U.S. patent application number 09/876202 was filed with the patent office on 2001-11-29 for disease resistance in vitis.
Invention is credited to Gray, Dennis J., Scorza, Ralph.
Application Number | 20010047522 09/876202 |
Document ID | / |
Family ID | 21799347 |
Filed Date | 2001-11-29 |
United States Patent
Application |
20010047522 |
Kind Code |
A1 |
Scorza, Ralph ; et
al. |
November 29, 2001 |
Disease resistance in vitis
Abstract
Disclosed are methods for producing transgenic grape plants
having resistance to bunch rot, powdery mildew, and downy mildew.
Also disclosed are grape plants having resistance to bunch rot,
powdery mildew, and downy mildew, wherein the plants express a
Shiva lytic peptide.
Inventors: |
Scorza, Ralph;
(Sheperdstown, WV) ; Gray, Dennis J.;
(Howey-in-the-Hills, FL) |
Correspondence
Address: |
CLARK & ELBING LLP
176 FEDERAL STREET
BOSTON
MA
02110-2214
US
|
Family ID: |
21799347 |
Appl. No.: |
09/876202 |
Filed: |
June 6, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09876202 |
Jun 6, 2001 |
|
|
|
09511606 |
Feb 23, 2000 |
|
|
|
09511606 |
Feb 23, 2000 |
|
|
|
09452577 |
Dec 1, 1999 |
|
|
|
09452577 |
Dec 1, 1999 |
|
|
|
08878750 |
Jun 19, 1997 |
|
|
|
6232528 |
|
|
|
|
60020569 |
Jun 26, 1996 |
|
|
|
Current U.S.
Class: |
800/279 ;
435/419; 435/469; 435/470; 800/288; 800/293; 800/294; 800/298;
800/301 |
Current CPC
Class: |
C12N 15/8282 20130101;
C12N 2770/00022 20130101; C07K 14/005 20130101; C12N 15/8281
20130101; A01H 6/88 20180501; C12N 15/8205 20130101; C12N
2770/18022 20130101; C12N 15/8283 20130101 |
Class at
Publication: |
800/279 ;
435/419; 800/298; 800/288; 800/301; 800/294; 800/293; 435/469;
435/470 |
International
Class: |
A01H 005/00; C12N
015/82; C12N 015/84 |
Claims
1. A method for producing a transgenic grape cell with resistance
to a fungal pathogen, said method comprising: transforming a grape
cell with a nucleic acid molecule which expresses a lytic peptide,
where expression of said lytic peptide provides resistance to a
fungal pathogen that causes bunch rot, powdery mildew, or downy
mildew.
2. The method of claim 1, further comprising propagating a grape
plant from said transformed grape cell.
3. The method of claim 1, wherein said grape cell is part of a
somatic embryo.
4. The method of claim 3, further comprising propagating a grape
plant from said somatic embryo.
5. The method of claim 1, wherein said transformation comprises
infecting said grape cell with Agrobacterium tumefaciens comprising
said nucleic acid molecule which expresses said lytic peptide.
6. The method of claim 1, wherein said transformation comprises
bombarding said grape cell with microprojectiles comprising said
nucleic acid molecule which expresses said lytic peptide.
7. The method of claim 1, wherein said transformation comprises
bombarding said grape cell with microprojectiles, followed by
infecting said bombarded cells with Agrobacterium tumefaciens
comprising said nucleic acid molecule which expresses said lytic
peptide.
8. the method of claim 1, wherein said lytic peptide is
Shiva-1.
9. The method of claim 1, wherein said grape cell is a Vitis
vinifera `Thompson Seedless` grape cell.
10. The method of claim 1, wherein said grape cell is a scion
cell.
11. The method of claim 1, wherein said grape cell is a rootstock
cell.
12. The method of claim 1, wherein expression of said lytic peptide
provides resistance to bunch rot.
13. The method of claim 1, wherein expression of said lytic peptide
provides resistance to powdery mildew.
14. The method of claim 1, wherein expression of said lytic peptide
provides resistance to downy mildew.
15. A grape cell comprising a nucleic acid molecule which expresses
a lytic peptide, wherein expression of said lytic peptide provides
resistance to a fungal pathogen that causes bunch rot, powdery
mildew, or downy mildew.
16. The cell of claim 15, wherein said nucleic acid molecule
comprises an expression vector.
17. The cell of claim 15, wherein said cell is a scion cell.
18. The cell of claim 15, wherein said cell is a rootstock
cell.
19. The cell of claim 15, wherein said cell is a somatic embryo
cell.
20. The cell of claim 15, wherein said lytic peptide is
Shiva-1.
21. The cell of claim 15, wherein said grape cell is a Vitis
vinifera `Thompson Seedless` grape cell.
22. The cell of claim 15, wherein expression of said lytic peptide
provides resistance to bunch rot.
23. The cell of claim 15, wherein expression of said lytic peptide
provides resistance to powdery mildew.
24. The cell of claim 15, wherein expression of said lytic peptide
provides resistance to downy mildew.
25. A grape plant comprising a nucleic acid molecule which
expresses a lytic peptide, wherein expression of said lytic peptide
provides resistance to a fungal pathogen that causes bunch rot,
powdery mildew, or downy mildew.
26. The plant of claim 25, wherein said grape plant is a scion.
27. The plant of claim 25, wherein said transgenic plant is a
rootstock.
28. The plant of claim 25, wherein said grape plant is a somatic
embryo.
29. The transgenic plant of claim 25, wherein said grape plant is
Vitis vinifera `Thompson Seedless.`
30. The plant of claim 25, wherein said nucleic acid molecule is
expressed from an expression vector.
31. The plant of claim 25, wherein said lytic peptide is
Shiva-1.
32. The plant of claim 25, wherein expression of said lytic peptide
provides resistance to bunch rot.
33. The plant of claim 25, wherein expression of said lytic peptide
provides resistance to powdery mildew.
34. The plant of claim 25, wherein expression of said lytic peptide
provides resistance to downy mildew.
35. A scion from a plant of claim 32.
36. A scion from a plant of claim 33.
37. A scion from a plant of claim 34.
38. A rootstock from a plant of claim 32.
39. A rootstock from a plant of claim 33.
40. A rootstock from a plant of claim 34.
41. A somatic embryo from a plant of claim 32.
42. A somatic embryo from a plant of claim 33.
43. A somatic embryo from a plant of claim 34.
44. A cell from a plant of claim 32.
45. A cell from a plant of claim 33.
46. A cell from a plant of claim 34.
47. A somatic embryo from a plant of claim 32.
48. A somatic embryo from a plant of claim 33.
49. A somatic embryo from a plant of claim 34.
50. A seed from a plant of claim 32.
51. A seed from a plant of claim 33.
52. A seed from a plant of claim 34.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims benefit from provisional application
60/020,569, filed Jun. 26, 1996.
BACKGROUND OF THE INVENTION
[0002] This application relates to disease resistance in Vitis.
[0003] Grape (Vitis spp.) is a deciduous temperate fruit crop of
ancient origin. Grape production (65.times.10.sup.6 metic tons)
exceeds that of any other temperate fruit crop and ranks after
Citrus and banana among all fruit crops worldwide (FAO Production
Yearbook, 1990). Grape surpasses all other fruit crops in value due
to its multiple uses for fresh fruit, juice, jelly, raisins, and
wine. For example, in the United States, seedless grapes represent
about 80% and 98% of the total table and raisin grape production,
respectively (In: 1994-95: The Distribution and Per Capita
Consumption of California Table Grapes By Major Varieties in the
United States and Canada, California Table Grape Commission,
Fresno, Calif. 1995). Only a few seedless cultivars make up this
production, of which `Thompson Seedless` is the most important.
This cultivar accounts for the most production of any single grape
variety in the United States. In 1992, `Thompson Seedless` was
grown on 263,621 acres in California (In: California Grape Acreage,
California Agricultural Statistics Service, Sacramento, Calif.,
1993). Thirty-five percent of the table grape production in
California in 1994 was `Thompson Seedless` (23,244,683 boxes, 10
kg/box). In 1993, 97% of the grapes grown for raisin production was
`Thompson Seedless` (In: Raisin Committee Marketing Policy 1994-95,
Raisin Administrative Committee, Fresno, Calif., 1994).
[0004] Although Vitis spp. is generally considered to have
desirable fruit quality, it is susceptible to many pests and
diseases, including anthracnose, black rot, botrytis bunch rot,
crown gall, downy mildew, eutypa dieback, various nematodes,
phomopsis cane and leaf spot, phylloxera, Pierce's disease, and
powdery mildew. Hybridization with resistant species has been the
only method available to produce resistant cultivars (Galet and
Morton, In: Compendium of Grape Diseases, R. C. Pearson and A. C.
Goheen, eds., APS Press, St. Paul, 1990, pp. 2-3). While improving
grape is possible by conventional breeding, it is difficult and
time consuming due to the two- to three-year generation cycle, the
long period of time required for reliable progeny testing and
selection, and inbreeding depression that prohibits selfing (Gray
and Meredith, In: Biotechnology of Perennial Fruit Crops, F. A.
Hammerschlag and R. E. Litz, eds., C.A.B. Intl., Wallingford, U.K.
1992). These characteristics make introgression of desirable traits
into existing grape cultivars difficult if not impossible to
achieve in an individual breeder's lifetime. Thus, the alternative,
and potentially less time-consuming, approach of using gene
transfer to insert desirable genes is one approach for improving
grapevine cultivars, even considering the time necessary for field
testing transgenic lines. The ability to improve the disease or
pest resistance or both of a major grape cultivar (e.g., `Thompson
Seedless`) offers the possibility of improving a large portion of
the grape production in a relatively short time, assuming that
cultivar integrity would not be compromised by the transgene or the
insertion event. Such a change could also reduce pesticide use for
a significant portion of grape production.
SUMMARY OF THE INVENTION
[0005] In one aspect, the invention features a method for producing
a transgenic plant of the genus Vitis having resistance to a plant
pathogen. The method, in general, includes the step of transforming
a plant cell with a nucleic acid which expresses a lytic peptide,
where the expression of such a lytic peptide provides resistance to
a plant pathogen. In preferred embodiments, the method further
includes propagating a grape plant from the transformed plant cell.
In other preferred embodiments, the method involves transforming a
plant cell that is a part of a somatic embryo and propagating or
regenerating a transgenic grape plant from the transformed somatic
embryo. Expression of the lytic peptide confers disease resistance
or tolerance or both to grapevine pathogens and pests including,
without limitation, bacterial, fungal, and viral pathogens.
[0006] In general, Vitis is transformed by introducing into a plant
cell or somatic or zygotic embryos a nucleic acid that includes a
lytic peptide by using A. tumefaciens, microprojectile bombardment,
or any combination of these methods (for example, by bombarding the
plant cell with microprojectiles, followed by infecting the
bombarded cells with Agrobacterium tumefaciens including a nucleic
acid which expresses the lytic peptide).
[0007] In preferred embodiments, the method of the invention
involves the use of the lytic peptides Shiva-1 or cecropin B or
both.
[0008] The methods of the invention are useful for providing
disease resistance or tolerance or both to a variety of grape
plants (for example, Vitis spp., Vitis spp. hybrids, and all
members of the subgenera Euvitis and Muscadinia), including scion
or rootstock cultivars. Exemplary scion cultivars include, without
limitation, those which are referred to as table or raisin grapes
and those used in wine production such as Cabernet Franc, Cabernet
Sauvignon, Chardonnay (e.g., CH 01, CH 02, CH Dijon), Merlot, Pinot
Noir (PN, PN Dijon), Semillon, White Riesling, Lambrusco, Thompson
Seedless, Autumn Seedless, Niagrara Seedless, and Seval Blanc.
Rootstock cultivars that are useful in the invention include,
without limitation, Vitis rupestris Constantia, Vitis rupestris St.
George, Vitis california, Vitis girdiana, Vitis rotundifolia, Vitis
rotundifolia Carlos, Richter 110 (Vitis berlandieri x rupestris),
101-14 Millarder et de Grasset (Vitis riparia x rupestris), Teleki
5C (Vitis berlandieri x riparia), 3309 Courderc (Vitis riparia x
rupestris), Riparia Gloire de Montpellier (Vitis riparia), 5BB
Teleki (selection Kober, Vitis berlandieri x riparia), SO.sub.4
(Vitis berlandieri x rupestris), 41B Millardet (Vitis vinifera x
berlandieri), and 039-16 (Vitis vinifera x Muscadinia).
[0009] In another aspect, the invention features a transgenic plant
or plant cell of the genus Vitis transformed with a nucleic acid
which expresses a lytic peptide, wherein expression of the lytic
peptide provides resistance to a plant pathogen. In preferred
embodiments, the transgenic grapevine or cell with the nucleic acid
includes an expression vector. Preferably, the transgenic grapevine
or cell is Vitis vinifera `Thompson Seedless` and the expression of
the lytic peptide provides resistance to the bacterium Xylella
fastidiosa, the causative agent of Pierce's Disease. In other
preferred embodiments, the transgenic grapevine is a somatic
embryo, a scion, or a rootstock.
[0010] In still another aspect, the invention features a method of
transforming Vitis with a nucleic acid which expresses a tomato
ringspot virus coat protein (TomRSV-CP) gene, where the expression
of such a coat protein gene provides resistance to a plant
pathogen.
[0011] In still another aspect, the invention features a method of
transforming Vitis with a nucleic acid which expresses a TomRSV-CP
gene and a lytic peptide gene, where the expression of such genes
in a grapevine provides resistance to a plant pathogen.
[0012] The invention also features scions, rootstocks, somatic or
zygotic embryos, cells, or seeds that are produced from any of the
transgenic grape plants described herein.
[0013] By "lytic peptide" is meant a gene encoding a polypeptide
capable of lysing a cell. Exemplary lytic peptides include, without
limitation, apidaceins, attacins, cercropins (e.g., cercropin B),
caerulins, bombinins, lysozyme, magainins, melittins, sapecin,
sarcotoxins, and xenopsins.
[0014] By "peptide" is meant any chain of amino acids, regardless
of length or post-translational modification (for example,
glycosylation or phosphorylation).
[0015] By "positioned for expression" is meant that the DNA
molecule is positioned adjacent to a DNA sequence which directs
transcription and translation of the sequence (i.e., facilitates
the production of, for example, a lytic peptide).
[0016] By "operably linked" is meant that a gene and a regulatory
sequence(s) are connected in such a way as to permit gene
expression when the appropriate molecules (for example,
transcriptional activator proteins) are bound to the regulatory
sequence(s).
[0017] By "plant cell" is meant any self-propagating cell bounded
by a semi-permeable membrane and containing a plastid. Such a cell
also requires a cell wall if further propagation is desired. A
plant cell, as used herein, is obtained from, without limitation,
seeds, suspension cultures, embryos, meristematic regions, callus
tissue, leaves, roots, shoots, somatic and zygotic embryos, as well
as any part of a reproductive or vegetative tissue or organ.
[0018] By "transgenic" is meant any cell which includes a DNA
sequence which is inserted by artifice into a cell and becomes part
of the genome of the organism which develops from that cell. As
used herein, the transgenic organisms are generally transgenic
grapevines and the DNA (transgene) is inserted by artifice into the
nuclear or plastidic genome. A transgenic grapevine according to
the invention contains at least one lytic peptide or TomRSV-CP or
both.
[0019] By "transgene" is meant any piece of DNA which is inserted
by artifice into a cell, and becomes part of the genome of the
organism which develops from that cell. Such a transgene may
include a gene which is partly or entirely heterologous (i.e.,
foreign) to the transgenic organism, or may represent a gene
homologous to an endogenous gene of the organism.
[0020] As discussed above, we have discovered that the expression
of a lytic peptide provides grapevines with resistance against
disease caused by plant pathogens and pests. Accordingly, the
invention provides a number of important advances and advantages
for viticulturists. For example, by demonstrating that the lytic
peptide Shiva-1 is effective against Xyellela fastidiosa, the
invention facilitates an effective and economical means for
protection against Pierce's Disease. Such protection reduces or
minimizes the need for traditional chemical practices that are
typically used by viticulturists for controlling the spread of
plant pathogens and providing protection against disease-causing
pathogens in vineyards. In addition, because grape plants
expressing one or more lytic peptide gene(s) described herein are
less vulnerable to pathogens and their diseases, the invention
further provides for increased production efficiency, as well as
for improvements in quality, color, flavor, and yield of grapes.
Furthermore, because the invention reduces the necessity for
chemical protection against plant pathogens, the invention benefits
the environment where the crops are grown. In addition, the
expression of a lytic peptide gene or TomRSV-CP or both in a
grapevine provides resistance to plant pathogens and can be used to
protect grapevines from pathogen infestation that reduces
productivity and viability. The methods of the invention are useful
for producing grapevines having resistance to diseases including,
without limitation, Pierce's disease, crown gall, bunch rot, downy
and powdery mildews, and viral diseases caused by arabis mosaic
virus, grapevine fanleaf virus, tomato ringspot virus, grapevine
leafroll associated virus.
[0021] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
DETAILED DESCRIPTION
[0022] The drawings will first be described.
DRAWINGS
[0023] FIG. 1A shows the partial map of the T-DNA region of
pBRS1.
[0024] FIG. 1B shows the partial map of the T-DNA region of
pGA482GG/cpTomRSV.
[0025] FIG. 2 is a photograph showing the results of PCR amplified
TomRSV-CP and Shiva-1 fragments from transgenic `Thompson Seedless`
grape plants. PCR analysis using TomRSV-CP primers are as follows:
pGA482GG transformant (without the TomRSV-CP gene); lane 2,
transformant 3-2; lane 3, transformant 3-3; lane 4, transformant
3S-2; lane 5, transformant 3S-6; lane 6, transformant 3SB-X. PCR
analysis using Shiva-1 primers are as follows: lane 7,
untransformed `Thompson Seedless` plant; lane 8, transformant 4-3;
lane 9, transformant 4S-2. Transgenic plants 3-2, 3-3, and 4-3,
were obtained from A. tumefaciens infection alone. Plants 3S-2,
3S-3, 3SB-X, and 4S-2 were obtained from A. tumefaciens infection
after microprojectile bombardment.
[0026] FIG. 3 is a photograph showing the results of a Southern
analysis of transgenic `Thompson Seedless` grape plants. DNA
extracted from TomRSV-CP transformants that was digested with EcoRI
and probed with a NOS/NPTII fragment is shown in lanes 1 through 9.
Lane 1, pGA482GG transformant (control without the TomRSV-CP gene);
lane 2, transformant 3-2; lane 3, transformant 3-3 from tissue
culture; lane 4, transformant 3-3 from greenhouse leaves (DNA runs
slower on gel); lane 5, transformant 3S-2; lane 6, transformant
3S-3; lane 7, transformant 3SB-X; lane 8, untransformed control
`Thompson Seedless`; lane 9, pGA482GG/cpTomRSV plasmid. Shiva-1
transformants digested with BamHI and probed with a NOS/NPTII
fragment are shown in lanes a-c. Lane a, transformant 4-3; lane b,
transformant 4S-2; lane c, untransformed control `Thompson
Seedless`. Transgenic plants 3-2, 3-3, and 4-3 were obtained from
A. tumefaciens infection alone. Plants 3S-2, 3S-3, 3SB-X, and 4S-2
were obtained from A. tumefaciens infection after microprojectile
bombardment.
[0027] FIG. 4 is a photograph showing a transgenic `Thompson
Seedless` grape plant four months after transfer to the
greenhouse.
[0028] A description for the production of disease resistant
transgenic Vitis now follows. Transgenic grape plants expressing
either lytic peptide or TomRSV coat protein genes were regenerated
from somatic embryos derived from leaves of in vitro-grown plants
of `Thompson Seedless` grape (Vitis vinifera L.) plants. Somatic
embryos were either exposed directly to engineered A. tumefaciens
or they were bombarded twice with 1-.mu.m gold particles and then
exposed to A. tumefaciens. Somatic embryos were transformed with
either the lytic peptide Shiva-1 gene or the tomato ringspot virus
coat protein (TomRSV-CP) gene. Integration of the foreign genes
into these grapevines was verified by growth in the presence of
kanamycin (kan), positive .beta.-glucuronidase (GUS) and polymerase
chain-reaction (PCR) assays, and Southern analysis. Resistance to
Pierce's disease in transgenic plants expressing a lytic peptide
was also examined. These examples are provided for the purpose of
illustrating the invention, and should not be construed as
limiting.
Plant Materials and Culture
[0029] Leaves from `Thompson Seedless` in vitro cultures were used
to produce somatic embryos following the method of Stamp et al. (J.
Amer. Hort. Sci. 115:1038-1042, 1990). Expanding leaves
(approximately 0.5 cm long) excised from in vitro-grown shoots were
cultured on a modified Nitsch and Nitsch (Science 163:85-87, 1969)
(NN) medium containing 5 .mu.M of 2,4-D, 1 .mu.M of BA, 60
grams/liter of sucrose, 2 grams/liter of activated charcoal, and 7
grams/liter of agar, pH 5.7. After a three- to twelve-week culture
period, somatic embryos formed. These were transferred to a
modified Murashige and Skoog (Plant Physiol. 15:473-497, 1962) (MS)
medium containing 120 grams/liter of sucrose, 2 grams/liter of
activated charcoal, and 7 grams/liter of agar, pH 5.7. After three
years of continual culture on the modified MS medium with transfers
each four to six weeks, somatic embryos were transferred to
Emershad and Ramming proliferation (ERP) medium (Emershad and
Ramming, Plant Cell Rpt. 14:6-12, 1994) for several transfers and
then exposed to transformation treatments.
Agrobacterium Strain and Plasmid Descriptions
[0030] For the transformation treatments described below, A.
tumefaciens strains were EHA101 and EHA105 (Hood et al., J.
Bacterial. 168:1283-1290, 1986) containing plasmid
pGA482GG/cpTomRSV (Slightom, Gene 100:252-255, 1991; Slightom et
al., In: Plant Mol. Biol. Man., S. B. Gelivn, R. A. Schilperoot,
and D. P. S. Verma, eds., Kluwer, Dordrecht, The Netherlands) or
pBPRS1, respectively, were used (FIGS. 1A-1B). Both plasmids
contained chimeric gusA (.beta.-glucuronidase (GUS)) and kanamycin
(Kan) (neomycin phosphotransferase II (NPT II)) genes. Plasmid
pGA482GG/cpTomRSV contained the tomato ringsport virus coat protein
(TomRSV-CP) gene and pBRPS contained the Shiva-1 lytic peptide gene
(Destefano-Beltran et al., In: The Molecular and Cellular Biology
of the Potato, M. Vayada and W. Parks, eds., C.A.B. Int'l
Wallingford, U.K.; Jaynes et al., Acta Hort. 336:33-39, 1993).
Cocultivation and Selection
[0031] Putative A. tumefaciens transformants were cocultivated and
selected as described by Scorza et al. (Plant Cell Rpt. 14:589-592,
1995). Briefly, A. tumefaciens cultures were grown overnight at
28.degree. C. in LB medium containing selective antibiotics for
each plasmid. These cultures were centrifuged (5,000.times.g, 10
minutes) and resuspended in a medium consisting of MS salts
containing 20 grams/liter of sucrose, 100 .mu.M of acetosyringone,
and 1.0 .mu.M of betaine phosphate. The cultures were then shaken
for about six hours at 20.degree. C. before use in the
transformation treatments that are described below.
Transformation
[0032] Somatic embryos were either bombarded with gold
microprojectiles and then exposed to A. tumefaciens as described by
Scorza et al. (J. Amer. Soc. Hort. Sci. 119:1091-1098, 1994) or
they were exposed to A. tumefaciens without prior bombardment as
follows. Microprojectile bombardment was accomplished using the
Biolistic PDS-1000/He device (Bio-Rad Laboratories). A total of 700
somatic embryos were separated into groups of 100. Each group was
placed onto a 25-mm polycarbonate membrane in the center of a
100-mm petri plate containing ERP medium twenty-four hours before
bombardment. Somatic embryos were shot with 1.0-.mu.m diameter gold
particles following the general procedures of Sanford et al. (Meth.
Enzmol. 217:483-509, 1991) using the parameters described by Scorza
et al. (Plant Cell Rpt. 14:589-592, 1995). All plates were
bombarded twice. Within two hours of bombardment, embryos were
cocultivated with A. tumefaciens. After bombardment, somatic
embryos were immersed in the resuspended A. tumefaciens culture
that was prepared as described above. After fifteen to twenty
minutes, the A. tumefaciens culture medium was removed and somatic
embryos were placed onto cocultivation medium (ERP medium
containing 100 .mu.m acetosyringone). Somatic embryos were
cocultivated for two days and then washed with liquid ERP medium
(without charcoal) containing 300 .mu.g/ml of cefotaxime and 200
.mu.g/ml of carbenicillin. Somatic embryos were then plated on
agar-solidified ERP medium (0.75% agar) with the above-mentioned
selective antibiotics. All somatic embryo cultures were allowed to
proliferate for two passages (3 weeks each) before being placed
onto selection medium. Selection was carried out on ERP medium
containing the above specified amounts of cefotaxime and
carbenicillin, and 40 .mu.g/ml of kanamycin.
[0033] In a second series of transformation experiments, an
additional 700 somatic embryos were exposed to A. tumefaciens
without prior bombardment according to the methods described
above.
[0034] After cocultivation and selection on ERP medium, putatively
transformed embryos were induced to germinate and root on woody
plant medium (Lloyd and McCown, Proc. Intl. Plant Prop. Soc.
30:421-427, 1981) containing 15 grams/liter of sucrose, 1 .mu.M of
BA, 3 grams/liter of agar, pH 6.0 following the protocol of
Emershad and Ramming (Plant Cell Rpt. 14:6-12, 1994).
Transformation Confirmation
[0035] Transformed somatic embryos and shoots produced after
somatic embryo germination were assayed by growth on
kanamycin-containing medium and through a histological GUS assay
(Jefferson, Plant Mol. Biol. Rpt. 5:387-405, 1987). Leaf samples of
the plants surviving kanamycin selection were observed to produce
the characteristic blue GUS positive reaction, indicating the
presence and activity of the GUS gene in these plants. Leaves from
untransformed control plants showed no blue staining.
[0036] Leaves sampled from plants growing in vitro were also
cultured for one week in liquid LB medium to assay for the presence
of contaminating A. lumefaciens. Excised leaves from putative
transformants cultured in liquid LB medium were negative for the
presence of contaminating A. tumefaciens.
[0037] After rooting and transfer to the greenhouse, transformed
plants were subjected to PCR and Southern analysis. PCR
amplification was conducted on DNA isolated from leaves of
putatively transformed grape plants. Specific oligonucleotide
primers from TomnRSV-CP and Shiva-1 gene sequences were used to
identify the presence of these genes in DNA from the different
clones. For the TomRSV-CP gene, these sequences were the 5' primer
5'-GGTTCAGGGCGGGTCCTGGAAG-3' (SEQ ID NO: 1) and 3' primer
5'-GTAAAAGCTAATTAAGAGGCCACC-3' (SEQ ID NO: 2); for Shiva-1 gene,
the sequences were the 5' primer 5'-ATCAAACAGGGTATCCTGCG-3' (SEQ ID
NO: 3) and 3' primer 5'-TTCCCACCAACGCTGATC-3' (SEQ ID NO: 4). PCR
reactions were run using the GeneAmp kit components (Perkin Elmer,
Norwalk, Conn.) using the following parameters: 1 minute at
94.degree. C., 1.5 minutes at 65.degree. C., and 2 minutes at
72.degree. C. The first cycle used an additional 3 minutes melt at
95.degree. C. and the last five cycles had a 4 minute extension
time period at 72.degree. C. After thirty-five amplification
cycles, the PCR products were analyzed by agarose gel
electrophoresis and stained with ethidium bromide. PCR analysis
using TomRSV-CP and Shiva-1 primers indicated that the thirteen
plants that survived kanamycin selection after being exposed to
TomRSV-CP or Shiva-1 transformation treatments contained the
predicted gene sequences (FIG. 2).
[0038] In addition, Southern analysis was used to demonstrate the
incorporation of the foreign genes into the grape genome. Southern
analysis was carried out using a PCR-generated 1.1 -kb NOS/NPTII
probe. Digestion with EcoRI was then used to test for unique
insertion events that would include segments of grape DNA in
pGA482GG/cpTomRSV transformants. BamHI restriction digestion was
used for the pBPRS1 (Shiva-1) transformants. Extraction of DNA from
transformants followed the procedures of Callahan et al. (Plant
Physiol. 100:482-488, 1992). Conditions for Southern analysis were
described by Scorza et al. (In Vitro Cell Dev. Biol. 26:829-834,
1990). The NOS/NPTII probe was radioactively labeled according to
standard methods using random primers according to the instructions
with the BioRad Random Primer DNA Labeling Kit (BioRad, Hercules,
Calif.).
[0039] While Southern analysis directly showed only the
incorporation of the NPTII gene into the genomes of the assayed
grape plants, the close linkage of the TomRSV-CP or the Shiva-1
genes to the NPTII gene coupled with the positive PCR assays for
the presence of these genes leads to the conclusion that these
plants also contained the TomRSV or Shiva-1 genes. This analysis
also indicated that most TomRSV-CP transformants contained multiple
copies of the gene insert. Shiva-1 transformants, however, appeared
to contain a single insert. Plasmid pGA482GG was used for
transferring the TomRSV-CP gene. Previous work using plasmid
pGA482GG for transforming grape and other species suggested that
multiple copy transformants are common (Scorza et al., J. Amer.
Soc. Hort. Sci. 119:1091-1098, 1994; Scorza et al., Plant Cell Rpt.
14:589-592, 1995).
[0040] Previous work examined the use of microprojectile
bombardment with A. tumefaciens to produce transgenic grape plants.
Here we used both microprojectile bombardment and A. tumefaciens
infection. Although microprojectile bombardment before A.
tumefaciens infection improved the yield of transformants, the
numbers of transformants obtained in this study were too low to be
compared with infection with A. tumefaciens infection alone. It is
apparent, however, that both microprojectile bombardment followed
by exposure to A. tumefaciens and A. tumefaciens infection alone
are effective for transforming grape somatic embryos. The overall
transformation rate in terms of transgenic plants produced per
somatic embryo treated was about 1% (Table 1).
1TABLE 1 Somatic Putative Treatment Embryos Tranformants
Transformation Agrobacterium tumefaciens alone Control plasmid 100
1 1.00 TomRSV-CP 300 2 0.67 Shiva-1 300 2 0.67 Particle bombardment
plus A. tumefaciens Control plasmid 100 1 1.00 TomRSV-CP 300 7 2.30
Shiva-1 300 2 0.67
[0041] The results described here differ from our previous report
in that we now report transforming grape from somatic embryos
derived from leaves, while previously we reported producing
transgenic plants from somatic embryos derived from zygotic
embryos. The genes transferred include a viral coat protein gene
and a lytic peptide gene. To date there have been few reports of
transgenic grapevine production, and our results document the
successful transformation of a major Vitis vinifera scion
cultivar.
Disease Resistance
[0042] Resistance to Pierce's Disease (PD) has been evaluated in
transgenic `Thompson Seedless` grapevines expressing the lytic
peptide Shiva-1. PD is a fatal disease of grapevine known
throughout the world. PD kills grapevines by blocking the plant's
water-transporting tissue, the xylem. The disease is caused by the
bacterium, Xyllella fastidiosa, and is spread by a leafhopper, the
blue-green sharpshooter that feeds on the xylem fluid of grape. The
sharpshooter transmits the bacteria from vine to vine. As the
bacteria multiply inside the plant, they plug the xylem vessels,
inhibiting water and nutrient transport throughout the plant.
Infected vines die for reasons related to water uptake. The
symptoms of PD therefore resemble those of water stress and include
the drying, marginal burning, or scorching of leaves due to initial
clogging of fine vessel elements, and eventual dieback of the vine
due to total occlusion of the vessels in the trunk. Other symptoms
include the shriveling and dying of fruit clusters.
[0043] Three transgenic grapevines expressing the Shiva-1 construct
have been evaluated for resistance to X fastidiosa. These included
a non-transformed control; a transformed grapevine containing one
Shiva-1 insert (designated clone B); and a transgenic grapevine
containing four Shiva-1 inserts (designated clone A). Each of these
plants were vegetatively propagated and then inoculated with X
fastidiosa according to the methods described by Hopkins
(Phytopathology 75:713-717, 1985). While replicate plants of all
three clones eventually succumbed to PD, clone A was observed to
exhibit milder PD symptomology, which did not include the typical
signs of marginal leaf burn when compared to the non-transformed
control plant. Instead the leaves of clone A slowly became
chlorotic, without signs of marginal burn. A second series of
inoculations were performed with the same results. In addition, the
growth of bacteria in the transgenic clones was evaluated and
compared to the non-transformed control plant. Although bacteria
were eventually found in the leaves of both transgenic and
non-transformed plants, the spread of bacteria was slower in clone
A. Our results therefore indicate that transgenic grapevine
expressing the lytic peptide Shiva-1 are effective at inhibiting
PD.
[0044] The methods of the invention are also useful for providing
resistance to other grapevine diseases. Transgenic grapevines
expressing a transgene containing a lytic peptide (e.g., Shiva-1 or
cecropin B) or TomRSV-CP or both are operably linked to a
constitutive promoter or to a controllable promoter such as a
tissue-specific promoter, cell-type specific promoter, or to a
promoter that is induced by an external signal or agent such as a
pathogen- or wound-inducible control element, thus limiting the
temporal or tissue expression or both. Such transgenes may also be
expressed in roots, leaves, or fruits, or at a site of a grapevine
that is susceptible to pathogen penetration and infection. For
example, a lytic peptide gene may be engineered for constitutive
low level expression in xylem-tissue expression and then
transformed into a Vitis host plant. To achieve pathogen resistance
or disease resistance or both, it is important to express the
transgene at an effective level. Evaluation of the level of
pathogen protection conferred to a plant by expression of such a
transgene is determined according to conventional methods and
assays as described herein.
[0045] In one working example, expression of a lytic peptide (e.g.,
Shiva-1 or cecropin B) is used to control bacterial infection, for
example, to control Agrobacterium, the causative agent of crown
gall disease. Specifically, the Shiva-1 expression vector described
herein or a plant expression vector containing the cecropin B gene
is used to transform somatic embryos according to the methods
described above. To assess resistance to Agrobacterium infection
and crown gall formation, transformed plants and appropriate
controls are grown, and the stems are inoculated with a suspension
of Agrobacterium according to standard methods. Transformed grape
plants are subsequently incubated in a growth chamber, and the
inoculated stems are analyzed for signs of resistance to crown gall
formation according to standard methods. For example, the number of
galls per inoculation are recorded and evaluated after inoculation.
From a statistical analysis of these data, levels of resistance to
Agrobacterium and crown gall formation are determined. Transformed
grape plants that express a lytic peptide (e.g., Shiva-1 or
cecropin B or both) having an increased level of resistance to
Agrobacterium or crown gall disease or both relative to control
plants are taken as being useful in the invention.
[0046] By "increased level of resistance" is meant a greater level
of resistance or tolerance to a disease-causing pathogen or pest in
a transgenic grapevine (or scion, rootstock, cell, or seed thereof)
than the level of resistance or tolerance or both relative to a
control plant (for example, a non-transgenic grapevine). In
preferred embodiments, the level of resistance in a transgenic
plant of the invention is at least 5-10% (and preferably 30% or
40%) greater than the resistance of a control plant. In other
preferred embodiments, the level of resistance to a disease-causing
pathogen is 50% greater, 60% greater, and more preferably even 75%
or 90% greater than a control plant; with up to 100% above the
level of resistance as compared to a control plant being most
preferred. The level of resistance or tolerance is measured using
conventional methods. For example, the level of resistance to a
pathogen may be determined by comparing physical features and
characteristics (for example, plant height and weight, or by
comparing disease symptoms, for example, delayed lesion
development, reduced lesion size, leaf wilting, shriveling, and
curling, decay of fruit clusters, water-soaked spots, leaf
scorching and marginal burning, and discoloration of cells) of
transgenic grape plants.
[0047] In another working example, constitutive expression of a
lytic peptide (e.g., Shiva-1 or cecropin B) is used to control the
fungus Botrytis, the causative agent of bunch rot disease.
Specifically, a plant expression vector is constructed that
contains a transgene sequence that expresses the lytic peptide(s).
This expression vector is then used to transform somatic embryos
according to the methods described above. To assess resistance to
fungal infection, transformed plants and appropriate controls are
grown to approximately 30 cm vinelength, and young leaves and
shoots are inoculated with a mycelial suspension of Botrytis. For
example, plugs of Botrytis mycelia are inoculated on each side of
the leaf midvein of developing leaves. Plants are subsequently
incubated in a growth chamber at 30.degree. C. with constant
fluorescent light and high humidity. Leaves of transformed and
control grapevines are then evaluated for resistance to Botrytis
infection and disease according to conventional experimental
methods. For this evaluation, for example, the number of lesions
per leaf and percentage of leaf area infected are recorded every
twenty-four hours for seven days after inoculation. From these
data, levels of resistance to Botrytis are determined. In addition,
if desired, fruit clusters can be sprayed with a suspension of
Botrytis and infection monitored at 15-20.degree. C. at 90%
relative humidity after fifteen to twenty-four hours. Transformed
grapevines that express a lytic peptide gene having an increased
level of resistance to Botrytis and infection and disease relative
to control plants are taken as being useful in the invention.
[0048] Alternatively, to assess resistance at the whole plant
level, transformed and control grapevines are transplanted to
potting soil containing an inoculum of Botrytis. Plants are then
evaluated for symptoms of fungal infection (for example, wilting or
decayed leaves) over a period of time lasting from several days to
weeks. Again, transformed grapevines expressing the lytic peptide
gene(s) having an increased level of resistance to the fungal
pathogen, Botrytis, relative to control plants are taken as being
useful in the invention.
Other Embodiments
[0049] The invention further includes analogs of any
naturally-occurring lytic peptide. Analogs can differ from the
naturally-occurring lytic peptide by amino acid sequence
differences, by post-translational modifications, or by both. In
preferred embodiments, lytic peptide analogs used in the invention
will generally exhibit about 30%, more preferably 50%, and most
preferably 60% or even having 70%, 80%, or 90% identity with all or
part of a naturally-occurring lytic peptide amino acid sequence.
The length of sequence comparison is at least 10 to 15 amino acid
residues, preferably at least 25 amino acid residues, and more
preferably more than 35 amino acid residues. Modifications include
chemical derivatization of polypeptides, e.g., acetylation,
carboxylation, phosphorylation, or glycosylation; such
modifications may occur during polypeptide synthesis or processing
or following treatment with isolated modifying enzymes. Lytic
peptide analogs can also differ from the naturally-occurring by
alterations in primary sequence. These include genetic variants,
both natural and induced (for example, resulting from random
mutagenesis by irradiation or exposure to ethyl methylsulfate or by
site-specific mutagenesis as described in Sambrook, Fritsch and
Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH
Press, 1989, or Ausubel et al., supra). Also included are cyclized
peptides, molecules, and analogs which contain residues other than
L-amino acids, e.g., D-amino acids or non-naturally occurring or
synthetic amino acids, e.g., .beta. or .gamma. amino acids.
[0050] In addition to full-length lytic peptides, the invention
also includes peptide fragments. As used herein, the term
"fragment," means at least 10 contiguous amino acids, preferably at
least 15 contiguous amino acids, more preferably at least 20
contiguous amino acids, and most preferably at least 30 to 40 or
more contiguous amino acids. Fragments of lytic peptides can be
generated by methods known to those skilled in the art or may
result from normal protein processing (e.g., removal of amino acids
from the nascent polypeptide that are not required for biological
activity or removal of amino acids by alternative mRNA splicing or
alternative protein processing events).
[0051] All publications mentioned in this specification are herein
incorporated by reference to the same extent as if each independent
publication or patent application was specifically and individually
indicated to be incorporated by reference.
Sequence CWU 1
1
4 1 22 DNA Artificial Sequence Synthetic primer 1 ggttcagggc
gggtcctgga ag 22 2 24 DNA Artificial Sequence Synthetic primer 2
gtaaaagcta attaagaggc cacc 24 3 20 DNA Artificial Sequence
Synthetic primer 3 atcaaacagg gtatcctgcg 20 4 18 DNA Artificial
Sequence Synthetic primer 4 ttcccaccaa cgctgatc 18
* * * * *