcaacaccg gtaacaccgg
caacgcgccg gcgaaggacg gcaatgccaa cgcgggcgcc 240aacgacccga
gcaagaacga cccgagcaag agccaggctc cgcagtcggc caacaagacc
300ggcaacgtcg acgacgccaa caaccaggat ccgatgcaag cgctgatgca
gctgctggaa 360gacctggtga agctgctgaa ggcggccctg cacatgcagc
agcccggcgg caatgacaag 420ggcaacggcg tgggcggtgc caacggcgcc
aagggtgccg gcggccaggg cggcctggcc 480gaagcgctgc aggagatcga
gcagatcctc gcccagctcg gcggcggcgg tgctggcgcc 540ggcggcgcgg
gtggcggtgt cggcggtgct ggtggcgcgg atggcggctc cggtgcgggt
600ggcgcaggcg gtgcgaacgg cgccgacggc ggcaatggcg tgaacggcaa
ccaggcgaac 660ggcccgcaga acgcaggcga tgtcaacggt gccaacggcg
cggatgacgg cagcgaagac 720cagggcggcc tcaccggcgt gctgcaaaag
ctgatgaaga tcctgaacgc gctggtgcag 780atgatgcagc aaggcggcct
cggcggcggc aaccaggcgc agggcggctc gaagggtgcc 840ggcaacgcct
cgccggcttc cggcgcgaac ccgggcgcga accagcccgg ttcggcggat
900gatcaatcgt ccggccagaa caatctgcaa tcccagatca tggatgtggt
gaaggaggtc 960gtccagatcc tgcagcagat gctggcggcg cagaacggcg
gcagccagca gtccacctcg 1020acgcagccga tgtaa 1035926PRTXanthomonas
campestris pv. glycines 9Thr Leu Ile Glu Leu Met Ile Val Val Ala
Ile Ile Ala Ile Leu Ala1 5 10 15Ala Ile Ala Leu Pro Ala Tyr Gln Asp
Tyr 20 251020PRTXanthomonas campestris pv. pelargonii 10Ser Ser Gln
Gln Ser Pro Ser Ala Gly Ser Glu Gln Gln Leu Asp Gln1 5 10 15Leu Leu
Ala Met 20
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