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United States Patent |
5,650,387
|
Wei
,   et al.
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July 22, 1997
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Hypersensitive response induced resistance in plants
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 under
conditions where the polypeptide or protein contacts cells of the plant.
The present invention is also directed to a pathogen resistant plant and a
composition for imparting pathogen resistance to plants.
Inventors:
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Wei; Zhong-Min (Ithaca, NY);
Beer; Steven V. (Ithaca, NY)
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Assignee:
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Cornell Research Foundation, Inc. (Ithaca, NY)
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Appl. No.:
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475775 |
Filed:
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June 7, 1995 |
Intern'l Class: |
A01N 037/18; A01N 063/00; A01N 065/00; A61K 038/00 |
Field of Search: |
514/2
424/93
435/847,800
|
References Cited
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5173403 | Dec., 1992 | Tang | 435/6.
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5217950 | Jun., 1993 | Blackburn et al. | 514/2.
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5243038 | Sep., 1993 | Ferrari et al. | 536/23.
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5260271 | Nov., 1993 | Blackburn et al. | 514/2.
|
Foreign Patent Documents |
WO94/01546 | Jan., 1994 | WO.
| |
WO94/26782 | Nov., 1994 | WO.
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Primary Examiner: Robinson; Douglas W.
Assistant Examiner: Harle; Jennifer
Attorney, Agent or Firm: Nixon, Hargrave, Devans & Doyle LLP
Goverment Interests
This invention was made with support from the U.S. Government under USDA
NRI Competitive Research Grant No. 91-37303-6430.
Claims
What is claimed:
1. A method of imparting pathogen resistance to plants comprising:
applying a hypersensitive response elicitor polypeptide or protein in a
non-infectious form to a plant under conditions where the polypeptide or
protein contacts cells of the plant, wherein the hypersensitive response
elicitor polypeptide or protein corresponds to that derived from a
pathogen selected from the group consisting of Erwinia amylovora, Erwinia
chrysanthemi, Pseudomonas syringae, Pseudomonas solancearum, Xanthomonas
campestris, and mixtures thereof.
2. A method according to claim 1, wherein the hypersensitive response
elicitor polypeptide or protein corresponds to that derived from Erwinia
chrysanthemi.
3. A method according to claim 2, wherein the hypersensitive response
elicitor polypeptide or protein has an amino acid sequence corresponding
to SEQ. ID. No. 1.
4. A method according to claim 2, wherein the hypersensitive response
elicitor polypeptide or protein has a molecular weight of 34 kDa.
5. A method according to claim 1, wherein the hypersensitive response
elicitor polypeptide or protein corresponds to that derived from Erwinia
amylovora.
6. A method according to claim 5, wherein the hypersensitive response
elicitor polypeptide or protein has an amino acid sequence corresponding
to SEQ. ID. No. 3.
7. A method according to claim 5, wherein the hypersensitive response
elicitor polypeptide or protein has a molecular weight of 37 kDa.
8. A method according to claim 1, wherein the hypersensitive response
elicitor polypeptide or protein corresponds to that derived from
Pseudomonas syringae.
9. A method according to claim 8, wherein the hypersensitive response
elicitor polypeptide or protein has an amino acid sequence corresponding
to SEQ. ID. No. 5.
10. A method according to claim 8, wherein the hypersensitive response
elicitor polypeptide or protein has a molecular weight of 34-35 kDa.
11. A method according to claim 1, wherein the hypersensitive response
elicitor polypeptide or protein corresponds to that derived from
Pseudomonas solanacearum.
12. A method according to claim 11, wherein the hypersensitive response
elicitor polypeptide or protein has an amino acid sequence corresponding
to SEQ. ID. No. 7.
13. A method according to claim 1, wherein the hypersensitive response
elicitor polypeptide or protein corresponds to that derived from
Xanthomonas campestris.
14. A method according to claim 13, wherein the hypersensitive response
elicitor polypeptide or protein has an amino acid sequence corresponding
to SEQ. ID. No. 9.
15. A method according to claim 1, wherein the plant is selected from the
group consisting of dicots and monocots.
16. A method according to claim 15, wherein the plant is selected from the
group consisting of rice, wheat, barley, rye, cotton, sunflower, 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.
17. A method according to claim 15, wherein the plant is selected from the
group consisting of Arabidopsis thaliana, Saintpaulia, petunia,
pelargonium, poinsettia, chrysanthemum, carnation, and zinnia.
18. A method according to claim 1, wherein the pathogen to which the plant
is resistant is selected from the group consisting of a viruses, bacteria,
fungi, and combinations thereof.
19. A method according to claim 1, wherein said applying is carried out by
spraying, injection, or leaf abrasion at a time proximate to when said
applying takes place.
20. A method according to claim 1, wherein the hypersensitive response
elicitor polypeptide or protein is applied to plants as a composition
further comprising a carrier.
21. A method according to claim 20, wherein the carrier is selected from
the group consisting of water and aqueous solutions.
22. A method according to claim 20, wherein the composition contains
greater than 500 nM of the hypersensitive response elicitor polypeptide or
protein.
23. A method according to claim 20, wherein the composition further
contains additives selected from the group consisting of fertilizer,
insecticide, fungicide, and mixtures thereof.
24. A method according to claim 1, wherein the hypersensitive response
elicitor polypeptide or protein is in isolated form.
25. A method according to claim 1, wherein the hypersensitive response
elicitor polypeptide or protein is applied as bacteria which do not cause
disease and are transformed with a gene encoding the hypersensitive
response elicitor polypeptide or protein.
26. A method according to claim 1, wherein the hypersensitive response
elicitor polypeptide or protein is applied as bacteria which cause disease
in some plant species, but not in those subjected to said applying, and
contain a gene encoding the hypersensitive response elicitor polypeptide
or protein.
27. A method according to claim 1, wherein said applying causes
infiltration of the polypeptide or protein into the plant.
Description
FIELD OF THE INVENTION
The present invention relates to imparting hypersensitive response induced
resistance to plants.
BACKGROUND OF THE INVENTION
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).
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).
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
261:754-296 (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).
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 at., "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
resistance mechanisms against the pathogen.
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,"
Phytopatholoqy 67:1285-9 (1977), which is hereby incorporated by
reference).
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 P. tabaci (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 are 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).
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 involved 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, 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).
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%.
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.
Removal of the inducer leaf from immunized cucumber plants did not reduce
the level of immunization of pre-existing expanded leaves. Howevers 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).
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 95-99%, based on the necrotic area and
degree of sporulation, leading to a yield increase of 10-25% in cured
tobacco.
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).
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
The present invention relates to a method of imparting pathogen resistance
to plants. This method involves applying a hypersensitive response
elicitor polypeptide or protein in a non-infectious form to a plant under
conditions where the polypeptide or protein contacts cells of the plant.
Another aspect of the present invention relates to a pathogen-resistant
plant with cells in contact with non-infectious hypersensitive response
elicitor polypeptide or protein.
Yet another aspect of the present invention relates to a composition for
imparting pathogen resistance to plants. The composition includes a
non-infectious, hypersensitive response elicitor polypeptide or protein
and a carrier.
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 infectious
agents to treat diseases. The present invention can impart resistance
without using agents pathogenic to the plants being treated or to plants
situated nearby those treated. Since the present invention involves use of
a natural product that is fully biodegradable, the environment would not
be contaminated.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the genetic organization of the gene cluster encoding the
hypersensitive response elicitor polypeptide or protein for Erwinia
amylovora (i.e. hrpN). The top line shows the restriction enzyme map of
plasmid vector pCPP430, where E=Eco RI, B=Bam HI, and H=Hind III. The
rectangles represent transcriptional units, and the arrows under the
rectangles indicate the directions of transcription. The bigger arrow
indicates the region necessary for ultimate translation of the
hypersensitive response elicitor polypeptide or protein. pCPP430
hrpN.sup.- is the derivative of pCPP430 in which hrpN is mutated by the
insertion of transposor TnStac.
FIG. 2 is a map of plasmid vector pCPP9. Significant features are the
mobilization (mob) site for conjugation; the cohesive site of .lambda.
(cos); and the partition region (par) for stable inheritance of the
plasmid. B, BamHI; E, EcoRI; H, HindIII; P, PstI; S, SaII; Sm, SmaI; oriV,
origin of replication; Sp.sup.r, spectinomycin resistance; Sm.sup.r,
streptomycin resistance.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method of imparting pathogen resistance
to plants. This method involves applying a hypersensitive response
elicitor polypeptide or protein in a non-infectious form to all or part of
a plant under conditions where the polypeptide or protein contacts all or
part of the cells of the plant.
Another aspect of the present invention relates to a patbogen-resistant
plant with cells in contact with a non-infectious hypersensitive response
elicitor polypeptide or protein.
Yet another aspect of the present invention relates to a composition for
imparting pathogen resistance to plants. The composition includes a
non-infectious hypersensitive response elicitor polypeptide or protein and
a carrier.
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 pathogens. Such
polypeptides or proteins are able to elicit local necrosis in plant tissue
contacted by the elicitor. Preferred pathogens include Erwinia amylovora,
Erwinia chrysanthemi, Pseudomonas syringae, Pseudomonas solancearum,
Xanthomonas campestris, or mixtures thereof.
For purposes of the present invention, non-infectious forms of the
hypersensitive response elicitor polypeptide or protein can induce a
hypersensitive response without causing disease in the plant with which
the polypeptide or protein is contacted. This can be achieved 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 one embodiment of the present invention, the hypersensitive response
elicitor polypeptides or proteins 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.
In other embodiments of the present invention, the hypersensitive response
elicitor polypeptide or protein of the present invention can be applied to
plants 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 cells. In these embodiments, the hypersensitive response
elicitor polypeptide or protein is produced by the bacteria in planta or
just prior to introduction of the bacteria to the plants.
In one embodiment of the bacterial application mode of the present
invention, the bacteria 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 or protein and
then applied to plants. Bacterial species (other than E. coli) can also be
used in this embodiment of the present invention.
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 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 to impart pathogen resistance without causing disease in
that species.
The hypersensitive response elicitor potypeptide or protein from Erwinia
chrysanthemi has an amino acid sequence corresponding to SEQ. ID. No. 1 as
follows:
##STR1##
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:
__________________________________________________________________________
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
GGCTGTCGTC
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
__________________________________________________________________________
The hypersensitive response elicitor polypeptide or protein derived from
Erwinia amylovora has an amino acid sequence corresponding to SEQ. ID. No.
3 as follows:
##STR2##
This hypersensitive response elicitor polypeptide or protein has a
molecular weight of about 37 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 Patbogen
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:
__________________________________________________________________________
ATGAGTCTGA
ATACAAGTGG
GCTGGGAGCG
TCAACGATGC
AAATTTCTAT
CGGCGGTGCG
60
GGCGGAAATA
ACGGGTTGCT
GGGTACCAGT
CGCCAGAATG
CTGGGTTGGG
TGGCAATTCT
120
GCACTGGGGC
TGGGCGGCGG
TAATCAAAAT
GATACCGTCA
ATCAGCTGGC
TGGCTTACTC
180
ACCGGCATGA
TGATGATGAT
GAGCATGATG
GGCGGTGGTG
GGCTGATGGG
CGGTGGCTTA
240
GGCGGTGGCT
TAGGTAATGG
CTTGGGTGGC
TCAGGTGGCC
TGGGCGAAGG
ACTGTCGAAC
300
GCGCTGAACG
ATATGTTAGG
CGGTTCGCTG
AACACGCTGG
GCTCGAAAGG
CGGCAACAAT
360
ACCACTTCAA
CAACAAATTC
CCCGCTGGAC
CAGGCGCTGG
GTATTAACTC
AACGTCCCAA
420
AACGACGATT
CCACCTCCGG
CACAGATTCC
ACCTCAGACT
CCAGCGACCC
GATGCAGCAG
480
CTGCTGAAGA
TGTTCAGCGA
GATAATGCAA
AGCCTGTTTG
GTGATGGGCA
AGATGGCACC
540
CAGGGCAGTT
CCTCGGGGGG
CAAGCAGCCG
ACCGAAGGCG
AGCAGAACGC
CTATAAAAAA
600
GGAGTCACTG
ATGCGCTGTC
GGGCCTGATG
GGTAATGGTC
TGAGCCAGCT
CCTTGGCAAC
660
GGGGGACTGG
GAGGTGGTCA
GGGCGGTAAT
GCTGGCACGG
GCTGGCACGG
TTCGTCGCTG
720
GGCGGCAAAG
GGCTGCAAAA
CCTGAGCGGG
CCGGTGGACT
ACCAGCAGTT
AGGTAACGCC
780
GTGGGTACCG
GTATCGGTAT
GAAAGCGGGC
ATTCAGGCGC
TGAATGATAT
CGGTACGCAC
840
AGGCACAGTT
CAACCCGTTC
TTTCGTCAAT
AAAGGCGATC
GGGCGATGGC
GAAGGAAATC
900
GGTCAGTTCA
TGGACCAGTA
TCCTGAGGTG
TTTGGCAAGC
CGCAGTACCA
GAAAGGCCCG
960
GGTCAGGAGG
TGAAAACCGA
TGACAAATCA
TGGGCAAAAG
CACTGAGCAA
GCCAGATGAC
1020
GACGGAATGA
CACCAGCCAG
TATGGAGCAG
TTCAACAAAG
CCAAGGGCAT
GATCAAAAGG
1080
CCCATGGCGG
GTGATACCGG
CAACGGCAAC
CTGCAGCACG
CGGTGCCGGT
GGTTCTTCGC
1140
TGGGTATTGA
TGCCATGA 1158
__________________________________________________________________________
The hypersensitive response elicitor polypeptide or protein derived from
Pseudomonas syringae has an amino acid sequence corresponding to SEQ. ID.
No. 5 as follows:
##STR3##
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 Re, 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:
__________________________________________________________________________
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
__________________________________________________________________________
The hypersensitive response elicitor polypeptide or protein derived from
Pseudomonas solanacearum has an amino acid sequence corresponding to SEQ.
ID. No. 7 as follows:
##STR4##
It is encoded by a DNA molecule having a nucleotide sequence corresponding
SEQ. ID. No. 8 as follows:
__________________________________________________________________________
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.
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:
##STR5##
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 Tanthomouas campestris pathovars.
The above elicitors are exemplary. Other elicitors can be identified by
growing 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.
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.
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.
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.
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 increase and expression of a truncated
peptide or protein.
Variants may also (or alternatively) be modified 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.
The protein or polypeptide of the present invention is preferably produced
in purified form (preferably at least about 80%, more preferably 90%,
pure) by conventional techniques. Typically, the protein or polypeptide of
the present invention is secreted into the growth medium of recombinant E.
coli. 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 sequential
ammonium sulfate precipitation. The 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
HPLC.
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.
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.
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.
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, pACYC184, 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
Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs
Laboratory, Cold Springs Harbor, N.Y. (1982), which is hereby incorporated
by reference.
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.
Different genetic signals and processing events control many levels of gene
expression (e.g., DNA transcription and messenger RNA (mRNA) translation).
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.
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.
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
lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of
transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5
(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.
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.
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.
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.
The method of the present invention can be utilized to treat a wide variety
of plants to impart patbogen resistance. Suitable plants include dicots
and monocots. More particularly, useful crop plants can include: rice,
wheat, barley, rye, cotton, sunflower, 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: Arabidopsis
thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum,
carnation, and zinnia.
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.
Resistance, inter alia, to the following viruses can be achieved by the
method of the present invention: Tobacco mosaic virus and tomato mosaic
virus.
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.
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.
The method of the present invention can be carried out through a variety of
procedures for applying the hypersensitive response elicitor polypeptide
or protein to all or part of the plant being treated. This may (but need
not) involve infiltration of the hypersensitive response elicitor
polypeptide or protein into the plant. Suitable application methods
include high or low pressure spraying, injection, and leaf abrasion
proximate to when elicitor application takes place. Other suitable
application procedures can be envisioned by those skilled in the art
provided they are able to effect contact of the hypersensitive response
elicitor polypeptide or protein with cells of the plant.
The hypersensitive response elicitor polypeptide or protein can be applied
to plants in accordance with the present invention alone or in a mixture
with other materials.
One aspect of the present invention involves a composition for imparting
pathogen resistance to plants containing a hypersensitive response
elicitor polypeptide or protein in a carrier. Suitable carriers include
water or aqueous solutions. In this embodiment, the composition contains
greater than 500 nM hypersensitive response elicitor potypeptide or
protein.
Although not required, this composition may contain additional additives
including fertilizer, insecticide, fungicide, and mixtures thereof.
Suitable fertilizers include (NH.sub.4).sub.2 NO.sub.3. An example of a
suitable insecticide is Malathion. Useful fungicides include Captan.
Other suitable additives include buffering agents, wetting agents, and
abrading agents. These materials can be used to facilitate the process of
the present invention.
EXAMPLES
Example 1
Harpin-induced Resistance of Tomato Against the Southern Bacterial Wilt
Disease (Pseudomonas solanacearum)
Two-week-old tomato seedlings, grown in 8.times.15 cm flats in the
greenhouse were treated as follows: 20 plants were used for each of the
six treatments, which were designated A through F, and are described as
follows:
(A) About 100 .mu.l of a 200 .mu.g/ml crude harpin (i.e. hypersensitive
response elicitor polypeptide or protein) preparation (Z-M. Wei, "Harpin,
Elicitor of the Hypersensitive Response Produced by the Plant Pathogen
Erwinia amylovora," Science 257:85-88 (1992), which is hereby incorporated
by reference) was infiltrated into the lowest true leaf of each of the
seedlings.
(B) The same harpin preparation as used in (A) was sprayed with 400-mesh
carborundum onto the leaf surface of the seedlings and then gently rubbed
in with the thumb.
(C) E. coli DH5 (pCPP430)(See FIG. 1 for map of plasmid vector pCPP430) was
grown in LB medium to OD.sub.620 =0.7. The culture was centrifuged and
then resuspended in 5 mM of potassium phosphate buffer pH 6.5. About 100
.mu.l of cell suspension was infiltrated into each leaf of the seedlings.
(D) E. coli DH5 (pCPP430::hrpN.sup.-)(See FIG. 1 for map of plasmid vector
pCPP430::hrpN.sup.-) was used as in (C). The cells were grown, and the
suspension and the amount of inoculum used were the same as described in
(C).
(E) For E. coli DH5 (pCPP9) (See FIG. 2), the cells were grown and the
suspension and the amount of inoculum used were the same as described in
(C).
(F) Infiltration of leaves with 5 mM potassium phosphate buffer was as
described in (C).
The challenge pathogenic bacterium, Pseudomonas solanacearum strain K60,
was grown in King's medium B to OD.sub.620 =0.7 (about 10.sup.8 cfu/ml).
The culture was centrifuged and resuspended in 100 volume of 5 mM
potassium phosphate buffer to a final concentration of about
1.times.10.sup.6 cfu/ml.
Three days after the tomato seedlings were treated with harpin or bacteria,
they were pulled up and about one cm of roots were cut off with scissors.
The seedlings were then dipped into the suspension of P. solanacearum K60
for 3 min. The inoculated plants were replanted into the same pots. The
plants were left in a greenhouse, and the disease incidence was recorded 7
days after inoculation.
A. Effect of treatment with harpin
After 24 hours, only those leaf portions that had been infiltrated with
harpin or E. coli DH5(pCPP430) had collapsed. Leaves sprayed with harpin
and carborundum showed only spotty necrosis.
B. Effect of treatment with harpin on the development of Southern Bacterial
Wilt.
None of the 20 harpin-infiltrated plants showed any symptoms one week after
inoculation with P. solanacearum K60 (Table 1). One out of the 20 plants
showed stunting symptoms. However, 7 of the 20 buffer-infiltrated plants
showed stunting symptoms. Treatment with E. coli DH5 (pCPP430.sup.-) (a
transposon-induced mutant unable to elicit the hypersensitive collapse) or
E. coli DH5 (pCPP9) did not show significant difference compared to the
plants treated with buffer. These results suggest that harpin or E. coli
DH5 (pCPP430), which produces harpin, induced resistance in the tomato
plants to southern bacterial wilt caused by P. solanacearum K60.
TABLE 1
______________________________________
Disease incidence of tomato seedlings 7 and 14
days after inoculation with P. solanacearum K60.
Number of Plants
Day 7 Day 14
Treatment Stunted Healthy Stunted Healthy
______________________________________
A. Harpin infiltration
0 20 2 18
B. Harpin spray
1 19 3 17
C. E. coli DH5 (pCPP430)
2 18 3 17
D. E. coli DH5 (pCPP430.sup.-)
4 16 7 13
E. E. coli DH5 (pCPP9)
5 15 6 + 1 wilted
13
F. Buffer 7 13 8 + 1 wilted
11
No pathogen 0 20 0 20
______________________________________
Four weeks after inoculation, plants treated with the harpin or E. coli DH5
(pcPP430) were taller and broader as compared to those treated with
buffer. The average heights of 10 plants that had been infiltrated with
harpin or buffer are given in Table 2.
TABLE 2
______________________________________
Heights (cm) of tomato plants four weeks after
inoculation with Pseudomonas solanacearum K60,
following treatment with harpin or buffer.
Infiltrated
with Buffer Infiltrated with Harpin
Infiltrated with Buffer
Not inoculated
Inoculated with K60
Inoculated with K60
______________________________________
36 32 11
41 29 21
35 38 33
34 35 12
39 37 15
35 33 32
36 22 25
35 35 15
41 40 37
37 29 38
Average
36.9 33 23.9
______________________________________
Example 2
Harpin-induced Resistance of Tomato against Southern Bacterial Wilt Disease
Pseudomonas solanacearum
All the methods used for infiltration and inoculation were the same as
described in Example 1, except that the concentration of P. solanacearum
K60 was about 5.times.10.sup.4 cfu/ml.
The buffer-infiltrated plants showed symptoms 15 days after inoculation
with P. solanacearum K60. Six out of 20 plants showed stunting symptoms
after 15 days; 2 plants were wilted after 21 days. The wilted plants
eventually died. However, none of the 20 harpin-treated plants showed
stunting symptoms. Three weeks after inoculation, 3 of the 20
harpin-treated plants showed stunting symptoms. It is possible that after
three weeks, the plants may have lost their induced resistance. As in the
first experiment, the overall girth and heights of the harpin-treated
plants were greater than those treated with buffer.
Example 3
Harpin-induced Resistance of Tomato against Southern Bacterial Wilt Disease
Pseudomonas solanacearum
This experiment was similar to Example 1, except that additional inoculum
of Pseudomonas solanacearum K60 was added to the pots containing the
treated tomato plants.
Harpin was infiltrated into two-week-old tomato seedlings. Two panels of
each plant were infiltrated with about 200 .mu.l harpin suspended in 5 mM
of potassium phosphate buffer at the concentration about 200 .mu.g/ml. A
total of 20 tomato seedlings were infiltrated. The same number of tomato
seedlings were infiltrated with buffer. After two days, the plants were
inoculated with Pseudomonas solanacearum K60 by root-dipping. The harpin-
or buffer-infiltrated plants were pulled from the soil mix and small
amounts of roots were cut off with scissors and then the remaining roots
were dipped into a suspension of P. solanacearum K60 for three minutes.
The concentration of the bacterial cell suspension was about
5.times.10.sup.8 cfu/ml. The seedlings were replanted into the same pot.
An additional 3 ml of bacterial suspension was added to the soil of each
individual 4-inch diameter pot. Disease incidence was scored after one
week. All the experiments were done in the greenhouse with limited
temperature control.
After three weeks, 11 of the 20 buffer-infiltrated tomato plants had died
and 2 plants that had wilted recovered, but remained severely stunted.
Only 4 plants grew normally compared with non-inoculated tomatoes.
However, 15 of the harpited plants appeared healthy; three plants were
stunted and two plants were wilted 3 weeks after inoculation. These
results are summarized below in Table 3.
TABLE 3
______________________________________
Harpin-induced resistance of tomato against
bacterial wilt disease caused by P. solanacearum
Weeks After Inoculation
Treatment 1 2 3
______________________________________
Harpin
Healthy 20 17 15
Wilted 0 1 2
Stunted 0 2 3
Buffer
Healthy 8 5 4
Wilted 8 12 13
Stunted 4 3 3
______________________________________
Example 4
Harpin-induced Resistance of Tobacco to Tobacco Mosaic Virus
One panel of a lower leaf of four-week old tobacco seedlings (cultivar,
Xanthi, with N gene) were infiltrated with E. amylovora harpin at the
concentration of 200 .mu.g/ml. After three days, the plants were
challenged with tobacco mosaic virus ("TMV"). Two concentrations of the
virus (5 .mu.g and 100 .mu.g/ml) were used. About 50 .mu.l of the virus
suspension was deposited on one upper tobacco leaf. The leaf was dusted
with 400-mesh carborundum and the leaves gently rubbed. Each concentration
was tested on three plants. Necrotic lesions were counted 4 days after
inoculation and on two subsequent days and the mean number on three leaves
is reported (Table 4). It was difficult to distinguish the individual
lesions by Day 10 because some of the necrotic lesions had merged
together. Therefore, the number of lesions recorded seemed less than those
recorded on Day 7. The size of the necrotic lesions in buffer-treated
leaves was much larger than the harpin-treated leaves.
TABLE 4
______________________________________
Harpin-induced resistance of tomato against
TMB from inoculation with 5 .mu.g/ml of virus
Mean Number of Lesions/Leaf
Treatment Day 4 Day 7 Day 10
______________________________________
Harpin 21 32 35
Buffer 67 102 76
______________________________________
There was no significant difference in the number of local lesions that
developed on the harpin-treated and buffer-treated tobacco when the
tobacco mosaic virus inoculum concentration was 100 .mu.g/ml.
Example 5
Harpin-induced Resistance of Tomato to Fusarium Wilt Disease
Six-week-old tomato plants were treated with harpin as described for
Example 3. The fungal pathogen, Fusarium oxysporum, was grown on Lima Bean
Agar medium for 5 days at 27.degree. C. Two entire agar plates with
mycelia were blended for 2 minutes in 20 ml of 5 mM potassium phosphate
buffer. The roots of harpin- or buffer-treated tomato plants were wounded
by plunging a wooden stake into the soil of the pots. Then, 3 ml of the
fungal suspension was poured into the soil of each 4-inch pot. The
inoculated plants remained in a controlled environment chamber at
24.degree. C. with 16 hours of light per day. Disease incidence was
recorded 7 days after inoculation. Each treatment was applied to 10
plants. The results are shown below in Table 5.
TABLE 5
______________________________________
Effect of harpin or buffer treatment on
Fusarium wilt disease of tomato
Number of plants (of 10) showing wilt symptoms
at the indicated time post-inoculation
Treatment Day 7 Day 10 Day 15
Day 20
______________________________________
Harpin 1 2 4 4 (1 dead)
Buffer 3 6 7 7 (4 dead)
______________________________________
Example 6
Harpin-Induced Resistance of Tobacco Against Wildfire Disease (Pseudomonas
syringae pv. tabaci).
Harpin was infiltrated into single panels of the lower leaves of 4-week-old
tobacco plants (20 cm high). After three days, suspensions of Pseudomonas
syringe pv. tabaci were infiltrated into single panels of upper leaves.
Four days later, disease incidence was recorded, as set forth in Table 6.
TABLE 6
______________________________________
Symptoms of infection by Wildfire disease in
tobacco leaves inoculated with Pseudomonas
Syringe pv. tabaci following treatment of lower
leaves with harpin.
Concentration of
P.s. tabaci
Treated with Harpin
Not treated with Harpin
______________________________________
10.sup.4 cfu/ml
no symptoms necrosis and water-soaking
10.sup.5 cfu/ml
no symptoms necrosis and water-soaking
10.sup.6 cfu/ml
no symptoms necrosis and water-soaking
10.sup.7 cfu/ml
no symptoms necrosis and water-soaking
10.sup.8 cfu/ml
necrosis necrosis and water-soaking
______________________________________
Example 7
Harpin-induced Resistance of Geranium (Pelargonium hortorum) Against
Bacterial Leaf Spot (Xanthamonas campestris pv. pelargonii)
This experiment was done with rooted cuttings of geranium growing in
individual 4" or 6" pots in an artificial soil mix in a greenhouse. Two
lower leaves on each plant were infiltrated with either 0.05M potassium
phosphate buffer, pH 6.5 (control), or harpin or a suspension of
Escherichia coli DH5 (pCPP430) (the entire cloned hrp gene cluster of E.
amylovora). Two to seven days following infiltration, all the plants were
inoculated with a pure culture of the bacterial leaf spot pathogen,
Xanthamonas campestris pv. pelargonii. A suspension of the bacteria
(5.times.10.sup.6 cfu/ml) was atomized over both upper and lower leaf
surfaces of the plants at low pressure. Each treatment was applied to two
plants (designated "A" and "B" in Table 7). The plants were maintained in
a closed chamber for 48 hours with supplemental misting supplied by
cool-mist foggers. Then, the plants were maintained on the greenhouse
bench subject to ambient humidity and temperature of 23.degree. C. to
32.degree. C. for 10 days before disease development was assessed.
TABLE 7
______________________________________
Effect of harpin and the hrp gene cluster of
Erwinia amylovora on the development of bacterial leaf
spot of geranium.
Time between treatment and inoculation with
Xanthomonas campestris pv. pelargonii
7 Days 5 Days 4 Days 3 Days 2 days
Plant Plant Plant Plant Plant
Treatment A B A B A B A B A B
______________________________________
Buffer 3* 5 5 4 3 2 4 3 4 5
Harpin 0 0 0 0 0 0 1 0 0 0
DH5 (pCPP430)
0 0 NT NT 0 0 0 1 1 0
______________________________________
*Numbers in table are the number of leaves showing disease symptoms
(pronounced necrosis, chlorosis, or wilting) 10 days following
inoculation.
Example 8
Activity of several harpins in inducing resistance to Wildfire Disease
caused by Pseudomonas syringae pv. tabaci
Tobacco plants (Nicotiana tabacum var. Xanthi) were grown in the
greenhouse. At 4 weeks of age, harpin preparations were infiltrated into a
single panel of two lower leaves of each plant. Twelve plants were treated
with each harpin preparation, and three were treated with the same
potassium phosphate buffer that was used to prepare the harpins. The
hypersensitive necrosis developed within 24 hours in the panels of the
leaves infiltrated with the harpin preparations, but not with buffer.
At 7, 10, 11, and 12 days after harpin treatment, all plants were
inoculated with suspensions of 10.sup.4 to 10.sup.6 cells/ml of
Pseudomonas syringae pv. tabaci by infiltrating panels on upper leaves.
Plants were incubated in the greenhouse for 7 days before disease
development was evaluated. The results are tabulated as follows in Table
8:
TABLE 8
______________________________________
Days between treatment and inoculation
Harpin source
12 11 10 7
log [Inoc.]
4 5 6 4 5 6 4 5 6 4 5
6
______________________________________
None (buffer)
+ + ++ + + ++ + + ++ + +
++
P. syringae - - + - - + - - + - - +
E. - - + - - + - - + - - +
chrysanthemi
E. amylovora - - + - - + - - + - - +
______________________________________
- = No symptoms,
+ = Necrosis with yellow halo, typical of wildfire disease
++ = Severe necrosis with yellow halo, typical of wildfire disease
The results indicate that the harpin preparations from the three bacteria
are effective in inducing resistance to the wildfire pathogeno Plants
treated with either harpin exhibited no symptoms with the two lower
inoculum concentrations used. At the higher concentration, symptoms were
more severe on buffer-treated plants than harpin-treated plants.
Example 9
Harpin induced resistance against the Late Blight disease caused by
Phytophthora infestans.
The late blight pathogen affects potatoes and tomatoes primarily. It was
responsible for the infamous Irish potato famine. The activity of harpin
in inducing resistance to this pathogen was tested on tomato seedlings
grown in the greenhouse. Three-week old seedlings (cultivar `Mama Mia`,
about 6 to 8 inches high) were treated with harpin and subsequently
inoculated with Phythophthora infestans. Two panels of a lower leaf of
each plant were infiltrated with a solution of harpin, a suspension of
Escherichia coli DH5 (pCPP430), which produces and secretes harpin, or
potassium phosphate buffer.
Two, three, or four days following infiltration, the plants were inoculated
with a mycelial suspension of Phytophthora infestans. The strain U.S. 7
was used, which is highly virulent to tomato. The mycelial suspension was
made by blending gently the contents of two barley-meal agar plates on and
in which the fungus had grown for 2 weeks at 21.degree. C. The suspension
was brushed onto the top and undersides of one leaf per treated plant with
an artist's broad paint brush.
The treated and inoculated plants were incubated in a specially constructed
mist chamber designed to maintain a temperature of 20.degree.-23.degree.
C. in the greenhouse, while maintaining high relative humidity. The
moisture was provided by several cool-mist foggers operating at maximum
rate on purified water. Disease incidence was evaluated 13 days following
inoculation with Phytophthora infestans, and the results are tabulated in
Table 9. Each treatment was applied to four individual plants.
TABLE 9
______________________________________
Numbers of lesion of late blight that were
present on tomato leaves 13 days after inoculation.
Days between treatment and inoculation
Treatment
4 3 2
Plant A B C D A B C D A B C
D
______________________________________
Buffer 3 2 0 0 1 2 2 0 0 0 4
1
Harpin 0 0 1 0 0 0 0 1 2 1 0 0
DH5 0 0 0 1 0 2 2 1 0 1 1 0
(pCPP430)
______________________________________
Treatment with harpin reduced the number of lesions that developed on
plants at all intervals between treatment and inoculation. The number of
late blight lesions that developed also was reduced by prior treatment
with DH5 (pCPP430), which produces and secretes harpin.
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 LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 9
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 338 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
MetGlnIleThrIleLysAlaHisIleGlyGlyAspLeuGlyValSer
151015
GlyLeuGlyAlaGlnGlyLeuLysGlyLeuAsnSerAlaAlaSerSer
202530
LeuGlySerSerValAspLysLeuSerSerThrIleAspLysLeuThr
354045
SerAlaLeuThrSerMetMetPheGlyGlyAlaLeuAlaGlnGlyLeu
505560
GlyAlaSerSerLysGlyLeuGlyMetSerAsnGlnLeuGlyGlnSer
65707580
PheGlyAsnGlyAlaGlnGlyAlaSerAsnLeuLeuSerValProLys
859095
SerGlyGlyAspAlaLeuSerLysMetPheAspLysAlaLeuAspAsp
100105110
LeuLeuGlyHisAspThrValThrLysLeuThrAsnGlnSerAsnGln
115120125
LeuAlaAsnSerMetLeuAsnAlaSerGlnMetThrGlnGlyAsnMet
130135140
AsnAlaPheGlySerGlyValAsnAsnAlaLeuSerSerIleLeuGly
145150155160
AsnGlyLeuGlyGlnSerMetSerGlyPheSerGlnProSerLeuGly
165170175
AlaGlyGlyLeuGlnGlyLeuSerGlyAlaGlyAlaPheAsnGlnLeu
180185190
GlyAsnAlaIleGlyMetGlyValGlyGlnAsnAlaAlaLeuSerAla
195200205
LeuSerAsnValSerThrHisValAspGlyAsnAsnArgHisPheVal
210215220
AspLysGluAspArgGlyMetAlaLysGluIleGlyGlnPheMetAsp
225230235240
GlnTyrProGluIlePheGlyLysProGluTyrGlnLysAspGlyTrp
245250255
SerSerProLysThrAspAspLysSerTrpAlaLysAlaLeuSerLys
260265270
ProAspAspAspGlyMetThrGlyAlaSerMetAspLysPheArgGln
275280285
AlaMetGlyMetIleLysSerAlaValAlaGlyAspThrGlyAsnThr
290295300
AsnLeuAsnLeuArgGlyAlaGlyGlyAlaSerLeuGlyIleAspAla
305310315320
AlaValValGlyAspLysIleAlaAsnMetSerLeuGlyLysLeuAla
325330335
AsnAla
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2141 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CGATTTTACCCGGGTGAACGTGCTATGACCGACAGCATCACGGTATTCGACACCGTTACG60
GCGTTTATGGCCGCGATGAACCGGCATCAGGCGGCGCGCTGGTCGCCGCAATCCGGCGTC120
GATCTGGTATTTCAGTTTGGGGACACCGGGCGTGAACTCATGATGCAGATTCAGCCGGGG180
CAGCAATATCCCGGCATGTTGCGCACGCTGCTCGCTCGTCGTTATCAGCAGGCGGCAGAG240
TGCGATGGCTGCCATCTGTGCCTGAACGGCAGCGATGTATTGATCCTCTGGTGGCCGCTG300
CCGTCGGATCCCGGCAGTTATCCGCAGGTGATCGAACGTTTGTTTGAACTGGCGGGAATG360
ACGTTGCCGTCGCTATCCATAGCACCGACGGCGCGTCCGCAGACAGGGAACGGACGCGCC420
CGATCATTAAGATAAAGGCGGCTTTTTTTATTGCAAAACGGTAACGGTGAGGAACCGTTT480
CACCGTCGGCGTCACTCAGTAACAAGTATCCATCATGATGCCTACATCGGGATCGGCGTG540
GGCATCCGTTGCAGATACTTTTGCGAACACCTGACATGAATGAGGAAACGAAATTATGCA600
AATTACGATCAAAGCGCACATCGGCGGTGATTTGGGCGTCTCCGGTCTGGGGCTGGGTGC660
TCAGGGACTGAAAGGACTGAATTCCGCGGCTTCATCGCTGGGTTCCAGCGTGGATAAACT720
GAGCAGCACCATCGATAAGTTGACCTCCGCGCTGACTTCGATGATGTTTGGCGGCGCGCT780
GGCGCAGGGGCTGGGCGCCAGCTCGAAGGGGCTGGGGATGAGCAATCAACTGGGCCAGTC840
TTTCGGCAATGGCGCGCAGGGTGCGAGCAACCTGCTATCCGTACCGAAATCCGGCGGCGA900
TGCGTTGTCAAAAATGTTTGATAAAGCGCTGGACGATCTGCTGGGTCATGACACCGTGAC960
CAAGCTGACTAACCAGAGCAACCAACTGGCTAATTCAATGCTGAACGCCAGCCAGATGAC1020
CCAGGGTAATATGAATGCGTTCGGCAGCGGTGTGAACAACGCACTGTCGTCCATTCTCGG1080
CAACGGTCTCGGCCAGTCGATGAGTGGCTTCTCTCAGCCTTCTCTGGGGGCAGGCGGCTT1140
GCAGGGCCTGAGCGGCGCGGGTGCATTCAACCAGTTGGGTAATGCCATCGGCATGGGCGT1200
GGGGCAGAATGCTGCGCTGAGTGCGTTGAGTAACGTCAGCACCCACGTAGACGGTAACAA1260
CCGCCACTTTGTAGATAAAGAAGATCGCGGCATGGCGAAAGAGATCGGCCAGTTTATGGA1320
TCAGTATCCGGAAATATTCGGTAAACCGGAATACCAGAAAGATGGCTGGAGTTCGCCGAA1380
GACGGACGACAAATCCTGGGCTAAAGCGCTGAGTAAACCGGATGATGACGGTATGACCGG1440
CGCCAGCATGGACAAATTCCGTCAGGCGATGGGTATGATCAAAAGCGCGGTGGCGGGTGA1500
TACCGGCAATACCAACCTGAACCTGCGTGGCGCGGGCGGTGCATCGCTGGGTATCGATGC1560
GGCTGTCGTCGGCGATAAAATAGCCAACATGTCGCTGGGTAAGCTGGCCAACGCCTGATA1620
ATCTGTGCTGGCCTGATAAAGCGGAAACGAAAAAAGAGACGGGGAAGCCTGTCTCTTTTC1680
TTATTATGCGGTTTATGCGGTTACCTGGACCGGTTAATCATCGTCATCGATCTGGTACAA1740
ACGCACATTTTCCCGTTCATTCGCGTCGTTACGCGCCACAATCGCGATGGCATCTTCCTC1800
GTCGCTCAGATTGCGCGGCTGATGGGGAACGCCGGGTGGAATATAGAGAAACTCGCCGGC1860
CAGATGGAGACACGTCTGCGATAAATCTGTGCCGTAACGTGTTTCTATCCGCCCCTTTAG1920
CAGATAGATTGCGGTTTCGTAATCAACATGGTAATGCGGTTCCGCCTGTGCGCCGGCCGG1980
GATCACCACAATATTCATAGAAAGCTGTCTTGCACCTACCGTATCGCGGGAGATACCGAC2040
AAAATAGGGCAGTTTTTGCGTGGTATCCGTGGGGTGTTCCGGCCTGACAATCTTGAGTTG2100
GTTCGTCATCATCTTTCTCCATCTGGGCGACCTGATCGGTT2141
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 385 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
MetSerLeuAsnThrSerGlyLeuGlyAlaSerThrMetGlnIleSer
151015
IleGlyGlyAlaGlyGlyAsnAsnGlyLeuLeuGlyThrSerArgGln
202530
AsnAlaGlyLeuGlyGlyAsnSerAlaLeuGlyLeuGlyGlyGlyAsn
354045
GlnAsnAspThrValAsnGlnLeuAlaGlyLeuLeuThrGlyMetMet
505560
MetMetMetSerMetMetGlyGlyGlyGlyLeuMetGlyGlyGlyLeu
65707580
GlyGlyGlyLeuGlyAsnGlyLeuGlyGlySerGlyGlyLeuGlyGlu
859095
GlyLeuSerAsnAlaLeuAsnAspMetLeuGlyGlySerLeuAsnThr
100105110
LeuGlySerLysGlyGlyAsnAsnThrThrSerThrThrAsnSerPro
115120125
LeuAspGlnAlaLeuGlyIleAsnSerThrSerGlnAsnAspAspSer
130135140
ThrSerGlyThrAspSerThrSerAspSerSerAspProMetGlnGln
145150155160
LeuLeuLysMetPheSerGluIleMetGlnSerLeuPheGlyAspGly
165170175
GlnAspGlyThrGlnGlySerSerSerGlyGlyLysGlnProThrGlu
180185190
GlyGluGlnAsnAlaTyrLysLysGlyValThrAspAlaLeuSerGly
195200205
LeuMetGlyAsnGlyLeuSerGlnLeuLeuGlyAsnGlyGlyLeuGly
210215220
GlyGlyGlnGlyGlyAsnAlaGlyThrGlyLeuAspGlySerSerLeu
225230235240
GlyGlyLysGlyLeuGlnAsnLeuSerGlyProValAspTyrGlnGln
245250255
LeuGlyAsnAlaValGlyThrGlyIleGlyMetLysAlaGlyIleGln
260265270
AlaLeuAsnAspIleGlyThrHisArgHisSerSerThrArgSerPhe
275280285
ValAsnLysGlyAspArgAlaMetAlaLysGluIleGlyGlnPheMet
290295300
AspGlnTyrProGluValPheGlyLysProGlnTyrGlnLysGlyPro
305310315320
GlyGlnGluValLysThrAspAspLysSerTrpAlaLysAlaLeuSer
325330335
LysProAspAspAspGlyMetThrProAlaSerMetGluGlnPheAsn
340345350
LysAlaLysGlyMetIleLysArgProMetAlaGlyAspThrGlyAsn
355360365
GlyAsnLeuGlnHisAlaValProValValLeuArgTrpValLeuMet
370375380
Pro
385
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1158 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
ATGAGTCTGAATACAAGTGGGCTGGGAGCGTCAACGATGCAAATTTCTATCGGCGGTGCG60
GGCGGAAATAACGGGTTGCTGGGTACCAGTCGCCAGAATGCTGGGTTGGGTGGCAATTCT120
GCACTGGGGCTGGGCGGCGGTAATCAAAATGATACCGTCAATCAGCTGGCTGGCTTACTC180
ACCGGCATGATGATGATGATGAGCATGATGGGCGGTGGTGGGCTGATGGGCGGTGGCTTA240
GGCGGTGGCTTAGGTAATGGCTTGGGTGGCTCAGGTGGCCTGGGCGAAGGACTGTCGAAC300
GCGCTGAACGATATGTTAGGCGGTTCGCTGAACACGCTGGGCTCGAAAGGCGGCAACAAT360
ACCACTTCAACAACAAATTCCCCGCTGGACCAGGCGCTGGGTATTAACTCAACGTCCCAA420
AACGACGATTCCACCTCCGGCACAGATTCCACCTCAGACTCCAGCGACCCGATGCAGCAG480
CTGCTGAAGATGTTCAGCGAGATAATGCAAAGCCTGTTTGGTGATGGGCAAGATGGCACC540
CAGGGCAGTTCCTCTGGGGGCAAGCAGCCGACCGAAGGCGAGCAGAACGCCTATAAAAAA600
GGAGTCACTGATGCGCTGTCGGGCCTGATGGGTAATGGTCTGAGCCAGCTCCTTGGCAAC660
GGGGGACTGGGAGGTGGTCAGGGCGGTAATGCTGGCACGGGTCTTGACGGTTCGTCGCTG720
GGCGGCAAAGGGCTGCAAAACCTGAGCGGGCCGGTGGACTACCAGCAGTTAGGTAACGCC780
GTGGGTACCGGTATCGGTATGAAAGCGGGCATTCAGGCGCTGAATGATATCGGTACGCAC840
AGGCACAGTTCAACCCGTTCTTTCGTCAATAAAGGCGATCGGGCGATGGCGAAGGAAATC900
GGTCAGTTCATGGACCAGTATCCTGAGGTGTTTGGCAAGCCGCAGTACCAGAAAGGCCCG960
GGTCAGGAGGTGAAAACCGATGACAAATCATGGGCAAAAGCACTGAGCAAGCCAGATGAC1020
GACGGAATGACACCAGCCAGTATGGAGCAGTTCAACAAAGCCAAGGGCATGATCAAAAGG1080
CCCATGGCGGGTGATACCGGCAACGGCAACCTGCAGCACGCGGTGCCGGTGGTTCTTCGC1140
TGGGTATTGATGCCATGA1158
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 341 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
MetGlnSerLeuSerLeuAsnSerSerSerLeuGlnThrProAlaMet
151015
AlaLeuValLeuValArgProGluAlaGluThrThrGlySerThrSer
202530
SerLysAlaLeuGlnGluValValValLysLeuAlaGluGluLeuMet
354045
ArgAsnGlyGlnLeuAspAspSerSerProLeuGlyLysLeuLeuAla
505560
LysSerMetAlaAlaAspGlyLysAlaGlyGlyGlyIleGluAspVal
65707580
IleAlaAlaLeuAspLysLeuIleHisGluLysLeuGlyAspAsnPhe
859095
GlyAlaSerAlaAspSerAlaSerGlyThrGlyGlnGlnAspLeuMet
100105110
ThrGlnValLeuAsnGlyLeuAlaLysSerMetLeuAspAspLeuLeu
115120125
ThrLysGlnAspGlyGlyThrSerPheSerGluAspAspMetProMet
130135140
LeuAsnLysIleAlaGlnPheMetAspAspAsnProAlaGlnPhePro
145150155160
LysProAspSerGlySerTrpValAsnGluLeuLysGluAspAsnPhe
165170175
LeuAspGlyAspGluThrAlaAlaPheArgSerAlaLeuAspIleIle
180185190
GlyGlnGlnLeuGlyAsnGlnGlnSerAspAlaGlySerLeuAlaGly
195200205
ThrGlyGlyGlyLeuGlyThrProSerSerPheSerAsnAsnSerSer
210215220
ValMetGlyAspProLeuIleAspAlaAsnThrGlyProGlyAspSer
225230235240
GlyAsnThrArgGlyGluAlaGlyGlnLeuIleGlyGluLeuIleAsp
245250255
ArgGlyLeuGlnSerValLeuAlaGlyGlyGlyLeuGlyThrProVal
260265270
AsnThrProGlnThrGlyThrSerAlaAsnGlyGlyGlnSerAlaGln
275280285
AspLeuAspGlnLeuLeuGlyGlyLeuLeuLeuLysGlyLeuGluAla
290295300
ThrLeuLysAspAlaGlyGlnThrGlyThrAspValGlnSerSerAla
305310315320
AlaGlnIleAlaThrLeuLeuValSerThrLeuLeuGlnGlyThrArg
325330335
AsnGlnAlaAlaAla
340
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1026 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
ATGCAGAGTCTCAGTCTTAACAGCAGCTCGCTGCAAACCCCGGCAATGGCCCTTGTCCTG60
GTACGTCCTGAAGCCGAGACGACTGGCAGTACGTCGAGCAAGGCGCTTCAGGAAGTTGTC120
GTGAAGCTGGCCGAGGAACTGATGCGCAATGGTCAACTCGACGACAGCTCGCCATTGGGA180
AAACTGTTGGCCAAGTCGATGGCCGCAGATGGCAAGGCGGGCGGCGGTATTGAGGATGTC240
ATCGCTGCGCTGGACAAGCTGATCCATGAAAAGCTCGGTGACAACTTCGGCGCGTCTGCG300
GACAGCGCCTCGGGTACCGGACAGCAGGACCTGATGACTCAGGTGCTCAATGGCCTGGCC360
AAGTCGATGCTCGATGATCTTCTGACCAAGCAGGATGGCGGGACAAGCTTCTCCGAAGAC420
GATATGCCGATGCTGAACAAGATCGCGCAGTTCATGGATGACAATCCCGCACAGTTTCCC480
AAGCCGGACTCGGGCTCCTGGGTGAACGAACTCAAGGAAGACAACTTCCTTGATGGCGAC540
GAAACGGCTGCGTTCCGTTCGGCACTCGACATCATTGGCCAGCAACTGGGTAATCAGCAG600
AGTGACGCTGGCAGTCTGGCAGGGACGGGTGGAGGTCTGGGCACTCCGAGCAGTTTTTCC660
AACAACTCGTCCGTGATGGGTGATCCGCTGATCGACGCCAATACCGGTCCCGGTGACAGC720
GGCAATACCCGTGGTGAAGCGGGGCAACTGATCGGCGAGCTTATCGACCGTGGCCTGCAA780
TCGGTATTGGCCGGTGGTGGACTGGGCACACCCGTAAACACCCCGCAGACCGGTACGTCG840
GCGAATGGCGGACAGTCCGCTCAGGATCTTGATCAGTTGCTGGGCGGCTTGCTGCTCAAG900
GGCCTGGAGGCAACGCTCAAGGATGCCGGGCAAACAGGCACCGACGTGCAGTCGAGCGCT960
GCGCAAATCGCCACCTTGCTGGTCAGTACGCTGCTGCAAGGCACCCGCAATCAGGCTGCA1020
GCCTGA1026
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 344 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
MetSerValGlyAsnIleGlnSerProSerAsnLeuProGlyLeuGln
151015
AsnLeuAsnLeuAsnThrAsnThrAsnSerGlnGlnSerGlyGlnSer
202530
ValGlnAspLeuIleLysGlnValGluLysAspIleLeuAsnIleIle
354045
AlaAlaLeuValGlnLysAlaAlaGlnSerAlaGlyGlyAsnThrGly
505560
AsnThrGlyAsnAlaProAlaLysAspGlyAsnAlaAsnAlaGlyAla
65707580
AsnAspProSerLysAsnAspProSerLysSerGlnAlaProGlnSer
859095
AlaAsnLysThrGlyAsnValAspAspAlaAsnAsnGlnAspProMet
100105110
GlnAlaLeuMetGlnLeuLeuGluAspLeuValLysLeuLeuLysAla
115120125
AlaLeuHisMetGlnGlnProGlyGlyAsnAspLysGlyAsnGlyVal
130135140
GlyGlyAlaAsnGlyAlaLysGlyAlaGlyGlyGlnGlyGlyLeuAla
145150155160
GluAlaLeuGlnGluIleGluGlnIleLeuAlaGlnLeuGlyGlyGly
165170175
GlyAlaGlyAlaGlyGlyAlaGlyGlyGlyValGlyGlyAlaGlyGly
180185190
AlaAspGlyGlySerGlyAlaGlyGlyAlaGlyGlyAlaAsnGlyAla
195200205
AspGlyGlyAsnGlyValAsnGlyAsnGlnAlaAsnGlyProGlnAsn
210215220
AlaGlyAspValAsnGlyAlaAsnGlyAlaAspAspGlySerGluAsp
225230235240
GlnGlyGlyLeuThrGlyValLeuGlnLysLeuMetLysIleLeuAsn
245250255
AlaLeuValGlnMetMetGlnGlnGlyGlyLeuGlyGlyGlyAsnGln
260265270
AlaGlnGlyGlySerLysGlyAlaGlyAsnAlaSerProAlaSerGly
275280285
AlaAsnProGlyAlaAsnGlnProGlySerAlaAspAspGlnSerSer
290295300
GlyGlnAsnAsnLeuGlnSerGlnIleMetAspValValLysGluVal
305310315320
ValGlnIleLeuGlnGlnMetLeuAlaAlaGlnAsnGlyGlySerGln
325330335
GlnSerThrSerThrGlnProMet
340
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1035 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
ATGTCAGTCGGAAACATCCAGAGCCCGTCGAACCTCCCGGGTCTGCAGAACCTGAACCTC60
AACACCAACACCAACAGCCAGCAATCGGGCCAGTCCGTGCAAGACCTGATCAAGCAGGTC120
GAGAAGGACATCCTCAACATCATCGCAGCCCTCGTGCAGAAGGCCGCACAGTCGGCGGGC180
GGCAACACCGGTAACACCGGCAACGCGCCGGCGAAGGACGGCAATGCCAACGCGGGCGCC240
AACGACCCGAGCAAGAACGACCCGAGCAAGAGCCAGGCTCCGCAGTCGGCCAACAAGACC300
GGCAACGTCGACGACGCCAACAACCAGGATCCGATGCAAGCGCTGATGCAGCTGCTGGAA360
GACCTGGTGAAGCTGCTGAAGGCGGCCCTGCACATGCAGCAGCCCGGCGGCAATGACAAG420
GGCAACGGCGTGGGCGGTGCCAACGGCGCCAAGGGTGCCGGCGGCCAGGGCGGCCTGGCC480
GAAGCGCTGCAGGAGATCGAGCAGATCCTCGCCCAGCTCGGCGGCGGCGGTGCTGGCGCC540
GGCGGCGCGGGTGGCGGTGTCGGCGGTGCTGGTGGCGCGGATGGCGGCTCCGGTGCGGGT600
GGCGCAGGCGGTGCGAACGGCGCCGACGGCGGCAATGGCGTGAACGGCAACCAGGCGAAC660
GGCCCGCAGAACGCAGGCGATGTCAACGGTGCCAACGGCGCGGATGACGGCAGCGAAGAC720
CAGGGCGGCCTCACCGGCGTGCTGCAAAAGCTGATGAAGATCCTGAACGCGCTGGTGCAG780
ATGATGCAGCAAGGCGGCCTCGGCGGCGGCAACCAGGCGCAGGGCGGCTCGAAGGGTGCC840
GGCAACGCCTCGCCGGCTTCCGGCGCGAACCCGGGCGCGAACCAGCCCGGTTCGGCGGAT900
GATCAATCGTCCGGCCAGAACAATCTGCAATCCCAGATCATGGATGTGGTGAAGGAGGTC960
GTCCAGATCCTGCAGCAGATGCTGGCGGCGCAGAACGGCGGCAGCCAGCAGTCCACCTCG1020
ACGCAGCCGATGTAA1035
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ThrLeuIleGluLeuMetIleValValAlaIleIleAlaIleLeuAla
151015
AlaIleAlaLeuProAlaTyrGlnAspTyr
2025
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