U.S. patent application number 10/441736 was filed with the patent office on 2004-01-22 for hypersensitive response elicitor-induced stress resistance.
Invention is credited to Schading, Richard L., Wei, Zhong-Min.
Application Number | 20040016029 10/441736 |
Document ID | / |
Family ID | 22315630 |
Filed Date | 2004-01-22 |
United States Patent
Application |
20040016029 |
Kind Code |
A1 |
Wei, Zhong-Min ; et
al. |
January 22, 2004 |
Hypersensitive response elicitor-induced stress resistance
Abstract
The present invention is directed to imparting stress resistance
to plants. This can be achieved by applying a hypersensitive
response elicitor in a non-infectious form to plants or plant seeds
under conditions effective to impart stress resistance to plants or
plants grown from the plant seeds. Alternatively, transgenic plants
or plant seeds transformed with a DNA molecule encoding the
elicitor can be provided and the transgenic plants or plants
resulting from the transgenic plant seeds are grown under
conditions effective to impart stress resistance to plants or
plants grown from the plant seeds.
Inventors: |
Wei, Zhong-Min; (Kirkland,
WA) ; Schading, Richard L.; (West Melbourne,
FL) |
Correspondence
Address: |
Nixon Peabody LLP
Clinton Square
P.O. Box 31051
Rochester
NY
14603-1051
US
|
Family ID: |
22315630 |
Appl. No.: |
10/441736 |
Filed: |
May 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10441736 |
May 20, 2003 |
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09431614 |
Nov 2, 1999 |
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6624139 |
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60107243 |
Nov 5, 1998 |
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Current U.S.
Class: |
800/289 |
Current CPC
Class: |
Y10S 435/91 20130101;
A01N 37/46 20130101; C12N 15/8273 20130101; C12N 15/8274 20130101;
Y10S 435/847 20130101; C07K 14/21 20130101; C12N 15/8271 20130101;
C07K 14/27 20130101; A01N 63/50 20200101; Y10S 435/874 20130101;
A01N 63/50 20200101; A01N 63/20 20200101; A01N 63/50 20200101; A01N
63/27 20200101; A01N 63/50 20200101; A01N 63/30 20200101 |
Class at
Publication: |
800/289 |
International
Class: |
A01H 001/00; C12N
015/82 |
Claims
What is claimed:
1. A method of imparting stress resistance to plants comprising:
providing a transgenic plant or plant seed transformed with a DNA
molecule which encodes for a hypersensitive response elicitor
protein or polypeptide and growing the transgenic plant or plants
produced from the transgenic plant seeds under conditions effective
to impart stress resistance.
2. A method according to claim 1, wherein a transgenic plant is
provided.
3. A method according to claim 1, wherein a transgenic plant seed
is provided, said method further comprising: planting the
transgenic seeds in natural or artificial soil and propagating
plants from seeds planted in soil.
4. A method according to claim 1, wherein the stress resistance is
resistance to a stress selected from the group consisting of
climated related stress, air pollution stress, chemical stress, and
nutritional stress.
5. A method according to claim 4, wherein the stress is chemical
stress where the chemical is selected from the group consisting of
insecticides, fungicides, herbicides, and heavy metals.
6. A method according to claim 4, wherein the stress is
climate-related stress selected from the group consisting of
drought, water, frost, cold temperature, high temperature,
excessive light, and insufficient light.
7. A method according to claim 4, wherein the stress is air
pollution stress selected from the group consisting of carbon
dioxide, carbon monoxide, sulfur dioxide, NO.sub.x, hydrocarbons,
ozone, ultraviolet radiation, and acidic rain.
8. A method according to claim 4, wherein the stress is nutritional
stress where the nutritional stress is caused by fertilizer,
micronutrients, or macronutrients.
9. A method according to claim 4, wherein the hypersensitive
response elicitor protein or polypeptide is derived from Erwinia,
Pseudomonas, Xanthamonas, Phythophthera, or Clavibacter.
10. A method according to claim 9, wherein the hypersensitive
response elicitor protein or polypeptide is derived from Erwinia
amylovora, Erwinia carotovora, Erwinia chrysanthemi, and Erwinia
stewartii.
11. A method according to claim 9, wherein the hypersensitive
response elicitor protein or polypeptide is derived from
Pseudomonas syringae or Pseudomonas solancearum.
12. A method according to claim 9, wherein the hypersensitive
response elicitor protein or polypeptide is derived from a
Xanthamonas species.
13. A method according to claim 4, wherein the plant is selected
from the group consisting of alfalfa, rice, wheat, barley, rye,
cotton, sunflower, peanut, corn, potato, sweet potato, bean pea,
chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip,
cauliflower, broccoli, turnip, radish, spinach, onion, garlic,
eggplant, pepper, celery, carrot, squash, pumpkin, zucchini,
cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry,
pineapple, soybean, tobacco, tomato, sorghum, and sugarcane.
14. A method according to claim 4, wherein the plant is selected
from the group consisting of Arabidopsis thaliana, Saintpaulia,
petunia, pelargonium, poinsettia, chrysanthemum, carnation, and
zinnia.
Description
[0001] This application claims benefit of U.S. Provisional Patent
Application Serial No. 60/107,243, filed Nov. 5, 1999.
FIELD OF THE INVENTION
[0002] The present invention relates to imparting stress resistance
to plants with a hypersensitive response elicitor.
BACKGROUND OF THE INVENTION
[0003] Under both natural and agricultural conditions, plants are
exposed to various forms of environmental stress. Stress is mainly
measured with respect to growth (i.e. biomass accumulation) or with
respect to the primary assimilation processes (i.e. carbon dioxide
and mineral intake). Soil water deficits, suboptimal and
supraoptimal temperatures, salinity, and poor aeration of soils may
each cause some growth restrictions during the growing season, so
that the yield of plants at the end of the season expresses only a
small fraction of their genetic potential. Indeed, it is estimated
that in the United States the yield of field-grown crops is only
22% of genetic potential. The same physicochemical factors can
become extreme in some habitats, such as deserts or marshes, and
only specially adapted vegetation can complete its life cycle in
the unusually hostile conditions. In less extreme environments,
individual plants can become acclimated to changes in water
potential, temperature, salinity, and oxygen deficiency so that
their fitness for those environments improves. Some species are
better able to adapt than others, and various anatomical,
structural, and biochemical mechanisms account for acclimation.
[0004] Under natural and agriculture conditions, plants must
constantly endure stress. Some environmental factors can become
stressful in a very short period of time (e.g., high or low
temperature) or may take long periods of time to stress plants
(e.g., soil water content or mineral nutrients). Generally,
environmental stress effecting plants can be in the form of climate
related stress, air pollution stress, chemical stress, and
nutritional stress. Examples of climate related stress include
drought, water, frost, cold temperature, high temperature,
excessive light, and insufficient light. Air pollution stress can
be in the form of carbon dioxide, carbon monoxide, sulfur dioxide,
NO.sub.x, hydrocarbons, ozone, ultraviolet radiation, and acidic
rain. Chemical stress can result from application of insecticides,
fungicides, herbicides, and heavy metals. Nutritional stress can be
caused by fertilizers, micronutrients, and macronutrients.
[0005] For most plants, water is essential for growth. Some plants
are able to preserve some water in the soil for later use, while
others complete their life cycles during a wet season before the
onset of any drought. Other plants are able to aggressively consume
water to save themselves while causing water deprivation for other
plants in that location. Plants lacking any of these capabilities
are severely hampered by the absence of water.
[0006] Chilling injury occurs in sensitive species at temperatures
that are too low for normal growth but not sufficiently low to form
ice. Such injury typically occurs in species of tropical or
subtropical origin. When chilling occurs, discoloration or lesions
appear on leaves giving them a water-soaked appearance. If roots
are chilled, the plants may wilt. On the other hand, freezing
temperatures and the accompanying formation of ice crystals in
plants can be lethal if ice crystals extend into protoplasts or
remain for long periods.
[0007] Stress is also caused by the other temperature extremes with
few plants being able to survive high temperatures. When higher
plant cells or tissues are dehydrated or are not growing, they can
survive higher temperatures than cells which are hydrated,
vegetative, and growing. Tissues which are actively growing can
rarely survive at temperatures above 45.degree. C.
[0008] High salt concentrations are another form of environmental
stress which can afflict plants. In natural conditions, such high
concentrations of salt are found close to seashores and estuaries.
Farther inland, natural salt may seep from geological deposits
adjoining agricultural areas. In addition, salt can accumulate in
irrigation water when pure water is evaporated or transpired from
soil. About 1/3 of all irrigated farmland is effected by high salt
concentrations. High salt content not only injures plants but
degrades soil structure by decreasing porosity and water
permeability.
[0009] Air pollution in the form of ozone, carbon dioxide, carbon
monoxide, sulfur dioxide, NO.sub.x, and hydrocarbons can very
adversely effect plant growth by creating smog and environmental
warming.
[0010] The present invention is directed to overcoming various
forms of environmental stress and imparting resistance in plants to
such stress.
SUMMARY OF THE INVENTION
[0011] The present invention relates to the use of a hypersensitive
response elicitor protein or polypeptide to impart stress
resistance to plants. In one embodiment of the present invention,
the hypersensitive response elicitor protein or polypeptide is
applied to plants or plant seeds under conditions effective to
impart stress resistance. Alternatively, stress resistance is
imparted by providing a transgenic plant or plant seed transformed
with a DNA molecule which encodes for a hypersensitive response
elicitor protein or polypeptide and growing the transgenic plant or
plants produced from the transgenic plant seeds under conditions
effective to impart stress resistance.
[0012] Stress encompasses any environmental factor having an
adverse effect on plant physiology and development. Examples of
such environmental stress include climate-related stress (e.g.,
drought, water, frost, cold temperature, high temperature,
excessive light, and insufficient light), air pollution stress
(e.g., carbon dioxide, carbon monoxide, sulfur dioxide, NO.sub.x,
hydrocarbons, ozone, ultraviolet radiation, acidic rain), chemical
(e.g., insecticides, fungicides, herbicides, heavy metals), and
nutritional stress (e.g., fertilizer, micronutrients,
macronutrients). Applicants have found that use of hypersensitive
response elicitors in accordance with the present invention impart
resistance to plants against such forms of environmental
stress.
DETAILED DESCRIPTION OF THE INVENTION
[0013] The present invention relates to the use of a hypersensitive
response elicitor protein or polypeptide to impart stress
resistance to plants. In one embodiment of the present invention,
the hypersensitive response elicitor protein or polypeptide is
applied to plants or plant seeds under conditions effective to
impart stress resistance. Alternatively, the stress resistance is
imparted by providing a transgenic plant or plant seed transformed
with a DNA molecule which encodes for a hypersensitive response
elicitor protein or polypeptide and growing the transgenic plant or
plants produced from the transgenic plant seeds under conditions
effective to impart stress resistance.
[0014] The hypersensitive response elicitor polypeptides or
proteins according to the present invention are derived from
hypersensitive response elicitor polypeptides or proteins of 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. Examples of suitable bacterial sources
of polypeptide or protein elicitors include Erwinia, Pseudomonas,
and Xanthamonas species (e.g., the following bacteria: Erwinia
amylovora, Erwinia chrysanthemi, Erwinia stewartii, Erwinia
carotovora, Pseudomonas syringae, Pseudomonas solancearum,
Xanthomonas campestris, and mixtures thereof). In addition to
hypersensitive response elicitors from these Gram negative
bacteria, it is possible to use elicitors from Gram positive
bacteria. One example is Clavibacter michiganensis subsp.
sepedonicus.
[0015] An example of a fungal source of a hypersensitive response
elicitor protein or polypeptide is Phytophthora. Suitable species
of Phytophthora include Phytophthora parasitica, Phytophthora
cryptogea, Phytophthora cinnamomi, Phytophthora capsici,
Phytophthora megasperma, and Phytophthora citrophthora.
[0016] 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
[0017] 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 GCGTTTATGC CCGCGATGAA CCGGCATCAG GCGGCGCGCT GGTCGCCGCA
ATCCGGCGTC 120 GATCTGGTAT TTCAGTTTGG GGACACCGGG CGTGAACTCA
TGATOCAGAT 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 GATAAAGGCC 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 CCGGCGGCCA 900 TGCGTTGTCA AAAATGTTTG ATAAAGCGCT
CGACGATCTG 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 ACTCGCCGCC
1860 CAGATGGAGA CACGTCTGCG ATAAATCTGT GCCGTAACGT GTTTCTATCC
GCCCCTTTAG 1920 CAGATAGATT GCGGTTTCGT AATCAACATG CTAATGCGGT
TCCGCCTGTG CGCCGGCCGG 1980 GATCACCACA ATATTCATAG AAAGCTGTCT
TGCACCTACC GTATCGCGGG AGATACCGAC 2040 AAAATAGGGC AGTTTTTGCG
TCGTATCCGT GGGGTGTTCC GGCCTGACAA TCTTGAGTTG 2100 GTTCGTCATC
ATCTTTCTCC ATCTGGGCGA CCTGATCGGT T 2141
[0018] 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
[0019] 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 ATGCCACGTT TGACCGTTGG GTCGGCAGGG TACGTTTGAA TTATTCATAA
60 CAGGAATACG TTATGAGTCT GAATACAAGT GGGCTGGGAG CGTCAACGAT
GCAAATTTCT 120 ATCGGCGGTG CGGGCGGAAA TAACGGGTTG CTGGGTACCA
GTCGCCAGAA TGCTGGGTTG 180 GGTGCCAATT CTGCACTGGG GCTGGGCGGC
GGTAATCAAA ATGATACCGT CAATCAGCTG 240 GCTGGCTTAC TCACCGGCAT
GATGATGATG ATGAGCATGA TGGGCGGTGG TGGGCTGATG 300 GGCGGTGGCT
TAGGCGGTGG CTTAGGTAAT CGCTTGGGTG GCTCAGGTGG CCTGGGCGAA 360
GGACTGTCGA ACGCGCTGAA CGATATGTTA GGCGGTTCGC TGAACACGCT GGGCTCGAAA
420 GGCGCCAACA ATACCACTTC AACAACAAAT TCCCCGCTGG ACCAGGCGCT
GGGTATTAAC 480 TCAACGTCCC AAAACGACGA TTCCACCTCC GGCACAGATT
CCACCTCAGA CTCCAGCGAC 540 CCGATGCAGC AGCTGCTGAA GATGTTCAGC
GAGATAATGC AAAGCCTGTT TGCTGATGGG 600 CAAGATGGCA CCCAGGGCAG
TTCCTCTGGG GGCAAGCAGC CGACCGAAGG CGAGCAGAAC 660 GCCTATAAAA
AAGGAGTCAC TGATGCGCTG TCGGGCCTGA TGGGTAATGG TCTGAGCCAG 720
CTCCTTGGCA ACGGGGGACT GGGAGCTGGT CAGGGCGGTA ATGCTGGCAC GGGTCTTGAC
780 GGTTCGTCGC TGGGCGGCAA AGGGCTGCAA AACCTGAGCG GGCCGGTGGA
CTACCAGCAG 840 TTAGGTAACG CCGTGGGTAC CGGTATCGGT ATGAAAGCGG
GCATTCAGGC GCTGAATGAT 900 ATCGGTACGC ACAGCCACAG 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 CGCCCATGGC GGCTGATACC GGCAACGGCA ACCTGCAGGC
ACGCGGTGCC 1200 GGTGGTTCTT CGCTGGGTAT TGATGCCATG ATGGCCGGTG
ATGCCATTAA CAATATGGCA 1260 CTTGGCAAGC TGGGCGCGGC TTAAGCTT 1288
[0020] Another potentially suitable hypersensitive response
elicitor from Erwinia amylovora is disclosed in U.S. patent
application Ser. No. 09/120,927, which is hereby incorporated by
reference. The protein is encoded by a DNA molecule having a
nucleic acid sequence of SEQ ID NO: 5 as follows:
5 ATGTCAATTC TTACGCTTAA CAACAATACC TCGTCCTCGC CGGGTCTGTT CCAGTCCGGG
60 GGGGACAACG GGCTTGGTGG TCATAATGCA AATTCTGCGT TGGGGCAACA
ACCCATCGAT 120 CGGCAAACCA TTGAGCAAAT GGCTCAATTA TTGGCGGAAC
TGTTAAAGTC ACTGCTATCG 180 CCACAATCAG GTAATGCGGC AACCGGAGCC
GGTGGCAATG ACCAGACTAC AGGAGTTGGT 240 AACGCTGGCG GCCTGAACGG
ACGAAAAGGC ACAGCAGGAA CCACTCCGCA GTCTGACAGT 300 CAGAACATGC
TGAGTGAGAT GGGCAACAAC GGGCTGGATC AGGCCATCAC GCCCGATGGC 360
CAGGGCGGCG GGCAGATCGG CGATAATCCT TTACTGAAAG CCATGCTGAA GCTTATTGCA
420 CGCATGATGG ACGGCCAAAG CGATCAGTTT GGCCAACCTG GTACGGGCAA
CAACAGTGCC 480 TCTTCCGGTA CTTCTTCATC TGGCGGTTCC CCTTTTAACG
ATCTATCAGG GGGGAAGGCC 540 CCTTCCGGCA ACTCCCCTTC CGGCAACTAC
TCTCCCGTCA GTACCTTCTC ACCCCCATCC 600 ACGCCAACGT CCCCTACCTC
ACCGCTTGAT TTCCCTTCTT CTCCCACCAA AGCAGCCGGG 660 GGCAGCACGC
CGGTAACCGA TCATCCTGAC CCTGTTGGTA GCGCCGGCAT CGGGGCCGGA 720
AATTCGGTGG CCTTCACCAG CGCCGGCGCT AATCAGACCG TGCTGCATGA CACCATTACC
780 GTGAAAGCGG GTCAGGTGTT TGATGGCAAA GGACAAACCT TCACCGCCGG
TTCAGAATTA 840 GGCCATGGCG GCCAGTCTGA AAACCAGAAA CCGCTGTTTA
TACTGGAAGA CGGTGCCAGC 900 CTCAAAAACG TCACCATGGG CGACGACGGG
GCGGATGGTA TTCATCTTTA CGGTGATGCC 960 AAAATAGACA ATCTGCACGT
CACCAACGTG GGTGAGGACG CGATTACCGT TAAGCCAAAC 1020 AGCGCGGGCA
AAAAATCCCA CGTTGAAATC ACTAACAGTT CCTTCGAGCA CGCCTCTGAC 1080
AAGATCCTGC AGCTGAATGC CGATACTAAC CTGAGCGTTG ACAACGTGAA GGCCAAAGAC
1140 TTTGGTACTT TTGTACGCAC TAACGGCGGT CAACAGGGTA ACTGGGATCT
GAATCTGAGC 1200 CATATCAGCG CAGAAGACGG TAAGTTCTCG TTCGTTAAAA
GCGATAGCGA GGGGCTAAAC 1260 GTCAATACCA GTGATATCTC ACTGGGTGAT
GTTGAAAACC ACTACAAAGT GCCGATGTCC 1320 GCCAACCTGA AGGTGGCTGA ATGA
1344
[0021] See GenBank Accession No. U94513. The isolated DNA molecule
of the present invention encodes a hypersensitive response elicitor
protein or polypeptide having an amino acid sequence of SEQ ID NO:
6 as follows:
6 Met Ser Ile Leu Thr Leu Asn Asn Asn Thr Ser Ser Ser Pro Gly Leu 1
5 10 15 Phe Gln Ser Gly Gly Asp Asn Gly Leu Gly Gly His Asn Ala Asn
Ser 20 25 30 Ala Leu Gly Gln Gln Pro Ile Asp Arg Gln Thr Ile Glu
Gln Met Ala 35 40 45 Gln Leu Leu Ala Glu Leu Leu Lys Ser Leu Leu
Ser Pro Gln Ser Gly 50 55 60 Asn Ala Ala Thr Gly Ala Gly Gly Asn
Asp Gln Thr Thr Gly Val Gly 65 70 75 80 Asn Ala Gly Gly Leu Asn Gly
Arg Lys Gly Thr Ala Gly Thr Thr Pro 85 90 95 Gln Ser Asp Ser Gln
Asn Met Leu Ser Glu Met Gly Asn Asn Gly Leu 100 105 110 Asp Gln Ala
Ile Thr Pro Asp Gly Gln Gly Gly Gly Gln Ile Gly Asp 115 120 125 Asn
Pro Leu Leu Lys Ala Met Leu Lys Leu Ile Ala Arg Met Met Asp 130 135
140 Gly Gln Ser Asp Gln Phe Gly Gln Pro Gly Thr Gly Asn Asn Ser Ala
145 150 155 160 Ser Ser Gly Thr Ser Ser Ser Gly Gly Ser Pro Phe Asn
Asp Leu Ser 165 170 175 Gly Gly Lys Ala Pro Ser Gly Asn Ser Pro Ser
Gly Asn Tyr Ser Pro 180 185 190 Val Ser Thr Phe Ser Pro Pro Ser Thr
Pro Thr Ser Pro Thr Ser Pro 195 200 205 Leu Asp Phe Pro Ser Ser Pro
Thr Lys Ala Ala Gly Gly Ser Thr Pro 210 215 220 Val Thr Asp His Pro
Asp Pro Val Gly Ser Ala Gly Ile Gly Ala Gly 225 230 235 240 Asn Ser
Val Ala Phe Thr Ser Ala Gly Ala Asn Gln Thr Val Leu His 245 250 255
Asp Thr Ile Thr Val Lys Ala Gly Gln Val Phe Asp Gly Lys Gly Gln 260
265 270 Thr Phe Thr Ala Gly Ser Glu Leu Gly Asp Gly Gly Gln Ser Glu
Asn 275 280 285 Gln Lys Pro Leu Phe Ile Leu Glu Asp Gly Ala Ser Leu
Lys Asn Val 290 295 300 Thr Met Gly Asp Asp Gly Ala Asp Gly Ile His
Leu Tyr Gly Asp Ala 305 310 315 320 Lys Ile Asp Asn Leu His Val Thr
Asn Val Gly Glu Asp Ala Ile Thr 325 330 335 Val Lys Pro Asn Ser Ala
Gly Lys Lys Ser His Val Glu Ile Thr Asn 340 345 350 Ser Ser Phe Glu
His Ala Ser Asp Lys Ile Leu Gln Leu Asn Ala Asp 355 360 365 Thr Asn
Leu Ser Val Asp Asn Val Lys Ala Lys Asp Phe Gly Thr Phe 370 375 380
Val Arg Thr Asn Gly Gly Gln Gln Gly Asn Trp Asp Leu Asn Leu Ser 385
390 395 400 His Ile Ser Ala Glu Asp Gly Lys Phe Ser Phe Val Lys Ser
Asp Ser 405 410 415 Glu Gly Leu Asn Val Asn Thr Ser Asp Ile Ser Leu
Gly Asp Val Glu 420 425 430 Asn His Tyr Lys Val Pro Met Ser Ala Asn
Leu Lys Val Ala Glu 435 440 445
[0022] This protein or polypeptide is acidic, rich in glycine and
serine, and lacks cysteine. It is also heat stable, protease
sensitive, and suppressed by inhibitors of plant metabolism. The
protein or polypeptide of the present invention has a predicted
molecular size of ca. 4.5 kDa.
[0023] Another potentially suitable hypersensitive response
elicitor from Erwinia amylovora is disclosed in U.S. patent
application Ser. No. 09/120,663, which is hereby incorporated by
reference. The protein is encoded by a DNA molecule having a
nucleic acid sequence of SEQ ID NO: 7 as follows:
7 ATGGAATTAA AATCACTGGG AACTGAACAC AAGGCGGCAG TACACACAGC GGCGCACAAC
60 CCTGTGGGGC ATGGTGTTGC CTTACAGCAG GGCAGCAGCA GCAGCAGCCC
GCAAAATGCC 120 GCTGCATCAT TGGCGGCAGA AGCCAAAAAT CGTGGGAAAA
TGCCGAGAAT TCACCAGCCA 180 TCTACTGCGG CTGATGGTAT CAGCGCTGCT
CACCAGCAAA AGAAATCCTT CAGTCTCAGG 240 GGCTGTTTCG GGACGAAAAA
ATTTTCCAGA TCGGCACCGC AGGGCCAGCC AGGTACCACC 300 CACAGCAAAG
GGGCAACATT GCGCGATCTG CTGCGCGGG ACGACGGCGA AACGCAGCAT 360
GAGGCGGCCG CGCCAGATGC GGCGCGTTTG ACCCGTTCGG GCGGCGTCAA ACGCCGCAAT
420 ATGGACGACA TGGCCGGGCC GCCAATGGTG AAAGGTGGCA GCGGCGAAGA
TAAGGTACCA 480 ACGCAGCAAA AACGGCATCA GCTGAACAAT TTTGGCCAGA
TGCGCCAAAC GATGTTGAGC 540 AAAATGGCTC ACCCGGCTTC AGCCAACGCC
GGCGATCGCC TGCAGCATTC ACCGCCGCAC 600 ATCCCGGGTA GCCACCACGA
AATCAAGGAA GAACCGGTTG GCTCCACCAG CAAGGCAACA 660 ACGGCCCACG
CAGACAGAGT GGAAATCGCT CAGGAAGATG ACGACAGCGA ATTCCAGCAA 720
CTGCATCAAC AGCGGCTGGC GCGCGAACGG GAAAATCCAC CGCAGCCGCC CAAACTCGGC
780 GTTGCCACAC CGATTAGCGC CAGGTTTCAG CCCAAACTGA CTGCGGTTGC
GGAAACCGTC 840 CTTGAGGGGA CAGATACCAC GCAGTCACCC CTTAAGCCGC
AATCAATGCT GAAAGGAAGT 900 GGAGCCGGGG TAACGCCGCT GGCGGTAACG
CTGGATAAAG GCAAGTTGCA GCTGGCACCG 960 GATAATCCAC CCGCGCTCAA
TACGTTGTTG AAGCAGACAT TGGGTAAAGA CACCCAGCAC 1020 TATCTGGCGC
ACCATGCCAG CAGCCACGGT AGCCAGCATC TGCTGCTGGA CAACAAAGGC 1080
CACCTGTTTG ATATCAAAAG CACCGCCACC AGCTATAGCG TGCTGCACAA CAGCCACCCC
1140 GGTGAGATAA AGGGCAAGCT GGCGCAGGCG GGTACTGGCT CCGTCAGCGT
AGACGGTAAA 1200 AGCGGCAAGA TCTCGCTGGG GAGCGGTACG CAAAGTCACA
ACAAAACAAT GCTAAGCCAA 1260 CCGGGGGAAG CGCACCGTTC CTTATTAACC
GGCATTTGGC AGCATCCTGC TGGCGCAGCG 1320 CGGCCGCAGG GCGAGTCAAT
CCGCCTGCAT GACGACAAAA TTCATATCCT GCATCCGGAG 1380 CTGGGCGTAT
GGCAATCTGC GGATAAAGAT ACCCACAGCC AGCTGTCTCG CCAGGCAGAC 1440
GGTAAGCTCT ATGCGCTGAA AGACAACCGT ACCCTGCAAA ACCTCTCCGA TAATAAATCC
1500 TCAGAAAAGC TGGTCGATAA AATCAAATCG TATTCCGTTG ATCAGCGGGG
GCAGGTGGCG 1560 ATCCTGACGG ATACTCCCGG CCGCCATAAG ATGAGTATTA
TGCCCTCGCT GGATGCTTCC 1620 CCGGAGAGCC ATATTTCCCT CAGCCTGCAT
TTTGCCGATG CCCACCAGGG GTTATTGCAC 1680 GGGAAGTCGG AGCTTGAGGC
ACAATCTGTC GCGATCAGCC ATGGGCGACT GGTTGTGGCC 1740 GATAGCGAAG
GCAAGCTGTT TAGCGCCGCC ATTCCGAAGC AACGGGATGG AAACGAACTG 1800
AAAATGAAAG CCATGCCTCA GCATGCGCTC GATGAACATT TTGGTCATGA CCACCAGATT
1860 TCTGGATTTT TCCATGACGA CCACGGCCAG CTTAATGCGC TGGTGAAAAA
TAACTTCAGG 1920 CAGCAGCATG CCTGCCCGTT GGGTAACGAT CATCAGTTTC
ACCCCGGCTG GAACCTGACT 1980 GATGCGCTGG TTATCGACAA TCAGCTGGGG
CTGCATCATA CCAATCCTGA ACCGCATGAG 2040 ATTCTTGATA TGGGCATTT
AGGCAGCCTG GCGTTACAGG AGGGCAAGCT TCACTATTTT 2100 GACCAGCTGA
CCAAAGGGTG GACTGGCGCG GAGTCAGATT GTAAGCAGCT GAAAAAAGGC 2160
CTGGATGGAG CAGCTTATCT ACTGAAAGAC GGTGAAGTGA AACGCCTGAA TATTAATCAG
2220 AGCACCTCCT CTATCAAGCA CGGAACGGAA AACGTTTTTT CGCTGCCGCA
TGTGCGCAAT 2280 AAACCGGAGC CGGGAGATGC CCTGCAAGGG CTGAATAAAG
ACGATAAGGC CCAGGCCATC 2340 GCGGTGATTG GGGTAAATAA ATACCTGGCG
CTGACGGAAA AAGGGGACAT TCGCTCCTTC 2400 CAGATAAAAC CCGGCACCCA
GCAGTTGGAG CGGCCGGCAC AAACTCTCAG CCGCGAAGGT 2460 ATCAGCGGCG
AACTGAAAGA CATTCATGTC GACCACAAGC AGAACCTGTA TGCCTTGACC 2520
CACGAGGGAG AGGTGTTTCA TCAGCCGCGT GAAGCCTGGC AGAATGGTGC CGAAAGCAGC
2580 AGCTGGCACA AACTGGCGTT GCCACAGAGT GAAAGTAAGC TAAAAAGTCT
GCACATGAGC 2640 CATGAGCACA AACCGATTGC CACCTTTGAA GACGGTAGCC
AGCATCAGCT GAAGGCTGGC 2700 GGCTGGCACG CCTATGCGGC ACCTGAACGC
GGGCCGCTGG CGGTGGGTAC CAGCGGTTCA 2760 CAAACCGTCT TTAACCGACT
AATGCAGCGG GTGAAAGGCA AGGTGATCCC AGGCACCGGG 2820 TTGACGGTTA
AGCTCTCGGC TCAGACGGGG GGAATGACCG GCGCCGAAGG GCGCAAGGTC 2880
AGCAGTAAAT TTTCCGAAAG GATCCGCGCC TATGCGTTCA ACCCAACAAT GTCCACGCCG
2940 CGACCGATTA AAAATGCTGC TTATGCCACA CAGCACGGCT GGCAGGGGCG
TGAGGGGTTG 3000 AAGCCGTTGT ACGAGATGCA GGGAGCGCTG ATTAAACAAC
TGGATGCGCA TAACGTTCGT 3060 CATAACGCGC CACAGCCAGA TTTGCAGAGC
AAACTGGAAA CTCTGGATTT AGGCGAACAT 3120 GCCGCAGAAT TGCTTAACGA
CATGAAGCGC TTCCGCGACG AACTGGAGCA GAGTGCAACC 3180 CGTTCGGTGA
CCGTTTTAGG TCAACATCAG GGAGTGCTAA AAAGCAACGG TGAAATCAAT 3240
AGCGAATTTA AGCCATCGCC CGGCAAGGCG TTGGTCCAGA GCTTTAACGT CAATCGCTCT
3300 GGTCAGGATC TAAGCAAGTC ACTGCAACAG GCAGTACATG CCACGCCGCC
ATCCGCAGAG 3360 AGTAAACTGC AATCCATGCT GGGGCACTTT GTCAGTGCCG
GGGTGGATAT GAGTCATCAG 3420 AAGGGCGAGA TCCCGCTGGG CCCCCACCGC
GATCCGAATG ATAAAACCGC ACTGACCAAA 3480 TCGCGTTTAA TTTTAGATAC
CGTGACCATC GGTCAACTGC ATGAACTGGC CGATAAGCCG 3540 AAACTGGTAT
CTGACCATAA ACCCGATGCC GATCAGATAA AACAGCTGCG CCAGCAGTTC 3600
GATACGCTGC GTGAAAAGCG GTATGAGAGC AATCCGGTGA AGCATTACAC CGATATGGGC
3660 TTCACCCATA ATAAGGCGCT GGAAGCAAAC TATGATGCGG TCAAAGCCTT
TATCAATGCC 3720 TTTAAGAAAG AGCACCACGG CGTCAATCTG ACCACCCGTA
CCGTACTGCA ATCACAGGGC 3780 AGTGCGGAGC TGGCGAAGAA GCTCAAGAAT
ACGCTGTTGT CCCTGGACAG TGGTGAAAGT 3840 ATGAGCTTCA GCCGGTCATA
TGGCGGGGGC GTCAGCACTG TCTTTGTGCC TACCCTTAGC 3900 AAGAAGGTGC
CAGTTCCGGT GATCCCCGGA GCCCGCATCA CGCTGGATCG CGCCTATAAC 3960
CTGAGCTTCA GTCGTACCAG CGGCGGATTG AACGTCAGTT TTGGCCGCGA CGGCGGGGTG
4020 AGTGGTAACA TCATGGTCGC TACCGGCCAT GATGTGATGC CCTATATGAC
CGGTAAGAAA 4080 ACCAGTGCAG CTAACGCCAG TGACTGGTTG AGCGCAAAAC
ATAAAATCAG CCCGGACTTG 4140 CGTATCGGCG CTGCTGTGAG TGGCACCCTG
CAACGAACGC TACAAAACAG CCTGAAGTTT 4200 AAGCTGACAG AGGATGAGCT
GCCTGGCTTT ATCCATGGCT TGACGCATGG CACGTTGACC 4260 CCGCCAGAAC
TGTTGCAAAA GGGGATCGAA CATCAGATGA AGCAGGGCAG CAAACTGACG 4320
TTTAGCGTCG ATACCTCGGC AAATCTGGAT CTGCGTGCCG GTATCAATCT GAACGAAGAC
4380 GGCAGTAAAC CAAATGGTGT CACTGCCCGT GTTTCTGCCG GGCTAAGTGC
ATCGGCAAAC 4440 CTGGCCGCCG GCTCGCGTGA ACGCAGCACC ACCTCTGGCC
AGTTTGGCAG CACGACTTCG 4500 GCCAGCAATA ACCGCCCAAC CTTCCTCAAC
GGGGTCGGCG CGGGTGCTAA CCTGACGGCT 4560 GCTTTAGGGG TTGCCCATTC
ATCTACGCAT GAAGGGAAAC CGGTCGGGAT CTTCCCGGCA 4620 TTTACCTCGA
CCAATGTTTC GGCAGCGCTG GCGCTGGATA ACCGTACCTC ACAGAGTATC 4680
AGCCTGGAAT TGAAGCGCGC GGACCCGGTG ACCAGCAACG ATATCAGCGA GTTGACCTCC
4740 ACGCTGGGAA AACACTTTAA GGATAGCGCC ACAACGAAGA TGCTTGCCGC
TCTCAAAGAG 4800 TTAGATGACG CTAAGCCCCC TGAACAACTG CATATTTTAC
AGCAGCATTT CAGTGCAAAA 4860 GATGTCGTCG GTGATGAACG CTACGAGGCG
GTGCGCAACC TGAAAAAACT GGTGATACGT 4920 CAACAGGCTG CGGACAGCCA
CAGCATGGAA TTAGGATCTG CCAGTCACAG CACGACCTAC 4980 AATAATCTGT
CGAGAATAAA TAATGACGGC ATTGTCGAGC TGCTACACAA ACATTTCGAT 5040
GCGGCATTAC CAGCAAGCAC TGCCAAACGT CTTGGTCAAA TGATGAATAA CGATCCGGCA
5100 CTGAAAGATA TTATTAACCA GCTGCAAAGT ACGCCGTTCA GCAGCGCCAG
CGTGTCGATG 5160 GAGCTGAAAG ATGGTCTGCG TGAGCAGACG GAAAAAGCAA
TACTGGACGG TAAGGTCGGT 5220 CGTGAAGAAG TGGGAGTACT TTTCCAGGAT
CGTAACAACT TGCGTGTTAA ATCGGTCAGC 5280 GTCAGTCAGT CCGTCAGCAA
AAGCGAAGGC TTCAATACCC CAGCGCTGTT ACTGGGGACG 5340 AGCAACAGCG
CTGCTATGAG CATGGAGCGC AACATCGGAA CCATTAATTT TAAATACGGC 5400
CAGGATCAGA ACACCCCACG GCGATTTACC CTGGAGGGTG GAATAGCTCA GGCTAATCCG
5460 CAGGTCGCAT CTGCGCTTAC TCATTTGAAG AAGGAAGGGC TGGAAATGAA GAGCTAA
5517
[0024] This DNA molecule is known as the dspE gene for Erwinia
amylovora. This isolated DNA molecule of the present invention
encodes a protein or polypeptide which elicits a plant pathogen's
hypersensitive response having an amino acid sequence of SEQ ID NO:
8 as follows:
8 Met Glu Leu Lys Ser Leu Gly Thr Glu His Lys Ala Ala Val His Thr 1
5 10 15 Ala Ala His Asn Pro Val Gly His Gly Val Ala Leu Gln Gln Gly
Ser 20 25 30 Ser Ser Ser Ser Pro Gln Asn Ala Ala Ala Ser Leu Ala
Ala Glu Gly 35 40 45 Lys Asn Arg Gly Lys Met Pro Arg Ile His Gln
Pro Ser Thr Ala Ala 50 55 60 Asp Gly Ile Ser Ala Ala His Gln Gln
Lys Lys Ser Phe Ser Leu Ary 65 70 75 80 Gly Cys Leu Gly Thr Lys Lys
Phe Ser Arg Ser Ala Pro Gln Gly Gln 85 90 95 Pro Gly Thr Thr His
Ser Lys Gly Ala Thr Leu Arg Asp Leu Leu Ala 100 105 110 Arg Asp Asp
Gly Glu Thr Gln His Glu Ala Ala Ala Pro Asp Ala Ala 115 120 125 Arg
Leu Thr Ary Ser Gly Gly Val Lys Arg Arg Asn Met Asp Asp Met 130 135
140 Ala Gly Arg Pro Met Val Lys Gly Gly Ser Gly Glu Asp Lys Val Pro
145 150 155 160 Thr Gln Gln Lys Arg His Gln Leu Asn Asn Phe Gly Gln
Met Arg Gln 165 170 175 Thr Met Leu Ser Lys Met Ala His Pro Ala Ser
Ala Asn Ala Gly Asp 180 185 190 Arg Leu Gln His Ser Pro Pro His Ile
Pro Gly Ser His His Glu Ile 195 200 205 Lys Glu Glu Pro Val Gly Ser
Thr Ser Lys Ala Thr Thr Ala His Ala 210 215 220 Asp Arg Val Glu Ile
Ala Gln Glu Asp Asp Asp Ser Glu Phe Gln Gln 225 230 235 240 Leu His
Gln Gln Arg Leu Ala Arg Glu Arg Glu Asn Pro Pro Gln Pro 245 250 255
Pro Lys Leu Gly Val Ala Thr Pro Ile Ser Ala Arg Phe Gln Pro Lys 260
265 270 Leu Thr Ala Val Ala Glu Ser Val Leu Glu Gly Thr Asp Thr Thr
Gln 275 280 285 Ser Pro Leu Lys Pro Gln Ser Met Leu Lys Gly Ser Gly
Ala Gly Val 290 295 300 Thr Pro Leu Ala Val Thr Leu Asp Lys Gly Lys
Leu Gln Leu Ala Pro 305 310 315 320 Asp Asn Pro Pro Ala Leu Asn Thr
Leu Leu Lys Gln Thr Leu Gly Lys 325 330 335 Asp Thr Gln His Tyr Leu
Ala His His Ala Ser Ser Asp Gly Ser Gln 340 345 350 His Leu Leu Leu
Asp Asn Lys Gly His Leu Phe Asp Ile Lys Ser Thr 355 360 365 Ala Thr
Ser Tyr Ser Val Leu His Asn Ser His Pro Gly Glu Ile Lys 370 375 380
Gly Lys Leu Ala Gln Ala Gly Thr Gly Ser Val Ser Val Asp Gly Lys 385
390 395 400 Ser Gly Lys Ile Ser Leu Gly Ser Gly Thr Gln Ser His Asn
Lys Thr 405 410 415 Met Leu Ser Gln Pro Gly Glu Ala His Arg Ser Leu
Leu Thr Gly Ile 420 425 430 Trp Gln His Pro Ala Gly Ala Ala Arg Pro
Gln Gly Glu Ser Ile Arg 435 440 445 Leu His Asp Asp Lys Ile His Ile
Leu His Pro Glu Leu Gly Val Trp 450 455 460 Gln Ser Ala Asp Lys Asp
Thr His Ser Gln Leu Ser Arg Gln Ala Asp 465 470 475 480 Gly Lys Leu
Tyr Ala Leu Lys Asp Asn Arg Thr Leu Gln Asn Leu Ser 485 490 495 Asp
Asn Lys Ser Ser Glu Lys Leu Val Asp Lys Ile Lys Ser Tyr Ser 500 505
510 Val Asp Gln Arg Gly Gln Val Ala Ile Leu Thr Asp Thr Pro Gly Arg
515 520 525 His Lys Met Ser Ile Met Pro Ser Leu Asp Ala Ser Pro Glu
Ser His 530 535 540 Ile Ser Len Ser Leu His Phe Ala Asp Ala His Gln
Gly Leu Leu His 545 550 555 560 Gly Lys Ser Glu Leu Glu Ala Gln Ser
Val Ala Ile Ser His Gly Arg 565 570 575 Leu Val Val Ala Asp Ser Glu
Gly Lys Leu Phe Ser Ala Ala Ile Pro 580 585 590 Lys Gln Gly Asp Gly
Asn Glu Leu Lys Met Lys Ala Met Pro Gln His 595 600 605 Ala Leu Asp
Glu His Phe Gly His Asp His Gln Ile Ser Gly Phe Phe 610 615 620 His
Asp Asp His Gly Gln Leu Asn Ala Leu Val Lys Asn Asn Phe Arg 625 630
635 640 Gln Gln His Ala Cys Pro Leu Gly Asn Asp His Gln Phe His Pro
Gly 645 650 655 Trp Asn Leu Thr Asp Ala Leu Val Ile Asp Asn Gln Leu
Gly Leu His 660 665 670 His Thr Asn Pro Glu Pro His Glu Ile Leu Asp
Met Gly His Leu Gly 675 680 685 Ser Leu Ala Leu Gln Glu Gly Lys Leu
His Tyr Phe Asp Gln Leu Thr 690 695 700 Lys Gly Trp Thr Gly Ala Glu
Ser Asp Cys Lys Gln Leu Lys Lys Gly 705 710 715 720 Leu Asp Gly Ala
Ala Tyr Leu Leu Lys Asp Gly Glu Val Lys Arg Leu 725 730 735 Asn Ile
Asn Gln Ser Thr Ser Ser Ile Lys His Gly Thr Glu Asn Val 740 745 750
Phe Ser Leu Pro His Val Arg Asn Lys Pro Glu Pro Gly Asp Ala Leu 755
760 765 Gln Gly Len Asn Lys Asp Asp Lys Ala Gln Ala Met Ala Val Ile
Gly 770 775 780 Val Asn Lys Tyr Leu Ala Leu Thr Glu Lys Gly Asp Ile
Arg Ser Phe 785 790 795 800 Gln Ile Lys Pro Gly Thr Gln Gln Leu Glu
Arg Pro Ala Gln Thr Leu 805 810 815 Ser Arg Glu Gly Ile Ser Gly Glu
Leu Lys Asp Ile His Val Asp His 820 825 830 Lys Gln Asn Leu Tyr Ala
Leu Thr His Glu Gly Glu Val Phe His Gln 835 840 845 Pro Arg Glu Ala
Trp Gln Asn Gly Ala Glu Ser Ser Ser Trp His Lys 850 855 860 Leu Ala
Leu Pro Gln Ser Glu Ser Lys Leu Lys Ser Leu Asp Met Ser 865 870 875
880 His Glu His Lys Pro Ile Ala Thr Phe Glu Asp Gly Ser Gln His Gln
885 890 895 Leu Lys Ala Gly Gly Trp His Ala Tyr Ala Ala Pro Glu Arg
Gly Pro 900 905 910 Leu Ala Val Gly Thr Ser Gly Ser Gln Thr Val Phe
Asn Arg Leu Met 915 920 925 Gln Gly Val Lys Gly Lys Val Ile Pro Gly
Ser Gly Leu Thr Val Lys 930 935 940 Leu Ser Ala Gln Thr Gly Gly Met
Thr Gly Ala Glu Gly Arg Lys Val 945 950 955 960 Ser Ser Lys Phe Ser
Glu Arg Ile Arg Ala Tyr Ala Phe Asn Pro Thr 965 970 975 Met Ser Thr
Pro Arg Pro Ile Lys Asn Ala Ala Tyr Ala Thr Gln His 980 985 990 Gly
Trp Gln Gly Arg Glu Gly Leu Lys Pro Leu Tyr Glu Met Gln Gly 995
1000 1005 Ala Leu Ile Lys Gln Leu Asp Ala His Asn Val Arg His Asn
Ala Pro 1010 1015 1020 Gln Pro Asp Leu Gln Ser Lys Leu Glu Thr Leu
Asp Leu Gly Glu His 1025 1030 1035 1040 Gly Ala Glu Leu Leu Asn Asp
Met Lys Arg Phe Arg Asp Glu Leu Glu 1045 1050 1055 Gln Ser Ala Thr
Arg Ser Val Thr Val Leu Gly Gln His Gln Gly Val 1060 1065 1070 Leu
Lys Ser Asn Gly Glu Ile Asn Ser Glu Phe Lys Pro Ser Pro Gly 1075
1080 1085 Lys Ala Leu Val Gln Ser Phe Asn Val Asn Arg Ser Gly Gln
Asp Leu 1090 1095 1100 Ser Lys Ser Leu Gln Gln Ala Val His Ala Thr
Pro Pro Ser Ala Glu 1105 1110 1115 1120 Ser Lys Leu Gln Ser Met Leu
Gly His Phe Val Ser Ala Gly Val Asp 1125 1130 1135 Met Ser His Gln
Lys Gly Glu Ile Pro Leu Gly Arg Gln Arg Asp Pro 1140 1145 1150 Asn
Asp Lys Thr Ala Leu Thr Lys Ser Arg Leu Ile Leu Asp Thr Val 1155
1160 1165 Thr Ile Gly Glu Leu His Glu Leu Ala Asp Lys Ala Lys Leu
Val Ser 1170 1175 1180 Asp His Lys Pro Asp Ala Asp Gln Ile Lys Gln
Leu Arg Gln Gln Phe 1185 1190 1195 1200 Asp Thr Leu Arg Glu Lys Arg
Tyr Glu Ser Asn Pro Val Lys His Tyr 1205 1210 1215 Thr Asp Met Gly
Phe Thr His Asn Lys Ala Leu Glu Ala Asn Tyr Asp 1220 1225 1230 Ala
Val Lys Ala Phe Ile Asn Ala Phe Lys Lys Glu His His Gly Val 1235
1240 1245 Asn Leu Thr Thr Arg Thr Val Leu Glu Ser Gln Gly Ser Ala
Glu Leu 1250 1255 1260 Ala Lys Lys Leu Lys Asn Thr Leu Leu Ser Leu
Asp Ser Gly Glu Ser 1265 1270 1275 1280 Met Ser Phe Ser Arg Ser Tyr
Gly Gly Gly Val Ser Thr Val Phe Val 1285 1290 1295 Pro Thr Leu Ser
Lys Lys Val Pro Val Pro Val Ile Pro Gly Ala Gly 1300 1305 1310 Ile
Thr Leu Asp Arg Ala Tyr Asn Leu Ser Phe Ser Arg Thr Ser Gly 1315
1320 1325 Gly Leu Asn Val Ser Phe Gly Arg Asp Gly Gly Val Ser Gly
Asn Ile 1330 1335 1340 Met Val Ala Thr Gly His Asp Val Met Pro Tyr
Met Thr Gly Lys Lys 1345 1350 1355 1360 Thr Ser Ala Gly Asn Ala Ser
Asp Trp Leu Ser Ala Lys His Lys Ile 1365 1370 1375 Ser Pro Asp Leu
Arg Ile Gly Ala Ala Val Ser Gly Thr Leu Gln Gly 1380 1385 1390 Thr
Leu Gln Asn Ser Leu Lys Phe Lys Leu Thr Glu Asp Gln Leu Pro 1395
1400 1405 Gly Phe Ile His Gly Leu Thr His Gly Thr Leu Thr Pro Ala
Glu Leu 1410 1415 1420 Leu Gln Lys Gly Ile Glu His Glu Met Lys Gln
Gly Ser Lys Leu Thr 1425 1430 1435 1440 Phe Ser Val Asp Thr Ser Ala
Asn Leu Asp Leu Arg Ala Gly Ile Asn 1445 1450 1455 Leu Asn Glu Asp
Gly Ser Lys Pro Asn Gly Val Thr Ala Arg Val Ser 1460 1465 1470 Ala
Gly Leu Ser Ala Ser Ala Asn Leu Ala Ala Gly Ser Arg Glu Arg 1475
1480 1485 Ser Thr Thr Ser Gly Gln Phe Gly Ser Thr Thr Ser Ala Ser
Asn Asn 1490 1495 1500 Arg Pro Thr Phe Leu Asn Gly Val Gly Ala Gly
Ala Asn Leu Thr Ala 1505 1510 1515 1520 Ala Leu Gly Val Ala His Ser
Ser Thr His Glu Gly Lys Pro Val Gly 1525 1530 1535 Ile Phe Pro Ala
Phe Thr Ser Thr Asn Val Ser Ala Ala Leu Ala Leu 1540 1545 1550 Asp
Asn Arg Thr Ser Gln Ser Ile Ser Leu Glu Leu Lys Arg Ala Glu 1555
1560 1565 Pro Val Thr Ser Asn Asp Ile Ser Glu Leu Thr Ser Thr Leu
Gly Lys 1570 1575 1580 His Phe Lys Asp Ser Ala Thr Thr Lys Met Leu
Ala Ala Leu Lys Glu 1585 1590 1595 1600 Leu Asp Asp Ala Lys Pro Ala
Glu Gln Leu His Ile Leu Gln Gln His 1605 1610 1615 Phe Ser Ala Lys
Asp Val Val Gly Asp Glu Ary Tyr Glu Ala Val Arg 1620 1625 1630 Asn
Leu Lys Lys Leu Val Ile Arg Gln Gln Ala Ala Asp Ser His Ser 1635
1640 1645 Met Glu Leu Gly Ser Ala Ser His Ser Thr Thr Tyr Asn Asn
Leu Ser 1650 1655 1660 Arg Ile Asn Asn Asp Gly Ile Val Glu Leu Leu
His Lys His Phe Asp 1665 1670 1675 1680 Ala Ala Leu Pro Ala Ser Ser
Ala Lys Arg Leu Gly Glu Met Met Asn 1685 1690 1695 Asn Asp Pro Ala
Leu Lys Asp Ile Ile Lys Gln Leu Gln Ser Thr Pro 1700 1705 1710 Phe
Ser Ser Ala Ser Val Ser Met Glu Leu Lys Asp Gly Leu Arg Glu 1715
1720 1725 Gln Thr Glu Lys Ala Ile Leu Asp Gly Lys Val Gly Arg Glu
Glu Val 1730 1735 1740 Gly Val Leu Phe Gln Asp Arg Asn Asn Leu Arg
Val Lys Ser Val Ser 1745 1750 1755 1760 Val Ser Gln Ser Val Ser Lys
Ser Glu Gly Phe Asn Thr Pro Ala Leu 1765 1770 1775 Leu Leu Gly Thr
Ser Asn Ser Ala Ala Met Ser Met Glu Arg Asn Ile 1780 1785 1790 Gly
Thr Ile Asn Phe Lys Tyr Gly Gln Asp Gln Asn Thr Pro Arg Arg 1795
1800 1805 Phe Thr Leu Glu Gly Gly Ile Ala Gln Ala Asn Pro Gln Val
Ala Ser 1810 1815 1820 Ala Leu Thr Asp Leu Lys Lys Glu Gly Leu Glu
Met Lys Ser 1825 1830 1835
[0025] This protein or polypeptide is about 198 kDa and has a pI of
8.98.
[0026] The present invention relates to an isolated DNA molecule
having a nucleotide sequence of SEQ ID NO: 9 as follows:
9 ATGACATCGT CACAGCAGCG GGTTGAAAGG TTTTTACAGT ATTTCTCCGC CGGGTGTAAA
60 ACGCCCATAC ATCTGAAAGA CGGGGTGTGC GCCCTGTATA ACGAACAACA
TGAGGAGGCG 120 GCGGTGCTGG AAGTACCGCA ACACAGCGAC AGCCTGTTAC
TACACTGCCG AATCATTCAG 180 GCTGACCCAC AAACTTCAAT AACCCTGTAT
TCGATGCTAT TACAGCTGAA TTTTGAAATG 240 GCGGCCATGC GCGGCTGTTG
GCTGGCGCTG GATGAACTGC ACAACGTGCG TTTATGTTTT 300 CAGCAGTCGC
TGGAGCATCT GGATGAAGCA AGTTTTAGCG ATATCGTTAG CGGCTTCATC 360
GAACATGCGG CAGAAGTGCG TGAGTATATA GCGCAATTAG ACGAGAGTAG CGCGGCATAA
420
[0027] This is known as the dspF gene. This isolated DNA molecule
of the present invention encodes a hypersensitive response elicitor
protein or polypeptide having an amino acid sequence of SEQ ID NO:
10 as follows:
10 Met Thr Ser Ser Gln Glu Arg Val Glu Arg Phe Leu 1 5 10 Gln Tyr
Phe Ser Ala Gly Cys Lys Thr Pro Ile His 15 20 Leu Lys Asp Gly Val
Cys Ala Leu Tyr Asn Glu Gln 25 30 35 Asp Glu Glu Ala Ala Val Leu
Glu Val Pro Gln His 40 45 Ser Asp Ser Leu Leu Leu His Cys Arg Ile
Ile Glu 50 55 60 Ala Asp Pro Gln Thr Ser Ile Thr Leu Tyr Ser Met 65
70 Leu Leu Gln Leu Asn Phe Glu Met Ala Ala Met Arg 75 80 Gly Cys
Trp Leu Ala Leu Asp Glu Leu His Asn Val 85 90 95 Arg Leu Cys Phe
Gln Gln Ser Leu Glu His Leu Asp 100 105 Glu Ala Ser Phe Ser Asp Ile
Val Ser Gly Phe Ile 110 115 120 Glu His Ala Ala Glu Val Arg Glu Tyr
Ile Ala Gln 125 130 Leu Asp Glu Ser Ser Ala Ala 135
[0028] This protein or polypeptide is about 16 kDa and has a pI of
4.45.
[0029] The hypersensitive response elicitor polypeptide or protein
derived from Pseudomonas syringae has an amino acid sequence
corresponding to SEQ ID NO: 11 as follows:
11 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
Her 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 Her
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
[0030] 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.
[0031] 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: 12
as follows:
12 ATGCAGAGTC TCAGTCTTAA CACCAGCTCG CTGCAAACCC CGGCAATGGC
CCTTGTCCTG 60 GTACGTCCTG AAGCCGAGAC GACTGGCAGT ACGTCGAGCA
AGGCGCTTCA GGAAGTTGTC 120 GTCAAGCTGG CCGAGGAACT GATGCGCAAT
GGTCAACTCG ACGACAGCTC GCCATTGGGA 180 AAACTGTTGG CCAAGTCGAT
GGCCGCAGAT GGCAAGGCGG GCGCCGGTAT TCAGGATCTC 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 CGCGCTCCTG GGTGAACGAA
CTCAAGGAAG ACAACTTCCT TGATGGCGAC 540 GAAACGGCTG CGTTCCGTTC
GGCACTCGAC ATCATTGGCC AGCAACTGGG TAATCAGCAG 600 AGTGACGCTC
GCAGTCTGGC AGGGACGGGT GGAGGTCTGG GCACTCCGAG CAGTTTTTCC 660
AACAACTCGT CCGTGATGGG TGATCCGCTG ATCGACGCCA ATACCGGTCC CGGTGACAGC
720 GGCAATACCC GTGGTGAAGC GGGCAACTG ATCGGCGAGC TTATCGACCG
TGGCCTGCAA 780 TCGGTATTGG CCGGTGGTGG ACTGGCCACA CCCGTAAACA
CCCCGCAGAC CGCTACGTCG 840 GCGAATGGCG GACAGTCCGC TCAGGATCTT
GATCAGTTGC TGGGCGGCTT GCTGCTCAAG 900 GGCCTGGAGG CAACGCTCAA
GGATGCCGGG CAAACAGGCA CCGACGTGCA GTCGAGCGCT 960 GCGCAAATCG
CCACCTTGCT GGTCAGTACG CTGCTCCAAG GCACCCGCAA TCAGGCTGCA 1020 GCCTGA
1026
[0032] Another potentially suitable hypersensitive response
elicitor from Pseudomonas syringae is disclosed in U.S. patent
application Ser. No. 09/120,817, which is hereby incorporated by
reference. The protein has a nucleotide sequence of SEQ ID NO: 13
as follows:
13 TCCACTTCGC TGATTTTGAA ATTGGCAGAT TCATAGAAAC GTTCAGGTGT
GGAAATCAGG 60 CTGAGTGCGC AGATTTCGTT GATAAGGGTG TGGTACTGGT
CATTGTTGGT CATTTCAAGG 120 CCTCTGAGTG CGGTGCGGAG CAATACCAGT
CTTCCTGCTG GCGTGTGCAC ACTGAGTCGC 180 AGGCATAGGC ATTTCAGTTC
CTTGCGTTGG TTGGGCATAT AAAAAAAGGA ACTTTTAAAA 240 ACAGTGCAAT
GAGATGCCGG CAAAACGGGA ACCGGTCGCT GCGCTTTGCC ACTCACTTCG 300
AGCAAGCTCA ACCCCAAACA TCCACATCCC TATCGAACGG ACAGCGATAC GGCCACTTGC
360 TCTGGTAAAC CCTGGAGCTG GCGTCGGTCC AATTGCCCAC TTAGCGAGGT
AACGCAGCAT 420 GAGCATCGGC ATCACACCCC GGCCGCAACA GACCACCACG
CCACTCGATT TTTCGGCGCT 480 AAGCGGCAAG AGTCCTCAAC CAAACACGTT
CGGCGAGCAG AACACTCAGC AAGCGATCGA 540 CCCGAGTGCA CTGTTGTTCG
GCAGCGACAC ACAGAAAGAC GTCAACTTCG GCACGCCCGA 600 CAGCACCGTC
CAGAATCCGC AGGACOCCAC CAAGCCCAAC GACAGCCACT CCAACATCGC 660
TAAATTCATC AGTGCATTGA TCATCTCGTT CCTGCAGATG CTCACCAACT CCAATAAAAA
720 GCAGGACACC AATCAGGAAC AGCCTGATAG CCAGGCTCCT TTCCAGAACA
ACGGCGGGCT 780 CGGTACACCG TCGGCCGATA GCGGGGGCGG CGCTACACCG
GATGCGACAG GTGGCGGCGG 840 CGGTGATACG CCAAGCGCAA CAGGCGGTGG
CGGCGGTGAT ACTCCGACCG CAACAGGCGG 900 TGGCGGCAGC GGTGGCGGCG
GCACACCCAC TGCAACAGGT GGCGGCAGCG GTGGCACACC 960 CACTGCAACA
GGCGCTGCCC AGCGTCGCCT AACACCGCAA ATCACTCCCC AGTTCGCCAA 1020
CCCTAACCGT ACCTCAGGTA CTGGCTCGGT GTCGGACACC CCAGGTTCTA CCGAGCAAGC
1080 CGGCAAGATC AATGTGGTGA AAGACACCAT CAACGTCGGC GCTGGCGAAG
TCTTTGACGG 1140 CCACGGCGCA ACCTTCACTG CCGACAAATC TATGGGTAAC
GGAGACCAGG GCGAAAATCA 1200 GAAGCCCATC TTCGACCTGG CTCAACCCGC
TACGTTGAAC AATGTGAACC TGGGTGAGAA 1260 CGAGGTCGAT GGCATCCACG
TGAAACCCAA AAACGCTCAG GAAGTCACCA TTGACAACGT 1320 GCATGCCCAG
AACGTCGGTG AAGACCTGAT TACGGTCAAA GGCGAGGGAG GCGCAGCGGT 1380
CACTAATCTG AACATCAAGA ACAGCAGTGC CAAAGGTGCA GACGACAACG TTGTCCAGCT
1440 CAACGCCAAC ACTCACTTGA AAATCCACAA CTTCAAGCCC GACGATTTCG
GCACGATGGT 1500 TCGCACCAAC CCTGGCAAGC ACTTTGATGA CATGAGCATC
GAGCTGAACG GCATCCAACC 1560 TAACCACGGC AAGTTCGCCC TGGTGAAAAG
CGACAGTGAC GATCTGAAGC TGGCAACGGG 1620 CAACATCGCC ATGACCGACG
TCAAACACGC CTACGATAAA ACCCAGGCAT CGACCCAACA 1680 CACCGAGCTT
TGAATCCAGA CAAGTACCTT GAAAAAAGCG GGTGGACTC 1729
[0033] This DNA molecule is known as the dspE gene for Pseudomonas
syringae. This isolated DNA molecule of the present invention
encodes a protein or polypeptide which elicits a plant pathogen's
hypersensitive response having an amino acid sequence of SEQ ID NO:
14 as follows:
14 Met Ser Ile Gly Ile Thr Pro Arg Pro Gln Gln Thr Thr Thr Pro Leu
1 5 10 15 Asp Phe Ser Ala Leu Ser Gly Lys Ser Pro Gln Pro Asn Thr
Phe Gly 20 25 30 Glu Gln Asn Thr Gln Gln Ala Ile Asp Pro Ser Ala
Leu Leu Phe Gly 35 40 45 Ser Asp Thr Gln Lys Asp Val Asn Phe Gly
Thr Pro Asp Ser Thr Val 50 55 60 Gln Asn Pro Gln Asp Ala Ser Lys
Pro Asn Asp Ser Gln Ser Asn Ile 65 70 75 80 Ala Lys Leu Ile Ser Ala
Leu Ile Met Ser Leu Leu Gln Met Leu Thr 85 90 95 Asn Ser Asn Lys
Lys Gln Asp Thr Asn Gln Glu Gln Pro Asp Ser Gln 100 105 110 Ala Pro
Phe Gln Asn Asn Gly Gly Leu Gly Thr Pro Ser Ala Asp Ser 115 120 125
Gly Gly Gly Gly Thr Pro Asp Ala Thr Gly Gly Gly Gly Gly Asp Thr 130
135 140 Pro Ser Ala Thr Gly Gly Gly Gly Gly Asp Thr Pro Thr Ala Thr
Gly 145 150 155 160 Gly Gly Gly Ser Gly Gly Gly Gly Thr Pro Thr Ala
Thr Gly Gly Gly 165 170 175 Ser Gly Gly Thr Pro Thr Ala Thr Gly Gly
Gly Glu Gly Gly Val Thr 180 185 190 Pro Gln Ile Thr Pro Gln Leu Ala
Asn Pro Asn Arg Thr Ser Gly Thr 195 200 205 Gly Ser Val Ser Asp Thr
Ala Gly Ser Thr Glu Gln Ala Gly Lys Ile 210 215 220 Asn Val Val Lys
Asp Thr Ile Lys Val Gly Ala Gly Glu Val Phe Asp 225 230 235 240 Gly
His Gly Ala Thr Phe Thr Ala Asp Lys Ser Met Gly Asn Gly Asp 245 250
255 Gln Gly Glu Asn Gln Lys Pro Met Phe Gln Len Ala Glu Gly Ala Thr
260 265 270 Leu Lys Asn Val Asn Leu Gly Glu Asn Glu Val Asp Gly Ile
His Val 275 280 285 Lys Ala Lys Asn Ala Gln Glu Val Thr Ile Asp Asn
Val His Ala Gln 290 295 300 Asn Val Gly Glu Asp Leu Ile Thr Val Lys
Gly Glu Gly Gly Ala Ala 305 310 315 320 Val Thr Asn Leu Asn Ile Lys
Asn Ser Ser Ala Lys Gly Ala Asp Asp 325 330 335 Lys Val Val Gln Leu
Asn Ala Asn Thr His Leu Lys Ile Asp Asn Phe 340 345 350 Lys Ala Asp
Asp Phe Gly Thr Met Val Arg Thr Asn Gly Gly Lys Gln 355 360 365 Phe
Asp Asp Met Ser Ile Glu Leu Asn Gly Ile Glu Ala Asn His Gly 370 375
380 Lys Phe Ala Leu Val Lys Ser Asp Ser Asp Asp Leu Lys Leu Ala Thr
385 390 395 400 Gly Asn Ile Ala Met Thr Asp Val Lys His Ala Tyr Asp
Lys Thr Gln 405 410 415 Ala Ser Thr Gln His Thr Glu Leu 420
[0034] This protein or polypeptide is about 42.9 kDa.
[0035] The hypersensitive response elicitor polypeptide or protein
derived from Pseudomonas solanacearum has an amino acid sequence
corresponding to SEQ ID NO: 15 as follows:
15 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
[0036] It is encoded by a DNA molecule having a nucleotide sequence
corresponding SEQ ID NO: 16 as follows:
16 ATGTCAGTCG GAAACATCCA GAGCCCGTCG AACCTCCCGG GTCTGCAGAA
CCTGAACCTC 60 AACACCAACA CCAACAGCCA GCAATCGCGC CAGTCCGTGC
AAGACCTGAT CAAGCAGGTC 120 GAGAAGGACA TCCTCAACAT CATCGCAGCC
CTCGTGCACA AGGCCCCACA 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 CAACGCCGCC AAGGGTGCCG
GCGGCCAGGG CCGCCTCGCC 480 GAAGCGCTGC AGGAGATCGA GCAGATCCTC
GCCCAGCTCG GCGGCGGCGG TGCTGGCGCC 540 GGCGGCGCGG GTGGCGGTGT
CGGCGGTGCT GGTGGCGCGG ATGGCGCCTC CGGTCCGGGT 600 GGCGCAGGCG
GTGCGAACGG CGCCGACGGC GGCAATGGCG TGAACGGCAA CCAGGCGAAC 660
GGCCCGCAGA ACGCAGGCCA TGTCAACGGT GCCAACGGCG CGGATGACGG CAGCGAAGAC
720 CAGGGCCGCC TCACCGGCGT GCTGCAAAAG CTGATGAAGA TCCTGAACGC
GCTGGTGCAG 780 ATGATGCAGC AAGGCGGCCT CGGCGGCGGC AACCAGGCGC
AGGGCGGCTC GAAGGGTGCC 840 GGCAACGCCT CGCCGGCTTC CGGCGCGAAC
CCGGGCGCGA ACCAGCCCGG TTCGGCGGAT 900 GATCAATCGT CCCGCCAGAA
CAATCTGCAA TCCCAGATCA TGCATGTGGT GAAGGAGGTC 960 GTCCAGATCC
TGCAGCAGAT GCTGGCGGCG CAGAACGGCG GCAGCCAGCA GTCCACCTCG 1020
ACGCAGCCGA TGTAA 1035
[0037] 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.
[0038] The hypersensitive response elicitor polypeptide or protein
from Xanthomonas campestris pv. glycines has an amino acid sequence
corresponding to SEQ ID NO: 17 as follows:
17 Thr Leu Ile Glu Leu Met Ile Val Val Ala Ile Ile 1 5 10 Ala Ile
Leu Ala Ala Ile Ala Leu Pro Ala Tyr Gln 15 20 Asp Tyr 25
[0039] This sequence is an amino terminal sequence having only 26
residues 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.
[0040] 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: 18 as follows:
18 Ser Ser Gln Gln Ser Pro Ser Ala Gly Ser Glu Gln 1 5 10 Gln Leu
Asp Gln Leu Leu Ala Met 15 20
[0041] 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 protein or polypeptide of Erwinia stewartii is set forth
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.
[0042] 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(1):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.
[0043] Another hypersensitive response elicitor in accordance with
the present invention is from Clavibacter michiganensis subsp.
sepedonicus which is fully described in U.S. patent application
Ser. No. 09/136,625, which is hereby incorporated by reference.
[0044] The above elicitors are exemplary. Other elicitors can be
identified by growing fungi or bacteria that elicit a
hypersensitive response under conditions 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.
[0045] Fragments of the above hypersensitive response elicitor
polypeptides or proteins as well as fragments of full length
elicitors from other pathogens are encompassed by the method of the
present invention.
[0046] 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
peptide that can be tested for elicitor activity according to the
procedure described below.
[0047] 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.
[0048] 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 expression of a truncated peptide or protein.
[0049] 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).
[0050] An example of suitable fragments of a hypersensitive
response elicitor which do not elicit a hypersensitive response
include fragments of the Erwinia. Suitable fragments include a
C-terminal fragment of the amino acid sequence of SEQ ID NO: 3, an
N-terminal fragment of the amino acid sequence of SEQ ID NO: 3, or
an internal fragment of the amino acid sequence of SEQ ID NO: 3.
The C-terminal fragment of the amino acid sequence of SEQ ID NO: 3
can span the following amino acids of SEQ ID NO: 3: 169 and 403,
210 and 403, 267 and 403, or 343 and 403. The internal fragment of
the amino acid sequence of SEQ ID NO: 3 can span the following
amino acids of SEQ ID NO: 3: 105 and 179, 137 and 166, 121 and 150,
or 137 and 156. Other suitable fragments can be identified in
accordance with the present invention.
[0051] Another example of suitable fragments of a hypersensitive
response elicitor which do elicit a hypersensitive response are
Erwinia amylovora fragments including a C-terminal fragment of the
amino acid sequence of SEQ ID NO: 3, an N-terminal fragment of the
amino acid sequence of SEQ ID NO: 3, or an internal fragment of the
amino acid sequence of SEQ ID NO: 3. The C-terminal fragment of the
amino acid sequence of SEQ ID NO: 3 can span amino acids 105 and
403 of SEQ ID NO: 3. The N-terminal fragment of the amino acid
sequence of SEQ ID NO: 3 can span the following amino acids of SEQ
ID NO: 3: 1 and 98, 1 and 104, 1 and 122, 1 and 168, 1 and 218, 1
and 266, 1 and 342, 1 and 321, and 1 and 372. The internal fragment
of the amino acid sequence of SEQ ID NO: 3 can span the following
amino acids of SEQ ID NO: 3: 76 and 209, 105 and 209, 99 and 209,
137 and 204, 137 and 200, 109 and 204, 109 and 200, 137 and 180,
and 105 and 180.
[0052] Suitable DNA molecules are those that hybridize to the DNA
molecule comprising a nucleotide sequence of SEQ ID NOS: 2, 4, 5,
7, 9, 12, 13, and 16 under stringent conditions. An example of
suitable high stringency conditions is when hybridization is
carried out at 65.degree. C. for 20 hours in a medium containing 1M
NaCl, 50 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.1% sodium dodecyl
sulfate, 0.2% ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serum
albumin, 50 .mu.m g/ml E. coli DNA.
[0053] Variants may be made by, for example, the deletion or
addition of amino acids that have minimal influence on the
properties, secondary structure and hydropathic nature of the
polypeptide. For example, a polypeptide may be conjugated to a
signal (or leader) sequence at the N-terminal end of the protein
which co-translationally or post-translationally directs transfer
of the protein. The polypeptide may also be conjugated to a linker
or other sequence for ease of synthesis, purification, or
identification of the polypeptide.
[0054] The hypersensitive response elicitor of the present
invention is preferably in isolated form (i.e. separated from its
host organism) and more preferably produced in purified form
(preferably at least about 60%, more preferably 80%, pure) by
conventional techniques. Typically, the hypersensitive response
elicitor 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 is separated by centrifugation.
The supernatant fraction containing the hypersensitive response
elicitor is subjected to gel filtration in an appropriately sized
dextran or polyacrylamide column to separate the fragment. If
necessary, the protein fraction may be further purified by ion
exchange or HPLC.
[0055] 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 sense orientation and correct reading frame. The vector
contains the necessary elements for the transcription and
translation of the inserted protein-coding sequences.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] Different genetic signals and processing events control many
levels of gene expression (e.g., DNA transcription and messenger
RNA (mRNA) translation).
[0061] Transcription of DNA is dependent upon the presence of a
promotor which is a DNA sequence that directs the binding of RNA
polymerase and thereby promotes mRNA synthesis. The DNA sequences
of eucaryotic promotors differ from those of procaryotic promotors.
Furthermore, eucaryotic promotors and accompanying genetic signals
may not be recognized in or may not function in a procaryotic
system, and, further, procaryotic promotors are not recognized and
do not function in eucaryotic cells.
[0062] 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.
[0063] 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 promotor, lac promotor, trp promotor, recA promotor,
ribosomal RNA promotor, the PR and PL promoters of coliphage lambda
and others, including but not limited, to lacUV5, ompF, bla, lpp,
and the like, may be used to direct high levels of transcription of
adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac)
promotor or other E. coli promotors produced by recombinant DNA or
other synthetic DNA techniques may be used to provide for
transcription of the inserted gene.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] The present invention's method of imparting stress
resistance to plants can involve applying the hypersensitive
response elicitor polypeptide or protein in a non-infectious form
to all or part of a plant or a plant seed under conditions
effective for the elicitor to impart stress resistance.
Alternatively, the hypersensitive response elicitor protein or
polypeptide can be applied to plants such that seeds recovered from
such plants themselves are able to impart stress resistance in
plants.
[0068] As an alternative to applying a hypersensitive response
elicitor polypeptide or protein to plants or plant seeds in order
to impart stress resistance in plants or 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 impart
stress resistance to plants. 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 impart stress
resistance to plants.
[0069] 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 hypersensitive response
elicitor or 2) application of bacteria which do not cause disease
and are transformed with a genes encoding the elicitor. In the
latter embodiment, the elicitor can be applied to plants or plant
seeds by applying bacteria containing the DNA molecule encoding a
hypersensitive response elicitor polypeptide or protein. Such
bacteria must be capable of secreting or exporting the elicitor so
that the elicitor can contact plant or plant seed cells. In these
embodiments, the elicitor is produced by the bacteria in planta or
on seeds or just prior to introduction of the bacteria to the
plants or plant seeds.
[0070] The methods of the present invention can be utilized to
treat a wide variety of plants or their seeds to impart stress
resistance. Suitable plants include dicots and monocots. More
particularly, useful crop plants can include: alfalfa, rice, wheat,
barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato,
bean, pea, chicory, lettuce, endive, cabbage, brussel sprout, beet,
parsnip, cauliflower, broccoli, turnip, radish, spinach, onion,
garlic, eggplant, pepper, celery, carrot, squash, pumpkin,
zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape,
raspberry, pineapple, soybean, tobacco, tomato, sorghum, and
sugarcane. Examples of suitable ornamental plants are: Arabidopsis
thaliana, Saintpaulia, petunia, pelargonium, poinsettia,
chrysanthemum, carnation, and zinnia.
[0071] In accordance with the present invention, the term "stress"
refers to drought, salt, cold temperatures (e.g., frost), chemical
treatment (e.g., insecticides, fungicides, herbicides,
fertilizers), water, excessive light, and insufficient light.
[0072] 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, propagules (e.g.,
cuttings), etc. This may (but need not) involve infiltration of the
hypersensitive response elicitor polypeptide or protein into the
plant. Suitable application methods include high or low pressure
spraying, injection, and leaf abrasion proximate to when elicitor
application takes place. When treating plant seeds or propagules
(e.g., cuttings), in accordance with the application embodiment of
the present invention, the hypersensitive response elicitor protein
or polypeptide, in accordance with present invention, can be
applied by low or high pressure spraying, coating, immersion, or
injection. Other suitable application procedures can be envisioned
by those skilled in the art provided they are able to effect
contact of the elicitor 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 impart stress resistance to plants.
[0073] The hypersensitive response elicitor polypeptide or protein,
in accordance with the present invention, can be applied to plants
or plant seeds 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.
[0074] 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 500 nM of the elicitor.
[0075] Although not required, this composition may contain
additional additives including fertilizer, insecticide, fungicide,
nematacide, 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.
[0076] 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 can be applied to
plant seeds with other conventional seed formulation and treatment
materials, including clays and polysaccharides.
[0077] In the alternative embodiment of the present invention
involving the use of transgenic plants and transgenic seeds, a
hypersensitive response elicitor need not be applied topically to
the plants or seeds. Instead, transgenic plants transformed with a
DNA molecule encoding such an elicitor are produced according to
procedures well known in the art.
[0078] The vector described above can be microinjected directly
into plant cells by use of micropipettes to transfer mechanically
the recombinant DNA. Crossway, Mol. Gen. Genetics, 202:179-85
(1985), which is hereby incorporated by reference. The genetic
material may also be transferred into the plant cell using
polyethylene glycol. Krens, et al., Nature, 296:72-74 (1982), which
is hereby incorporated by reference.
[0079] Another approach to transforming plant cells with a gene is
particle bombardment (also known as biolistic transformation) of
the host cell. This can be accomplished in one of several ways. The
first involves propelling inert or biologically active particles at
cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050,
5,036,006, and 5,100,792, all to Sanford et al., which are hereby
incorporated by reference. Generally, this procedure involves
propelling inert or biologically active particles at the cells
under conditions effective to penetrate the outer surface of the
cell and to be incorporated within the interior thereof. When inert
particles are utilized, the vector can be introduced into the cell
by coating the particles with the vector containing the
heterologous DNA. Alternatively, the target cell can be surrounded
by the vector so that the vector is carried into the cell by the
wake of the particle. Biologically active particles (e.g., dried
bacterial cells containing the vector and heterologous DNA) can
also be propelled into plant cells.
[0080] Yet another method of introduction is fusion of protoplasts
with other entities, either minicells, cells, lysosomes, or other
fusible lipid-surfaced bodies. Fraley, et al., Proc. Natl. Acad.
Sci. USA, 79:1859-63 (1982), which is hereby incorporated by
reference.
[0081] The DNA molecule may also be introduced into the plant cells
by electroporation. Fromm et al., Proc. Natl. Acad. Sci. USA,
82:5824 (1985), which is hereby incorporated by reference. In this
technique, plant protoplasts are electroporated in the presence of
plasmids containing the expression cassette. Electrical impulses of
high field strength reversibly permeabilize biomembranes allowing
the introduction of the plasmids. Electroporated plant protoplasts
reform the cell wall, divide, and regenerate.
[0082] Another method of introducing the DNA molecule into plant
cells is to infect a plant cell with Agrobacterium tumefaciens or
A. rhizogenes previously transformed with the gene. Under
appropriate conditions known in the art, the transformed plant
cells are grown to form shoots or roots, and develop further into
plants. Generally, this procedure involves inoculating the plant
tissue with a suspension of bacteria and incubating the tissue for
48 to 72 hours on regeneration medium without antibiotics at
25-28.degree. C.
[0083] Agrobacterium is a representative genus of the Gram-negative
family Rhizobiaceae. Its species are responsible for crown gall (A.
tumefaciens) and hairy root disease (A. rhizogenes). The plant
cells in crown gall tumors and hairy roots are induced to produce
amino acid derivatives known as opines, which are catabolized only
by the bacteria. The bacterial genes responsible for expression of
opines are a convenient source of control elements for chimeric
expression cassettes. In addition, assaying for the presence of
opines can be used to identify transformed tissue.
[0084] Heterologous genetic sequences can be introduced into
appropriate plant cells, by means of the Ti plasmid of A.
tumefaciens or the Ri plasmid of A. rhizogenes. The Ti or Ri
plasmid is transmitted to plant cells on infection by Agrobacterium
and is stably integrated into the plant genome. J. Schell, Science,
237:1176-83 (1987), which is hereby incorporated by reference.
[0085] After transformation, the transformed plant cells must be
regenerated.
[0086] Plant regeneration from cultured protoplasts is described in
Evans et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan
Publishing Co., New York, 1983); and Vasil I. R. (ed.), Cell
Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando,
Vol. 1, 1984, and Vol. III (1986), which are hereby incorporated by
reference.
[0087] It is known that practically all plants can be regenerated
from cultured cells or tissues, including but not limited to, all
major species of sugarcane, sugar beets, cotton, fruit trees, and
legumes.
[0088] Means for regeneration vary from species to species of
plants, but generally a suspension of transformed protoplasts or a
petri plate containing transformed explants is first provided.
Callus tissue is formed and shoots may be induced from callus and
subsequently rooted. Alternatively, embryo formation can be induced
in the callus tissue. These embryos germinate as natural embryos to
form plants. The culture media will generally contain various amino
acids and hormones, such as auxin and cytokinins. It is also
advantageous to add glutamic acid and proline to the medium,
especially for such species as corn and alfalfa. Efficient
regeneration will depend on the medium, on the genotype, and on the
history of the culture. If these three variables are controlled,
then regeneration is usually reproducible and repeatable.
[0089] After the expression cassette is stably incorporated in
transgenic plants, it can be transferred to other plants by sexual
crossing. Any of a number of standard breeding techniques can be
used, depending upon the species to be crossed.
[0090] 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 stress resistance to the plant.
Alternatively, transgenic seeds or propagules (e.g., cuttings) are
recovered from the transgenic plants. The 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
stress resistance to plants. While not wishing to be bound by
theory, such stress resistance may be RNA mediated or may result
from expression of the elicitor polypeptide or protein.
[0091] 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 in accordance
with the present invention is applied. These other materials,
including a hypersensitive response elicitor in accordance with the
present invention, can be applied to the transgenic plants and
plant seeds by the above-noted procedures, including high or low
pressure spraying, injection, coating, 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 in accordance with the present
invention to impart stress resistance. Such plants may also be
treated with conventional plant treatment agents (e.g.,
insecticides, fertilizers, etc.).
EXAMPLES
Example 1
[0092] Hypersensitive Response Elicitor-Treated Cotton is More
Resistant to the Damage Caused by Insecticide Stress
[0093] Aphids (Aphids gossypii) infect cotton during the entire
growth season. The damage of aphid infection ranges from honeydew
deposit that contaminates the lint and reduces crop value to
defoliation that reduces or destroys crops. To protect plants from
aphid infection, cotton is usually sprayed with insecticides, for
example Asana XL when the infection pressure is not very high, and
Admire when the infestation pressure is high. The effect of a
hypersensitive response elicitor on aphids in cotton was studied by
a trial involving a randomized complete block design. This involved
treatment with Erwinia amylovora hypersensitive response elicitor
(i.e. HP-1000.TM.) at 20, 60, and 80 ppm and a chemical
insecticide, Asana XL, at 8 oz./ac. Each treatment involved foliar
application beginning at cotyledon to three true leaves and
thereafter at 14 day intervals using a backpack sprayer. Aphid
counts and overall growth of the cotton were made immediately prior
to spray application at 14, 28, 35, and 42 days after the first
treatment ("DAT 1"). Twenty-five randomly selected leaves per plot
were collected at the first three sampling dates and the leaves per
plot at the final sampling date.
[0094] Results
[0095] 1. Aphid control: The number of aphids in the hypersensitive
response elicitor-treated cotton were significantly reduced in
comparison to the chemical treated cotton (see Table 1).
19TABLE 1 Aphid count per leaf on cotton after treatment with Asana
XL .RTM. or HP-1000 .TM. Number of aphids per leaf.sup.1 No. sprays
applied/days after treatment Treatment Rate.sup.2 1/14DAT1 2/28DAT1
3/35DAT1 4/42DAT1 Asana XL .RTM. 8 oz/ac 0.2 a 32.2 a 110.0 a 546.9
a HP-1000 .TM. 20 .mu.g/ml 0.2 a 7.8 b 22.9 b 322.1 a HP-1000 .TM.
60 .mu.g/ml 0.1 a 4.9 b 34.6 b 168.3 a HP-1000 .TM. 80 .mu.g/ml 0.0
a 2.7 b 25.8 b 510.2 a .sup.1Means followed by different letters
are significantly different according to Duncan's MRT, P = 0.05.
.sup.2Rate for Asana XL .RTM. is for formulated product, rate for
HP-1000 .TM. is for active ingredient (a.i.).
[0096] At 14 days after DAT 1, aphid counts were relatively low
across all of the treatments, but by 28 days after DAT 1 (by which
time two sprayings had been applied), the number of aphids per leaf
were significantly greater in Asana XL-treated plants compared to
the hypersensitive response elicitor-treated cottons. By 35 days
after DAT 1 (by which time three sprayings had been applied), aphid
counts had risen for all treatments, yet aphid counts per leaf were
still significantly lower for hypersensitive response
elicitor-treated cotton compared to the Asana XL treatment.
Finally, at 42 days after DAT 1 (by which time four sprayings had
been applied), the number of aphids per leaf had increased to a
level that threatened to overwhelm the plants even when treated
with the standard chemical insecticide. To save the trial, another
chemical, Pravado (Admire), was applied to all plots to eradicate
aphids from the field.
[0097] 2. Hypersensitive response elicitor-treated cotton was more
resistant to the damage caused by Pravado (Admire) and Asana. After
the second chemical spraying, it was observed that cotton plants
were stress shocked by the insecticides. The cotton plants
previously treated with Asana and untreated control were
defoliated. On most of the chemical-treated cotton, there were no
leaves, or very few leaves, in the lower portion of plants.
However, the hypersensitive response elicitor-treated plants,
especially the plot where hypersensitive response elicitor was
applied at 80 ppm, had no defoliation and the cotton plants were
vigorous and healthy. By counting the number of mature balls, it
clearly showed that hypersensitive response elicitor-treated plants
(at 80 ppm) had more ball setting than chemical and untreated
control (Table 2), indicating that hypersensitive response
elicitor-treated plants were more tolerant to the stress caused by
insecticide.
20TABLE 2 Number of Formed Cotton Balls Counted on Ten Plants in
Each of Four Replicates Per Treatment. Treatment No. balls/10
plants/replicate UTC 28 Chemical standard 6 Hypersensitive Response
Elicitor 35
Example 2
[0098] Hypersensitive Response Elicitor-Treated Cucumbers are More
Resistant to Drought
[0099] A cucumber field trial was set up to test the effect of
Erwinia amylovora hypersensitive response elicitor on disease
control, tolerance to drought stress, and yield. Three different
rates were tested, there at 15, 30, and 60 .mu.g/ml. In addition to
hypersensitive response elicitor treatment, there was an untreated
control. Each treatment contained three replicate plots. When the
first true leaf emerges, hypersensitive response elicitor was
sprayed with a back bag sprayer. The second spray was applied ten
days after the first spray. The third application was right after
the recovery of cucumber seedlings after the transplanting to the
field. Individual treatment was randomly assigned in the field.
[0100] When the first true leaf emerged (Day 0), a first
application was sprayed. Usually cucumber seedlings are
transplanted when seedlings show two true leaves. It has been known
that the recovery rate after the transplanting is closely related
to the size of the seedlings. Because of the drought, the seedlings
were maintained in the nursery for an extra ten days and the second
spray was applied on Day 10. Two days after the second spray, the
plants were transplanted into fields and covered with plastic
sheets. The plants had 4-5 true leaves.
[0101] Result
[0102] The recovery rate of the transplanted cucumber seedlings was
higher for the hypersensitive response elicitor-treated plants than
for the untreated control. More than 80% of the hypersensitive
response elicitor-treated cucumber seedlings survived, while only
57% untreated plants survived.
[0103] Throughout the growth season, there was a serious drought
problem. Early field visits indicated that hypersensitive response
elicitor-treated plants had more root mass and better over-all
growth. Hypersensitive response elicitor-treated cucumber started
to flower 14 days earlier than untreated control cucumber. The
early flowering resulted in an earlier harvest. In the first
harvest, more than 0.4 kilograms of cucumber fruits per plant were
harvested from the hypersensitive response elicitor-treated
cucumbers; however, virtually no fruit was harvested from untreated
control. By the end of the season, untreated plants died due to
severe drought, but hypersensitive response elicitor-treated plants
were still alive and had one more harvest.
[0104] The final yield was significantly different between
hypersensitive response elicitor-treated and untreated plants.
Hypersensitive response elicitor administered at the rate of 30 ppm
produced three times greater yield than the control plants (Table
3).
21TABLE 3 Yield Increase of Cucumber Fruit from Hypersensitive
Response Elicitor Treated Plants % of the Yield Treatment Replicate
kg/plant Yield/Replicate Increase HP 15 I 1.25 37.5 II 1.00 30.0
103.8 241 III 1.21 36.3 HP 30 I 1.54 46.2 II 1.43 42.9 133.2 339
III 1.47 44.1 Control I 0.43 12.9 II 0.41 12.3 39.3 III 0.47
14.1
[0105] The increased yield was partially attributed to
hypersensitive response elicitor-induced growth enhancement and
partially resulted from more tolerance of hypersensitive response
elicitor-treated cucumber to drought, because usually the yield
increase from hypersensitive response elicitor-induced growth
enhancement is between 10-40%.
Example 3
[0106] Hypersensitive Response Elicitor-Treated Pepper is More
Tolerant to Herbicide Stress
[0107] Pepper seedlings were drenched with hypersensitive response
elicitor at 20 ppm seven days before transplanting, sprayed seven
days after the transplanting, and then, sprayed every fourteen
days. Standard chemicals, Brave, Maneb, Kocide, and Admire, were
used for the rest of the treatment. In addition to early growth
enhancement, which resulted in a higher yield, larger fruit, and
resistance to several diseases, hypersensitive response
elicitor-treated pepper was more tolerant to herbicide damage. The
pepper field was applied with the herbicide SENCOR which is not
labeled for pepper. This herbicide is known to cause severe foliar
damage to pepper in chemically-treated plants but not with
hypersensitive response elicitor-treated plants.
[0108] The difference between the adverse effect of the herbicide
on the hypersensitive response elicitor and non-hypersensitive
response elicitor treated plants is dramatic. See Table 4 below.
Thirty-nine of the 60 elicitor-treated plants showed only minor
damage by the herbicide, the damaged leaves were less than 20%. In
contrast, 53 out of the 60 chemically-treated pepper plants had
severe damage, 40-57% of the leaves were damaged, and 20 plants
were dead. The ability of hypersensitive response elicitors to help
crops withstand the phytotoxic effects of a herbicide is very
important benefit to in agricultural industry.
22TABLE 4 Hypersensitive Response Elicitor-Treated Peppers are More
Tolerant to Herbicide Damage. Treatment Damage Rating Damage Index
% 1 2 3 4 5 6 41 Hypersensitive 1 38 17 3 1 0 Response Elicitor
Chemicals 0 1 6 16 19 18 87 Damage Rating: 1. No damage; 2. 0-20%
leaves damaged; 3. 20-40% leaves damaged; 4. 40-50% leaves damaged;
6. More than 75% leaves damaged or entire plant dead. Damage index
= sum of each rating times the number of plants under the rating
scale, divided by total number of plants times 6. Damage index for
hypersensitive response elicitor-treated plants = 1 .times. 1 + 2
.times. 38 + 3 .times. 17 + 4 .times. 3 + 5 .times. 1 + 6 .times. 0
.times. 100% = 41%
Example 4
[0109] Hypersensitive Response Elicitor-Treated Pepper is More
Tolerant to Herbicide Stress under Controlled Experimental
Conditions
[0110] A field trial was conducted to test if hypersensitive
elicitor treated pepper would be more tolerant to herbicide stress.
The trial contains 6 treatments and 4 replicates for each
treatment. The treatments are described as follows:
[0111] 1. Control, the peppers were neither treated by a
hypersensitive response ("HR") elicitor nor by LEXONE.TM. herbicide
(DuPont Agricultural Products, Wilmington, Del.).
[0112] 2. Control pepper with application of 0.15 pound LEXONE.TM.
herbicide/acre.
[0113] 3. Control pepper with application of 0.3 pound LEXONE.TM.
herbicide/acre.
[0114] 4. HR elicitor treatment with no application of LEXONE.TM.
herbicide using a formulated product known as MESSENGER.TM.
biopesticide (Eden Bioscience Corporation, Bothell, Wash.)
containing 3% HR elicitor protein was used.
[0115] 5. HR elicitor treatment with application of 0.15 pound
LEXONE.TM. herbicide/acre.
[0116] 6. HR elicitor treatment with application of 0.3 pound
LEXONE.TM. herbicide/acre.
[0117] LEXONE.TM. contains the same active ingredient as SENCOR.TM.
herbicide (Bayer, Kansas City, Mo.) used in Example 3. Pepper
seedlings were drenched with MESSENGER.TM. solution at the
concentration of HR elicitor protein of about 20 ppm seven days
before transplanting into the field and then sprayed every 14 days
after the transplanting. LEXONE was applied at high (0.3
pound/acre) and low levels (0.15 pound/acre). 50 gallon water and
100 mL of the herbicide solution was introduced into the root zone
of each plant in the respective treatment five weeks after
transplant into the field.
[0118] The treatments were evaluated for the percent of chlorosis
caused by the LEXONE.TM. herbicide application and for the pepper
yield. HR elicitor-treated plants exposed to the high rate of
herbicide had significantly less chlorosis and produced 108% more
fruit in comparison to the non-hypersensitive response elicitor
treated plants exposed to the same amount of herbicide. See Tables
5 and 6 below. There was no significant difference in the reduction
of chlorosis at the low rate of herbicide between the HR elicitor
treated and non-HR elicitor treated peppers. However, the HR
elicitor treated plants produced 15% more fruit than the
corresponding control plants exposed to the same amount of
herbicide. There was no chlorosis in either the check or HR
elicitor-treated plants that did not receive LEXONE.TM. herbicide
treatment.
[0119] The HR elicitor treated plants were much less severely
affected by the herbicide application than the respective control
plants at the high rate of herbicide. However, the amount of visual
chlorosis was similar at the low rate for both the check and HR
elicitor-treated plants. More importantly, the yields from both the
high and low rate herbicide treatments of HR elicitor treated
plants were less severely effected by the herbicide than the
checks. These findings further confirm that HR elicitors can help
crops withstand the phytotoxic effects of herbicides and are very
beneficial to the agricultural industry.
23TABLE 5 Reduction of Foliar Chlorosis and Increase in Yield in
Hypersensitive Response Elicitor Treated Plants after Exposure to
LEXONE .TM.Herbicide Percent foliar chlorosis and yield of pepper %
difference from the Yield respective Treatment A B C D E (pound)
control 6 (MESSENGER .TM. + 13.75 30.00 37.50 36.25 40.00 8.31 108%
High rate LEXONE .TM.) 3 (High rate 26.25 43.75 51.25 50.00 51.25
4.00 -- LEXONE .TM.) 5 (MESSENGER .TM. + 16.25 22.50 28.75 23.75
27.50 8.00 15% low rate LENOXE .TM.) 2 (LENOXE .TM.) 12.50 20.00
25.00 25.00 23.75 6.81 --
[0120]
24TABLE 6 Weight of Harvested Peppers Increased in Hypersensitive
Response Elicitor Treated Plants after Exposure to LEXONE .TM.
Herbicide Compared to Check Plants. Weight of peppers Treatment
harvested Dec. 1, 1998 in pounds HP20 + high rate LEXONE .TM. 8.31
Check + high rate LEXONE .TM. 4.00 HP20 + low rate LEXONE .TM. 8.00
Check + low rate LEXONE .TM. 6.81
Example 5
[0121] Hypersensitive Response Elicitor-Treated Cotton is More
Tolerant to Drought Stress
[0122] A non-irrigated cotton trial experienced 26 consecutive days
of drought. The average daily heat index was near or over 100
degrees F., adding to the stress placed on the plants in the
field.
[0123] Observations in the field indicated that plants treated with
HR elicitor at the concentration of 15 ppm (2.2 oz formulated
product, MESSENGER.TM. containing 3% active ingredient HR elicitor
protein) were more vigorous and had less defoliation than the check
plants as a result of the heat and drought stress. Equal numbers of
plants from the MESSENGER.TM.-treated and the non-MESSENGER.TM.
treated plots were carefully removed from the field and mapped for
the number of nodes and bolls by position. The plants were also
weighed on a Metler analytical scale to determine whole plant, root
and shoot weights.
[0124] MESSENGER.TM. treated plants survived the heat and drought
stresses much better than the untreated plants did. Plants treated
with MESSENGER.TM. had 37.6% more root and shoot mass than the
check plants (Table 7). The MESSENGER.TM. treated plants also had
significantly more cotton bolls than the check plants (Table 8).
The number of cotton bolls from positions 1 and 2 have a
significant contribution to the overall yield. Table 8 showed that
MESSENGER.TM. treated plants had 47% more bolls in positions 1 and
2 and 57% more boll from a whole plant in comparison to the yield
achieved using a grower standard treatment (i.e. with no
MESSENGER.TM. treatment). A common reaction to stress in cotton is
for the plant to abort bolls. The results indicate that
MESSENGER.TM.-treated plants are more tolerant to the drought
stress.
25TABLE 7 Weight per Plant of Non-Irrigated Cotton Following 26
Days of Drought. Whole plant Root weight Shoot weight % weight %
Treatment (pond/plant) % Difference (pond/plant) difference
(pond/plant) difference MESSENGER .TM. 0.041 a* 37.6% 0.505 a 37.5%
0.546 37.5% 2.2 oz/acre Control (Grower 0.0298 b 0.367 b 0.397
standard) Level of P = 0.119 P = 0.034 P = 0.033 statistically
significant *Same letter indicates no statistical difference
between the two treatments at the defined level; different letter
indicates a statistical difference between the two treatments at
the defined level.
[0125]
26TABLE 8 Number of Bolls per 5 Plants at the Number 1 & 2
positions, and Total Number of Bolls from Whole Plants in
Non-irrigated Cotton Following 26 days of drought. Avg. # bolls in
Avg. # of the #1 & 2 Percent total bolls Percent Treatment
position difference per 5 plant difference MESSENGER .TM. 18.4 a
+46.0% 21.4 a +57.0% 2.2 OZ. Check 12.6 b 13.6 b -- Statistically P
= 0.032 P = 0.01 significant level *Same letter indicates no
statistical difference between the two treatments at the defined
level; different letter indicates a statistical difference between
the two treatments at the defined level.
Example 6
[0126] Hypersensitive Response Elicitor-Treated Tomato is More
Tolerant to Calcium Deficiency
[0127] Calcium is an important element for plant physiology and
development. A deficiency in calcium can cause several plant
diseases. For example, blossom-end rot is caused by a localized
calcium deficiency in the distal end of the tomato fruit. Because
calcium is not a highly mobile element, a deficiency can occur with
a fluctuation in water supply. In the past, tomato growers
experienced higher level of blossom-end rot during dry weather
conditions when infrequent rains storms dumped a lot of water and
then return to a hot and dry condition quickly. Lowering or raising
the irrigation water table erratically during a dry and hot growing
season can also increase the disease.
[0128] A field trial was designed to test if HR elicitor
protein-treated tomato can be more tolerant to the calcium
deficiency under a dry hot growing season. MESSENGER.TM., the
formulated product containing 3% HR elicitor, was used for the
trial. The application rate of the MESSENGER.TM. was 2.27 oz per
care. The first spray of MESSENGER.TM. was carried out 7 days
before the transplanting and then every 14-days after
transplanting. MESSENGER.TM.-treated tomatoes were compared with a
standard grower treatment not utilizing MESSENGER.TM.. Each
treatment had 4 replicates.
[0129] The number of infected fruit was counted from a 100 square
foot field. The rot typically begins with light tan water soaked
lesions, which then enlarge, and then turn black. In a survey,
about 20% of the fruits were infected. Severe end-rot symptoms
occurred in the standard treatment; however, an average of only
2.5% of the fruit was infected in the MESSENGER.TM.-treated plants.
The harvest data showed that MESSENGER.TM.-treated plants had 8%
more marketable fruit (Table 9). The test results demonstrated that
MESSENGER.TM.-treatment can reduce the stress resulting from
calcium deficiency and increase plant resistance to blossom-end
rot.
27TABLE 9 Hypersensitive Response Elicitor Treatment Reduced
Blossom-End Rot Infection, Increased Yield of Tomato Fruit
Blossom-End Infected Fruit* Tomato Fruit Yield Treatment Rep I Rep
II Rep III Rep IV Bin/Acre % Difference MESSENGER .TM. 0 9 0 1 35 8
Standard Treatment) 24 22 16 17 31.5 -- *The data were collected
from the fruits in 100 square foot plot
[0130] 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
18 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 1344 DNA
Erwinia amylovora 5 atgtcaattc ttacgcttaa caacaatacc tcgtcctcgc
cgggtctgtt ccagtccggg 60 ggggacaacg ggcttggtgg tcataatgca
aattctgcgt tggggcaaca acccatcgat 120 cggcaaacca ttgagcaaat
ggctcaatta ttggcggaac tgttaaagtc actgctatcg 180 ccacaatcag
gtaatgcggc aaccggagcc ggtggcaatg accagactac aggagttggt 240
aacgctggcg gcctgaacgg acgaaaaggc acagcaggaa ccactccgca gtctgacagt
300 cagaacatgc tgagtgagat gggcaacaac gggctggatc aggccatcac
gcccgatggc 360 cagggcggcg ggcagatcgg cgataatcct ttactgaaag
ccatgctgaa gcttattgca 420 cgcatgatgg acggccaaag cgatcagttt
ggccaacctg gtacgggcaa caacagtgcc 480 tcttccggta cttcttcatc
tggcggttcc ccttttaacg atctatcagg ggggaaggcc 540 ccttccggca
actccccttc cggcaactac tctcccgtca gtaccttctc acccccatcc 600
acgccaacgt cccctacctc accgcttgat ttcccttctt ctcccaccaa agcagccggg
660 ggcagcacgc cggtaaccga tcatcctgac cctgttggta gcgcgggcat
cggggccgga 720 aattcggtgg ccttcaccag cgccggcgct aatcagacgg
tgctgcatga caccattacc 780 gtgaaagcgg gtcaggtgtt tgatggcaaa
ggacaaacct tcaccgccgg ttcagaatta 840 ggcgatggcg gccagtctga
aaaccagaaa ccgctgttta tactggaaga cggtgccagc 900 ctgaaaaacg
tcaccatggg cgacgacggg gcggatggta ttcatcttta cggtgatgcc 960
aaaatagaca atctgcacgt caccaacgtg ggtgaggacg cgattaccgt taagccaaac
1020 agcgcgggca aaaaatccca cgttgaaatc actaacagtt ccttcgagca
cgcctctgac 1080 aagatcctgc agctgaatgc cgatactaac ctgagcgttg
acaacgtgaa ggccaaagac 1140 tttggtactt ttgtacgcac taacggcggt
caacagggta actgggatct gaatctgagc 1200 catatcagcg cagaagacgg
taagttctcg ttcgttaaaa gcgatagcga ggggctaaac 1260 gtcaatacca
gtgatatctc actgggtgat gttgaaaacc actacaaagt gccgatgtcc 1320
gccaacctga aggtggctga atga 1344 6 447 PRT Erwinia amylovora 6 Met
Ser Ile Leu Thr Leu Asn Asn Asn Thr Ser Ser Ser Pro Gly Leu 1 5 10
15 Phe Gln Ser Gly Gly Asp Asn Gly Leu Gly Gly His Asn Ala Asn Ser
20 25 30 Ala Leu Gly Gln Gln Pro Ile Asp Arg Gln Thr Ile Glu Gln
Met Ala 35 40 45 Gln Leu Leu Ala Glu Leu Leu Lys Ser Leu Leu Ser
Pro Gln Ser Gly 50 55 60 Asn Ala Ala Thr Gly Ala Gly Gly Asn Asp
Gln Thr Thr Gly Val Gly 65 70 75 80 Asn Ala Gly Gly Leu Asn Gly Arg
Lys Gly Thr Ala Gly Thr Thr Pro 85 90 95 Gln Ser Asp Ser Gln Asn
Met Leu Ser Glu Met Gly Asn Asn Gly Leu 100 105 110 Asp Gln Ala Ile
Thr Pro Asp Gly Gln Gly Gly Gly Gln Ile Gly Asp 115 120 125 Asn Pro
Leu Leu Lys Ala Met Leu Lys Leu Ile Ala Arg Met Met Asp 130 135 140
Gly Gln Ser Asp Gln Phe Gly Gln Pro Gly Thr Gly Asn Asn Ser Ala 145
150 155 160 Ser Ser Gly Thr Ser Ser Ser Gly Gly Ser Pro Phe Asn Asp
Leu Ser 165 170 175 Gly Gly Lys Ala Pro Ser Gly Asn Ser Pro Ser Gly
Asn Tyr Ser Pro 180 185 190 Val Ser Thr Phe Ser Pro Pro Ser Thr Pro
Thr Ser Pro Thr Ser Pro 195 200 205 Leu Asp Phe Pro Ser Ser Pro Thr
Lys Ala Ala Gly Gly Ser Thr Pro 210 215 220 Val Thr Asp His Pro Asp
Pro Val Gly Ser Ala Gly Ile Gly Ala Gly 225 230 235 240 Asn Ser Val
Ala Phe Thr Ser Ala Gly Ala Asn Gln Thr Val Leu His 245 250 255 Asp
Thr Ile Thr Val Lys Ala Gly Gln Val Phe Asp Gly Lys Gly Gln 260 265
270 Thr Phe Thr Ala Gly Ser Glu Leu Gly Asp Gly Gly Gln Ser Glu Asn
275 280 285 Gln Lys Pro Leu Phe Ile Leu Glu Asp Gly Ala Ser Leu Lys
Asn Val 290 295 300 Thr Met Gly Asp Asp Gly Ala Asp Gly Ile His Leu
Tyr Gly Asp Ala 305 310 315 320 Lys Ile Asp Asn Leu His Val Thr Asn
Val Gly Glu Asp Ala Ile Thr 325 330 335 Val Lys Pro Asn Ser Ala Gly
Lys Lys Ser His Val Glu Ile Thr Asn 340 345 350 Ser Ser Phe Glu His
Ala Ser Asp Lys Ile Leu Gln Leu Asn Ala Asp 355 360 365 Thr Asn Leu
Ser Val Asp Asn Val Lys Ala Lys Asp Phe Gly Thr Phe 370 375 380 Val
Arg Thr Asn Gly Gly Gln Gln Gly Asn Trp Asp Leu Asn Leu Ser 385 390
395 400 His Ile Ser Ala Glu Asp Gly Lys Phe Ser Phe Val Lys Ser Asp
Ser 405 410 415 Glu Gly Leu Asn Val Asn Thr Ser Asp Ile Ser Leu Gly
Asp Val Glu 420 425 430 Asn His Tyr Lys Val Pro Met Ser Ala Asn Leu
Lys Val Ala Glu 435 440 445 7 5517 DNA Erwinia amylovora 7
atggaattaa aatcactggg aactgaacac aaggcggcag tacacacagc ggcgcacaac
60 cctgtggggc atggtgttgc cttacagcag ggcagcagca gcagcagccc
gcaaaatgcc 120 gctgcatcat tggcggcaga aggcaaaaat cgtgggaaaa
tgccgagaat tcaccagcca 180 tctactgcgg ctgatggtat cagcgctgct
caccagcaaa agaaatcctt cagtctcagg 240 ggctgtttgg ggacgaaaaa
attttccaga tcggcaccgc agggccagcc aggtaccacc 300 cacagcaaag
gggcaacatt gcgcgatctg ctggcgcggg acgacggcga aacgcagcat 360
gaggcggccg cgccagatgc ggcgcgtttg acccgttcgg gcggcgtcaa acgccgcaat
420 atggacgaca tggccgggcg gccaatggtg aaaggtggca gcggcgaaga
taaggtacca 480 acgcagcaaa aacggcatca gctgaacaat tttggccaga
tgcgccaaac gatgttgagc 540 aaaatggctc acccggcttc agccaacgcc
ggcgatcgcc tgcagcattc accgccgcac 600 atcccgggta gccaccacga
aatcaaggaa gaaccggttg gctccaccag caaggcaaca 660 acggcccacg
cagacagagt ggaaatcgct caggaagatg acgacagcga attccagcaa 720
ctgcatcaac agcggctggc gcgcgaacgg gaaaatccac cgcagccgcc caaactcggc
780 gttgccacac cgattagcgc caggtttcag cccaaactga ctgcggttgc
ggaaagcgtc 840 cttgagggga cagataccac gcagtcaccc cttaagccgc
aatcaatgct gaaaggaagt 900 ggagccgggg taacgccgct ggcggtaacg
ctggataaag gcaagttgca gctggcaccg 960 gataatccac ccgcgctcaa
tacgttgttg aagcagacat tgggtaaaga cacccagcac 1020 tatctggcgc
accatgccag cagcgacggt agccagcatc tgctgctgga caacaaaggc 1080
cacctgtttg atatcaaaag caccgccacc agctatagcg tgctgcacaa cagccacccc
1140 ggtgagataa agggcaagct ggcgcaggcg ggtactggct ccgtcagcgt
agacggtaaa 1200 agcggcaaga tctcgctggg gagcggtacg caaagtcaca
acaaaacaat gctaagccaa 1260 ccgggggaag cgcaccgttc cttattaacc
ggcatttggc agcatcctgc tggcgcagcg 1320 cggccgcagg gcgagtcaat
ccgcctgcat gacgacaaaa ttcatatcct gcatccggag 1380 ctgggcgtat
ggcaatctgc ggataaagat acccacagcc agctgtctcg ccaggcagac 1440
ggtaagctct atgcgctgaa agacaaccgt accctgcaaa acctctccga taataaatcc
1500 tcagaaaagc tggtcgataa aatcaaatcg tattccgttg atcagcgggg
gcaggtggcg 1560 atcctgacgg atactcccgg ccgccataag atgagtatta
tgccctcgct ggatgcttcc 1620 ccggagagcc atatttccct cagcctgcat
tttgccgatg cccaccaggg gttattgcac 1680 gggaagtcgg agcttgaggc
acaatctgtc gcgatcagcc atgggcgact ggttgtggcc 1740 gatagcgaag
gcaagctgtt tagcgccgcc attccgaagc aaggggatgg aaacgaactg 1800
aaaatgaaag ccatgcctca gcatgcgctc gatgaacatt ttggtcatga ccaccagatt
1860 tctggatttt tccatgacga ccacggccag cttaatgcgc tggtgaaaaa
taacttcagg 1920 cagcagcatg cctgcccgtt gggtaacgat catcagtttc
accccggctg gaacctgact 1980 gatgcgctgg ttatcgacaa tcagctgggg
ctgcatcata ccaatcctga accgcatgag 2040 attcttgata tggggcattt
aggcagcctg gcgttacagg agggcaagct tcactatttt 2100 gaccagctga
ccaaagggtg gactggcgcg gagtcagatt gtaagcagct gaaaaaaggc 2160
ctggatggag cagcttatct actgaaagac ggtgaagtga aacgcctgaa tattaatcag
2220 agcacctcct ctatcaagca cggaacggaa aacgtttttt cgctgccgca
tgtgcgcaat 2280 aaaccggagc cgggagatgc cctgcaaggg ctgaataaag
acgataaggc ccaggccatg 2340 gcggtgattg gggtaaataa atacctggcg
ctgacggaaa aaggggacat tcgctccttc 2400 cagataaaac ccggcaccca
gcagttggag cggccggcac aaactctcag ccgcgaaggt 2460 atcagcggcg
aactgaaaga cattcatgtc gaccacaagc agaacctgta tgccttgacc 2520
cacgagggag aggtgtttca tcagccgcgt gaagcctggc agaatggtgc cgaaagcagc
2580 agctggcaca aactggcgtt gccacagagt gaaagtaagc taaaaagtct
ggacatgagc 2640 catgagcaca aaccgattgc cacctttgaa gacggtagcc
agcatcagct gaaggctggc 2700 ggctggcacg
cctatgcggc acctgaacgc gggccgctgg cggtgggtac cagcggttca 2760
caaaccgtct ttaaccgact aatgcagggg gtgaaaggca aggtgatccc aggcagcggg
2820 ttgacggtta agctctcggc tcagacgggg ggaatgaccg gcgccgaagg
gcgcaaggtc 2880 agcagtaaat tttccgaaag gatccgcgcc tatgcgttca
acccaacaat gtccacgccg 2940 cgaccgatta aaaatgctgc ttatgccaca
cagcacggct ggcaggggcg tgaggggttg 3000 aagccgttgt acgagatgca
gggagcgctg attaaacaac tggatgcgca taacgttcgt 3060 cataacgcgc
cacagccaga tttgcagagc aaactggaaa ctctggattt aggcgaacat 3120
ggcgcagaat tgcttaacga catgaagcgc ttccgcgacg aactggagca gagtgcaacc
3180 cgttcggtga ccgttttagg tcaacatcag ggagtgctaa aaagcaacgg
tgaaatcaat 3240 agcgaattta agccatcgcc cggcaaggcg ttggtccaga
gctttaacgt caatcgctct 3300 ggtcaggatc taagcaagtc actgcaacag
gcagtacatg ccacgccgcc atccgcagag 3360 agtaaactgc aatccatgct
ggggcacttt gtcagtgccg gggtggatat gagtcatcag 3420 aagggcgaga
tcccgctggg ccgccagcgc gatccgaatg ataaaaccgc actgaccaaa 3480
tcgcgtttaa ttttagatac cgtgaccatc ggtgaactgc atgaactggc cgataaggcg
3540 aaactggtat ctgaccataa acccgatgcc gatcagataa aacagctgcg
ccagcagttc 3600 gatacgctgc gtgaaaagcg gtatgagagc aatccggtga
agcattacac cgatatgggc 3660 ttcacccata ataaggcgct ggaagcaaac
tatgatgcgg tcaaagcctt tatcaatgcc 3720 tttaagaaag agcaccacgg
cgtcaatctg accacgcgta ccgtactgga atcacagggc 3780 agtgcggagc
tggcgaagaa gctcaagaat acgctgttgt ccctggacag tggtgaaagt 3840
atgagcttca gccggtcata tggcgggggc gtcagcactg tctttgtgcc tacccttagc
3900 aagaaggtgc cagttccggt gatccccgga gccggcatca cgctggatcg
cgcctataac 3960 ctgagcttca gtcgtaccag cggcggattg aacgtcagtt
ttggccgcga cggcggggtg 4020 agtggtaaca tcatggtcgc taccggccat
gatgtgatgc cctatatgac cggtaagaaa 4080 accagtgcag gtaacgccag
tgactggttg agcgcaaaac ataaaatcag cccggacttg 4140 cgtatcggcg
ctgctgtgag tggcaccctg caaggaacgc tacaaaacag cctgaagttt 4200
aagctgacag aggatgagct gcctggcttt atccatggct tgacgcatgg cacgttgacc
4260 ccggcagaac tgttgcaaaa ggggatcgaa catcagatga agcagggcag
caaactgacg 4320 tttagcgtcg atacctcggc aaatctggat ctgcgtgccg
gtatcaatct gaacgaagac 4380 ggcagtaaac caaatggtgt cactgcccgt
gtttctgccg ggctaagtgc atcggcaaac 4440 ctggccgccg gctcgcgtga
acgcagcacc acctctggcc agtttggcag cacgacttcg 4500 gccagcaata
accgcccaac cttcctcaac ggggtcggcg cgggtgctaa cctgacggct 4560
gctttagggg ttgcccattc atctacgcat gaagggaaac cggtcgggat cttcccggca
4620 tttacctcga ccaatgtttc ggcagcgctg gcgctggata accgtacctc
acagagtatc 4680 agcctggaat tgaagcgcgc ggagccggtg accagcaacg
atatcagcga gttgacctcc 4740 acgctgggaa aacactttaa ggatagcgcc
acaacgaaga tgcttgccgc tctcaaagag 4800 ttagatgacg ctaagcccgc
tgaacaactg catattttac agcagcattt cagtgcaaaa 4860 gatgtcgtcg
gtgatgaacg ctacgaggcg gtgcgcaacc tgaaaaaact ggtgatacgt 4920
caacaggctg cggacagcca cagcatggaa ttaggatctg ccagtcacag cacgacctac
4980 aataatctgt cgagaataaa taatgacggc attgtcgagc tgctacacaa
acatttcgat 5040 gcggcattac cagcaagcag tgccaaacgt cttggtgaaa
tgatgaataa cgatccggca 5100 ctgaaagata ttattaagca gctgcaaagt
acgccgttca gcagcgccag cgtgtcgatg 5160 gagctgaaag atggtctgcg
tgagcagacg gaaaaagcaa tactggacgg taaggtcggt 5220 cgtgaagaag
tgggagtact tttccaggat cgtaacaact tgcgtgttaa atcggtcagc 5280
gtcagtcagt ccgtcagcaa aagcgaaggc ttcaataccc cagcgctgtt actggggacg
5340 agcaacagcg ctgctatgag catggagcgc aacatcggaa ccattaattt
taaatacggc 5400 caggatcaga acaccccacg gcgatttacc ctggagggtg
gaatagctca ggctaatccg 5460 caggtcgcat ctgcgcttac tgatttgaag
aaggaagggc tggaaatgaa gagctaa 5517 8 1838 PRT Erwinia amylovora 8
Met Glu Leu Lys Ser Leu Gly Thr Glu His Lys Ala Ala Val His Thr 1 5
10 15 Ala Ala His Asn Pro Val Gly His Gly Val Ala Leu Gln Gln Gly
Ser 20 25 30 Ser Ser Ser Ser Pro Gln Asn Ala Ala Ala Ser Leu Ala
Ala Glu Gly 35 40 45 Lys Asn Arg Gly Lys Met Pro Arg Ile His Gln
Pro Ser Thr Ala Ala 50 55 60 Asp Gly Ile Ser Ala Ala His Gln Gln
Lys Lys Ser Phe Ser Leu Arg 65 70 75 80 Gly Cys Leu Gly Thr Lys Lys
Phe Ser Arg Ser Ala Pro Gln Gly Gln 85 90 95 Pro Gly Thr Thr His
Ser Lys Gly Ala Thr Leu Arg Asp Leu Leu Ala 100 105 110 Arg Asp Asp
Gly Glu Thr Gln His Glu Ala Ala Ala Pro Asp Ala Ala 115 120 125 Arg
Leu Thr Arg Ser Gly Gly Val Lys Arg Arg Asn Met Asp Asp Met 130 135
140 Ala Gly Arg Pro Met Val Lys Gly Gly Ser Gly Glu Asp Lys Val Pro
145 150 155 160 Thr Gln Gln Lys Arg His Gln Leu Asn Asn Phe Gly Gln
Met Arg Gln 165 170 175 Thr Met Leu Ser Lys Met Ala His Pro Ala Ser
Ala Asn Ala Gly Asp 180 185 190 Arg Leu Gln His Ser Pro Pro His Ile
Pro Gly Ser His His Glu Ile 195 200 205 Lys Glu Glu Pro Val Gly Ser
Thr Ser Lys Ala Thr Thr Ala His Ala 210 215 220 Asp Arg Val Glu Ile
Ala Gln Glu Asp Asp Asp Ser Glu Phe Gln Gln 225 230 235 240 Leu His
Gln Gln Arg Leu Ala Arg Glu Arg Glu Asn Pro Pro Gln Pro 245 250 255
Pro Lys Leu Gly Val Ala Thr Pro Ile Ser Ala Arg Phe Gln Pro Lys 260
265 270 Leu Thr Ala Val Ala Glu Ser Val Leu Glu Gly Thr Asp Thr Thr
Gln 275 280 285 Ser Pro Leu Lys Pro Gln Ser Met Leu Lys Gly Ser Gly
Ala Gly Val 290 295 300 Thr Pro Leu Ala Val Thr Leu Asp Lys Gly Lys
Leu Gln Leu Ala Pro 305 310 315 320 Asp Asn Pro Pro Ala Leu Asn Thr
Leu Leu Lys Gln Thr Leu Gly Lys 325 330 335 Asp Thr Gln His Tyr Leu
Ala His His Ala Ser Ser Asp Gly Ser Gln 340 345 350 His Leu Leu Leu
Asp Asn Lys Gly His Leu Phe Asp Ile Lys Ser Thr 355 360 365 Ala Thr
Ser Tyr Ser Val Leu His Asn Ser His Pro Gly Glu Ile Lys 370 375 380
Gly Lys Leu Ala Gln Ala Gly Thr Gly Ser Val Ser Val Asp Gly Lys 385
390 395 400 Ser Gly Lys Ile Ser Leu Gly Ser Gly Thr Gln Ser His Asn
Lys Thr 405 410 415 Met Leu Ser Gln Pro Gly Glu Ala His Arg Ser Leu
Leu Thr Gly Ile 420 425 430 Trp Gln His Pro Ala Gly Ala Ala Arg Pro
Gln Gly Glu Ser Ile Arg 435 440 445 Leu His Asp Asp Lys Ile His Ile
Leu His Pro Glu Leu Gly Val Trp 450 455 460 Gln Ser Ala Asp Lys Asp
Thr His Ser Gln Leu Ser Arg Gln Ala Asp 465 470 475 480 Gly Lys Leu
Tyr Ala Leu Lys Asp Asn Arg Thr Leu Gln Asn Leu Ser 485 490 495 Asp
Asn Lys Ser Ser Glu Lys Leu Val Asp Lys Ile Lys Ser Tyr Ser 500 505
510 Val Asp Gln Arg Gly Gln Val Ala Ile Leu Thr Asp Thr Pro Gly Arg
515 520 525 His Lys Met Ser Ile Met Pro Ser Leu Asp Ala Ser Pro Glu
Ser His 530 535 540 Ile Ser Leu Ser Leu His Phe Ala Asp Ala His Gln
Gly Leu Leu His 545 550 555 560 Gly Lys Ser Glu Leu Glu Ala Gln Ser
Val Ala Ile Ser His Gly Arg 565 570 575 Leu Val Val Ala Asp Ser Glu
Gly Lys Leu Phe Ser Ala Ala Ile Pro 580 585 590 Lys Gln Gly Asp Gly
Asn Glu Leu Lys Met Lys Ala Met Pro Gln His 595 600 605 Ala Leu Asp
Glu His Phe Gly His Asp His Gln Ile Ser Gly Phe Phe 610 615 620 His
Asp Asp His Gly Gln Leu Asn Ala Leu Val Lys Asn Asn Phe Arg 625 630
635 640 Gln Gln His Ala Cys Pro Leu Gly Asn Asp His Gln Phe His Pro
Gly 645 650 655 Trp Asn Leu Thr Asp Ala Leu Val Ile Asp Asn Gln Leu
Gly Leu His 660 665 670 His Thr Asn Pro Glu Pro His Glu Ile Leu Asp
Met Gly His Leu Gly 675 680 685 Ser Leu Ala Leu Gln Glu Gly Lys Leu
His Tyr Phe Asp Gln Leu Thr 690 695 700 Lys Gly Trp Thr Gly Ala Glu
Ser Asp Cys Lys Gln Leu Lys Lys Gly 705 710 715 720 Leu Asp Gly Ala
Ala Tyr Leu Leu Lys Asp Gly Glu Val Lys Arg Leu 725 730 735 Asn Ile
Asn Gln Ser Thr Ser Ser Ile Lys His Gly Thr Glu Asn Val 740 745 750
Phe Ser Leu Pro His Val Arg Asn Lys Pro Glu Pro Gly Asp Ala Leu 755
760 765 Gln Gly Leu Asn Lys Asp Asp Lys Ala Gln Ala Met Ala Val Ile
Gly 770 775 780 Val Asn Lys Tyr Leu Ala Leu Thr Glu Lys Gly Asp Ile
Arg Ser Phe 785 790 795 800 Gln Ile Lys Pro Gly Thr Gln Gln Leu Glu
Arg Pro Ala Gln Thr Leu 805 810 815 Ser Arg Glu Gly Ile Ser Gly Glu
Leu Lys Asp Ile His Val Asp His 820 825 830 Lys Gln Asn Leu Tyr Ala
Leu Thr His Glu Gly Glu Val Phe His Gln 835 840 845 Pro Arg Glu Ala
Trp Gln Asn Gly Ala Glu Ser Ser Ser Trp His Lys 850 855 860 Leu Ala
Leu Pro Gln Ser Glu Ser Lys Leu Lys Ser Leu Asp Met Ser 865 870 875
880 His Glu His Lys Pro Ile Ala Thr Phe Glu Asp Gly Ser Gln His Gln
885 890 895 Leu Lys Ala Gly Gly Trp His Ala Tyr Ala Ala Pro Glu Arg
Gly Pro 900 905 910 Leu Ala Val Gly Thr Ser Gly Ser Gln Thr Val Phe
Asn Arg Leu Met 915 920 925 Gln Gly Val Lys Gly Lys Val Ile Pro Gly
Ser Gly Leu Thr Val Lys 930 935 940 Leu Ser Ala Gln Thr Gly Gly Met
Thr Gly Ala Glu Gly Arg Lys Val 945 950 955 960 Ser Ser Lys Phe Ser
Glu Arg Ile Arg Ala Tyr Ala Phe Asn Pro Thr 965 970 975 Met Ser Thr
Pro Arg Pro Ile Lys Asn Ala Ala Tyr Ala Thr Gln His 980 985 990 Gly
Trp Gln Gly Arg Glu Gly Leu Lys Pro Leu Tyr Glu Met Gln Gly 995
1000 1005 Ala Leu Ile Lys Gln Leu Asp Ala His Asn Val Arg His Asn
Ala Pro 1010 1015 1020 Gln Pro Asp Leu Gln Ser Lys Leu Glu Thr Leu
Asp Leu Gly Glu His 1025 1030 1035 1040 Gly Ala Glu Leu Leu Asn Asp
Met Lys Arg Phe Arg Asp Glu Leu Glu 1045 1050 1055 Gln Ser Ala Thr
Arg Ser Val Thr Val Leu Gly Gln His Gln Gly Val 1060 1065 1070 Leu
Lys Ser Asn Gly Glu Ile Asn Ser Glu Phe Lys Pro Ser Pro Gly 1075
1080 1085 Lys Ala Leu Val Gln Ser Phe Asn Val Asn Arg Ser Gly Gln
Asp Leu 1090 1095 1100 Ser Lys Ser Leu Gln Gln Ala Val His Ala Thr
Pro Pro Ser Ala Glu 1105 1110 1115 1120 Ser Lys Leu Gln Ser Met Leu
Gly His Phe Val Ser Ala Gly Val Asp 1125 1130 1135 Met Ser His Gln
Lys Gly Glu Ile Pro Leu Gly Arg Gln Arg Asp Pro 1140 1145 1150 Asn
Asp Lys Thr Ala Leu Thr Lys Ser Arg Leu Ile Leu Asp Thr Val 1155
1160 1165 Thr Ile Gly Glu Leu His Glu Leu Ala Asp Lys Ala Lys Leu
Val Ser 1170 1175 1180 Asp His Lys Pro Asp Ala Asp Gln Ile Lys Gln
Leu Arg Gln Gln Phe 1185 1190 1195 1200 Asp Thr Leu Arg Glu Lys Arg
Tyr Glu Ser Asn Pro Val Lys His Tyr 1205 1210 1215 Thr Asp Met Gly
Phe Thr His Asn Lys Ala Leu Glu Ala Asn Tyr Asp 1220 1225 1230 Ala
Val Lys Ala Phe Ile Asn Ala Phe Lys Lys Glu His His Gly Val 1235
1240 1245 Asn Leu Thr Thr Arg Thr Val Leu Glu Ser Gln Gly Ser Ala
Glu Leu 1250 1255 1260 Ala Lys Lys Leu Lys Asn Thr Leu Leu Ser Leu
Asp Ser Gly Glu Ser 1265 1270 1275 1280 Met Ser Phe Ser Arg Ser Tyr
Gly Gly Gly Val Ser Thr Val Phe Val 1285 1290 1295 Pro Thr Leu Ser
Lys Lys Val Pro Val Pro Val Ile Pro Gly Ala Gly 1300 1305 1310 Ile
Thr Leu Asp Arg Ala Tyr Asn Leu Ser Phe Ser Arg Thr Ser Gly 1315
1320 1325 Gly Leu Asn Val Ser Phe Gly Arg Asp Gly Gly Val Ser Gly
Asn Ile 1330 1335 1340 Met Val Ala Thr Gly His Asp Val Met Pro Tyr
Met Thr Gly Lys Lys 1345 1350 1355 1360 Thr Ser Ala Gly Asn Ala Ser
Asp Trp Leu Ser Ala Lys His Lys Ile 1365 1370 1375 Ser Pro Asp Leu
Arg Ile Gly Ala Ala Val Ser Gly Thr Leu Gln Gly 1380 1385 1390 Thr
Leu Gln Asn Ser Leu Lys Phe Lys Leu Thr Glu Asp Glu Leu Pro 1395
1400 1405 Gly Phe Ile His Gly Leu Thr His Gly Thr Leu Thr Pro Ala
Glu Leu 1410 1415 1420 Leu Gln Lys Gly Ile Glu His Gln Met Lys Gln
Gly Ser Lys Leu Thr 1425 1430 1435 1440 Phe Ser Val Asp Thr Ser Ala
Asn Leu Asp Leu Arg Ala Gly Ile Asn 1445 1450 1455 Leu Asn Glu Asp
Gly Ser Lys Pro Asn Gly Val Thr Ala Arg Val Ser 1460 1465 1470 Ala
Gly Leu Ser Ala Ser Ala Asn Leu Ala Ala Gly Ser Arg Glu Arg 1475
1480 1485 Ser Thr Thr Ser Gly Gln Phe Gly Ser Thr Thr Ser Ala Ser
Asn Asn 1490 1495 1500 Arg Pro Thr Phe Leu Asn Gly Val Gly Ala Gly
Ala Asn Leu Thr Ala 1505 1510 1515 1520 Ala Leu Gly Val Ala His Ser
Ser Thr His Glu Gly Lys Pro Val Gly 1525 1530 1535 Ile Phe Pro Ala
Phe Thr Ser Thr Asn Val Ser Ala Ala Leu Ala Leu 1540 1545 1550 Asp
Asn Arg Thr Ser Gln Ser Ile Ser Leu Glu Leu Lys Arg Ala Glu 1555
1560 1565 Pro Val Thr Ser Asn Asp Ile Ser Glu Leu Thr Ser Thr Leu
Gly Lys 1570 1575 1580 His Phe Lys Asp Ser Ala Thr Thr Lys Met Leu
Ala Ala Leu Lys Glu 1585 1590 1595 1600 Leu Asp Asp Ala Lys Pro Ala
Glu Gln Leu His Ile Leu Gln Gln His 1605 1610 1615 Phe Ser Ala Lys
Asp Val Val Gly Asp Glu Arg Tyr Glu Ala Val Arg 1620 1625 1630 Asn
Leu Lys Lys Leu Val Ile Arg Gln Gln Ala Ala Asp Ser His Ser 1635
1640 1645 Met Glu Leu Gly Ser Ala Ser His Ser Thr Thr Tyr Asn Asn
Leu Ser 1650 1655 1660 Arg Ile Asn Asn Asp Gly Ile Val Glu Leu Leu
His Lys His Phe Asp 1665 1670 1675 1680 Ala Ala Leu Pro Ala Ser Ser
Ala Lys Arg Leu Gly Glu Met Met Asn 1685 1690 1695 Asn Asp Pro Ala
Leu Lys Asp Ile Ile Lys Gln Leu Gln Ser Thr Pro 1700 1705 1710 Phe
Ser Ser Ala Ser Val Ser Met Glu Leu Lys Asp Gly Leu Arg Glu 1715
1720 1725 Gln Thr Glu Lys Ala Ile Leu Asp Gly Lys Val Gly Arg Glu
Glu Val 1730 1735 1740 Gly Val Leu Phe Gln Asp Arg Asn Asn Leu Arg
Val Lys Ser Val Ser 1745 1750 1755 1760 Val Ser Gln Ser Val Ser Lys
Ser Glu Gly Phe Asn Thr Pro Ala Leu 1765 1770 1775 Leu Leu Gly Thr
Ser Asn Ser Ala Ala Met Ser Met Glu Arg Asn Ile 1780 1785 1790 Gly
Thr Ile Asn Phe Lys Tyr Gly Gln Asp Gln Asn Thr Pro Arg Arg 1795
1800 1805 Phe Thr Leu Glu Gly Gly Ile Ala Gln Ala Asn Pro Gln Val
Ala Ser 1810 1815 1820 Ala Leu Thr Asp Leu Lys Lys Glu Gly Leu Glu
Met Lys Ser 1825 1830 1835 9 420 DNA Erwinia amylovora 9 atgacatcgt
cacagcagcg ggttgaaagg tttttacagt atttctccgc cgggtgtaaa 60
acgcccatac atctgaaaga cggggtgtgc gccctgtata acgaacaaga tgaggaggcg
120 gcggtgctgg aagtaccgca acacagcgac agcctgttac tacactgccg
aatcattgag 180 gctgacccac aaacttcaat aaccctgtat tcgatgctat
tacagctgaa ttttgaaatg 240 gcggccatgc gcggctgttg gctggcgctg
gatgaactgc acaacgtgcg tttatgtttt 300 cagcagtcgc tggagcatct
ggatgaagca agttttagcg atatcgttag cggcttcatc 360 gaacatgcgg
cagaagtgcg tgagtatata gcgcaattag acgagagtag cgcggcataa 420 10 139
PRT Erwinia amylovora 10 Met Thr Ser Ser Gln Gln Arg Val Glu Arg
Phe Leu Gln Tyr Phe Ser 1 5 10 15 Ala Gly Cys Lys Thr Pro Ile His
Leu Lys Asp Gly Val Cys Ala Leu 20 25 30 Tyr Asn Glu Gln Asp Glu
Glu Ala Ala Val Leu Glu Val Pro Gln His 35 40 45 Ser Asp Ser Leu
Leu Leu His Cys Arg Ile Ile Glu Ala Asp Pro Gln 50 55
60 Thr Ser Ile Thr Leu Tyr Ser Met Leu Leu Gln Leu Asn Phe Glu Met
65 70 75 80 Ala Ala Met Arg Gly Cys Trp Leu Ala Leu Asp Glu Leu His
Asn Val 85 90 95 Arg Leu Cys Phe Gln Gln Ser Leu Glu His Leu Asp
Glu Ala Ser Phe 100 105 110 Ser Asp Ile Val Ser Gly Phe Ile Glu His
Ala Ala Glu Val Arg Glu 115 120 125 Tyr Ile Ala Gln Leu Asp Glu Ser
Ser Ala Ala 130 135 11 341 PRT Pseudomonas syringae 11 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 12 1026 DNA
Pseudomonas syringae 12 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 13 1729 DNA Pseudomonas syringae 13 tccacttcgc
tgattttgaa attggcagat tcatagaaac gttcaggtgt ggaaatcagg 60
ctgagtgcgc agatttcgtt gataagggtg tggtactggt cattgttggt catttcaagg
120 cctctgagtg cggtgcggag caataccagt cttcctgctg gcgtgtgcac
actgagtcgc 180 aggcataggc atttcagttc cttgcgttgg ttgggcatat
aaaaaaagga acttttaaaa 240 acagtgcaat gagatgccgg caaaacggga
accggtcgct gcgctttgcc actcacttcg 300 agcaagctca accccaaaca
tccacatccc tatcgaacgg acagcgatac ggccacttgc 360 tctggtaaac
cctggagctg gcgtcggtcc aattgcccac ttagcgaggt aacgcagcat 420
gagcatcggc atcacacccc ggccgcaaca gaccaccacg ccactcgatt tttcggcgct
480 aagcggcaag agtcctcaac caaacacgtt cggcgagcag aacactcagc
aagcgatcga 540 cccgagtgca ctgttgttcg gcagcgacac acagaaagac
gtcaacttcg gcacgcccga 600 cagcaccgtc cagaatccgc aggacgccag
caagcccaac gacagccagt ccaacatcgc 660 taaattgatc agtgcattga
tcatgtcgtt gctgcagatg ctcaccaact ccaataaaaa 720 gcaggacacc
aatcaggaac agcctgatag ccaggctcct ttccagaaca acggcgggct 780
cggtacaccg tcggccgata gcgggggcgg cggtacaccg gatgcgacag gtggcggcgg
840 cggtgatacg ccaagcgcaa caggcggtgg cggcggtgat actccgaccg
caacaggcgg 900 tggcggcagc ggtggcggcg gcacacccac tgcaacaggt
ggcggcagcg gtggcacacc 960 cactgcaaca ggcggtggcg agggtggcgt
aacaccgcaa atcactccgc agttggccaa 1020 ccctaaccgt acctcaggta
ctggctcggt gtcggacacc gcaggttcta ccgagcaagc 1080 cggcaagatc
aatgtggtga aagacaccat caaggtcggc gctggcgaag tctttgacgg 1140
ccacggcgca accttcactg ccgacaaatc tatgggtaac ggagaccagg gcgaaaatca
1200 gaagcccatg ttcgagctgg ctgaaggcgc tacgttgaag aatgtgaacc
tgggtgagaa 1260 cgaggtcgat ggcatccacg tgaaagccaa aaacgctcag
gaagtcacca ttgacaacgt 1320 gcatgcccag aacgtcggtg aagacctgat
tacggtcaaa ggcgagggag gcgcagcggt 1380 cactaatctg aacatcaaga
acagcagtgc caaaggtgca gacgacaagg ttgtccagct 1440 caacgccaac
actcacttga aaatcgacaa cttcaaggcc gacgatttcg gcacgatggt 1500
tcgcaccaac ggtggcaagc agtttgatga catgagcatc gagctgaacg gcatcgaagc
1560 taaccacggc aagttcgccc tggtgaaaag cgacagtgac gatctgaagc
tggcaacggg 1620 caacatcgcc atgaccgacg tcaaacacgc ctacgataaa
acccaggcat cgacccaaca 1680 caccgagctt tgaatccaga caagtagctt
gaaaaaaggg ggtggactc 1729 14 424 PRT Pseudomonas syringae 14 Met
Ser Ile Gly Ile Thr Pro Arg Pro Gln Gln Thr Thr Thr Pro Leu 1 5 10
15 Asp Phe Ser Ala Leu Ser Gly Lys Ser Pro Gln Pro Asn Thr Phe Gly
20 25 30 Glu Gln Asn Thr Gln Gln Ala Ile Asp Pro Ser Ala Leu Leu
Phe Gly 35 40 45 Ser Asp Thr Gln Lys Asp Val Asn Phe Gly Thr Pro
Asp Ser Thr Val 50 55 60 Gln Asn Pro Gln Asp Ala Ser Lys Pro Asn
Asp Ser Gln Ser Asn Ile 65 70 75 80 Ala Lys Leu Ile Ser Ala Leu Ile
Met Ser Leu Leu Gln Met Leu Thr 85 90 95 Asn Ser Asn Lys Lys Gln
Asp Thr Asn Gln Glu Gln Pro Asp Ser Gln 100 105 110 Ala Pro Phe Gln
Asn Asn Gly Gly Leu Gly Thr Pro Ser Ala Asp Ser 115 120 125 Gly Gly
Gly Gly Thr Pro Asp Ala Thr Gly Gly Gly Gly Gly Asp Thr 130 135 140
Pro Ser Ala Thr Gly Gly Gly Gly Gly Asp Thr Pro Thr Ala Thr Gly 145
150 155 160 Gly Gly Gly Ser Gly Gly Gly Gly Thr Pro Thr Ala Thr Gly
Gly Gly 165 170 175 Ser Gly Gly Thr Pro Thr Ala Thr Gly Gly Gly Glu
Gly Gly Val Thr 180 185 190 Pro Gln Ile Thr Pro Gln Leu Ala Asn Pro
Asn Arg Thr Ser Gly Thr 195 200 205 Gly Ser Val Ser Asp Thr Ala Gly
Ser Thr Glu Gln Ala Gly Lys Ile 210 215 220 Asn Val Val Lys Asp Thr
Ile Lys Val Gly Ala Gly Glu Val Phe Asp 225 230 235 240 Gly His Gly
Ala Thr Phe Thr Ala Asp Lys Ser Met Gly Asn Gly Asp 245 250 255 Gln
Gly Glu Asn Gln Lys Pro Met Phe Glu Leu Ala Glu Gly Ala Thr 260 265
270 Leu Lys Asn Val Asn Leu Gly Glu Asn Glu Val Asp Gly Ile His Val
275 280 285 Lys Ala Lys Asn Ala Gln Glu Val Thr Ile Asp Asn Val His
Ala Gln 290 295 300 Asn Val Gly Glu Asp Leu Ile Thr Val Lys Gly Glu
Gly Gly Ala Ala 305 310 315 320 Val Thr Asn Leu Asn Ile Lys Asn Ser
Ser Ala Lys Gly Ala Asp Asp 325 330 335 Lys Val Val Gln Leu Asn Ala
Asn Thr His Leu Lys Ile Asp Asn Phe 340 345 350 Lys Ala Asp Asp Phe
Gly Thr Met Val Arg Thr Asn Gly Gly Lys Gln 355 360 365 Phe Asp Asp
Met Ser Ile Glu Leu Asn Gly Ile Glu Ala Asn His Gly 370 375 380 Lys
Phe Ala Leu Val Lys Ser Asp Ser Asp Asp Leu Lys Leu Ala Thr 385 390
395 400 Gly Asn Ile Ala Met Thr Asp Val Lys His Ala Tyr Asp Lys Thr
Gln 405 410 415 Ala Ser Thr Gln His Thr Glu Leu 420 15 344 PRT
Pseudomonas solanacearum 15 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 16 1035 DNA Pseudomonas
solanacearum 16 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 17 26 PRT Xanthomonas campestris pv.
glycines 17 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 18
20 PRT Xanthomonas campestris pv. pelargonii 18 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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