U.S. patent application number 10/034158 was filed with the patent office on 2003-02-06 for method of imparting drought resistance to plants.
Invention is credited to Wei, Zhong-Min.
Application Number | 20030028918 10/034158 |
Document ID | / |
Family ID | 27359896 |
Filed Date | 2003-02-06 |
United States Patent
Application |
20030028918 |
Kind Code |
A1 |
Wei, Zhong-Min |
February 6, 2003 |
Method of imparting drought resistance to plants
Abstract
The present invention relates to a method of enhancing growth of
plants. This involves applying a hypersensitive response elicitor
polypeptide or protein in a non-infectious form to a plant or plant
seed under conditions effective to enhance growth of the plant or
plants produced from the plant seed. Alternatively, transgenic
plants or transgenic plant seeds transformed with a DNA molecule
encoding a hypersensitive response elicitor polypeptide or protein
can be provided and the transgenic plants or plants resulting from
the transgenic plant seeds are grown under conditions effective to
enhance plant growth.
Inventors: |
Wei, Zhong-Min; (Kirkland,
WA) |
Correspondence
Address: |
Michael L. Goldman
NIXON PEABODY LLP
Clinton Square
P.O. Box 31051
Rochester
NY
14603
US
|
Family ID: |
27359896 |
Appl. No.: |
10/034158 |
Filed: |
December 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10034158 |
Dec 20, 2001 |
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09597840 |
Jun 20, 2000 |
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09597840 |
Jun 20, 2000 |
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09013587 |
Jan 26, 1998 |
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6277814 |
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60036048 |
Jan 27, 1997 |
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Current U.S.
Class: |
800/278 ;
800/288 |
Current CPC
Class: |
C12N 15/8261 20130101;
C07K 14/27 20130101; Y02A 40/146 20180101; C07K 14/21 20130101;
A01N 37/46 20130101; A01N 63/50 20200101; A01N 63/50 20200101; A01N
63/20 20200101; A01N 63/50 20200101; A01N 63/27 20200101 |
Class at
Publication: |
800/278 ;
800/288 |
International
Class: |
A01H 005/00 |
Goverment Interests
[0002] This invention was made with support from the U.S.
Government under USDA NRI Competitive Research Grant No.
91-37303-6430.
Claims
What is claimed:
1. A method of imparting drought resistance to plants comprising:
applying a hypersensitive response elicitor protein or polypeptide
in a non-infectious form to a plant under conditions effective to
impart drought resistance.
2. A method according to claim 1, wherein the hypersensitive
response elicitor protein or polypeptide is derived from Erwinia,
Pseudomonas, Xanthomonas, or Phytophthera.
3. A method according to claim 2, wherein the hypersensitive
response elicitor protein or polypeptide is derived from Erwinia
amylovora, Erwinia carotovora, Erwinia chrysanthemi, and Erwinia
stewartii.
4. A method according to claim 2, wherein the hypersensitive
response elicitor protein or polypeptide is derived from
Pseudomonas syringae or Pseudomonas solancearum.
5. A method according to claim 2, wherein the hypersensitive
response elicitor protein or polypeptide is derived from a
Xanthomonas species.
6. A method according to claim 2, wherein the hypersensitive
response elicitor protein or polypeptide is derived from a
Phytophthera species.
7. A method according to claim 1, 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.
8. A method according to claim 1, wherein the plant is selected
from the group consisting of rose, Saintpaulia, petunia,
pelargonium, poinsettia, chrysanthemum, carnation, and zinnia.
9. A method according to claim 1, wherein said applying is carried
out prior to a drought.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/597,840, filed Jun. 20, 2000, which is a
division of U.S. patent application Ser. No. 09/013,587, filed Jan.
26, 1998, now U.S. Pat. No.6,227,814, and claims the benefit of
U.S. Provisional Patent Application Serial No. 60/036,048, filed
Jan. 27, 1997.
FIELD OF THE INVENTION
[0003] The present invention relates to the enhancement of growth
in plants.
BACKGROUND OF THE INVENTION
[0004] The improvement of plant growth by the application of
organic fertilizers has been known and carried out for centuries
(H. Marschner, "Mineral Nutrition of Higher Plants," Academic
Press: New York pg. 674 (1986). Modern man has developed a complex
inorganic fertilizer production system to produce an easy product
that growers and farmers can apply to soils or growing crops to
improve performance by way of growth enhancement. Plant size,
coloration, maturation, and yield may all be improved by the
application of fertilizer products. Inorganic fertilizers include
such commonly applied chemicals as ammonium nitrate. Organic
fertilizers may include animal manures and composted lawn debris,
among many other sources.
[0005] In most recent years, researchers have sought to improve
plant growth through the use of biological products. Insect and
disease control agents such as Beauveria bassiana and Trichoderma
harizamum have been registered for the control of insect and
disease problems and thereby indirectly improve plant growth and
performance (Fravel et al., "Formulation of Microorganisms to
Control Plant Diseases," Formulation of Microbial Biopesticides,
Beneficial Microorganisms, and Nematodes, H. D. Burges, ed. Chapman
and Hall: London (1996).
[0006] There is some indication of direct plant growth enhancement
by way of microbial application or microbial by-products.
Nodulating bacteria have been added to seeds of leguminous crops
when introduced to a new site (Weaver et al., "Rhizobium," Methods
of Soil Analysis, Part 2, Chemical and Microbiological Properties,
2nd ed., American Society of Agronomy: Madison (1982)). These
bacteria may improve the nodulation efficiency of the plant and
thereby improve the plant's ability to convert free nitrogen into a
usable form, a process called nitrogen fixation. Non-leguminous
crops do not, as a rule, benefit from such treatment. Added
bacteria such as Rhizobium directly parasitize the root hairs, then
begin a mutualistic relationship by providing benefit to the plant
while receiving protection and sustenance.
[0007] Mycorrhizal fungi have also been recognized as necessary
microorganisms for optional growth of many crops, especially
conifers in nutrient-depleted soils. Mechanisms including
biosynthesis of plant hormones (Frankenberger et al., "Biosynthesis
of Indole-3-Acetic Acid by the Pine Ectomycorrhizal Fungas
Pisolithus tinctorius," Appl. Environ. Microbiol. 53:2908-13
(1987)), increased uptake of minerals (Harley et al., "The Uptake
of Phosphate by Excised Mycorrhizal Roots of Beech," New
Phytologist 49:388-97 (1950) and Harley et al., "The Uptake of
Phosphate by Excised Mycorrhizal Roots of Beech. IV. The Effect of
Oxygen Concentration Upon Host and Fungus," New Phytologist
52:124-32 (1953)), and water (A. B. Hatch, "The Physical Basis of
Mycotrophy in Pinus," Black Rock Forest Bull. No. 6, 168 pp.
(1937)) have been postulated. Mycorrhizal fungi have not achieved
the common frequency of use that nodulating bacteria have due to
variable and inconsistent results with any given mycorrhizal strain
and the difficulty of study of the organisms.
[0008] Plant growth-promoting rhizobacteria ("PGPR") have been
recognized in recent years for improving plant growth and
development. Hypothetical mechanisms range from direct influences
(e.g., increased nutrient uptake) to indirect mechanisms (e.g.,
pathogen displacement). Growth enhancement by application of a PGPR
generally refers to inoculation with a live bacterium to the root
system and achieving improved growth through bacterium-produced
hormonal effects, siderophores, or by prevention of disease through
antibiotic production, or competition. In all of the above cases,
the result is effected through root colonization, sometimes through
the application of seed coatings. There is limited information to
suggest that some PGPR strains may be direct growth promoters that
enhance root elongation under gnotobiotic conditions (Anderson et
al., "Responses of Bean to Root Colonization With Pseudomonas
putida in a Hydroponic System," Phytopathology 75:992-95 (1985),
Lifshitz et al., "Growth Promotion of Canola (rapeseed) Seedlings
by a Strain of Pseudomonasputida Under Gnotobiotic Conditions,"
Can. J. Microbiol. 33:390-95 (1987), Young et al., "PGPR: Is There
Relationship Between Plant Growth Regulators and the Stimulation of
Plant Growth or Biological Activity?," Promoting Rhizobacteria:
Progress and Prospects, Second International Workshop on Plant
Growth-promoting Rhizobacteria, pp. 182-86 (1991), Loper et al.,
"Influence of Bacterial Sources of Indole-3-Acetic Acid on Root
Elongation of Sugar Beet," Phytopathology 76:386-89 (1986), and
Muller et al., "Hormonal Interactions in the Rhizosphere of Maize
(Zea mays L.) and Their Effect on Plant Development," Z.
Pflanzenernhrung Bodenkunde 152:247-54 (1989); however, the
production of plant growth regulators has been proposed as the
mechanism mediating these effects. Many bacteria produce various
plant growth regulators in vitro (Atzorn et al., "Production of
Gibberellins and Indole-3-Acetic Acid by Rhizobium phaseoli in
Relation to Nodulation of Phaseolus vulgaris Roots," Planta
175:532-38 (1988) and M. E. Brown, "Plant Growth Substances
Produced by Micro-Organism of Solid and Rhizosphere," J. Appl.
Bact. 35:443-51 (1972)) or antibiotics (Gardner et al., "Growth
Promotion and Inhibition by Antibiotic-Producing Fluorescent
Pseudomonads on Citrus Roots," Plant Soil 77:103-13 (1984)).
Siderphore production is another mechanism proposed for some PGPR
strains (Ahl et al., "Iron Bound-Siderophores, Cyanic Acid, and
Antibiotics Involved in Suppression of Thievaliopsis basicola by a
Pseudomonas fluorescens Strain," J. Phytopathol. 116:121-34 (1986),
Kloepper et al., "Enhanced Plant Growth by Siderophores Produced by
Plant Growth-Promoting Rhizobacteria," Nature 286:885-86 (1980),
and Kloepper et al., "Pseudomonas siderophores: A Mechanism
Explaining Disease-Suppressive Soils," Curr. Microbiol. 4:317-20
(1980)). The colonization of root surfaces and thus the direct
competition with pathogenic bacteria on the surfaces is another
mechanism of action (Kloepper et al., "Relationship of in vitro
Antibiosis of Plant Growth-Promoting Rhizobacteria to Plant Growth
and the Displacement of Root Microflora," Phytopathology 71:1020-24
(1981), Weller, et al., "Increased Growth of Wheat by Seed
Treatments With Fluorescent Pseudomonads, and Implications of
Pythium Control," Can. J. Microbiol. 8:328-34 (1986), and Suslow et
al., "Rhizobacteria of Sugar Beets: Effects of Seed Application and
Root Colonization on Yield," Phytopathology 72:199-206 (1982)).
Canola (rapeseed) studies have indicated PGPR increased plant
growth parameters including yields, seedling emergence and vigor,
early-season plant growth (number of leaves and length of main
runner), and leaf area (Kloepper et al., "Plant Growth-Promoting
Rhizobacteria on Canola (rapeseed)," Plant Disease 72:42-46
(1988)). Studies with potato indicated greater yields when
Pseudomonas strains were applied to seed potatoes (Burr et al.,
"Increased Potato Yields by Treatment of Seed Pieces With Specific
Strains of Pseudomonas Fluorescens and P. putida," Phytopathology
68:1377-83 (1978), Kloepper et al., "Effect of Seed Piece
Inoculation With Plant Growth-Promoting Rhizobacteria on
Populations of Erwinia carotovora on Potato Roots and in Daughter
Tubers," Phytopathology 73:217-19 (1983), Geels et al., "Reduction
of Yield Depressions in High Frequency Potato Cropping Soil After
Seed Tuber Treatments With Antagonistic Fluorescent Pseudomonas
spp.," Phytopathol. Z. 108:207-38 (1983), Howie et al.,
"Rhizobacteria: Influence of Cultivar and Soil Type on Plant Growth
and Yield of Potato," Soil Biol. Biochem. 15:127-32 (1983), and
Vrany et al., "Growth and Yield of Potato Plants Inoculated With
Rhizosphere Bacteria," Folia Microbiol. 29:248-53 (1984)). Yield
increase was apparently due to the competitive effects of the PGPR
to eliminate pathogenic bacteria on the seed tuber, possibly by
antibiosis (Kloepper et al., "Effect of Seed Piece Inoculation With
Plant Growth-Promoting Rhizobacteria on Populations of Erwinia
carotovora on Potato Roots and in Daughter Tubers," Phytopathology
73:217-19 (1983), Kloepper et al., "Effects of Rhizosphere
Colonization by Plant Growth-Promoting Rhizobacteria on Potato
Plant Development and Yield," Phytopathology 70:1078-82(1980),
Kloepper et al., "Emergence-Promoting Rhizobacteria: Description
and Implications for Agriculture," pp. 155-164, Iron, Siderophores,
and Plant Disease, T. R. Swinbume, ed. Plenum, New York (1986), and
Kloepper et al., "Relationship of in vitro Antibiosis of Plant
Growth-Promoting Rhizobacteria to Plant Growth and the Displacement
of Root Microflora," Phytopathology 71:1020-24 (1981)). In several
studies, plant emergence was improved using PGPR (Tipping et al.,
"Development of Emergence-Promoting Rhizobacteria for Supersweet
Corn," Phytopathology 76:938-41 (1990) (abstract) and Kloepper et
al., "Emergence-Promoting Rhizobacteria: Description and
Implications for Agriculture," pp. 155-164, Iron, Siderophores, and
Plant Disease, T. R. Swinbume, ed. Plenum, New York (1986)).
Numerous other studies indicated improved plant health upon
treatment with rhizobacteria, due to biocontrol of plant pathogens
(B. Schippers, "Biological Control of Pathogens With
Rhizobacteria," Phil. Trans. R. Soc. Lond. B. 318:283-93 (1988),
Schroth et al., "Disease-Suppressive Soil and Root-Colonizing
Bacteria," Science 216:1376-81 (1982), Stutz et al., "Naturally
Occurring Fluorescent Pseudomonads Involved in Suppression of Black
Root Rot of Tobacco," Phytopathology 76:181-85 (1986), and D. M.
Weller, "Biological Control of Soilborne Plant Pathogens in the
Rhizosphere With Bacteria," Annu. Rev. Phytopathol. 26:379-407
(1988)).
[0009] Pathogen-induced immunization of a plant has been found to
promote growth. Injection of Peronospora tabacina externally to
tobacco xylem not only alleviated stunting but also promoted growth
and development. Immunized tobacco plants, in both greenhouse and
field experiments, were approximately 40% taller, had a 40%
increase in dry weight, a 30% increase in fresh weight, and 4-6
more leaves than control plants (Tuzun, S., et al., "The Effect of
Stem Injection with Peronospora tabacina and Metalaxyl Treatment on
Growth of Tobacco and Protection Against Blue Mould in the Field,"
Phytopathology, 74:804 (1984). 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)).
[0010] The present invention is directed to an improvement over
prior plant growth enhancement procedures.
SUMMARY OF THE INVENTION
[0011] The present invention relates to a method of enhancing
growth in plants. This method involves applying a hypersensitive
response elicitor polypeptide or protein in a non-infectious form
to plants or plant seeds under conditions to impart enhanced growth
to the plants or to plants grown from the plant seeds.
[0012] As an alternative to applying a hypersensitive response
elicitor polypeptide or protein to plants or plant seeds in order
to impart enhanced growth to the plants or to plants grown from the
seeds, transgenic plants or plant seeds can be utilized. When
utilizing transgenic plants, this involves providing a transgenic
plant transformed with a DNA molecule encoding a hypersensitive
response elicitor polypeptide or protein and growing the plant
under conditions effective to permit that DNA molecule to enhance
growth. Alternatively, a transgenic plant seed transformed with a
DNA molecule encoding a hypersensitive response elicitor
polypeptide or protein can be provided and planted in soil. A plant
is then propagated from the planted seed under conditions effective
to permit that DNA molecule to enhance growth.
[0013] The present invention is directed to effecting any form of
plant growth enhancement or promotion. This can occur as early as
when plant growth begins from seeds or later in the life of a
plant. For example, plant growth according to the present invention
encompasses greater yield, increased quantity of seeds produced,
increased percentage of seeds germinated, increased plant size,
greater biomass, more and bigger fruit, earlier fruit coloration,
and earlier fruit and plant maturation. As a result, the present
invention provides significant economic benefit to growers. For
example, early germination and early maturation permit crops to be
grown in areas where short growing seasons would otherwise preclude
their growth in that locale. Increased percentage of seed
germination results in improved crop stands and more efficient seed
use. Greater yield, increased size, and enhanced biomass production
allow greater revenue generation from a given plot of land. It is
thus apparent that the present invention constitutes a significant
advance in agricultural efficiency.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a map of plasmid vector pCPP2139 which contains
the Erwinia amylovora hypersensitive response elicitor gene.
[0015] FIG. 2 is a map of plasmid vector pCPP50 which does not
contain the Erwinia amylovora hypersensitive response elicitor gene
but is otherwise the same as plasmid vector pCPP2139 shown in FIG.
1. See Masui, et al., Bio/Technology 2:81-85 (1984), which is
hereby incorporated by reference.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention relates to a method of enhancing
growth in plants. This method involves applying a hypersensitive
response elicitor polypeptide or protein in a non-infectious form
to all or part of a plant or a plant seed under conditions to
impart enhanced growth to the plant or to a plant grown from the
plant seed. Alternatively, plants can be treated in this manner to
produce seeds, which when planted, impart enhanced growth in
progeny plants.
[0017] As an alternative to applying a hypersensitive response
elicitor polypeptide or protein to plants or plant seeds in order
to impart enhanced growth to the plants or to plants grown from the
seeds, transgenic plants or plant seeds can be utilized. When
utilizing transgenic plants, this involves providing a transgenic
plant transformed with a DNA molecule encoding a hypersensitive
response elicitor polypeptide or protein and growing the plant
under conditions effective to permit that DNA molecule to enhance
growth. Alternatively, a transgenic plant seed transformed with a
DNA molecule encoding a hypersensitive response elicitor
polypeptide or protein can be provided and planted in soil. A plant
is then propagated from the planted seed under conditions effective
to permit that DNA molecule to enhance growth.
[0018] The hypersensitive response elicitor polypeptide or protein
utilized in the present invention can correspond to hypersensitive
response elicitor polypeptides or proteins derived from a wide
variety of fungal and bacterial pathogens. Such polypeptides or
proteins are able to elicit local necrosis in plant tissue
contacted by the elicitor.
[0019] Examples of suitable bacterial sources of polypeptide or
protein elicitors include Erwinia, Pseudomonas, and Xanthamonas
species (e.g., the following bacteria: Erwinia amylovora, Erwinia
chrysanthemi, Erwinia stewartii, Erwinia earotovora, Pseudomonas
syringae, Pseudomonas solancearum, Xanthomonas campestris, and
mixtures thereof).
[0020] An example of a fungal source of a hypersensitive response
elicitor protein or polypeptide is Phytophthora. Suitable species
of Phytophthora include Phytophthora pythium, Phytophthora
cryptogea, Phytophthora cinnamomi, Phytophthora capsici,
Phytophthora megasperma, and Phytophthora citrophthora.
[0021] The embodiment of the present invention where the
hypersensitive response elicitor polypeptide or protein is applied
to the plant or plant seed can be carried out in a number of ways,
including: 1) application of an isolated elicitor polypeptide or
protein; 2) application of bacteria which do not cause disease and
are transformed with genes encoding a hypersensitive response
elicitor polypeptide or protein; and 3) application of bacteria
which cause disease in some plant species (but not in those to
which they are applied) and naturally contain a gene encoding the
hypersensitive response elicitor polypeptide or protein. In
addition, seeds in accordance with the present invention can be
recovered from plants which have been treated with a hypersensitive
response elicitor protein or polypeptide in accordance with the
present invention.
[0022] 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
or plant seeds. 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.
[0023] In other embodiments of the present invention, the
hypersensitive response elicitor polypeptide or protein of the
present invention can be applied to plants or plant seeds by
applying bacteria containing genes encoding the hypersensitive
response elicitor polypeptide or protein. Such bacteria must be
capable of secreting or exporting the polypeptide or protein so
that the elicitor can contact plant or plant seeds cells. In these
embodiments, the hypersensitive response elicitor polypeptide or
protein is produced by the bacteria in planta or on seeds or just
prior to introduction of the bacteria to the plants or plant
seeds.
[0024] 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 does 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.
[0025] 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
or their seeds 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 plants or seeds to enhance growth without
causing disease in that species.
[0026] The hypersensitive response elicitor polypeptide or protein
from Erwinia chrysanthemi has an amino acid sequence corresponding
to SEQ. ID. No. 1 as follows:
1 Met Gln Ile Thr Ile Lys Ala His Ile Gly Gly Asp Leu Gly Val Ser 1
5 10 15 Gly Leu Gly Ala Gln Gly Leu Lys Gly Leu Asn Ser Ala Ala Ser
Ser 20 25 30 Leu Gly Ser Ser Val Asp Lys Leu Ser Ser Thr Ile Asp
Lys Leu Thr 35 40 45 Ser Ala Leu Thr Ser Met Met Phe Gly Gly Ala
Leu Ala Gln Gly Leu 50 55 60 Gly Ala Ser Ser Lys Gly Leu Gly Met
Ser Asn Gln Leu Gly Gln Ser 65 70 75 80 Phe Gly Asn Gly Ala Gln Gly
Ala Ser Asn Leu Leu Ser Val Pro Lys 85 90 95 Ser Gly Gly Asp Ala
Leu Ser Lys Met Phe Asp Lys Ala Leu Asp Asp 100 105 110 Leu Leu Gly
His Asp Thr Val Thr Lys Leu Thr Asn Gln Ser Asn Gln 115 120 125 Leu
Ala Asn Ser Met Leu Asn Ala Ser Gln Met Thr Gln Gly Asn Met 130 135
140 Asn Ala Phe Gly Ser Gly Val Asn Asn Ala Leu Ser Ser Ile Leu Gly
145 150 155 160 Asn Gly Leu Gly Gln Ser Met Ser Gly Phe Ser Gln Pro
Ser Leu Gly 165 170 175 Ala Gly Gly Leu Gln Gly Leu Ser Gly Ala Gly
Ala Phe Asn Gln Leu 180 185 190 Gly Asn Ala Ile Gly Met Gly Val Gly
Gln Asn Ala Ala Leu Ser Ala 195 200 205 Leu Ser Asn Val Ser Thr His
Val Asp Gly Asn Asn Arg His Phe Val 210 215 220 Asp Lys Glu Asp Arg
Gly Met Ala Lys Glu Ile Gly Gln Phe Met Asp 225 230 235 240 Gln Tyr
Pro Glu Ile Phe Gly Lys Pro Glu Tyr Gln Lys Asp Gly Trp 245 250 255
Ser Ser Pro Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser Lys 260
265 270 Pro Asp Asp Asp Gly Met Thr Gly Ala Ser Met Asp Lys Phe Arg
Gln 275 280 285 Ala Met Gly Met Ile Lys Ser Ala Val Ala Gly Asp Thr
Gly Asn Thr 290 295 300 Asn Leu Asn Leu Arg Gly Ala Gly Gly Ala Ser
Leu Gly Ile Asp Ala 305 310 315 320 Ala Val Val Gly Asp Lys Ile Ala
Asn Met Ser Leu Gly Lys Leu Ala 325 330 335 Asn Ala
[0027] 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:
2 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
[0028] The hypersensitive response elicitor polypeptide or protein
derived from Erwinia amylovora has an amino acid sequence
corresponding to SEQ. ID. No. 3 as follows:
3 Met Ser Leu Asn Thr Ser Gly Leu Gly Ala Ser Thr Met Gln Ile Ser 1
5 10 15 Ile Gly Gly Ala Gly Gly Asn Asn Gly Leu Leu Gly Thr Ser Arg
Gln 20 25 30 Asn Ala Gly Leu Gly Gly Asn Ser Ala Leu Gly Leu Gly
Gly Gly Asn 35 40 45 Gln Asn Asp Thr Val Asn Gln Leu Ala Gly Leu
Leu Thr Gly Met Met 50 55 60 Met Met Met Ser Met Met Gly Gly Gly
Gly Leu Met Gly Gly Gly Leu 65 70 75 80 Gly Gly Gly Leu Gly Asn Gly
Leu Gly Gly Ser Gly Gly Leu Gly Glu 85 90 95 Gly Leu Ser Asn Ala
Leu Asn Asp Met Leu Gly Gly Ser Leu Asn Thr 100 105 110 Leu Gly Ser
Lys Gly Gly Asn Asn Thr Thr Ser Thr Thr Asn Ser Pro 115 120 125 Leu
Asp Gln Ala Leu Gly Ile Asn Ser Thr Ser Gln Asn Asp Asp Ser 130 135
140 Thr Ser Gly Thr Asp Ser Thr Ser Asp Ser Ser Asp Pro Met Gln Gln
145 150 155 160 Leu Leu Lys Met Phe Ser Glu Ile Met Gln Ser Leu Phe
Gly Asp Gly 165 170 175 Gln Asp Gly Thr Gln Gly Ser Ser Ser Gly Gly
Lys Gln Pro Thr Glu 180 185 190 Gly Glu Gln Asn Ala Tyr Lys Lys Gly
Val Thr Asp Ala Leu Ser Gly 195 200 205 Leu Met Gly Asn Gly Leu Ser
Gln Leu Leu Gly Asn Gly Gly Leu Gly 210 215 220 Gly Gly Gln Gly Gly
Asn Ala Gly Thr Gly Leu Asp Gly Ser Ser Leu 225 230 235 240 Gly Gly
Lys Gly Leu Gln Asn Leu Ser Gly Pro Val Asp Tyr Gln Gln 245 250 255
Leu Gly Asn Ala Val Gly Thr Gly Ile Gly Met Lys Ala Gly Ile Gln 260
265 270 Ala Leu Asn Asp Ile Gly Thr His Arg His Ser Ser Thr Arg Ser
Phe 275 280 285 Val Asn Lys Gly Asp Arg Ala Met Ala Lys Glu Ile Gly
Gln Phe Met 290 295 300 Asp Gln Tyr Pro Glu Val Phe Gly Lys Pro Gln
Tyr Gln Lys Gly Pro 305 310 315 320 Gly Gln Glu Val Lys Thr Asp Asp
Lys Ser Trp Ala Lys Ala Leu Ser 325 330 335 Lys Pro Asp Asp Asp Gly
Met Thr Pro Ala Ser Met Glu Gln Phe Asn 340 345 350 Lys Ala Lys Gly
Met Ile Lys Arg Pro Met Ala Gly Asp Thr Gly Asn 355 360 365 Gly Asn
Leu Gln Ala Arg Gly Ala Gly Gly Ser Ser Leu Gly Ile Asp 370 375 380
Ala Met Met Ala Gly Asp Ala Ile Asn Asn Met Ala Leu Gly Lys Leu 385
390 395 400 Gly Ala Ala
[0029] This hypersensitive response elicitor polypeptide or protein
has a molecular weight of about 39 kDa, has a pI of approximately
4.3, and is heat stable at 100.degree. C. for at least 10 minutes.
This hypersensitive response elicitor polypeptide or protein has
substantially no cysteine. The hypersensitive response elicitor
polypeptide or protein derived from Erwinia amylovora is more fully
described in Wei, Z. -M., R. J. Laby, C. H. Zumoff, D. W. Bauer, S.
-Y. He, A. Collmer, and S. V. Beer, "Harpin, Elicitor of the
Hypersensitive Response Produced by the Plant Pathogen Erwinia
amylovora," Science 257:85-88 (1992), which is hereby incorporated
by reference. The DNA molecule encoding this polypeptide or protein
has a nucleotide sequence corresponding to SEQ. ID. No. 4 as
follows:
4 AAGCTTCGGC ATGGCACGTT TGACCGTTGG GTCGGCAGGG TACGTTTGAA TTATTCATAA
60 GAGGAATACG TTATGAGTCT GAATACAAGT GGGCTGGGAG CGTCAACGAT
GCAAATTTCT 120 ATCGGCGGTG CGGGCGGAAA TAACGGGTTG CTGGGTACCA
GTCGCCAGAA TGCTGGGTTG 180 GGTGGCAATT CTGCACTGGG GCTGGGCGGC
GGTAATCAAA ATGATACCGT CAATCAGCTG 240 GCTGGCTTAC TCACCGGCAT
GATGATGATG ATGAGCATGA TGGGCGGTGG TGGGCTGATG 300 GGCGGTGGCT
TAGGCGGTGG CTTAGGTAAT GGCTTGGGTG GCTCAGGTGG CCTGGGCGAA 360
GGACTGTCGA ACGCGCTGAA CGATATGTTA GGCGGTTCGC TGAACACGCT GGGCTCGAAA
420 GGCGGCAACA ATACCACTTC AACAACAAAT TCCCCGCTGG ACCAGGCGCT
GGGTATTAAC 480 TCAACGTCCC AAAACGACGA TTCCACCTCC GGCACAGATT
CCACCTCAGA CTCCAGCGAC 540 CCGATGCAGC AGCTGCTGAA GATGTTCAGC
GAGATAATGC AAAGCCTGTT TGGTGATGGG 600 CAAGATGGCA CCCAGGGCAG
TTCCTCTGGG GGCAAGCAGC CGACCGAAGG CGAGCAGAAC 660 GCCTATAAAA
AAGGAGTCAC TGATGCGCTG TCGGGCCTGA TGGGTAATGG TCTGAGCCAG 720
CTCCTTGGCA ACGGGGGACT GGGAGGTGGT CAGGGCGGTA ATGCTGGCAC GGGTCTTGAC
780 GGTTCGTCGC TGGGCGGCAA AGGGCTGCAA AACCTGAGCG GGCCGGTGGA
CTACCAGCAG 840 TTAGGTAACG CCGTGGGTAC CGGTATCGGT ATGAAAGCGG
GCATTCAGGC GCTGAATGAT 900 ATCGGTACGC ACAGGCACAG TTCAACCCGT
TCTTTCGTCA ATAAAGGCGA TCGGGCGATG 960 GCGAAGGAAA TCGGTCAGTT
CATGGACCAG TATCCTGAGG TGTTTGGCAA GCCGCAGTAC 1020 CAGAAAGGCC
CGGGTCAGGA GGTGAAAACC GATGACAAAT CATGGGCAAA AGCACTGAGC 1080
AAGCCAGATG ACGACGGAAT GACACCAGCC AGTATGGAGC AGTTCAACAA AGCCAAGGGC
1140 ATGATCAAAA GGCCCATGGC GGGTGATACC GGCAACGGCA ACCTGCAGGC
ACGCGGTGCC 1200 GGTGGTTCTT CGCTGGGTAT TGATGCCATG ATGGCCGGTG
ATGCCATTAA CAATATGGCA 1260 CTTGGCAAGC TGGGCGCGGC TTAAGCTT 1288
[0030] The hypersensitive response elicitor polypeptide or protein
derived from Pseudomonas syringae has an amino acid sequence
corresponding to SEQ. ID. No. 5 as follows:
5 Met Gln Ser Leu Ser Leu Asn Ser Ser Ser Leu Gln Thr Pro Ala Met 1
5 10 15 Ala Leu Val Leu Val Arg Pro Glu Ala Glu Thr Thr Gly Ser Thr
Ser 20 25 30 Ser Lys Ala Leu Gln Glu Val Val Val Lys Leu Ala Glu
Glu Leu Met 35 40 45 Arg Asn Gly Gln Leu Asp Asp Ser Ser Pro Leu
Gly Lys Leu Leu Ala 50 55 60 Lys Ser Met Ala Ala Asp Gly Lys Ala
Gly Gly Gly Ile Glu Asp Val 65 70 75 80 Ile Ala Ala Leu Asp Lys Leu
Ile His Glu Lys Leu Gly Asp Asn Phe 85 90 95 Gly Ala Ser Ala Asp
Ser Ala Ser Gly Thr Gly Gln Gln Asp Leu Met 100 105 110 Thr Gln Val
Leu Asn Gly Leu Ala Lys Ser Met Leu Asp Asp Leu Leu 115 120 125 Thr
Lys Gln Asp Gly Gly Thr Ser Phe Ser Glu Asp Asp Met Pro Met 130 135
140 Leu Asn Lys Ile Ala Gln Phe Met Asp Asp Asn Pro Ala Gln Phe Pro
145 150 155 160 Lys Pro Asp Ser Gly Ser Trp Val Asn Glu Leu Lys Glu
Asp Asn Phe 165 170 175 Leu Asp Gly Asp Glu Thr Ala Ala Phe Arg Ser
Ala Leu Asp Ile Ile 180 185 190 Gly Gln Gln Leu Gly Asn Gln Gln Ser
Asp Ala Gly Ser Leu Ala Gly 195 200 205 Thr Gly Gly Gly Leu Gly Thr
Pro Ser Ser Phe Ser Asn Asn Ser Ser 210 215 220 Val Met Gly Asp Pro
Leu Ile Asp Ala Asn Thr Gly Pro Gly Asp Ser 225 230 235 240 Gly Asn
Thr Arg Gly Glu Ala Gly Gln Leu Ile Gly Glu Leu Ile Asp 245 250 225
Arg Gly Leu Gln Ser Val Leu Ala Gly Gly Gly Leu Gly Thr Pro Val 260
265 270 Asn Thr Pro Gln Thr Gly Thr Ser Ala Asn Gly Gly Gln Ser Ala
Gln 275 280 285 Asp Leu Asp Gln Leu Leu Gly Gly Leu Leu Leu Lys Gly
Leu Glu Ala 290 295 300 Thr Leu Lys Asp Ala Gly Gln Thr Gly Thr Asp
Val Gln Ser Ser Ala 305 310 315 320 Ala Gln Ile Ala Thr Leu Leu Val
Ser Thr Leu Leu Gln Gly Thr Arg 325 330 335 Asn Gln Ala Ala Ala
340
[0031] This hypersensitive response elicitor polypeptide or protein
has a molecular weight of 34-35 kDa. It is rich in glycine (about
13.5%) and lacks cysteine and tyrosine. Further information about
the hypersensitive response elicitor derived from Pseudomonas
syringae is found in He, S. Y., H. C. Huang, and A. Collmer,
"Pseudomonas syringae pv. syringae Harpin.sub.Pss: a Protein that
is Secreted via the Hrp Pathway and Elicits the Hypersensitive
Response in Plants," Cell 73:1255-1266 (1993), which is hereby
incorporated by reference. The DNA molecule encoding the
hypersensitive response elicitor from Pseudomonas syringae has a
nucleotide sequence corresponding to SEQ. ID. No. 6 as follows:
6 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
[0032] The hypersensitive response elicitor polypeptide or protein
derived from Pseudomonas solanacearum has an amino acid sequence
corresponding to SEQ. ID. No. 7 as follows:
7 Met Ser Val Gly Asn Ile Gln Ser Pro Ser Asn Leu Pro Gly Leu Gln 1
5 10 15 Asn Leu Asn Leu Asn Thr Asn Thr Asn Ser Gln Gln Ser Gly Gln
Ser 20 25 30 Val Gln Asp Leu Ile Lys Gln Val Glu Lys Asp Ile Leu
Asn Ile Ile 35 40 45 Ala Ala Leu Val Gln Lys Ala Ala Gln Ser Ala
Gly Gly Asn Thr Gly 50 55 60 Asn Thr Gly Asn Ala Pro Ala Lys Asp
Gly Asn Ala Asn Ala Gly Ala 65 70 75 80 Asn Asp Pro Ser Lys Asn Asp
Pro Ser Lys Ser Gln Ala Pro Gln Ser 85 90 95 Ala Asn Lys Thr Gly
Asn Val Asp Asp Ala Asn Asn Gln Asp Pro Met 100 105 110 Gln Ala Leu
Met Gln Leu Leu Glu Asp Leu Val Lys Leu Leu Lys Ala 115 120 125 Ala
Leu His Met Gln Gln Pro Gly Gly Asn Asp Lys Gly Asn Gly Val 130 135
140 Gly Gly Ala Asn Gly Ala Lys Gly Ala Gly Gly Gln Gly Gly Leu Ala
145 150 155 160 Glu Ala Leu Gln Glu Ile Glu Gln Ile Leu Ala Gln Leu
Gly Gly Gly 165 170 175 Gly Ala Gly Ala Gly Gly Ala Gly Gly Gly Val
Gly Gly Ala Gly Gly 180 185 190 Ala Asp Gly Gly Ser Gly Ala Gly Gly
Ala Gly Gly Ala Asn Gly Ala 195 200 205 Asp Gly Gly Asn Gly Val Asn
Gly Asn Gln Ala Asn Gly Pro Gln Asn 210 215 220 Ala Gly Asp Val Asn
Gly Ala Asn Gly Ala Asp Asp Gly Ser Glu Asp 225 230 235 240 Gln Gly
Gly Leu Thr Gly Val Leu Gln Lys Leu Met Lys Ile Leu Asn 245 250 255
Ala Leu Val Gln Met Met Gln Gln Gly Gly Leu Gly Gly Gly Asn Gln 260
265 270 Ala Gln Gly Gly Ser Lys Gly Ala Gly Asn Ala Ser Pro Ala Ser
Gly 275 280 285 Ala Asn Pro Gly Ala Asn Gln Pro Gly Ser Ala Asp Asp
Gln Ser Ser 290 295 300 Gly Gln Asn Asn Leu Gln Ser Gln Ile Met Asp
Val Val Lys Glu Val 305 310 315 320 Val Gln Ile Leu Gln Gln Met Leu
Ala Ala Gln Asn Gly Gly Ser Gln 325 330 335 Gln Ser Thr Ser Thr Gln
Pro Met 340
[0033] It is encoded by a DNA molecule having a nucleotide sequence
corresponding SEQ. ID. No. 8 as follows:
8 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
[0034] 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.
[0035] 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:
9 Thr Leu Ile Glu Leu Met Ile Val Val Ala Ile Ile Ala Ile Leu Ala 1
5 10 15 Ala Ile Ala Leu Pro Ala Tyr Gln Asp Tyr 20 25
[0036] This sequence is an amino terminal sequence having 26
residues only from the hypersensitive response elicitor polypeptide
or protein of Xanthomonas campestris pv. glycines. It matches with
fimbrial subunit proteins determined in other Xanthomonas
campestris pathovars.
[0037] The hypersensitive response elicitor polypeptide or protein
from Xanthomonas campestris pv. pelargonii is heat stable, protease
sensitive, and has a molecular weight of 20 kDa. It includes an
amino acid sequence corresponding to SEQ. ID. No. 10 as
follows:
10 Ser Ser Gln Gln Ser Pro Ser Ala Gly Ser Glu Gln Gln Leu Asp Gln
1 5 10 15 Leu Leu Ala Met 20
[0038] Isolation of Erwinia carotovora hypersensitive response
elictor protein or polypeptide is described in Cui et al., "The
RsmA Mutants of Erwinia carotovora subsp. carotovora Strain Ecc71
Overexpress hrp N.sub.Ecc and Elicit a Hypersensitive Reaction-like
Response in Tobacco Leaves," MPMI, 9(7):565-73 (1996), which is
hereby incorporated by reference. The hypersensitive response
elicitor proptein or polypeptide is shown in Ahmad et al., "Harpin
is Not Necessary for the Pathogenicity of Erwinia stewartii on
Maize," 8th Int'l. Cong. Molec. Plant-Microbe Interact., Jul.
14-19, 1996 and Ahmad, et al., "Harpin is Not Necessary for the
Pathogenicity of Erwinia stewartii on Maize," Ann. Mtg. Am.
Phytopath. Soc., Jul. 27-31, 1996, which are hereby incorporated by
reference.
[0039] Hypersensitive response elicitor proteins or polypeptides
from Phytophthora parasitica, Phytophthora cryptogea, Phytophthora
cinnamoni, Phytophthora capsici, Phytophthora megasperma, and
Phytophora citrophthora are described in Kaman, et al.,
"Extracellular Protein Elicitors from Phytophthora: Most
Specificity and Induction of Resistance to Bacterial and Fungal
Phytopathogens," Molec. Plant-Microbe Interact., 6(l):15-25 (1993),
Ricci et al., "Structure and Activity of Proteins from Pathogenic
Fungi Phytophthora Eliciting Necrosis and Acquired Resistance in
Tobacco," Eur. J. Biochem., 183:555-63 (1989), Ricci et al.,
"Differential Production of Parasiticein, and Elicitor of Necrosis
and Resistance in Tobacco, by Isolates of Phytophthora parasitica,"
Plant Path. 41:298-307 (1992), Baillreul et al, "A New Elicitor of
the Hypersensitive Response in Tobacco: A Fungal Glycoprotein
Elicits Cell Death, Expression of Defence Genes, Production of
Salicylic Acid, and Induction of Systemic Acquired Resistance,"
Plant J., 8(4):551-60 (1995), and Bonnet et al., "Acquired
Resistance Triggered by Elicitors in Tobacco and Other Plants,"
Eur. J. Plant Path., 102:181-92 (1996), which are hereby
incorporated by reference.
[0040] The above elicitors are exemplary. Other elicitors can be
identified by growing fungi or bacteria that elicit a
hypersensitive response under which genes encoding an elicitor are
expressed. Cell-free preparations from culture supernatants can be
tested for elicitor activity (i.e. local necrosis) by using them to
infiltrate appropriate plant tissues.
[0041] It is also possible to use fragments of the above
hypersensitive response elicitor polypeptides or proteins as well
as fragments of full length elicitors from other pathogens, in the
method of the present invention.
[0042] Suitable fragments can be produced by several means. In the
first, subclones of the gene encoding a known elicitor protein are
produced by conventional molecular genetic manipulation by
subcloning gene fragments. The subclones then are expressed in
vitro or in vivo in bacterial cells to yield a smaller protein or a
peptide that can be tested for elicitor activity according to the
procedure described below.
[0043] As an alternative, fragments of an elicitor protein can be
produced by digestion of a full-length elicitor protein with
proteolytic enzymes like chymotrypsin or Staphylococcus proteinase
A, or trypsin. Different proteolytic enzymes are likely to cleave
elicitor proteins at different sites based on the amino acid
sequence of the elicitor protein. Some of the fragments that result
from proteolysis may be active elicitors of resistance.
[0044] In another approach, based on knowledge of the primary
structure of the protein, fragments of the elicitor protein gene
may be synthesized by using the PCR technique together with
specific sets of primers chosen to represent particular portions of
the protein. These then would be cloned into an appropriate vector
for increase and expression of a truncated peptide or protein.
[0045] Chemical synthesis can also be used to make suitable
fragments. Such a synthesis is carried out using known amino acid
sequences for the elicitor being produced. Alternatively,
subjecting a full length elicitor to high temperatures and
pressures will produce fragments. These fragments can then be
separated by conventional procedures (e.g., chromatography,
SDS-PAGE).
[0046] An example of a useful fragment is the popA1 fragment of the
hypersensitive response elicitor polypeptide or protein from
Pseudomonas solanacearum. See Arlat, M., F. Van Gijsegem, J. C.
Huet, J. C. Pemollet, and C. A. Boucher, "PopA1, a Protein Which
Induces a Hypersensitive-like Response in Specific Petunia
Genotypes is Secreted via the Hrp Pathway of Pseudomonas
solanacearum," EMBO J. 13:543-53 (1994), which is hereby
incorporated by reference. As to Erwinia amylovora, a suitable
fragment can be, for example, either or both the polypeptide
extending between and including amino acids 1 and 98 of SEQ. ID.
No. 3 and the polypeptide extending between and including amino
acids 137 and 204 of SEQ. ID. No. 3.
[0047] 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.
[0048] The protein or polypeptide of the present invention is
preferably produced in purified form (preferably at least about
60%, more preferably 80%, pure) by conventional techniques.
Typically, the protein or polypeptide of the present invention is
produced but not secreted into the growth medium of recombinant
host cells. Alternatively, the protein or polypeptide of the
present invention is secreted into growth medium. In the case of
unsecreted protein, to isolate the protein, the host cell (e.g., E.
coli) carrying a recombinant plasmid is propagated, lysed by
sonication, heat, or chemical treatment, and the homogenate is
centrifuged to remove bacterial debris. The supernatant is then
subjected to heat treatment and the hypersensitive response
elicitor protein is separated by centrifugation. The supernatant
fraction containing the polypeptide or protein of the present
invention is subjected to gel filtration in an appropriately sized
dextran or polyacrylamide column to separate the proteins. If
necessary, the protein fraction may be further purified by ion
exchange or HPLC. 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.
[0049] 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.
[0050] Recombinant genes may also be introduced into viruses, such
as vaccina virus. Recombinant viruses can be generated by
transfection of plasmids into cells infected with virus.
[0051] Suitable vectors include, but are not limited to, the
following viral vectors such as lambda vector system gt11, gt
WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325,
pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290,
pKC37, pKC101, SV 40, pBluescript II SK +/- or KS +/- (see
"Stratagene Cloning Systems" Catalog (1993) from Stratagene, La
Jolla, Calif. which is hereby incorporated by reference), pQE,
pIH821, pGEX, pET series (see F. W. Studier et. al., "Use of T7 RNA
Polymerase to Direct Expression of Cloned Genes," Gene Expression
Technology vol. 185 (1990), which is hereby incorporated by
reference), and any derivatives thereof. Recombinant molecules can
be introduced into cells via transformation, particularly
transduction, conjugation, mobilization, or electroporation. The
DNA sequences are cloned into the vector using standard cloning
procedures in the art, as described by Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs
Harbor, N.Y. (1989), which is hereby incorporated by reference.
[0052] 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.
[0053] Different genetic signals and processing events control many
levels of gene expression (e.g., DNA transcription and messenger
RNA (mRNA) translation).
[0054] 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 promoters are not recognized and
do not function in eucaryotic cells.
[0055] 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-Dalgamo ("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.
[0056] 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 promoters 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 promoters 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 promoters produced by
recombinant DNA or other synthetic DNA techniques may be used to
provide for transcription of the inserted gene.
[0057] 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-galac- toside). A variety of other
operons, such as trp, pro, etc., are under different controls.
[0058] 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 an
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.
[0059] 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.
[0060] The method of the present invention can be utilized to treat
a wide variety of plants or their seeds to enhance growth. 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: rose,
Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum,
carnation, and zinnia.
[0061] The method of the present invention involving application of
the hypersensitive response elicitor polypeptide or protein can be
carried out through a variety of procedures when all or part of the
plant is treated, including leaves, stems, roots, etc. This may
(but need not) involve infiltration of the hypersensitive response
elicitor polypeptide or protein into the plant. Suitable
application methods include topical application (e.g., high or low
pressure spraying), injection, dusting, and leaf abrasion proximate
to when elicitor application takes place. When treating plant
seeds, in accordance with the application embodiment of the present
invention, the hypersensitive response elicitor protein or
polypeptide can be applied by topical application (low or high
pressure spraying), coating, immersion, dusting, or injection.
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 or plant seed. Once treated with the hypersensitive
response elicitor of the present invention, the seeds can be
planted in natural or artificial soil and cultivated using
conventional procedures to produce plants. After plants have been
propagated from seeds treated in accordance with the present
invention, the plants may be treated with one or more applications
of the hypersensitive response elicitor protein or polypeptide to
enhance growth in the plants. Such propagated plants may, in turn,
be useful in producing seeds or propagules (e.g., cuttings) that
produce plants capable of enhanced growth.
[0062] The hypersensitive response elicitor polypeptide or protein
can be applied to plants or plant seeds in accordance with the
present invention alone or in a mixture with other materials.
Alternatively, the hypersensitive response elicitor polypeptide or
protein can be applied separately to plants with other materials
being applied at different times.
[0063] A composition suitable for treating plants or plant seeds in
accordance with the application embodiment of the present invention
contains a hypersensitive response elicitor polypeptide or protein
in a carrier. Suitable carriers include water, aqueous solutions,
slurries, or dry powders. In this embodiment, the composition
contains greater than 0.5 nM hypersensitive response elicitor
polypeptide or protein.
[0064] Although not required, this composition may contain
additional additives including fertilizer, insecticide, fungicide,
nematacide, herbicide, and mixtures thereof. Suitable fertilizers
include (NH.sub.4).sub.2NO.sub.3. An example of a suitable
insecticide is Malathion. Useful fungicides include Captan.
[0065] Other suitable additives include buffering agents, wetting
agents, coating agents, and abrading agents. These materials can be
used to facilitate the process of the present invention. In
addition, the hypersensitive response elicitor polypeptide or
protein can be applied to plant seeds with other conventional seed
formulation and treatment materials, including clays and
polysaccharides.
[0066] In the alternative embodiment of the present invention
involving the use of transgenic plants and transgenic seeds, a
hypersensitive response elicitor polypeptide or protein need not be
applied topically to the plants or seeds. Instead, transgenic
plants transformed with a DNA molecule encoding a hypersensitive
response elicitor polypeptide or protein are produced according to
procedures well known in the art, such as by biolistics or
Agrobacterium mediated transformation. Examples of suitable
hypersensitive response elicitor polypeptides or proteins and the
nucleic acid sequences for their encoding DNA are disclosed supra.
Once transgenic plants of this type are produced, the plants
themselves can be cultivated in accordance with conventional
procedure with the presence of the gene encoding the hypersensitive
response elicitor resulting in enhanced growth of the plant.
Alternatively, transgenic seeds are recovered from the transgenic
plants. These seeds can then be planted in the soil and cultivated
using conventional procedures to produce transgenic plants. The
transgenic plants are propagated from the planted transgenic seeds
under conditions effective to impart enhanced growth. While not
wishing to be bound by theory, such growth enhancement may be RNA
mediated or may result from expression of the elicitor polypeptide
or protein.
[0067] When transgenic plants and plant seeds are used in
accordance with the present invention, they additionally can be
treated with the same materials as are used to treat the plants and
seeds to which a hypersensitive response elicitor polypeptide or
protein is applied. These other materials, including hypersensitive
response elicitors, can be applied to the transgenic plants and
plant seeds by the above-noted procedures, including high or low
pressure spraying, injection, coating, dusting, and immersion.
Similarly, after plants have been propagated from the transgenic
plant seeds, the plants may be treated with one or more
applications of the hypersensitive response elicitor to enhance
plant growth. Such plants may also be treated with conventional
plant treatment agents (e.g., insecticides, fertilizers, etc.). The
transgenic plants of the present invention are useful in producing
seeds or propagules (e.g., cuttings) from which plants capable of
enhanced growth would be produced.
EXAMPLES
Example 1
Effect of Treating Tomato Seeds with Erwinia amylovora
[0068] Hypersensitive Response Elicitor on Germination Percentage
Seeds of the Marglobe Tomato Variety were submerged in 40 ml of
Erwinia amylovora hypersensitive response elicitor solution
("harpin"). Harpin was prepared by growing E. coli strain DH5
containing the plasmid pCPP2139 (see FIG. 1), lysing the cells by
sonication, heat treating by holding in boiling water for 5 minutes
before centrifuging to remove cellular debris, and precipitating
proteins and other heat-labile components. The resulting
preparation ("CFEP") was diluted serially. These dilutions (1:40,
1:80, 1:160, 1:320 and 1:640) contained 20, 10, 5, 2.5, and 1.25
.mu.gm/ml, respectively, of harpin based on Western Blot assay.
Seeds were soaked in harpin or buffer in beakers on day 0 for 24
hours at 28.degree. C. in a growth chamber. After soaking, the
seeds were sown in germination pots with artificial soil on day 1.
This procedure was carried out on 100 seeds per treatment.
[0069] Treatments:
[0070] 1. Seeds in harpin (1:40) (20 .mu.gm/ml).
[0071] 2. Seeds in harpin (1:80)(10 .mu.gm/ml).
[0072] 3. Seeds in harpin (1:160) (5 .mu.gm/ml).
[0073] 4. Seeds in harpin (1:320) (2.5 .mu.gm/ml).
[0074] 5. Seeds in harpin (1:640) (1.25 .mu.gm/ml).
[0075] 6. Seeds in buffer (5 mM KPO.sub.4, pH 6.8).
11TABLE 1 Number of Seedlings After Seed Treatment Treatment Number
of seeds germinated Day 0 Day 1 Day 5 Day 7 Day 9 Harpin seed soak
(20 .mu.gm/ml) sowing 43 57 59 Harpin seed soak (10 .mu.gm/ml)
sowing 43 52 52 Harpin seed soak (5 .mu.gm/ml) sowing 40 47 51
Harpin seed soak (2.5 .mu.gm/ml) sowing 43 56 58 Harpin seed soak
(1.25 .mu.gm/ml) sowing 38 53 57 Buffer seed soak sowing 27 37
40
[0076] As shown in Table 1, the treatment of tomato seeds with
Erwinia amylovora hypersensitive response elicitor reduced the time
needed for germination and greatly increased the percentage of
germination.
Example 2
Effect of Treating Tomato Seeds with Erwinia amylovora
[0077] Hypersensitive Response Elicitor on Tomato Plant Height
Seeds of the Marglobe Tomato Variety were submerged in Erwinia
amylovora harpin (1 :15, 1:30, 1:60, and 1: 120) or buffer in
beakers on day 0 for 24 hours at 28.degree. C. in a growth chamber.
After soaking, the seeds were sown in germination pots with
artificial soil on day 1.
[0078] Ten uniform appearing plants per treatment were chosen
randomly and measured. The seedlings were measured by ruler from
the surface of soil to the top of plant.
[0079] Treatments:
[0080] 1. Harpin (1:15) (52 .mu.gm/ml).
[0081] 2. Harpin (1:30) (26 .mu.gm/ml).
[0082] 3. Harpin (1:60)(13 .mu.gm/ml).
[0083] 4. Harpin (1:120) (6.5 .mu.gm/ml).
[0084] 5. Buffer (5 mM KPO.sub.4, pH 6.8).
12TABLE 2 Seedling Height (cm) 15 Days After Seed Treatment. Treat
Plants 1 2 3 4 5 6 7 8 9 10 Mean 52 10 5.6 5.8 5.8 5.6 6.0 6.0 5.8
5.4 5.8 5.6 5.7 .mu.gm/ml 26 10 6.8 7.2 6.6 7.0 6.8 6.8 7.0 7.4 7.2
7.0 7.0 .mu.gm/ml 13 10 5.8 5.6 6.0 5.6 5.8 5.8 5.6 5.8 6.0 5.6 5.9
.mu.gm/ml 6.5 10 5.4 5.2 5.6 5.4 5.2 5.4 5.6 5.6 5.4 5.2 5.4
.mu.gm/ml Buffer 10 5.6 5.4 5.2 5.2 5.4 5.2 5.0 5.2 5.4 5.6 5.3
[0085]
13TABLE 3 Seedling Height (cm) 21 Days After Seed Treatment. Treat
Plants 1 2 3 4 5 6 7 8 9 10 Mean 52 10 7.6 7.8 7.6 7.6 7.8 7.8 7.8
7.4 7.6 7.6 7.7 .mu.gm/ml 26 10 8.2 8.2 8.0 9.0 8.4 8.6 8.6 9.0 9.2
9.0 8.6 .mu.gm/ml 13 10 6.8 6.6 6.8 6.8 6.8 6.8 6.6 7.2 7.0 7.2 6.9
.mu.gm/ml 6.5 10 6.8 6.6 6.6 6.4 6.8 6.6 6.8 6.6 6.6 6.8 6.7
.mu.gm/ml Buffer 10 6.6 6.4 6.2 6.6 6.4 6.6 6.8 6.4 6.4 6.6 6.5
[0086]
14TABLE 4 Seedling Height (cm) 27 Days After Seed Treatment. Treat
1 2 3 4 5 6 7 8 9 10 Mean 52 .mu.gm/ml 10.2 10.6 10.4 10.6 10.4
10.6 10.8 10.4 10.8 10.6 10.5 26 .mu.gm/ml 11.6 11.4 11.6 11.8 11.8
11.8 11.6 11.4 11.6 11.4 11.6 13 .mu.gm/ml 9.8 9.6 9.8 9.6 9.8 9.8
9.6 9.4 9.6 9.8 9.7 6.5 .mu.gm/ml 9.4 9.4 9.6 9.4 9.6 9.4 9.6 9.6
9.4 9.2 9.5 Buffer 9.6 10.2 10.0 9.8 10.0 10.2 10.0 10.2 10.4 9.6
10.0
[0087]
15TABLE 5 Summary--Mean Height of Tomato Plants after Treatment.
Treatment Mean height of tomato plants (cm) Day 0 Day 1 Day 15 Day
21 Day 27 Harpin seed soak (1:15) sowing 5.7 7.7 10.5 Harpin seed
soak (1:30) sowing 7.0 8.6 11.6 Harpin seed soak (1:60) sowing 5.9
6.9 9.7 Harpin seed soak (1:120) sowing 5.4 6.7 9.5 Buffer seed
soak sowing 5.3 6.5 10.0
[0088] As shown in Tables 2-5, the treatment of tomato seeds with
Erwinia amylovora hypersensitive response elicitor increased plant
growth. A 1:30 dilution had the greatest effect--a 16% increase in
seedling height.
Example 3
Effect of Treating Tomato Plants with Erwinia amylovora
[0089] Hypersensitive Response Elicitor on Tomato Plant Height When
Marglobe tomato plants were 4 weeks old, they were sprayed with 6
ml/plant of Erwinia amylovora harpin solution containing 13
.mu.gm/ml (1:60) or 8.7 .mu.gm/ml (1:90) of harpin or buffer (5 mM
KPO.sub.4) in a growth chamber at 28.degree. C. The heights of
tomato plants were measured 2 weeks after spraying harpin
(6-week-old tomato plants) and 2 weeks plus 5 days after spraying.
Ten uniform appearing plants per treatment were chosen randomly and
measured. The seedlings were measured by ruler from the surface of
soil to the top of plant.
[0090] Treatments:
[0091] 1. Harpin (1:60)(13 .mu.gm/ml).
[0092] 2. Harpin (1:90) (8.7 .mu.gm/ml).
[0093] 3. Buffer (5 mM KPO.sub.4, pH 6.8).
16TABLE 6 Mean Height of Tomato Plants after Treatment With Harpin.
Mean height (cm) Operation and Treatment of tomato plants Day 0 Day
14 Day 28 Day 42 Day 47 sowing transplant harpin 1:60 35.5 36.0 (13
.mu.gm/ml) sowing transplant harpin 1:90 35.7 36.5 (8.7 .mu.gm/ml)
sowing transplant buffer 32.5 33.0
[0094] As shown in Table 6, spraying tomato seedlings with Erwinia
amylovora hypersensitive response elicitor can increase growth of
tomato plants. Similar increases in growth were noted for the two
doses of the hypersensitive response elicitor tested compared with
the buffer-treated control.
Example 4
Effect of Treating Tomato Seeds with Erwinia amylovora
[0095] Hypersensitive Response Elicitor on Tomato Plant Height
Marglobe tomato seeds were submerged in Erwinia amylovora
hypersensitive response elicitor solution ("harpin") (1:40, 1:80,
1:160, 1:320, and 1:640) or buffer in beakers on day 0 for 24 hours
at 28.degree. C. in the growth chamber. After soaking seeds in
harpin or buffer, they were sown in germination pots with
artificial soil on day 1. Ten uniform appearing plants per
treatment were chosen randomly and measured. The seedlings were
measured by ruler from the surface of soil to the top of plant.
[0096] Treatments:
[0097] 1. Harpin (1:40) (20 .mu.gm/ml).
[0098] 2. Harpin (1:80) (10 .mu.gm/ml).
[0099] 3. Harpin (1:160) (5 .mu.gm/ml).
[0100] 4. Harpin (1:320) (2.5 .mu.gm/ml).
[0101] 5. Harpin (1:640) (1.25 .mu.gm/ml).
[0102] 6. Buffer (5 mM KPO.sub.4, pH 6.8).
17TABLE 7 Seedling Height (cm) 12 Days After Seed Treatment. Treat
Plants 1 2 3 4 5 6 7 8 9 10 Mean 20 10 6.5 6.8 6.8 6.5 6.4 6.4 6.8
6.4 6.8 6.6 6.6 .mu.gm/ml 10 10 6.8 6.2 6.6 6.4 6.8 6.8 6.6 6.4 6.8
6.4 6.6 .mu.gm/ml 5 10 6.2 6.6 6.0 6.6 6.4 6.2 6.6 6.2 6.0 6.6 6.3
.mu.gm/ml 2.5 10 6.4 6.2 6.6 6.0 6.2 6.4 6.0 6.0 6.2 6.2 6.2
.mu.gm/ml 1.25 10 6.2 6.2 6.0 6.4 6.0 6.0 6.4 6.2 6.4 6.2 6.2
.mu.gm/ml Buffer 10 5.8 6.0 6.2 6.2 5.8 5.8 6.0 6.2 6.0 6.0 6.0
[0103]
18TABLE 8 Seedling Height (cm) 14 Days After Seed Treatment. Treat
Plants 1 2 3 4 5 6 7 8 9 10 Mean 20 10 7.8 7.8 8.2 8.0 8.2 8.4 7.8
8.4 7.6 7.8 8.0 .mu.gm/ml 10 10 8.6 8.8 8.4 9.2 8.4 8.6 7.8 7.8 8.4
8.4 8.4 .mu.gm/ml 5 10 9.8 9.2 9.8 9.6 9.2 9.4 8.6 9.2 9.0 8.6 9.2
.mu.gm/ml 2.5 10 8.8 8.6 8.6 8.4 7.8 8.6 8.4 9.0 8.0 7.8 8.4
.mu.gm/ml 1.25 10 8.4 7.8 8.4 8.0 8.6 8.4 8.0 8.2 8.4 8.2 8.2
.mu.gm/ml Buffer 10 7.2 8.2 7.4 7.6 7.8 7.6 7.8 7.4 7.8 7.6 7.6
[0104]
19TABLE 9 Seedling Height (cm) 17 Days After Seed Treatment. Treat
Plants 1 2 3 4 5 6 7 8 9 10 Mean 20 .mu.gm/ml0 10 11.2 11.6 11.4
11.6 11.4 11.2 11.8 11.4 11.8 11.6 11.5 10 .mu.gm/ml 10 13.4 13.4
13.8 13.2 13.4 12.6 12.4 13.4 13.2 13.4 13.2 5 .mu.gm/ml 10 13.6
12.8 13.6 13.2 14.2 13.8 12.6 13.4 13.8 13.6 13.5 2.5 .mu.gm/ml 10
11.6 12.4 12.4 11.8 11.6 12.2 12.6 11.8 12.0 11.6 12.0 1.25
.mu.gm/ml 10 12.8 12.6 12.0 12.4 11.6 11.8 12.2 11.4 11.2 11.4 11.9
Buffer 10 10.0 10.4 10.6 10.6 10.4 10.4 10.8 10.2 10.4 10.0
10.4
[0105]
20TABLE 10 Summary - Mean Height of Tomato Plants after Treatment
Operation and Treatment Mean height of tomato plants (cm) Day 0 Day
1 Day 12 Day 14 Day 17 Harpin seed soak (20 .mu.gm/ml) sowing 6.6
8.0 11.5 Harpin seed soak (10 .mu.gm/ml) sowing 6.6 8.4 13.2 Harpin
seed soak (5 .mu.gm/ml) sowing 6.3 9.2 13.5 Harpin seed soak (2.5
.mu.gm/ml) sowing 6.2 8.4 12.0 Harpin seed soak (1.25 .mu.gm/ml)
sowing 6.2 8.2 11.9 Buffer seed soak sowing 6.0 7.6 10.4
[0106] As shown in Tables 7-10, the treatment of tomato seeds with
Erwinia amylovora hypersensitive response elicitor can increase
growth of tomato plants. A 1:160 dilution (5 .mu.g/ml harpin) had
the greatest effect--seedling height was increased more than 20%
over the buffer treated plants.
Example 5
Effect of Treating Tomato Seeds with Erwinia amylovora
[0107] Hypersensitive Response Elicitor on Seed Germination
Percentage Marglobe tomato seeds were submerged in 40 ml of Erwinia
amylovora hypersensitive response elicitor ("harpin") solution
(dilutions of CFEP from E. coli DH5 (pCPP2139) of 1:50 or 1:100
which contained, respectively, 8 .mu.gm/ml and 4 .mu.gm/ml of
hypersensitive response elicitor) and buffer in beakers on day 0
for 24 hours at 28.degree. C. in a growth chamber. After soaking,
the seeds were sown in germination pots with artificial soil on day
1. This treatment was carried out on 20 seeds per pot and 4 pots
per treatment.
[0108] Treatments:
[0109] 1. Harpin (8 .mu.gm/ml).
[0110] 2. Harpin (8 .mu.gm/ml).
[0111] 3. Harpin (8 .mu.gm/ml).
[0112] 4. Harpin (8 .mu.gm/ml).
[0113] 5. Harpin (4 .mu.gm/ml).
[0114] 6. Harpin (4 .mu.gm/ml).
[0115] 7. Harpin (4 .mu.gm/ml).
[0116] 8. Harpin (4 .mu.gm/ml).
[0117] 9. Buffer (5 mM KPO.sub.4, pH 6.8).
[0118] 10. Buffer (5 mM KPO.sub.4, pH 6.8).
[0119] 11. Buffer (5 mM KPO.sub.4, pH 6.8).
[0120] 12. Buffer (5 mM KPO.sub.4, pH 6.8).
21TABLE 11 Number of Seedlings After Seed Treatment With Harpin
Operation and Number of seeds germinated Treatment (out of a total
of 20) Day Day Day Day Day 0 1 5 Mean 42 Mean 47 Mean Harpin (8
.mu.gm/ml) sowing 11 15 19 Harpin (8 .mu.gm/ml) sowing 13 17 20
Harpin (8 .mu.gm/ml) sowing 10 13 16 Harpin (8 .mu.gm/ml) sowing 9
10.8 15 15.0 16 17.8 Harpin (4 .mu.gm/ml) sowing 11 17 17 Harpin (4
.mu.gm/ml) sowing 15 17 18 Harpin (4 .mu.gm/ml) sowing 9 12 14
Harpin (4 .mu.gm/ml) sowing 9 11.0 14 15.0 16 16.3 Buffer sowing 11
11 14 Buffer sowing 9 14 15 Buffer sowing 10 14 14 Buffer sowing 10
10.0 12 12.8 14 14.3
[0121] As shown in Table 11, treatment of tomato seeds with Erwinia
amylovora hypersensitive response elicitor can increase germination
rate and level of tomato seeds. The higher dose used appeared to be
more effective than buffer at the end of the experiment.
Example 6
Effect on Plant Growth of Treating Tomato Seeds with Proteins
Prepared from E. coli Containing a Hypersensitive Response Elicitor
Encoding Construct, pCPP2139, or Plasmid Vector pCPP50
[0122] Marglobe tomato seeds were submerged in Erwinia amylovora
hypersensitive response elicitor ("harpin") (from E. coli
DH5.alpha.(pCPP2139) (FIG. 1) or vector preparation (from
DH5.alpha.(pCPP50) (FIG. 2) with added BSA protein as control. The
control vector preparation contained, per ml, 33.6 .mu.l of BSA (10
mg/ml) to provide about the same amount of protein as contained in
the pCPP2139 preparation due to harpin. Dilutions of 1:50 (8.0
.mu.g/ml), 1:100 (4.0 .mu.g/ml), and 1:200 (2.0 .mu.g/ml) were
prepared in beakers on day 1, and seed was submerged for 24 hours
at 28.degree. C. in a controlled environment chamber. After
soaking, seeds were sown in germination pots with artificial soil
on day 2. Ten uniform appearing plants per treatment were chosen
randomly and measured at three times after transplanting. The
seedlings were measured by ruler from the surface of soil to the
top of plant.
22 Treatments: 1. Harpin 1:50 (8.0 .mu.g/ml) 2. Harpin 1:100 (4.0
.mu.g/ml) 3. Harpin 1:200 (2.0 .mu.g/ml) 4. Vector + BSA 1:50 (0
harpin) 5. Vector + BSA 1:100 (0 harpin) 6. Vector + BSA 1:200 (0
harpin)
[0123]
23TABLE 12 Seedling Height (cm) 18 Days After Seed Treatment Treat
Harpin 1 2 3 4 5 6 7 8 9 10 Mean H1:50 8.0 3.6 5.0 4.8 5.0 4.2 5.2
5.8 4.6 4.0 4.8 4.7 H1:100 4.0 4.6 5.8 6.2 6.0 5.6 6.8 6.0 4.8 5.6
6.2 5.8 H1:200 2.0 4.0 5.8 5.8 4.6 5.4 5.0 5.8 4.6 4.6 5.8 5.1
V1:50 0 3.8 5.0 4.6 5.4 5.6 4.6 5.0 5.2 4.6 4.8 4.9 V1:100 0 4.4
5.2 4.6 4.4 5.4 4.8 5.0 4.6 4.4 5.2 4.8 V1:200 0 4.2 4.8 5.4 4.6
5.0 4.8 4.8 5.4 4.6 5.0 4.9
[0124]
24TABLE 13 Seedling Height (cm) 22 Days After Seed Treatment. Treat
Harpin 1 2 3 4 5 6 7 8 9 10 Mean H1:50 8.0 4.2 5.6 5.2 6.0 4.8 5.4
5.0 5.2 5.4 5.0 5.2 H1:100 4.0 7.6 6.8 7.0 7.2 6.8 7.4 7.6 7.0 6.8
7.4 7.2 H1:200 2.0 7.0 6.6 6.8 7.2 7.4 6.8 7.0 7.2 6.8 7.2 7.0
V1:50 0 5.6 5.8 6.2 6.4 5.6 5.2 5.6 5.8 6.0 5.8 5.8 V1:100 0 5.4
6.0 5.8 6.2 5.8 5.6 5.4 5.2 6.0 5.6 5.7 V1:200 0 5.2 6.2 5.8 5.4
6.2 6.0 5.6 6.4 5.8 6.0 5.9
[0125]
25TABLE 14 Seedling Height (cm) 26 Days After Seed Treatment.
Treat. Harpin 1 2 3 4 5 6 7 8 9 10 Mean H1:50 8.0 7.6 8.4 8.8 6.8
9.6 8.2 7.4 9.8 9.2 9.0 8.5 H1:100 4.0 12.0 11.4 11.2 11.0 10.8
12.0 11.2 11.6 10.4 10.2 11.2 H1:200 2.0 10.6 11.2 11.6 10.2 11.0
10.8 10.0 11.8 10.2 10.6 10.8 V1:50 0 9.0 9.4 8.8 8.4 9.6 9.2 9.2
8.6 8.0 9.4 9.2 V1:100 0 9.2 10.0 9.8 9.6 8.4 9.4 9.6 9.8 8.0 9.6
9.3 V1:200 0 8.8 9.6 8.2 9.2 8.4 8.0 9.8 9.0 9.4 9.2 9.0
[0126]
26TABLE 15 Mean Height of Tomato Plants after Treatment Operation
and Treatment Mean height of tomato plants (cm) Day 1 Day 2 Day 18
Day 22 Day 26 Harpin (1:50) (8.0 .mu.gm/ml) sowing 4.7 5.2 8.5
Harpin (1:100) (4.0 .mu.gm/ml) sowing 5.8 7.2 11.2 Harpin (1:200)
(2.0 .mu.gm/ml) sowing 5.1 7.0 10.8 Vector + BSA (1:50) (0) sowing
4.9 5.8 9.2 Vector + BSA (1:100) (0) sowing 4.8 5.7 9.3 Vector +
BSA (1:200) (0) sowing 4.9 5.9 9.0
[0127] As shown in Tables 12-15, treatment with E. coli containing
the gene encoding the Erwinia amylovora hypersensitive response
elicitor can increase growth of tomato plants. The 1:100 dilution
(4.0 .mu.g/ml) had the greatest effect, while higher and lower
concentrations had less effect. Mean seedling height for treatment
with 4.0 .mu.g/ml of harpin was increased about 20% relative to
vector control preparation, which contained a similar amount of
non-harpin protein. Components of the lysed cell preparation from
the strain E. coli DH5.alpha.(pCPP50), which harbors the vector of
the hrpN gene in E. coli strain DH5.alpha.(pCPP2139), do not have
the same growth-promoting effect as the harpin-containing
preparation, even given that it is supplemented with BSA protein to
the same extent as the DH5.alpha.(pCPP2139) preparation, which
contains large amounts of harpin protein.
Example 7
Effect on Tomato Plant Growth of Treating Tomato Seeds with
Proteins Prepared from E. coli Containing a Hypersensitive Response
Elicitor Encoding Construct, pCPP2139, or its Plasmid Vector
pCPP50
[0128] Marglobe tomato seeds were submerged in Erwinia amylovora
hypersensitive response elicitor solution ("harpin") (from the
harpin encoding plasmid pCPP2139 vector) and from pCPP50
vector-containing solution at dilutions of 1:25, 1:50, and 1:100 in
beakers on day 1 for 24 hours at 28.degree. C. in a growth chamber.
After soaking seeds, they were sown in germination pots with
artificial soil on day 2. Ten uniform appearing plants per
treatment were chosen randomly and measured. The seedlings were
measured by ruler from the surface of soil to the top of plant.
[0129] Treatments:
[0130] 1. Harpin 16 .mu.gm/ml
[0131] 2. Harpin 8 .mu.gm/ml
[0132] 3. Harpin 4 .mu.gm/ml
[0133] 4. Vector 16 .mu.gm/ml
[0134] 5. Vector 8 .mu.gm/ml
[0135] 6. Vector 4 .mu.gm/ml
27TABLE 16 Seedling Height (cm) 11 Days After Seed Treatment Treat.
Harpin Plants 1 2 3 4 5 6 7 8 9 10 Mean H1:25 16 .mu.gm/ml 10 5.0
5.2 4.8 4.6 4.4 4.6 3.8 4.2 3.8 4.2 4.5 H1:50 8 .mu.gm/ml 10 5.6
5.4 6.0 5.8 4.8 6.8 5.8 5.0 5.2 4.8 5.5 H1:100 4 .mu.gm/ml 10 5.2
5.6 5.0 5.0 5.0 4.8 5.0 5.6 4.8 5.2 5.1 V1:25 0 10 4.4 4.4 4.8 4.6
4.8 4.6 4.0 4.8 4.4 4.6 4.5 V1:50 0 10 4.8 4.4 4.6 4.0 4.4 4.2 4.6
4.0 4.4 4.2 4.4 V1:100 0 10 4.6 4.2 4.8 4.4 4.4 4.0 4.2 4.0 4.4 4.0
4.3
[0136]
28TABLE 17 Seedling Height (cm) 14 Days After Seed Treatment Treat.
Harpin Plants 1 2 3 4 5 6 7 8 9 10 Mean H1:25 16 .mu.gm/ml 10 7.6
7.6 7.2 7.4 7.8 7.8 7.6 7.0 7.4 7.0 7.4 H1:50 8 .mu.gm/ml 10 8.5
8.2 8.4 7.6 7.8 8.4 8.6 9.0 7.6 8.2 8.2 H1:100 4 .mu.gm/ml 10 7.2
8.4 8.2 7.4 8.0 7.6 7.6 8.0 8.6 7.6 7.9 V1:25 0 10 6.8 6.4 7.8 6.6
6.6 6.8 7.4 6.0 6.4 6.4 6.7 V1:50 0 10 6.6 5.8 6.4 7.6 7.4 7.2 6.8
6.6 6.4 5.8 6.7 V1:100 0 10 6.2 6.0 6.8 6.6 6.4 5.8 6.6 7.0 5.8 6.4
6.4
[0137]
29TABLE 18 Mean Height of Tomato Plants After Treatment. Mean
height of Operation and Treatment tomato plants (cm) Day 1 Day 2
Day 11 Day 14 Harpin seed soak (16 .mu.gm/ml) sowing 4.5 7.4 Harpin
seed soak (8 .mu.gm/ml) sowing 5.5 8.2 Harpin seed soak (4
.mu.gm/ml) sowing 5.1 7.9 Vector seed soak (16 .mu.gm/ml) sowing
4.5 6.7 Vector seed soak (8 .mu.gm/ml) sowing 4.4 6.7 Vector seed
soak (4 .mu.gm/ml) sowing 4.3 6.4
[0138] As shown in Tables 16-18, treatment with Erwinia amylovora
hypersensitive response elicitor can increase growth of tomato
plants. A 1:50 dilution (8 .mu.g/ml hypersensitive response
elicitor) had the greatest effect with seedling height being
increased by about 20% over the control.
Example 8
Effect of Cell-Free Erwinia amylovora Hypersensitive Response
Elicitor on Growth of Potato
[0139] Three-week-old potato plants, variety Norchip, were grown
from tuber pieces in individual containers. The foliage of each
plant was sprayed with a solution containing Erwinia amylovora
hypersensitive response elicitor ("harpin"), or a control solution
containing proteins of E. coli and those of the vector pCPP50
("vector"), diluted 1:50, 1 :100, and 1:200. On day 20, 12 uniform
appearing plants were chosen randomly for each of the following
treatments. One plant from each treatment was maintained at
16.degree. C., in a growth chamber, while two plants from each
treatment were maintained on a greenhouse bench at 18-25.degree. C.
Twenty-five days after treatment, the shoots (stems) on all plants
were measured individually.
30 Treatments: 1. Harpin 1:50 16 .mu.gm/ml 2. Harpin 1:100 8
.mu.gm/ml 3. Harpin 1:200 4 .mu.gm/ml 4. Vector 1:50 0 harpin 5.
Vector 1:100 0 harpin 6. Vector 1:200 0 harpin
[0140]
31TABLE 19 Length of Potato Stems of Plants at 16.degree. C.
Treatment on day 20 Length of potato stems (cm) on day 45 stem 1
stem 2 stem 3 stem 4 stem 5 stem 6 Plant Mean Harpin 1:50 43.0 39.5
42.5 34.0 38.0 39.5 39.4 Harpin 1:100 42.0 38.5 (2 branch) 40.3
Harpin 1:200 35.5 30.5 31.5 (3 branch) 32.5 Vector 1:50 34.0 32.0
31.5 28.0 27.5 (5 branch) 30.6 Vector 1:100 30.0 33.5 33.0 30.0
28.0 33.0 31.3 Vector 1:200 33.5 31.5 32.5 (3 branch) 32.5
[0141]
32TABLE 20 Length of Potato Stems of Plants on a Greenhouse Bench
Treatment on day 20 Length of potato stems (cm) on day 45 stem 1
stem 2 stem 3 stem 4 stem 5 stem 6 Plant Treat. Mean Harpin 1:50
65.5 58.5 57.5 62.5 68.5 (5 branch) 62.5 Harpin 1:50 62.5 67.0 65.0
69.0 (4 branch) 65.9 64.2 Harpin 1:100 70.5 73.5 74.0 80.5 (4
branch) 74.6 Harpin 1:100 83.0 80.5 76.5 76.0 81.5 (5 branch) 79.5
77.1 Harpin 1:200 56.5 59.5 50.5 53.0 55.5 48.0 53.9 Harpin 1:200
57.0 59.5 69.5 (3 branch) 62.0 58.0 Vector 1:50 53.0 62.0 59.5 62.5
(4 branch) 59.3 Vector 1:50 52.0 46.0 61.5 56.5 61.5 57.0 55.8 57.6
Vector 1:100 62.0 51.5 66.0 67.5 62.0 63.0 62.0 Vector 1:100 61.5
62.5 59.0 65.5 63.0 63.5 62.5 62.3 Vector 1:200 62.0 66.0 (2
branch) 64.0 Vector 1:200 61.0 60.0 63.5 (3 branch) 61.5 62.8
[0142] As shown in Tables 19 and 20, treatment of potato plants
with Erwinia amylovora hypersensitive response elicitor enhanced
shoot (stem) growth. Thus, overall growth, as judged by both the
number and mean lengths of stems, were greater in the
harpin-treated plants in both the greenhouse and growth
chamber-grown plants. The potato plants treated with the medium
dose of harpin (8 .mu.gm/ml) seemed enhanced in their stem growth
more than those treated with either higher or lower doses.
Treatment with the medium dose of harpin resulted in greater growth
under both growing conditions.
Example 9
Effect of Spraying Tomatoes With a Cell-Free Elicitor Preparation
Containing the Erwinia amylovora Harpin
[0143] Marglobe tomato plants were sprayed with harpin preparation
(from E. coli DH5.alpha.(pCPP2139)) or vector preparation (from E.
coli DH5.alpha.(pCPP50)) with added BSA protein as control 8 days
after transplanting. The control vector preparation contained, per
ml, 33.6 .mu.l of BSA (10 mg/ml) to provide about the same amount
of protein as contained in the pCPP2139 preparation due to harpin.
Dilutions of 1:50 (8.0 .mu.g/ml), 1:100 (4.0 .mu.g/ml), and 1:200
(2.0 .mu.g/ml) were prepared and sprayed on the plants to runoff
with an electricity-powered atomizer. Fifteen uniform appearing
plants per treatment were chosen randomly and assigned to
treatment. The plants were maintained at 28.degree. C. in a
controlled environment chamber before and after treatment.
[0144] Overall heights were measured several times after treatment
from the surface of soil to the top of the plant. The tops of the
tomato plants were weighed immediately after cutting the stems near
the surface of the soil.
33 Treatments: (Dilutions and harpin content) 1. Harpin 1:50 (8.0
.mu.g/ml) 2. Harpin 1:100 (4.0 .mu.g/ml) 3. Harpin 1:200 (2.0
.mu.g/ml) 4. Vector + BSA 1:50 (0 harpin) 5. Vector + BSA 1:100 (0
harpin) 6. Vector + BSA 1:200 (0 harpin)
[0145]
34TABLE 21 Tomato plant height (cm) 1 day after spray treatment
Treat 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mean H 50 5.4 5.0 5.6 5.0
5.2 4.8 5.0 5.2 5.4 5.0 5.6 4.8 4.6 5.0 5.8 5.16 H 100 5.0 5.2 5.0
5.4 5.4 5.0 5.2 4.8 5.6 5.2 5.4 5.0 4.8 5.0 5.2 5.15 H 200 5.0 4.6
5.4 4.6 5.0 5.2 5.4 4.8 5.0 5.2 5.4 5.2 5.0 5.2 5.0 5.13 V 50 5.2
4.6 4.8 5.0 5.6 4.8 5.0 5.2 5.6 5.4 5.2 5.8 5.0 4.8 5.2 5.15 V 100
5.2 4.8 5.2 5.0 5.6 4.8 5.4 5.2 5.0 4.8 5.0 4.8 5.6 5.2 5.4 5.13 V
200 5.2 5.4 5.0 5.4 5.2 5.4 5.0 5.2 5.4 5.2 4.6 4.8 5.2 5.0 5.4
5.16
[0146]
35TABLE 22 Tomato plant height (cm) 15 days after spray treatment
Treat 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mean H 50 22.0 21.0 22.0
21.5 23.0 22.0 23.5 25.0 22.0 20.5 21.0 23.5 22.0 22.5 21.0 22.2 H
100 26.0 26.5 27.0 29.0 27.5 26.0 28.0 29.0 28.5 26.0 27.5 28.0
28.0 29.0 26.0 27.5 H 200 24.5 26.0 25.0 26.0 26.5 27.5 28.5 28.0
26.0 24.0 26.5 24.5 26.0 24.0 27.5 26.0 V 50 23.5 21.5 20.5 22.5
20.5 21.0 22.0 23.5 22.0 20.5 22.0 21.0 20.5 22.5 21.5 21.7 V 100
22.5 21.0 20.5 23.0 22.0 20.0 20.5 20.0 21.0 22.0 23.0 20.0 22.0
21.0 22.5 21.4 V 200 21.5 20.5 23.5 20.5 22.0 22.0 22.5 20.0 22.0
23.5 23.5 22.0 20.0 23.0 21.0 21.8
[0147]
36TABLE 23 Tomato plant height (cm) 21 days after spray treatment
Treat 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mean H 50 28.5 28.0 27.5
26.0 27.0 28.5 28.5 29.0 30.0 28.5 29.0 27.0 28.5 28.0 27.0 28.1 H
100 37.0 38.0 37.5 39.0 37.0 38.5 36.0 38.0 37.0 38.5 37.0 36.0
37.0 37.0 38.5 37.5 H 200 34.5 34.0 36.0 33.5 32.0 34.5 32.5 34.0
32.0 36.5 30.5 32.0 30.0 32.5 34.0 33.2 V 50 30.0 28.0 28.0 28.5
30.0 27.0 26.5 28.0 29.5 28.5 26.5 28.5 27.0 29.5 28.5 28.3 V 100
28.0 27.5 30.0 29.5 28.5 29.0 30.0 26.5 27.5 28.0 30.0 29.0 28.5
28.0 29.5 28.6 V 200 28.5 30.5 27.0 29.0 28.5 27.5 29.0 30.0 28.0
28.5 29.0 30.5 27.5 28.5 28.0 28.7
[0148]
37TABLE 24 Mean Height of Tomato Plants After Spraying Mean height
of tomato plants (cm) Days After Treatment Treatment (Dil. &
harpin) Day 1 Day 11 Day 14 Harpin 1:50 (8.0 .mu.g/ml) 5.16 22.2
28.1 Harpin 1:100 (4.0 .mu.g/ml) 5.15 27.5 37.5 Harpin 1:200 (2.0
.mu.g/ml) 5.13 26.0 33.2 Vector + BSA 1:50 (0) 5.15 21.7 28.5
Vector + BSA 1:100 (0) 5.13 21.4 28.6 Vector + BSA 1:200 (0) 5.16
21.8 28.7
[0149]
38TABLE 25 Fresh Weight of Tomato Plants (g/plant) 21 Days After
Spray Treatment Treat 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mean H 50
65.4 60.3 58.9 73.2 63.8 70.1 58.4 60.1 62.7 55.6 58.3 68.9 58.2
64.2 56.4 62.3 H 100 84.3 68.8 74.6 66.7 78.5 58.9 76.4 78.6 84.8
78.4 86.4 66.5 76.5 82.4 80.5 76.2 H 200 80.1 76.5 68.4 79.5 64.8
79.6 76.4 80.2 66.8 72.5 78.8 72.3 62.8 76.4 73.2 73.9 V 50 64.0
56.8 69.4 72.3 56.7 66.8 71.2 62.3 61.0 62.5 63.4 58.3 72.1 67.8
67.0 64.7 V 100 62.8 58.4 70.2 64.2 58.1 72.7 68.4 53.6 67.5 66.3
59.3 68.2 71.2 65.2 59.2 64.4 V 200 64.2 59.6 70.2 66.6 64.3 60.4
60.8 56.7 71.8 60.6 63.6 58.9 68.3 57.2 60.0 62.9
[0150] A single spray of tomato seedlings with harpin, in general,
resulted in greater subsequent growth than spray treatment with the
control (vector) preparation, which had been supplemented with BSA
protein. Enhanced growth in the harpin-treated plants was seen in
both plant height and fresh weight measurements. Of the three
concentrations tested, the two lower ones resulted in more plant
growth (based on either measure) than the higher dose (8.0
.mu.g/ml). There was little difference in the growth of plants
treated with the two lower (2 and 4 .mu.g/ml) concentrations.
Components of the lysed cell preparation from the strain E. coli
DH5.alpha.(pCPP50), which harbors the vector of the hrpn gene in E.
coli strain DH5.alpha.(pCPP2139), do not have the same
growth-promoting effect as the harpin-containing preparation, even
though it is supplemented with BSA protein to the same extent as
the DH5.alpha.(pCPP2139) preparation, which contains large amounts
of harpin protein. Thus, this experiment demonstrates that harpin
is responsible for enhanced plant growth.
Example 10
Early Coloration and Early Ripening of Raspberry Fruits
[0151] A field trial was conducted to evaluate the effect of
hypersensitive response elicitor ("harpin") treatment on yield and
ripening parameters of raspberry cv. Canby. Established plants were
treated with harpin at 2.5 mg/100 square feet in plots 40 feet
long.times.3 feet wide (1 plant wide), untreated ("Check"), or
treated with the industry standard chemical Ronilan at recommended
rates ("Ronilan"). Treatments were replicated four times and
arranged by rep in an experimental field site. Treatments were made
beginning at 5-10% bloom followed by two applications at 7-10 day
intervals. The first two harvests were used to evaluate disease
control and fruit yield data was collected from the last two
harvests. Observations indicated harpin-treated fruits were larger
and exhibited more redness than untreated fruits, indicating
ripening was accelerated by 1-2 weeks. The number of ripe fruits
per cluster bearing a minimum of ten fruits was determined at this
time and is summarized in Table 26. Harpin treated plots had more
ripe fruits per 10-berry cluster than either the check or Ronilan
treatments. Combined yields from the last two harvests indicated
increased yield in harpin and Ronilan treated plots over the
untreated control (Table 27).
39TABLE 26 Number of Ripe Raspberry Fruits Per Clusters With Ten
Berries or More on June 20, 1996. Treatment Ripe fruit/10 berry
clusters % of Control Check 2.75 100.0 Ronilan 2.75 100.0 Harpin
7.25 263.6
[0152]
40TABLE 27 Mean Raspberry Fruit Yield by weight (lbs.) Combined in
Last Two Harvest. Treatment Total Yield % of Control Check 32.5
100.0 Ronilan 37.5 115.4 Harpin 39.5 121.5
Example 11
Growth Enhancement For Snap Beans
[0153] Snap beans of the variety Bush Blue Lake were treated by
various methods, planted in 25-cm-d plastic pots filled with
commercial potting mix, and placed in an open greenhouse for the
evaluation of growth parameters. Treatments included untreated bean
seeds ("Check"), seeds treated with a slurry of 1.5% methyl
cellulose prepared with water as diluent ("M/C"), seeds treated
with 1.5% methyl cellulose followed by a foliar application of
hypersensitive response elicitor ("harpin") at 0.125 mg/ml
("M/C+H"), and seeds treated with 1.5% methyl cellulose plus harpin
spray dried at 5.0 .mu.g harpin per 50 seeds followed by a foliar
application of harpin at 0.125 mg/ml ("M/C-SD+H"). Seeds were sown
on day 0, planted 3 per pot, and thinned to 1 plant per pot upon
germination. Treatments were replicated 10 times and randomized by
rep in an open greenhouse. Bean pods were harvested after 64 days,
and fresh weights of bean pods of marketable size (E10 cm.times.5
cm in size) were collected as yield. Data were analyzed by analysis
of variance with Fisher's LSD used to separate treatment means.
41TABLE 28 Effect of Erwinia amylovora Harpin Treatment by Various
Methods on Yield of Market Sized Snap Bean Pods Treatment
Marketable Yield, g.sup.1 % of Untreated (Check) M/C-SD + H 70.6 a
452 M/C-H 58.5 ab 375 M/C 46.3 bc 297 M/C + H 42.3 bc 271 M/C-SD
40.0 cd 256 Check 15.6 e 100 .sup.1Marketable yield included all
bean pods 10 cm .times. 0.5 cm or larger. Means followed by the
same letter are not significantly different at P = 0.05 according
to Fisher's LSD.
[0154] As shown in Table 28, the application of Erwinia amylovora
harpin by various methods of application resulted in an increase in
the yield of marketable size snap bean pods. Treatment with methyl
cellulose alone also results in an increase in bean yield but was
substantially increased when combined with harpin as seed (spray
dried) and foliar treatments.
Example 12
Yield Increase in Cucumbers from Foliar Application of HP-1000.TM.
to Cucumbers
[0155] Cucumber seedlings and transplants were treated with foliar
sprays of HP-1000.TM. (EDEN Bioscience, Bothell, Wash.) (Erwinia
amylovora hypersensitive response elicitor formulation) at rates of
15, 30, or 60 .mu.g/ml active ingredient (a.i.). The first spray
was applied when the first true leaves were fully expanded. The
second application was made 10 days after the first spray. All
sprays were applied using a back-pack sprayer, and an untreated
control (UTC) was also included in the trial. Three days after the
second application of HP-1000.TM., ten plants from each treatment
were transplanted into randomized field plots replicated three
times. This yielded a total of thirty plants per treatment. Seven
days after transplanting, a third foliar spray of HP-1000.TM. was
applied. Although severe drought followed resulting in significant
water stress, a total of six harvests were made following a
standard commercial harvesting pattern. The total weight of fruit
harvested from each treatment is presented in Table 29. Results
indicate that plants treated with HP-1000.TM. at rates of 15 and 30
.mu.g/ml yielded significantly more fruit than the UTC. Plants
treated with HP-1000.TM. yielded a moderate yield increase.
[0156] These results indicated that HP-1000.TM. treated plants were
significantly more tolerant to drought stress conditions than
untreated plants.
42TABLE 29 Increase yield of cucumbers after treatment with HP-1000
.TM. Treatment Rate.sup.1 Yield,.sup.2 lbs./10 plants % above UTC
UTC -- 9.7 a -- HP-1000 .TM. 15 .mu.g/ml 25.4 b 161.4 HP-1000 .TM.
30 .mu.g/ml 32.6 c 236.4 HP-1000 .TM. 60 .mu.g/ml 11.2 a 15.9
.sup.1Active ingredient (a.i.). .sup.2Means followed by different
letters are significantly different according to Duncan's MRT, P =
0.05.
Example 13
Yield Increase in Cotton from Treatment with HP-1000.TM.
[0157] Cotton was planted in four, 12.times.20 foot replicate field
plots in a randomized complete block (RCB) field trial. Plants were
treated with HP-1000.TM. (EDEN Bioscience) (Erwinia amylovora
hypersensitive response elicitor formulation), HP-1000.TM.+Pix.RTM.
(Pix.RTM. (BASF Corp., Mount Olive, N.J.) is a growth regulator
applied to keep cotton plants compact in height) or Early
Harvest.RTM. (Griffen Corp., Valdosta, Ga.) (a competitive growth
enhancing agent). An untreated control (UTC) was also included in
the trial. Using a back-pack sprayer, foliar applications were made
of all treatments at three crop growth stages; first true leaves,
pre-bloom, and early bloom. All fertilizers and weed control
products were applied according to conventional farming practices
for all treatments. The number of cotton bolls per plant ten weeks
before harvest was significantly higher for the HP-1000.TM. treated
plants compared to other treatments. By harvest, HP-1000.TM.
treatment was shown to have a significantly increased lint yield
(43%) compared to UTC (Table 30). When HP-1000.TM. was combined
with Pix.RTM., lint yield was increased 20% over UTC. Since Pixt is
commonly applied to large acreages of cotton, this result indicates
that HP-1000.TM. may be successfully tank-mixed with Pix.RTM..
Application of the competitive growth enhancing agent, Early
Harvest.RTM. only produced a 9% increase in lint yield vs. UTC.
43TABLE 30 Increased lint yield from cotton after treatment with
HP-1000 .TM., HP-1000 .TM. + PiX .RTM., or Early Harvest .RTM.. %
above Treatment Rate.sup.1 Lint Yield (lbs./ac) UTC UTC -- 942.1 --
Early Harvest .RTM. 2 oz./ac. 1,077.4* 14.3 HP-1000 .TM. + Pix
.RTM. 40 .mu.g/ml + 8 oz./ac. 1,133.1* 20.4 HP-1000 .TM. 40
.mu.g/ml 1,350.0* 43.3 (*significant at P = 0.05) lsd = 122.4
.sup.1Rates for HP-1000 .TM. are for active ingredient (a.i.);
rates for Early Harvest .RTM. and Pix .RTM. are formulated
product.
Example 14
Yield Increase of Chinese Egg Plant from Treatment with
HP-100.TM.
[0158] Nursery grown Chinese egg plant seedlings were sprayed once
with HP-1000.TM. at (EDEN Bioscience) (Erwinia amylovora
hypersensitive response elicitor formulation) 15, 30, or 60
.mu.g/ml (a.i.), then transplanted into field plots replicated
three times for each treatment. Two weeks after transplanting, a
second application of HP-1000.TM. was made. A third and final
application of HP-1000.TM. was applied approximately two weeks
after the second spray. All sprays were applied using a back-pack
sprayer; an untreated control (UTC) was also included in the trial.
As the season progressed, a total of eight harvests from each
treatment were made. Data from these harvests indicate that
treatment with HP-1000.TM. resulted in greater yield of fruit per
plant.
44TABLE 31 Increased yield for Chinese egg plant after treatment
with HP-1000 .TM.. Treatment Rate (a.i.) Yield(lbs./plant) % above
UTC UTC -- 1.45 -- HP-1000 .TM. 15 .mu.g/ml 2.03 40.0 HP-1000 .TM.
30 .mu.g/mi 1.90 31.0 HP-1000 .TM. 60 .mu.g/ml 1.95 34.5
Example 15
Yield Increase of Rice From Treatment with HP-1000.TM.
[0159] Rice seedlings were transplanted into field plots replicated
three times, then treated with foliar sprays of HP-1000.TM. (EDEN
Bioscience) (Erwinia amylovora hypersensitive response elicitor
formulation) at three different rates using a back-pack sprayer. An
untreated control (UTC) was also included in the trial. The first
application of HP-1000.TM. was made one week after transplanting,
the second three weeks after the first. A third and final spray was
made just before rice grains began to fill the heads. Results at
harvest demonstrated that foliar applications of HP-1000.TM. at
both 30 and 60 g/ml significantly increased yield by 47 and 56%,
respectively (Table 32).
45TABLE 32 Increase yield of rice after foliar treatment with
HP-1000 .TM.. Treatment Rate (a.i.) Yield.sup.1 (lbs./ac.) % above
UTC UTC -- 3,853 a -- HP-1000 .TM. 15 .mu.g/ml 5,265 ab 35.9
HP-1000 .TM. 30 .mu.g/ml 5,710 b 47.3 HP-1000 .TM. 60 .mu.g/ml
6,043 b 56.1 .sup.1Means followed by different letters are
significantly different according to Duncan's MRT, P = 0.05.
Example 16
Yield Increase of Soybeans From Treatment with HP-1000.TM.
[0160] Soybeans were planted into randomized field plots replicated
three times for each treatment. A back-pack sprayer was used to
apply foliar sprays of HP-1000.TM. (EDEN Bioscience) (Erwinia
amylovora hypersensitive response elicitor formulation) and an
untreated control (UTC) was also included in the trial. Three rates
of HP-1000.TM. were applied beginning at four true leaves when
plants were approximately eight inches tall. A second spray of
HP-1000.TM. was applied ten days after the first spray and a third
spray ten days after the second. Plant height measured ten days
after the first spray treatment indicated that application of
HP-1000.TM. resulted in significant growth enhancement (Table 33).
In addition, plants treated with HP-1000.TM. at the rate of 60
.mu.g/ml began to flower five days earlier than the other
treatments. Approximately ten days after application of the third
spray, the number of soybean pods per plant was counted from ten
randomly selected plants per replication. These results indicated
that the growth enhancement from treatment with HP-1000.TM.
resulted in significantly greater yield (Table 34).
46TABLE 33 Increased plant height of soybeans after foliar
treatment with HP-1000 .TM.. Treatment Rate (a.i.) Plant Ht..sup.1
(in.) % above UTC UTC -- 12.2 a -- HP-1000 .TM. 15 .mu.g/ml 13.2 b
8.3 HP-1000 .TM. 30 .mu.g/ml 14.1 c 16.2 HP-1000 .TM. 60 .mu.g/ml
14.3 c 17.3 .sup.1Means followed by different letters are
significantly different according to Duncan's MRT, P = 0.05.
[0161]
47TABLE 34 Increased pod set of soybeans after foliar treatment
with HP-1000 .TM.. Treatment Rate (a.i.) No. Pods/plant.sup.1 %
above UTC UTC -- 41.1 a -- HP-1000 .TM. 15 .mu.g/ml 45.4 ab 10.4
HP-1000 .TM. 30 .mu.g/ml 47.4 b 15.4 HP-1000 .TM. 60 .mu.g/ml 48.4
b 17.7 .sup.1Means followed by different letters are significantly
different according to Duncan's MRT, P = 0.05.
Example 17
Yield Increase of Strawberries From Treatment With HP-100.TM.
[0162] Two field trials with HP-1000.TM. (EDEN Bioscience) (Erwinia
amylovora hypersensitive response elicitor formulation) were
conducted on two strawberry varieties, Camarosa and Selva. For each
variety, a randomized complete block (RCB) design was established
having four replicate plots (5.33.times.10 feet) per treatment in a
commercially producing strawberry field. Within each plot,
strawberry plants were planted in a double row layout. An untreated
control (UTC) was also included in the trial. Before applications
began, all plants were picked clean of any flowers and berries.
Sprays of HP-1000.TM. at the rate of 40 .mu.g/ml were applied as
six weekly using a back-pack sprayer. Just prior to application of
each spray, all ripe fruit from each treatment was harvested,
weighed, and graded according to commercial standards. Within three
weeks of the first application of HP-1000.TM. to Selva strawberry
plants, growth enhancement was discernible as visibly greater
above-ground biomass and a more vigorous, greener and healthier
appearance. After six harvests (i.e. the scheduled life-span for
these plants), all yield data were summed and analyzed. For the
Camarosa variety, yield of marketable fruit from HP-1000.TM.
treated plants was significantly increased (27%) over the UTC when
averaged over the last four pickings (Table 35). Significant
differences between treatments were not apparent for this variety
for the first two pickings. The Selva variety was more responsive
to the growth enhancing effects from treatment with HP-1000.TM.;
Selva strawberry plants yielded a statistically significant 64%
more marketable fruit vs. the UTC when averaged over six pickings
(Table 35).
48TABLE 35 Increased yield of strawberries after foliar treatment
with HP-1000 .TM.. Treatment Rate (a.i.) Yield.sup.1 (lbs./rep) %
above UTC Variety: Camarosa UTC -- 1.71 a -- HP-1000 .TM. 40
.mu.g/ml 2.17 b 27 Variety: Selva UTC -- 0.88 a -- HP-1000 .TM. 40
.mu.g/ml 1.44 b 64 .sup.1Means followed by different letters are
significantly different according to Duncan's MRT, P = 0.05.
Example 18
Earlier Maturity and Increased Yield of Tomatoes from Treatment
with HP-1000.TM.
[0163] Fresh market tomatoes (var. Solar Set) were grown in plots
(2.times.30 feet) replicated 5 times in a randomized complete block
(RCB) field trial within a commercial tomato production field.
Treatments included HP-1000.TM. (EDEN Bioscience) (Erwinia
amylovora hypersensitive response elicitor formulation), an
experimental competitive product (Actigard.TM. (Novartis,
Greensboro, N.C.)) and a chemical standard (Kocide.RTM. (Griffen
Corp., Valdosta, Ga.)) +Manebg (DuPont Agricultural Products,
Wilmington, Del.)) for disease control. The initial application of
HP-1000.TM. was made as a 50 ml drench (of 30 .mu.g/ml a.i.) poured
directly over the seedling immediately after transplanting.
Thereafter, eleven weekly foliar sprays were applied using a
back-pack sprayer. The first harvest from all treatments was made
approximately six weeks after transplanting and only fully red,
ripe tomatoes were harvested from each treatment. Results indicated
that HP-1000.TM. treated plants had a significantly greater amount
of tomatoes ready for the first harvest (Table 36). The tomatoes
harvested from the HP-1000.TM. treated plants were estimated to be
10-14 days ahead other treatments.
49TABLE 36 Increased yield of tomatoes at first harvest after
foliar treatment with of HP-1000 .TM.. Treatment Rate (a.i.).sup.1
Yield.sup.2 (lbs./rep) % above UTC UTC -- 0.61 a -- HP-1000 .TM. 30
.mu.g/ml 2.87 b 375 Actigard .TM. 14 g/ac 0.45 a -25.1 Kocide .RTM.
+ 2 lbs./ac. 0.31 a -49.1 Maneb .RTM. 1 lb./ac .sup.1Rates for
Kocide .RTM. and Maneb .RTM. are for formulated product.
.sup.2Means followed by different letters are significantly
different according to Duncan's MRT, P = 0.05.
Example 19
Earlier Flowering and Growth Enhancement of Strawberries
[0164] From Treatment with HP-1000.TM. When Planted in
Non-Fumigated Soil
[0165] Strawberry plants ("plugs" and "bare-root"), cv. Commander
were transplanted into plots (2.times.30 feet) replicated 5 times
in a randomized complete block field trial. Approximately sixty
individual plants were transplanted into each replicate. Treatments
applied in this field trial are listed below:
50 Treatment Application method HP-1000 .TM. 50-ml drench solution
of HP-1000 .TM. (plug plants) (EDEN Bioscience) (Erwinia amylovora
hypersensitive response elicitor formulation) at 40 .mu.g/ml(a.i.)
poured directly over the individual plants immediately after
transplanting into non-fumigated soil.sup.1, followed by foliar
applications of HP-1000 .TM. at 40 .mu.g/ml every 14 days. HP-1000
.TM. root soak in solution of HP-1000 .TM. at 40 (bare- .mu.g/ml
(a.i.) for 1 hour, immediately root plants) before transplanting
into non-fumigated soil,.sup.1 followed by foliar applications of
HP-1000 .TM. at 40 .mu.g/ml every 14 days. methyl soil fumigation
at 300 lbs./ac via bromide/ injection prior to transplanting, no
chlorpicrin HP-1000 .TM. treatments applied. 75/25 Telone/ soil
fumigation at 45 gal./ac via chlorpicrin injection prior to
transplanting, no 70/30 HP-1000 .TM. treatments applied. untreated
no fumigation, no HP-1000 .TM. treatments control (UTC)
.sup.1Non-fumigated soil had been cropped to vetch for the two
previous years.
[0166] Transplanting was done in late fall when cool weather tended
to slow plant growth. Two weeks after transplanting, the first
foliar application of HP-1000.TM. was made at 40 .mu.g/ml (a.i.)
with a back-pack sprayer. Three weeks after transplanting,
preliminary results were gathered comparing HP-1000.TM. treatment
against methyl bromide and UTC by counting the number of flowers on
all strawberry "plug" plants in each replication. Since flowering
had not yet occurred in the "bare-root" plants, each plant in
replicates for this treatment was assessed for early leaf growth by
measuring the distance from leaf tip to stem on the middle leaf of
3-leaf cluster. Results (Tables 37 and 38) indicated that treatment
with HP-1000.TM. provided early enhanced flower growth and leaf
size for "plug" and "bare-root" strawberry plants,
respectively.
51TABLE 37 Earlier flowering of "plug" strawberry transplants after
foliar treatment with HP-1000 .TM.. Treatment Rate (a.i.) No.
flowers/rep.sup.1 % above UTC UTC -- 2.0 a -- HP-1000 .TM. 40
.mu.g/ml 7.5 b 275 Methyl bromide/ 300 lbs./ac 5.3 b 163
chlorpicrin .sup.1Means followed by different letters are
significantly different according to Duncan's MRT, P = 0.05.
[0167]
52TABLE 38 Increased leaf growth in "bare-root" strawberry
transplants after foliar treatment with HP-1000 .TM.. Treatment
Rate (a.i.) Leaf length.sup.1 (in.) % above UTC UTC -- 1.26 a --
HP-1000 .TM. 40 .mu.g/ml 1.81 b 44 .sup.1Means followed by
different letters are significantly different according to Duncan's
MRT, P = 0.05.
Example 20
Early Growth Enhancement of Jalapeo Peppers from Application of
HP-1000.TM.
[0168] Jalapeo pepper (cv. Mittlya) transplants were treated with a
root drench of HP-1000 (EDEN Bioscience) (Erwinia amylovora
hypersensitive response elicitor formulation) (30 .mu.g/ml a.i.)
for 1 hour, then transplanted into randomized field plots
replicated four times. An untreated control (UTC) was also
included. Beginning 14 days after transplanting, treated plants
received three foliar sprays of HP-1000.TM. at 14 day intervals
using a back-pack sprayer. One week after the third application of
HP-1000.TM. (54 days after transplanting), plant height was
measured from four randomly selected plants per replication.
Results from these measurements indicated that the HP-1000.TM.
treated plants were approximately 26% taller than the UTC plants
(Table 39). In addition, the number of buds, flowers or fruit on
each plant was counted. These results indicated that the
HP-1000.TM. treated plants had over 61% more flowers, fruit or buds
compared to UTC plants (Table 40).
53TABLE 39 Increased plant height in Jalapeo peppers after
treatment with HP-1000 .TM.. Treatment Rate (a.i.) Plant Ht.
(in).sup.1 % above UTC UTC -- a 7.0 -- HP-1000 .TM. 30 .mu.g/ml 8.6
b 23.6 .sup.1Means followed by different letters are significantly
different according to Duncan's MRT, P = 0.05.
[0169]
54TABLE 40 Increased number of flowers, fruit or buds in Jalapeno
peppers after treatment with HP-1000 .TM.. No. flowers, fruit
Treatment Rate (a.i.) or buds/plant.sup.1 % above UTC UTC -- 20.6 a
-- HP-1000 .TM. 30 .mu.g/ml 12.8 b 61.3 .sup.1Means followed by
different letters are significantly different according to Duncan's
MRT, P = 0.05.
Example 21
Growth Enhancement of Tobacco from Application of HP-1000.TM.
[0170] Tobacco seedlings were transplanted into randomized field
plots replicated three times. A foliar spray of HP-1000.TM. (EDEN
Bioscience) (Erwinia amylovora hypersensitive response elicitor
formulation) was applied after transplanting at one of three rates:
15, 30, or 60 .mu.g/ml a.i. Sixty days later, a second foliar
application of HP-1000 was made. Two days after the second
application, plant height, number of leaves per plant, and the leaf
size (area) were measured from ten, randomly selected plants per
treatment. Results from these measurements indicated treatment with
HP-1000.TM. enhanced tobacco plant growth significantly (Tables 41,
42, and 43). Plant height was increased by 6-13%, while plants
treated with HP-1000.TM. at 30 and 60 .mu.g/ml averaged just over 1
more leaf per plant than UTC. Most significantly, however,
treatment with HP-1000.TM. at 15, 30, and 60 .mu.g/ml resulted in
corresponding increases in leaf area. Tobacco plants with an extra
leaf per plant and an increase in average leaf size (area)
represent a commercially significant response.
55TABLE 41 Increased plant height in tobacco after treatment with
HP-1000 .TM.. Treatment Rate (a.i.) Plant Ht. (cm) % above UTC UTC
-- 72.0 -- HP-1000 .TM. 15 .mu.g/ml 76.4 5.3 HP-1000 .TM. 30
.mu.g/ml 79.2 9.0 HP-1000 .TM. 60 .mu.g/ml 81.3 6.9
[0171]
56TABLE 42 Increased number of tobacco leaves per plant after
treatment with HP-1000 .TM.. Treatment Rate (a.i.)
Leaves/plant.sup.1 % above UTC UTC -- 16.8 -- HP-1000 .TM. 15
.mu.g/ml 17.4 3.6 HP-1000 .TM. 30 .mu.g/ml 18.1 7.7 HP-1000 .TM. 60
.mu.g/ml 17.9 6.5
[0172]
57TABLE 43 Increased leaf area in tobacco after treatment with
HP-1000 .TM.. Treatment Rate (a.i.) Leaf area (cm.sup.2) % above
UTC UTC -- 1,246 -- HP-1000 .TM. 15 .mu.g/ml 1,441 16 HP-1000 .TM.
30 .mu.g/ml 1,543 24 HP-1000 .TM. 60 .mu.g/ml 1,649 32
Example 22
Growth Enhancement of Winter Wheat from Application of
HP-1000.TM.
[0173] Winter wheat seed was "dusted" with dry HP-1000.TM. (EDEN
Bioscience) (Erwinia amylovora hypersensitive response elicitor
formulation) powder at the rate of 3 ounces of formulated product
(3% a.i.) per 100 lbs. seed, then planted 40 using conventional
seeding equipment into randomized test plots 11.7 feet by 100 feet
long. Additional treatments included a seed "dusting" with
HP-1000.TM. powder (3% a.i.) at 1 oz. formulated product per 100
lbs. seed, a seed-soak in a solution of HP-1000.TM. at a
concentration of 20 .mu.g/ml, a.i., for four hours, then air-dried
before planting, a standard chemical (Dividend.RTM.) fungicide
"dusting", and an untreated control (UTC). Eight days after
planting, HP-1000.TM. treated seeds began to emerge, whereas the
UTC and chemical standard-treated seed did not emerge until
approximately 14 days after planting, the normal time expected. At
41 days after planting, seedlings were removed from the ground and
evaluated. Root mass for wheat treated with HP-1000.TM. as a
"dusting" at 3 oz./100 lb. was visually inspected and judged to be
approximately twice as great as any of the other treatments.
[0174] Following the field trial, a greenhouse experiment was
designed to gain confirmation of these results. Treatments included
wheat seed dusted with dry HP-1000.TM. (10% a.i.) at a rate of 3
ounces per 100 lbs. of seed, seed soaking of HP-1000.TM. in
solution concentration of 20 mg/ml for four hours before planting,
and an untreated control (UTC). Wheat seeds from each treatment
were planted at the rate of 25 seeds per pot, with five pots
serving as replicates for each treatment. Fifteen days after
planting, ten randomly selected seedlings from each treatment pot
were removed, carefully cleaned, and measured for root length.
Since the above-ground portion of individual seedlings did not
exhibit any treatment effect, increased root growth from treatment
with HP-1000.TM. did not influence the selection of samples. The
increase in root growth from either HP-1000.TM. treatment was
significantly greater than UTC (Table 49); however, the seed
dusting treatment appeared to give slightly better results.
58TABLE 44 Increased root growth in wheat seedlings after treatment
with HP-1000 .TM.. Treatment Rate Root length. (cm).sup.1 % above
UTC UTC -- 35.6 a -- HP-1000 .TM. 3 oz./100 lbs. 41.0 b 17.4
(dusting) HP-1000 .TM. 20 .mu.g/ml 40.8 b 14.6 (soaking)
.sup.1Means followed by different letters are significantly
different according to Duncan's MRT, P = 0.05.
Example 23
Growth Enhancement of Cucumbers from Application of HP-100.TM.
[0175] A field trial of commercially produced cucumbers consisted
of four treatments, HP-1000.TM. (EDEN Bioscience) (Erwinia
amylovora hypersensitive response elicitor formulation) at two
rates (20 or 40 .mu.g/ml), a chemical standard for disease control
(Bravo.RTM. (Zeneca Ag Products, Wilmington, Del.)+Maneb(g) and an
untreated control (UTC). Each treatment was replicated four times
in 3.times.75 foot plots with a plant spacing of approximately 2
feet for each treatment. Foliar sprays of HP-1000.TM. were applied
beginning at first true leaf and repeated at 14 day intervals until
the last harvest for a total of six applications. The standard
fungicide mix was applied every seven days or sooner if conditions
warranted. Commercial harvesting began approximately two months
after first application of HP-1000.TM. (after five sprays of
HP-1000.TM. had been applied), and a final harvest was made
approximately 14 days after the first harvest.
[0176] Results from the first harvest indicated that treatment with
HP-1000.TM. enhanced the average cucumber yield by increasing the
total number of cucumbers harvested and not the average weight of
individual cucumbers (Tables 45-47). The same trend was noted at
the final harvest (Tables 48-49). It was commercially important
that the yield increase resulting from treatment with HP-1000.TM.
was not achieved by significantly increasing average cucumber
size.
59TABLE 45 Increased cucumber yield after treatment with HP-1000
.TM., first harvest. Treatment Rate (a.i.) Yield/trt.sup.1(kg.) %
above UTC UTC -- 10.0 a -- Bravo + Maneb label 10.8 a 8.4 HP-1000
.TM. 20 .mu.g/ml 12.3 ab 22.8 HP-1000 .TM. 40 .mu.g/ml 13.8 b 38.0
.sup.1Means followed by different letters are significantly
different according to Duncan's MRT, P = 0.05.
[0177]
60TABLE 46 Increased number of fruit in cucumbers after treatment
with HP-1000 .TM., first harvest. Treatment Rate (a.i.) No.
fruit/trt.sup.1 % above UTC UTC -- 24.5 a -- Bravo + Maneb label
27.6 ab 12.8 HP-1000 .TM. 20 .mu.g/ml 31.2 b 27.0 HP-1000 .TM. 40
.mu.g/ml 34.3 b 39.8 .sup.1Means followed by different letters are
significantly different according to Duncan's MRT, P = 0.05.
[0178]
61TABLE 47 Average weight of cucumbers after treatment with HP-1000
.TM., first harvest. Treatment Rate (a.i.) Weight/fruit (g) %
change vs. UTC UTC -- 406 -- Bravo + Maneb label 390 -4 HP-1000
.TM. 20 .mu.g/ml 395 -3 HP-1000 .TM. 40 .mu.g/ml 403 -1
[0179]
62TABLE 48 Increased cucumber yield after treatment with HP-1000
.TM., third harvest. Treatment Rate (a.i.) Yield/trt.sup.1(kg.) %
above UTC UTC -- 17.5 a -- Bravo + Maneb label 14.0 b -20.1 HP-1000
.TM. 20 .mu.g/ml 20.1 a 15.3 HP-1000 .TM. 40 .mu.g/ml 20.2 a 15.6
.sup.1Means followed by different letters are significantly
different according to Duncan's MRT, P = 0.05.
[0180]
63TABLE 49 Increased number of fruit in cucumbers after treatment
with HP-1000 .TM., third harvest. Treatment Rate (a.i.) No.
fruit/trt.sup.1 % change vs. UTC UTC -- 68.8 ab -- Bravo + Maneb
label 60.0 a -12.7 HP-1000 .TM. 20 .mu.g/ml 82.3 b 19.6 HP-1000
.TM. 40 .mu.g/ml 85.3 b 24.0 .sup.1Means followed by different
letters are significantly different according to Duncan's MRT, P =
0.05.
[0181]
64TABLE 50 Average weight of cucumbers after treatment with HP-1000
.TM., third harvest. Treatment Rate (a.i.) Weight/fruit (g) %
change vs. UTC UTC -- 255 -- Bravo + Maneb label 232 -9 HP-1000
.TM. 20 .mu.g/ml 247 -3 HP-1000 .TM. 40 .mu.g/ml 237 -7
Example 24
HarPin.sub.Pss from Pseudomonas syringae pv syringae Induces Growth
Enhancement in Tomato
[0182] To test if harpin.sub.Pss (i.e. the hypersensitive response
elicitor from Pseudomonas syringae pv syringae) (He, S. Y., et al.,
"Pseudomonas syringae pv syringae Harpin.sub.Pss. A Protein that is
Secreted via the Hrp Pathway and Elicits the Hypersensitive
Response in Plants," Cell 73:1255-66 (1993), which is hereby
incorporated by reference) also stimulates plant growth, tomato
seeds (Marglobe variety) were sowed in 8 inches pots with
artificial soil. 10 days after sowing, the seedlings were
transplanted into individual pots. Throughout the experiment,
fertilizer, irrigation of water, temperature, and soil moisture
were maintained uniformly among plants. 16 days after
transplanting, the initial plant height was measured and the first
application of harpin.sub.Pss was made, this is referred to as day
0. A second application was made on day 15. Additional growth data
was collected on day 10 and day 30. The final data collection on
day 30 included both plant height and fresh weight.
[0183] The harpin.sub.Pss used for application during the
experiment was produced by fermenting E. coli DH5 containing the
plasmid with the gene encoding harpin.sub.Pss (i.e. hrpZ). The
cells were harvested, resuspended in 5 mM potassium phosphate
buffer, and disrupted by sonication. The sonicated material was
boiled for 5 minutes and then centrifugated for 10 min. at 10,000
rpm. The supernantant was considered as Cell-Free Elicitor
Preparation (CFEP). 20 and 50 .mu.g/ml harpin.sub.Pss solution was
made with the same buffer used to make cell suspension. CFEP
prepared from the same strain containing the same plasmid but
without hrpZ gene was used as the material for control
treatment.
[0184] The wetting agent, Pinene II (Drexel Chemical Co., Memphis,
Tenn.) was added to the harpin.sub.Pss solution at the
concentration of 0. 1%, then harpin.sub.Pss was sprayed onto tomato
plant until there was run off.
[0185] Table 51 shows that there was a significant difference
between the harpin.sub.Pss treatment groups and the control group.
Harpin.sub.Pss treated tomato increased more than 10% in height.
The data supports the claim that harpin.sub.Pss does act similar to
the hypersensitive response elicitor from Erwinia amylovora, in
that when applied to tomato and many other species of plants, there
is a growth enhancement effect. In addition to a significant
increase of tomato height harpin.sub.Pss-treated tomato had more
biomass, big leaves, early flower setting, and over all healthier
appearance.
65TABLE 51 Harpin.sub.pss enhances the growth of tomato plant Plant
Height (cm.sup.1) Treatment Day 0 Day 10 Day 30 CFEP Control
8.5.sup.2 (0.87) a.sup.3 23.9 (1.90) a 68.2 (8.60) a Harpinpss 20
.mu.g/ml 8.8 (0.98) a 27.3 (1.75) b 74.2 (6.38) b Harpinpss 50
.mu.g/ml 8.8 (1.13) a 26.8 (2.31) b 75.4 6.30) b .sup.1Plant height
was measured to the nearest 0.5 cm. Day 0 refers to the day the
initial plant heights were recorded and the first application was
made. .sup.2Means are given with SD in parenthesis (n = 20 for all
treatment groups). .sup.3Different letters (a and b) indicates
significant differences (P 0.05) among means. Differences were
evaluated by ANOVA followed by Fisher LSD.
[0186] Although the invention has been described in detail for the
purpose of illustration, it is understood that such detail is
solely for that purpose, and variations can be made therein by
those skilled in the art without departing from the spirit and
scope of the invention which is defined by the following claims.
Sequence CWU 1
1
10 1 338 PRT Erwinia chrysanthemi 1 Met Gln Ile Thr Ile Lys Ala His
Ile Gly Gly Asp Leu Gly Val Ser 1 5 10 15 Gly Leu Gly Ala Gln Gly
Leu Lys Gly Leu Asn Ser Ala Ala Ser Ser 20 25 30 Leu Gly Ser Ser
Val Asp Lys Leu Ser Ser Thr Ile Asp Lys Leu Thr 35 40 45 Ser Ala
Leu Thr Ser Met Met Phe Gly Gly Ala Leu Ala Gln Gly Leu 50 55 60
Gly Ala Ser Ser Lys Gly Leu Gly Met Ser Asn Gln Leu Gly Gln Ser 65
70 75 80 Phe Gly Asn Gly Ala Gln Gly Ala Ser Asn Leu Leu Ser Val
Pro Lys 85 90 95 Ser Gly Gly Asp Ala Leu Ser Lys Met Phe Asp Lys
Ala Leu Asp Asp 100 105 110 Leu Leu Gly His Asp Thr Val Thr Lys Leu
Thr Asn Gln Ser Asn Gln 115 120 125 Leu Ala Asn Ser Met Leu Asn Ala
Ser Gln Met Thr Gln Gly Asn Met 130 135 140 Asn Ala Phe Gly Ser Gly
Val Asn Asn Ala Leu Ser Ser Ile Leu Gly 145 150 155 160 Asn Gly Leu
Gly Gln Ser Met Ser Gly Phe Ser Gln Pro Ser Leu Gly 165 170 175 Ala
Gly Gly Leu Gln Gly Leu Ser Gly Ala Gly Ala Phe Asn Gln Leu 180 185
190 Gly Asn Ala Ile Gly Met Gly Val Gly Gln Asn Ala Ala Leu Ser Ala
195 200 205 Leu Ser Asn Val Ser Thr His Val Asp Gly Asn Asn Arg His
Phe Val 210 215 220 Asp Lys Glu Asp Arg Gly Met Ala Lys Glu Ile Gly
Gln Phe Met Asp 225 230 235 240 Gln Tyr Pro Glu Ile Phe Gly Lys Pro
Glu Tyr Gln Lys Asp Gly Trp 245 250 255 Ser Ser Pro Lys Thr Asp Asp
Lys Ser Trp Ala Lys Ala Leu Ser Lys 260 265 270 Pro Asp Asp Asp Gly
Met Thr Gly Ala Ser Met Asp Lys Phe Arg Gln 275 280 285 Ala Met Gly
Met Ile Lys Ser Ala Val Ala Gly Asp Thr Gly Asn Thr 290 295 300 Asn
Leu Asn Leu Arg Gly Ala Gly Gly Ala Ser Leu Gly Ile Asp Ala 305 310
315 320 Ala Val Val Gly Asp Lys Ile Ala Asn Met Ser Leu Gly Lys Leu
Ala 325 330 335 Asn Ala 2 2141 DNA Erwinia chrysanthemi 2
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 3 403 PRT Erwinia amylovora
3 Met Ser Leu Asn Thr Ser Gly Leu Gly Ala Ser Thr Met Gln Ile Ser 1
5 10 15 Ile Gly Gly Ala Gly Gly Asn Asn Gly Leu Leu Gly Thr Ser Arg
Gln 20 25 30 Asn Ala Gly Leu Gly Gly Asn Ser Ala Leu Gly Leu Gly
Gly Gly Asn 35 40 45 Gln Asn Asp Thr Val Asn Gln Leu Ala Gly Leu
Leu Thr Gly Met Met 50 55 60 Met Met Met Ser Met Met Gly Gly Gly
Gly Leu Met Gly Gly Gly Leu 65 70 75 80 Gly Gly Gly Leu Gly Asn Gly
Leu Gly Gly Ser Gly Gly Leu Gly Glu 85 90 95 Gly Leu Ser Asn Ala
Leu Asn Asp Met Leu Gly Gly Ser Leu Asn Thr 100 105 110 Leu Gly Ser
Lys Gly Gly Asn Asn Thr Thr Ser Thr Thr Asn Ser Pro 115 120 125 Leu
Asp Gln Ala Leu Gly Ile Asn Ser Thr Ser Gln Asn Asp Asp Ser 130 135
140 Thr Ser Gly Thr Asp Ser Thr Ser Asp Ser Ser Asp Pro Met Gln Gln
145 150 155 160 Leu Leu Lys Met Phe Ser Glu Ile Met Gln Ser Leu Phe
Gly Asp Gly 165 170 175 Gln Asp Gly Thr Gln Gly Ser Ser Ser Gly Gly
Lys Gln Pro Thr Glu 180 185 190 Gly Glu Gln Asn Ala Tyr Lys Lys Gly
Val Thr Asp Ala Leu Ser Gly 195 200 205 Leu Met Gly Asn Gly Leu Ser
Gln Leu Leu Gly Asn Gly Gly Leu Gly 210 215 220 Gly Gly Gln Gly Gly
Asn Ala Gly Thr Gly Leu Asp Gly Ser Ser Leu 225 230 235 240 Gly Gly
Lys Gly Leu Gln Asn Leu Ser Gly Pro Val Asp Tyr Gln Gln 245 250 255
Leu Gly Asn Ala Val Gly Thr Gly Ile Gly Met Lys Ala Gly Ile Gln 260
265 270 Ala Leu Asn Asp Ile Gly Thr His Arg His Ser Ser Thr Arg Ser
Phe 275 280 285 Val Asn Lys Gly Asp Arg Ala Met Ala Lys Glu Ile Gly
Gln Phe Met 290 295 300 Asp Gln Tyr Pro Glu Val Phe Gly Lys Pro Gln
Tyr Gln Lys Gly Pro 305 310 315 320 Gly Gln Glu Val Lys Thr Asp Asp
Lys Ser Trp Ala Lys Ala Leu Ser 325 330 335 Lys Pro Asp Asp Asp Gly
Met Thr Pro Ala Ser Met Glu Gln Phe Asn 340 345 350 Lys Ala Lys Gly
Met Ile Lys Arg Pro Met Ala Gly Asp Thr Gly Asn 355 360 365 Gly Asn
Leu Gln Ala Arg Gly Ala Gly Gly Ser Ser Leu Gly Ile Asp 370 375 380
Ala Met Met Ala Gly Asp Ala Ile Asn Asn Met Ala Leu Gly Lys Leu 385
390 395 400 Gly Ala Ala 4 1288 DNA Erwinia amylovora 4 aagcttcggc
atggcacgtt tgaccgttgg gtcggcaggg tacgtttgaa ttattcataa 60
gaggaatacg ttatgagtct gaatacaagt gggctgggag cgtcaacgat gcaaatttct
120 atcggcggtg cgggcggaaa taacgggttg ctgggtacca gtcgccagaa
tgctgggttg 180 ggtggcaatt ctgcactggg gctgggcggc ggtaatcaaa
atgataccgt caatcagctg 240 gctggcttac tcaccggcat gatgatgatg
atgagcatga tgggcggtgg tgggctgatg 300 ggcggtggct taggcggtgg
cttaggtaat ggcttgggtg gctcaggtgg cctgggcgaa 360 ggactgtcga
acgcgctgaa cgatatgtta ggcggttcgc tgaacacgct gggctcgaaa 420
ggcggcaaca ataccacttc aacaacaaat tccccgctgg accaggcgct gggtattaac
480 tcaacgtccc aaaacgacga ttccacctcc ggcacagatt ccacctcaga
ctccagcgac 540 ccgatgcagc agctgctgaa gatgttcagc gagataatgc
aaagcctgtt tggtgatggg 600 caagatggca cccagggcag ttcctctggg
ggcaagcagc cgaccgaagg cgagcagaac 660 gcctataaaa aaggagtcac
tgatgcgctg tcgggcctga tgggtaatgg tctgagccag 720 ctccttggca
acgggggact gggaggtggt cagggcggta atgctggcac gggtcttgac 780
ggttcgtcgc tgggcggcaa agggctgcaa aacctgagcg ggccggtgga ctaccagcag
840 ttaggtaacg ccgtgggtac cggtatcggt atgaaagcgg gcattcaggc
gctgaatgat 900 atcggtacgc acaggcacag ttcaacccgt tctttcgtca
ataaaggcga tcgggcgatg 960 gcgaaggaaa tcggtcagtt catggaccag
tatcctgagg tgtttggcaa gccgcagtac 1020 cagaaaggcc cgggtcagga
ggtgaaaacc gatgacaaat catgggcaaa agcactgagc 1080 aagccagatg
acgacggaat gacaccagcc agtatggagc agttcaacaa agccaagggc 1140
atgatcaaaa ggcccatggc gggtgatacc ggcaacggca acctgcaggc acgcggtgcc
1200 ggtggttctt cgctgggtat tgatgccatg atggccggtg atgccattaa
caatatggca 1260 cttggcaagc tgggcgcggc ttaagctt 1288 5 341 PRT
Pseudomonas syringae 5 Met Gln Ser Leu Ser Leu Asn Ser Ser Ser Leu
Gln Thr Pro Ala Met 1 5 10 15 Ala Leu Val Leu Val Arg Pro Glu Ala
Glu Thr Thr Gly Ser Thr Ser 20 25 30 Ser Lys Ala Leu Gln Glu Val
Val Val Lys Leu Ala Glu Glu Leu Met 35 40 45 Arg Asn Gly Gln Leu
Asp Asp Ser Ser Pro Leu Gly Lys Leu Leu Ala 50 55 60 Lys Ser Met
Ala Ala Asp Gly Lys Ala Gly Gly Gly Ile Glu Asp Val 65 70 75 80 Ile
Ala Ala Leu Asp Lys Leu Ile His Glu Lys Leu Gly Asp Asn Phe 85 90
95 Gly Ala Ser Ala Asp Ser Ala Ser Gly Thr Gly Gln Gln Asp Leu Met
100 105 110 Thr Gln Val Leu Asn Gly Leu Ala Lys Ser Met Leu Asp Asp
Leu Leu 115 120 125 Thr Lys Gln Asp Gly Gly Thr Ser Phe Ser Glu Asp
Asp Met Pro Met 130 135 140 Leu Asn Lys Ile Ala Gln Phe Met Asp Asp
Asn Pro Ala Gln Phe Pro 145 150 155 160 Lys Pro Asp Ser Gly Ser Trp
Val Asn Glu Leu Lys Glu Asp Asn Phe 165 170 175 Leu Asp Gly Asp Glu
Thr Ala Ala Phe Arg Ser Ala Leu Asp Ile Ile 180 185 190 Gly Gln Gln
Leu Gly Asn Gln Gln Ser Asp Ala Gly Ser Leu Ala Gly 195 200 205 Thr
Gly Gly Gly Leu Gly Thr Pro Ser Ser Phe Ser Asn Asn Ser Ser 210 215
220 Val Met Gly Asp Pro Leu Ile Asp Ala Asn Thr Gly Pro Gly Asp Ser
225 230 235 240 Gly Asn Thr Arg Gly Glu Ala Gly Gln Leu Ile Gly Glu
Leu Ile Asp 245 250 255 Arg Gly Leu Gln Ser Val Leu Ala Gly Gly Gly
Leu Gly Thr Pro Val 260 265 270 Asn Thr Pro Gln Thr Gly Thr Ser Ala
Asn Gly Gly Gln Ser Ala Gln 275 280 285 Asp Leu Asp Gln Leu Leu Gly
Gly Leu Leu Leu Lys Gly Leu Glu Ala 290 295 300 Thr Leu Lys Asp Ala
Gly Gln Thr Gly Thr Asp Val Gln Ser Ser Ala 305 310 315 320 Ala Gln
Ile Ala Thr Leu Leu Val Ser Thr Leu Leu Gln Gly Thr Arg 325 330 335
Asn Gln Ala Ala Ala 340 6 1026 DNA Pseudomonas syringae 6
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 7 344
PRT Pseudomonas solanacearum 7 Met Ser Val Gly Asn Ile Gln Ser Pro
Ser Asn Leu Pro Gly Leu Gln 1 5 10 15 Asn Leu Asn Leu Asn Thr Asn
Thr Asn Ser Gln Gln Ser Gly Gln Ser 20 25 30 Val Gln Asp Leu Ile
Lys Gln Val Glu Lys Asp Ile Leu Asn Ile Ile 35 40 45 Ala Ala Leu
Val Gln Lys Ala Ala Gln Ser Ala Gly Gly Asn Thr Gly 50 55 60 Asn
Thr Gly Asn Ala Pro Ala Lys Asp Gly Asn Ala Asn Ala Gly Ala 65 70
75 80 Asn Asp Pro Ser Lys Asn Asp Pro Ser Lys Ser Gln Ala Pro Gln
Ser 85 90 95 Ala Asn Lys Thr Gly Asn Val Asp Asp Ala Asn Asn Gln
Asp Pro Met 100 105 110 Gln Ala Leu Met Gln Leu Leu Glu Asp Leu Val
Lys Leu Leu Lys Ala 115 120 125 Ala Leu His Met Gln Gln Pro Gly Gly
Asn Asp Lys Gly Asn Gly Val 130 135 140 Gly Gly Ala Asn Gly Ala Lys
Gly Ala Gly Gly Gln Gly Gly Leu Ala 145 150 155 160 Glu Ala Leu Gln
Glu Ile Glu Gln Ile Leu Ala Gln Leu Gly Gly Gly 165 170 175 Gly Ala
Gly Ala Gly Gly Ala Gly Gly Gly Val Gly Gly Ala Gly Gly 180 185 190
Ala Asp Gly Gly Ser Gly Ala Gly Gly Ala Gly Gly Ala Asn Gly Ala 195
200 205 Asp Gly Gly Asn Gly Val Asn Gly Asn Gln Ala Asn Gly Pro Gln
Asn 210 215 220 Ala Gly Asp Val Asn Gly Ala Asn Gly Ala Asp Asp Gly
Ser Glu Asp 225 230 235 240 Gln Gly Gly Leu Thr Gly Val Leu Gln Lys
Leu Met Lys Ile Leu Asn 245 250 255 Ala Leu Val Gln Met Met Gln Gln
Gly Gly Leu Gly Gly Gly Asn Gln 260 265 270 Ala Gln Gly Gly Ser Lys
Gly Ala Gly Asn Ala Ser Pro Ala Ser Gly 275 280 285 Ala Asn Pro Gly
Ala Asn Gln Pro Gly Ser Ala Asp Asp Gln Ser Ser 290 295 300 Gly Gln
Asn Asn Leu Gln Ser Gln Ile Met Asp Val Val Lys Glu Val 305 310 315
320 Val Gln Ile Leu Gln Gln Met Leu Ala Ala Gln Asn Gly Gly Ser Gln
325 330 335 Gln Ser Thr Ser Thr Gln Pro Met 340 8 1035 DNA
Pseudomonas solanacearum 8 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 9 26 PRT Xanthomonas
campestris pv. glycines 9 Thr Leu Ile Glu Leu Met Ile Val Val Ala
Ile Ile Ala Ile Leu Ala 1 5 10 15 Ala Ile Ala Leu Pro Ala Tyr Gln
Asp Tyr 20 25 10 20 PRT Xanthomonas campestris pv. pelargonii 10
Ser Ser Gln Gln Ser Pro Ser Ala Gly Ser Glu Gln Gln Leu Asp Gln 1 5
10 15 Leu Leu Ala Met 20
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