U.S. patent application number 09/835684 was filed with the patent office on 2002-02-14 for treatment of fruits or vegetables with hypersensitive response elicitor to inhibit postharvest disease or desiccation.
Invention is credited to Qiu, Dewen, Remick, Dean, Wei, Zhong-Min.
Application Number | 20020019337 09/835684 |
Document ID | / |
Family ID | 22733063 |
Filed Date | 2002-02-14 |
United States Patent
Application |
20020019337 |
Kind Code |
A1 |
Wei, Zhong-Min ; et
al. |
February 14, 2002 |
Treatment of fruits or vegetables with hypersensitive response
elicitor to inhibit postharvest disease or desiccation
Abstract
The present invention relates to a methods of inhibiting
postharvest disease or desiccation in a fruit or vegetable, either
by treating a fruit or vegetable with a hypersensitive response
elicitor protein or polypeptide under conditions effective to
inhibit postharvest disease or desiccation, or by providing a
transgenic plant or plant seed transformed with a DNA molecule
encoding a hypersensitive response elicitor polypeptide or protein
and growing the transgenic plant or transgenic plant produced from
the transgenic plant seed under conditions effective to inhibit a
postharvest disease or desiccation in a fruit or vegetable
harvested from the transgenic plant. Also disclosed are DNA
constructs and expression systems, host cells, and transgenic
plants containing the DNA construct.
Inventors: |
Wei, Zhong-Min; (Kirkland,
WA) ; Qiu, Dewen; (Seattle, WA) ; Remick,
Dean; (Lake Placid, FL) |
Correspondence
Address: |
Michael L. Goldman
NIXON PEABODY LLP
Clinton Square
P.O. Box 31051
Rochester
NY
14603
US
|
Family ID: |
22733063 |
Appl. No.: |
09/835684 |
Filed: |
April 16, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60198359 |
Apr 19, 2000 |
|
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|
Current U.S.
Class: |
800/279 ;
514/1.1 |
Current CPC
Class: |
C07K 14/21 20130101;
C12N 15/8282 20130101; C07K 14/27 20130101; C12N 15/8249 20130101;
C12N 15/8273 20130101; A01N 63/50 20200101; A01N 37/46 20130101;
Y10S 435/849 20130101; A01N 63/50 20200101; A01N 63/27 20200101;
A01N 63/20 20200101 |
Class at
Publication: |
514/2 ;
800/279 |
International
Class: |
C12N 015/82; A01N
037/18 |
Claims
What is claimed:
1. A method of inhibiting postharvest disease or desiccation in a
fruit or vegetable, said method comprising: treating a fruit or
vegetable with a hypersensitive response elicitor protein or
polypeptide under conditions effective to inhibit postharvest
disease or desiccation.
2. The method according to claim 1, wherein hypersensitive response
elicitor protein or polypeptide is in isolated form.
3. The method according to claim 2, wherein said treating is
carried out prior to harvest of the fruit or vegetable.
4. The method according to claim 3, wherein said treating is
carried out by spraying the fruit or vegetable with the
hypersensitive response elicitor protein or polypeptide.
5. The method according to claim 4, wherein the hypersensitive
response elicitor protein or polypeptide is in liquid or powder
form.
6. The method according to claim 1, wherein said treating is
carried out after harvest of the fruit or vegetable.
7. The method according to claim 6, wherein said treating is
carried out by spraying the fruit or vegetable with the
hypersensitive response elicitor protein or polypeptide.
8. The method according to claim 7, wherein the hypersensitive
response elicitor protein or polypeptide is in liquid or powder
form.
9. The method according to claim 6, wherein said treating is
carried out by immersing the fruit or vegetable in the
hypersensitive response elicitor protein or polypeptide.
10. The method according to claim 1, wherein the hypersensitive
response elicitor protein or polypeptide is derived from a species
of pathogen selected from the group consisting of Erwinia,
Xanthomonas, Pseudomonas, Phytophthora, and Clavibacter.
11. The method according to claim 10, wherein the hypersensitive
response elicitor protein or polypeptide is derived from Erwinia
amylovora.
12. The method according to claim 10, wherein the hypersensitive
response elicitor protein or polypeptide is derived from Erwinia
carotovora.
13. The method according to claim 10, wherein the hypersensitive
response elicitor protein or polypeptide is derived from Erwinia
stewartii.
14. The method according to claim 10, wherein the hypersensitive
response elicitor protein or polypeptide is derived from Erwinia
chrysanthemi.
15. The method according to claim 10, wherein the hypersensitive
response elicitor protein or polypeptide is derived from
Pseudomonas syringae.
16. The method according to claim 10, wherein the hypersensitive
response elicitor protein or polypeptide is derived from
Pseudomonas solanacearum.
17. The method according to claim 1, wherein the hypersensitive
response elicitor protein or polypeptide is derived from a species
of Phytophthora.
18. The method according to claim 1, wherein said treating inhibits
desiccation in a fruit or vegetable.
19. The method according to claim 1, wherein said treating inhibits
a postharvest disease in a fruit or vegetable.
20. The method according to claim 19, wherein the postharvest
disease is caused by Penicillium, Botrytis, Phytophthora, or
Erwinia.
21. A method of inhibiting postharvest disease or desiccation in a
fruit or vegetable, said method comprising: providing a transgenic
plant or plant seed transformed with a DNA molecule encoding a
hypersensitive response elicitor polypeptide or protein and growing
the transgenic plant or transgenic plant produced from the
transgenic plant seed under conditions effective to inhibit a
postharvest disease or desiccation in a fruit or vegetable
harvested from the transgenic plant.
22. The method according to claim 21, wherein a transgenic plant is
provided.
23. The method according to claim 21, wherein a transgenic plant
seed is provided.
24. The method according to claim 21, wherein the transgenic plant
is a dicot or a monocot.
25. The method according to claim 21, further comprising: applying
the hypersensitive response elicitor polypeptide or protein to the
fruit or vegetable to inhibit postharvest disease or
desiccation.
26. The method according to claim 25, wherein said applying is
carried out prior to harvest of the fruit or vegetable.
27. The method according to claim 25, wherein said applying is
carried out after harvest of the fruit or vegetable.
28. The method according to claim 21, wherein the hypersensitive
response elicitor protein or polypeptide is derived from a species
of pathogen selected from the group consisting of Erwinia,
Xanthomonas, Pseudomonas, Phytophthora, and Clavibacter.
29. The method according to claim 28, wherein the hypersensitive
response elicitor protein or polypeptide is derived from Erwinia
amylovora.
30. The method according to claim 28, wherein the hypersensitive
response elicitor protein or polypeptide is derived from Erwinia
carotovora.
31. The method according to claim 28, wherein the hypersensitive
response elicitor protein or polypeptide is derived from Erwinia
stewartii.
32. The method according to claim 28, wherein the hypersensitive
response elicitor protein or polypeptide is derived from Erwinia
chrysanthemi.
33. The method according to claim 28, wherein the hypersensitive
response elicitor protein or polypeptide is derived from
Pseudomonas syringae.
34. The method according to claim 28, wherein the hypersensitive
response elicitor protein or polypeptide is derived from
Pseudomonas solanacearum.
35. The method according to claim 28, wherein the hypersensitive
response elicitor protein or polypeptide is derived from a species
of Phytophthora.
36. The method according to claim 21, wherein the postharvest
disease is caused by Penicillium, Botrytis, Phytophthora, or
Erwinia.
37. A DNA construct comprising: a DNA molecule encoding a
hypersensitive response elicitor protein or polypeptide; a
plant-expressible promoter operably coupled 5' to the DNA molecule,
the promoter being effective to transcribe the DNA molecule in
fruit or vegetable tissue; and a 3' regulatory region operably
coupled to the DNA molecule, wherein expression of the DNA molecule
in fruit or vegetable tissue imparts to a fruit or vegetable
resistance against postharvest disease or desiccation.
38. An expression system comprising a vector into which is inserted
a heterologous DNA construct according to claim 37.
39. A host cell comprising a heterologous DNA construct according
to claim 37.
40. The host cell according to claim 39, wherein the host cell is a
plant cell or a bacteria cell.
41. The host cell according to claim 40, wherein the bacteria cell
is an Agrobacterium cell.
42. A transgenic plant comprising a heterologous DNA construct
according to claim 37.
43. A method of enhancing the longevity of fruit or vegetable
ripeness comprising: treating a fruit or vegetable with a
hypersensitive response elicitor protein or polypeptide under
conditions effective to enhance the longevity of fruit or vegetable
ripeness.
44. The method according to claim 43, wherein hypersensitive
response elicitor protein or polypeptide is in isolated form.
45. The method according to claim 43, wherein said treating is
carried out prior to harvest of the fruit or vegetable.
46. The method according to claim 43, wherein said treating is
carried out after harvest of the fruit or vegetable.
47. A method of enhancing the longevity of fruit or vegetable
ripeness comprising: providing a transgenic plant or plant seed
transformed with a DNA molecule encoding a hypersensitive response
elicitor polypeptide or protein and growing the transgenic plant or
transgenic plant produced from the transgenic plant seed under
conditions effective to enhance the longevity of fruit or vegetable
ripeness in a fruit or vegetable harvested from the transgenic
plant.
48. The method according to claim 47, further comprising: applying
the hypersensitive response elicitor polypeptide or protein to the
fruit or vegetable to enhance the longevity of fruit or vegetable
ripeness.
49. The method according to claim 48, wherein said applying is
carried out prior to harvest of the fruit or vegetable.
50. The method according to claim 48, wherein said applying is
carried out after harvest of the fruit or vegetable.
Description
[0001] This application claims benefit of U.S. Provisional Patent
Application Serial No. 60/198,359, filed Apr. 19, 2000, which is
hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to methods of treating fruits
or vegetables to inhibit postharvest diseases and/or desiccation of
harvested fruits or vegetables.
BACKGROUND OF THE INVENTION
[0003] Postharvest diseases are often extensions of disease
occurring in the field or orchard. Brown rot of stone fruits
(Monilinia fructicola (Wint.) Honey), for example, may cause
blossom and twig blighting in the orchard. Infections in the
orchard may not be visible at harvest if fruits are not
refrigerated. Colletotrichum gloeosporioiders (Penz.) Arx may
attack blossoms or leaves and young fruit of citrus, avocados,
mangos, papayas, and a wide range of other tropical and subtropical
species; infections in developing fruit are usually latent, and rot
lesions appear only at the onset of fruit ripening. Pezicula
malicorticis (Jacks.) Nannfld. causes cankers of limbs of apples
and pears; infections in developing fruit are latent, and active
rotting usually commences only after the fruit has spent several
months in storage and proceeds during -1.degree. C. storage because
the organism is able to grow at very low temperatures. These fungi
used as examples are able to penetrate the cuticle and epidermis of
the fruit.
[0004] Whether capable of being penetrated directly or not, wounds
are often the usual means by which the fungus enters fruit. Cuts,
punctures, bruises, and abrasions cannot be avoided completely
during harvest and handling. If the cuticle and epidermis are
broken, spores find nutrients and humidity in fresh wounds ideal
for spore germination and colonization. Separation of fruits from
the parent plant at harvest creates an unavoidable wound that
encourages stem-end rots.
[0005] Rots developing at the blossom end usually involve prior
colonization of floral parts. For example, Botrytis blossom-end rot
(B. cinerea) sometimes occurs in Bartlett pears after a month or
two in storage at -1.degree. C. Initiation of rot in fruit flesh is
associated with old styles and stamens retained within the fruit.
Floral infections occur in the senescing floral parts at the end of
blossoming. Mostly these floral parts are invaded by Alternaria
spp. and common saprophytic fungi, but B. cinerea also is found
occasionally. Not all fruits having B. cinerea-invaded floral parts
rot in storage, but a significant percentage do. By contrast, test
fruits remain free from Botrytis blossom-end rot if the old floral
parts of developing fruits are free from B. cinerea. Rotting of
fruits in storage is greatly reduced by a single orchard spray with
a fungicide at the end of blossoming.
[0006] Contact infection, by which mycelia grow from a rotting
fruit to contact and penetrate nearby fruit, is an especially
serious aspect of some very common postharvest pathogens. The
ever-enlarging "nest" of rotting fruit tied together by fungus
mycelia will involve all fruit in a container, if given sufficient
time.
[0007] Disease or threat of disease dictates in large measure the
manner in which perishable fruits are handled. In recent decades,
fruits have been shipped to increasingly greater distances from
points of production. Exploitation of these distant markets,
however, may offer large economic benefits only if the life of the
commodity is stretched to its limit. Diseases and disorders
ordinarily manageable during handling and transcontinental transit
and marketing may be excessive when transoceanic marine transport
of longer duration is involved. Similarly, the extension of
marketing periods by storing fruits until they near the end of
their physiological life may cause additional disease problems.
Losses are especially serious if they occur in market areas,
because the costs of sorting, packaging, cooling, storage, and
transportation, which may greatly exceed production costs, have
already been incurred. Of even greater long-term importance may be
an impaired reputation leading to reduced future sales.
[0008] Postharvest diseases of fruit cause 15 to 25% losses yearly
in the fruit industry worldwide and much of this is due to rot
caused by microorganisms. Fungicides, which have been the primary
means of controlling postharvest diseases, have come under scrutiny
as posing potential oncogenic risks when applied to processed
foods. Thus, research efforts have been intensified to develop
biological control procedures for postharvest diseases of fruits
and vegetables that pose less risk to human health and the
environment.
[0009] Considerable attention has been placed on assessing the use
of antagonistic microorganisms as a viable alternative to the use
of synthetic fungicides. Two basic approaches are available for
using antagonistic microorganisms to control postharvest diseases.
Naturally occurring antagonists that already exist on fruit and
vegetable surfaces have been shown to control several rot pathogens
on diverse commodities. Alternatively, artificially introduced
antagonists have been shown to be effective in biologically
controlling postharvest pathogens.
[0010] Since 1983, an explosion of research has occurred in the
area of biological control of postharvest diseases by artificially
introduced antagonists, mostly on fruit diseases (Janisiewicz,
"Biological Control of Diseases of Fruit," In Biocontrol of Plant
Diseases II, Mukergie et al. (ed.), CRC Press, Boca Raton, pp.
153-165 (1988) and Wilson et al., "Potential for Biological Control
of Postharvest Plant Diseases," Plant Disease 69:375-378 (1985)).
For example, rot on apples was controlled with yeast (Wisniewski et
al., "Biological Control of Postharvest Diseases of Fruit:
Inhibition of Botrytis Rot on Apples by an Antagonistic Yeast,"
Proc. Electron Microsc. Soc. Am. 46:290-91 (1988)), while brown rot
in apricots was controlled with Bacillus subtilis (Pusey et al.,
"Postharvest Biological Control of Stone Fruit Brown Rot by
Bacillus subtilis," Plant Dis. 68:753-56 (1984)). Mold incidence
was reduced from 35% to 8% in lemon peel by a species of
Trichoderma (De Matos, "Chemical and Microbiological Factors
Influencing the Infection of Lemons by Geotrichum candidum and
Penicillium digitatum," Ph.D. dissertation, University of
California, Riverside, 106 pp. (1983)). Biocontrol of citrus rot
pathogens was demonstrated with Bacillus subtilis (Singh et al.,
"Bacillus subtilis as a Control Agent Against Fungal Pathogens of
Citrus Fruit," Trans. Br. Mycol. Soc. 83:487-90 (1984)). Such
antagonists have various modes of action: antibiosis or competition
for nutrients and space or both, induction of resistance in the
host tissue, and direct interaction with the pathogen (Wilson et
al., "Biological Control of Postharvest Diseases of Fruits and
Vegetables: An Emerging Technology," Annu. Rev. Phytopathol.
27:425-441 (1989)).
[0011] While treatment with antagonistic bacterial or fungal
species may be, at least to some extent, effective in controlling
postharvest diseases, there are a number of factors which must be
considered before this approach is used in commercial applications.
First, the antagonists must be grown and maintained for use in
treatments. This may result in significant expense and regulatory
burdens depending on when and how frequently such antagonists would
be applied. Also, it is questionable whether growers would want to
maintain bioreactors for growing and propagating particular
antagonist strains. Second, the efficacy of those antagonists may
depend on storage conditions during shipment of harvested fruit.
Some antagonists may not be able to tolerate variations in
conditions during shipment, thereby allowing the pathogens to
overcome any inhibitory effects of the antagonists. Given the above
problems, it is not surprising that few of the antagonists reported
to control plant pathogens have been successfully transferred from
the laboratory into the field or postharvest environment.
[0012] Thus, there still exists a need to provide an effective,
commercially viable method for treating fruits and vegetables to
control postharvest diseases which avoids entirely or otherwise
significantly reduces the need for fungicide treatments. In
particular, it would be desirable to provide an effective,
practicable treatment which presents little or no harm to humans or
the environment.
[0013] The present invention is directed to overcoming these and
other deficiencies in the art.
SUMMARY OF THE INVENTION
[0014] The present invention relates to a method of inhibiting
postharvest disease or desiccation in a fruit or vegetable. This
method is carried out treating a fruit or vegetable with a
hypersensitive response elicitor protein or polypeptide under
conditions effective to inhibit postharvest disease or
desiccation.
[0015] A further aspect of the present invention relates to another
method of inhibiting postharvest disease or desiccation in a fruit
or vegetable. This method is carried out by providing a transgenic
plant or plant seed transformed with a DNA molecule encoding a
hypersensitive response elicitor polypeptide or protein and growing
the transgenic plant or transgenic plant produced from the
transgenic plant seed under conditions effective to inhibit a
postharvest disease or desiccation in a fruit or vegetable
harvested from the transgenic plant.
[0016] Another aspect of the present invention relates to a DNA
construct that includes a DNA molecule encoding a hypersensitive
response elicitor protein or polypeptide, a plant-expressible
promoter operably coupled 5' to the DNA molecule, the promoter
being effective to transcribe the DNA molecule in fruit or
vegetable tissue, and a 3' regulatory region operably coupled to
the DNA molecule, wherein expression of the DNA molecule in fruit
or vegetable tissue imparts to a fruit or vegetable resistance
against postharvest disease or desiccation. Also disclosed are
expression systems, host cells, and transgenic plants which contain
a heterologous DNA construct of the present invention.
[0017] By the present invention, the hypersensitive response
elicitor protein or polypeptide can be used to inhibit or otherwise
control postharvest diseases (i.e., caused by pathogens) in fruits
or vegetables. Likewise, such treatment can also inhibit
postharvest desiccation of treated fruits or vegetables. In
achieving these objectives, the present invention enables produce
growers, warehouse packers, shippers, and suppliers to process,
handle, and store fruits and vegetables with reduced losses caused
by postharvest disease and desiccation. As a result, the cost of
bringing fruits and vegetables from the field to the consumer can
be reduced. Importantly, the quality of the treated fruits or
vegetables is improved.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention relates to a method of inhibiting
postharvest disease or desiccation in a fruit or vegetable. This
method is carried out treating a fruit or vegetable with a
hypersensitive response elicitor protein or polypeptide under
conditions effective to inhibit postharvest disease or
desiccation.
[0019] A further aspect of the present invention relates to another
method of inhibiting postharvest disease or desiccation in a fruit
or vegetable. This method is carried out by providing a transgenic
plant or plant seed transformed with a DNA molecule encoding a
hypersensitive response elicitor polypeptide or protein and growing
the transgenic plant or transgenic plant produced from the
transgenic plant seed under conditions effective to inhibit a
postharvest disease or desiccation in a fruit or vegetable
harvested from the transgenic plant.
[0020] For use in accordance with these methods, suitable
hypersensitive response elicitor proteins or polypeptides are those
derived from a wide variety of bacterial and fungal pathogens,
preferably bacterial pathogens.
[0021] Exemplary hypersensitive response elicitor proteins and
polypeptides from bacterial sources include, without limitation,
the hypersensitive response elicitors derived from Erwinia species
(e.g., Erwinia amylovora, Erwinia chrysanthemi, Erwinia stewartii,
Erwinia carotovora, etc.), Pseudomonas species (e.g., Pseudomonas
syringae, Pseudomonas solanacearum, etc.), and Xanthomonas species
(e.g., Xanthomonas campestris). In addition to hypersensitive
response elicitors from these Gram-negative bacteria, it is
possible to use elicitors derived from Gram-positive bacteria. One
example is the hypersensitive response elicitor derived from
Clavibacter michiganensis subsp. sepedonicus.
[0022] Exemplary hypersensitive response elicitor proteins or
polypeptides from fungal sources include, without limitation, the
hypersensitive response elicitors (i.e., elicitins) from various
Phytophthora species (e.g., Phytophthora parasitica, Phytophthora
cryptogea, Phytophthora cinnamomi, Phytophthora capsici,
Phytophthora megasperma, Phytophthora citrophthora, etc.).
[0023] Preferably, the hypersensitive response elicitor protein or
polypeptide is derived from Erwinia chrysanthemi, Erwinia
amylovora, Pseudomonas syringae, or Pseudomonas solanacearum.
[0024] A hypersensitive response elicitor protein or polypeptide
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
[0025] This hypersensitive response elicitor protein or polypeptide
has a molecular weight of 34 kDa, is heat stable, has a glycine
content of greater than 16%, and contains substantially no
cysteine. This Erwinia chrysanthemi hypersensitive response
elicitor protein or polypeptide is encoded by a DNA molecule having
a nucleotide sequence corresponding to SEQ. ID. No. 2 as
follows:
2 cgattttacc cgggtgaacg tgctatgacc gacagcatca cggtattcga caccgttacg
60 gcgtttatgg ccgcgatgaa ccggcatcag gcggcgcgct ggtcgccgca
atccggcgtc 120 gatctggtat ttcagtttgg ggacaccggg cgtgaactca
tgatgcagat tcagccgggg 180 cagcaatatc ccggcatgtt gcgcacgctg
ctcgctcgtc gttatcagca ggcggcagag 240 tgcgatggct gccatctgtg
cctgaacggc agcgatgtat tgatcctctg gtggccgctg 300 ccgtcggatc
ccggcagtta tccgcaggtg atcgaacgtt tgtttgaact ggcgggaatg 360
acgttgccgt cgctatccat agcaccgacg gcgcgtccgc agacagggaa cggacgcgcc
420 cgatcattaa gataaaggcg gcttttttta ttgcaaaacg gtaacggtga
ggaaccgttt 480 caccgtcggc gtcactcagt aacaagtatc catcatgatg
cctacatcgg gatcggcgtg 540 ggcatccgtt gcagatactt ttgcgaacac
ctgacatgaa tgaggaaacg aaattatgca 600 aattacgatc aaagcgcaca
tcggcggtga tttgggcgtc tccggtctgg ggctgggtgc 660 tcagggactg
aaaggactga attccgcggc ttcatcgctg ggttccagcg tggataaact 720
gagcagcacc atcgataagt tgacctccgc gctgacttcg atgatgtttg gcggcgcgct
780 ggcgcagggg ctgggcgcca gctcgaaggg gctggggatg agcaatcaac
tgggccagtc 840 tttcggcaat ggcgcgcagg gtgcgagcaa cctgctatcc
gtaccgaaat ccggcggcga 900 tgcgttgtca aaaatgtttg ataaagcgct
ggacgatctg ctgggtcatg acaccgtgac 960 caagctgact aaccagagca
accaactggc taattcaatg ctgaacgcca gccagatgac 1020 ccagggtaat
atgaatgcgt tcggcagcgg tgtgaacaac gcactgtcgt ccattctcgg 1080
caacggtctc ggccagtcga tgagtggctt ctctcagcct tctctggggg caggcggctt
1140 gcagggcctg agcggcgcgg gtgcattcaa ccagttgggt aatgccatcg
gcatgggcgt 1200 ggggcagaat gctgcgctga gtgcgttgag taacgtcagc
acccacgtag acggtaacaa 1260 ccgccacttt gtagataaag aagatcgcgg
catggcgaaa gagatcggcc agtttatgga 1320 tcagtatccg gaaatattcg
gtaaaccgga ataccagaaa gatggctgga gttcgccgaa 1380 gacggacgac
aaatcctggg ctaaagcgct gagtaaaccg gatgatgacg gtatgaccgg 1440
cgccagcatg gacaaattcc gtcaggcgat gggtatgatc aaaagcgcgg tggcgggtga
1500 taccggcaat accaacctga acctgcgtgg cgcgggcggt gcatcgctgg
gtatcgatgc 1560 ggctgtcgtc ggcgataaaa tagccaacat gtcgctgggt
aagctggcca acgcctgata 1620 atctgtgctg gcctgataaa gcggaaacga
aaaaagagac ggggaagcct gtctcttttc 1680 ttattatgcg gtttatgcgg
ttacctggac cggttaatca tcgtcatcga tctggtacaa 1740 acgcacattt
tcccgttcat tcgcgtcgtt acgcgccaca atcgcgatgg catcttcctc 1800
gtcgctcaga ttgcgcggct gatggggaac gccgggtgga atatagagaa actcgccggc
1860 cagatggaga cacgtctgcg ataaatctgt gccgtaacgt gtttctatcc
gcccctttag 1920 cagatagatt gcggtttcgt aatcaacatg gtaatgcggt
tccgcctgtg cgccggccgg 1980 gatcaccaca atattcatag aaagctgtct
tgcacctacc gtatcgcggg agataccgac 2040 aaaatagggc agtttttgcg
tggtatccgt ggggtgttcc ggcctgacaa tcttgagttg 2100 gttcgtcatc
atctttctcc atctgggcga cctgatcggt t 2141
[0026] The above nucleotide and amino acid sequences are disclosed
and further described in U.S. Pat. No. 5,850,015 to Bauer et al.
and U.S. Pat. No. 5,776,889 to Wei et al., which are hereby
incorporated by reference in their entirety.
[0027] A hypersensitive response elicitor protein or polypeptide
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
[0028] This hypersensitive response elicitor protein or polypeptide
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 protein or polypeptide has
substantially no cysteine. The hypersensitive response elicitor
protein or polypeptide derived from Erwinia amylovora is more fully
described in Wei, Z-M., et al., "Harpin, Elicitor of the
Hypersensitive Response Produced by the Plant Pathogen Erwinia
amylovora," Science 257:85-88 (1992), which is hereby incorporated
by reference in its entirety. The DNA molecule encoding this
hypersensitive response elicitor protein or polypeptide has a
nucleotide sequence corresponding to SEQ. ID. No. 4 as follows:
4 aagcttcggc atggcacgtt tgaccgttgg gtcggcaggg tacgtttgaa ttattcataa
60 gaggaatacg ttatgagtct gaatacaagt gggctgggag cgtcaacgat
gcaaatttct 120 atcggcggtg cgggcggaaa taacgggttg ctgggtacca
gtcgccagaa tgctgggttg 180 ggtggcaatt ctgcactggg gctgggcggc
ggtaatcaaa atgataccgt caatcagctg 240 gctggcttac tcaccggcat
gatgatgatg atgagcatga tgggcggtgg tgggctgatg 300 ggcggtggct
taggcggtgg cttaggtaat ggcttgggtg gctcaggtgg cctgggcgaa 360
ggactgtcga acgcgctgaa cgatatgtta ggcggttcgc tgaacacgct gggctcgaaa
420 ggcggcaaca ataccacttc aacaacaaat tccccgctgg accaggcgct
gggtattaac 480 tcaacgtccc aaaacgacga ttccacctcc ggcacagatt
ccacctcaga ctccagcgac 540 ccgatgcagc agctgctgaa gatgttcagc
gagataatgc aaagcctgtt tggtgatggg 600 caagatggca cccagggcag
ttcctctggg ggcaagcagc cgaccgaagg cgagcagaac 660 gcctataaaa
aaggagtcac tgatgcgctg tcgggcctga tgggtaatgg tctgagccag 720
ctccttggca acgggggact gggaggtggt cagggcggta atgctggcac gggtcttgac
780 ggttcgtcgc tgggcggcaa agggctgcaa aacctgagcg ggccggtgga
ctaccagcag 840 ttaggtaacg ccgtgggtac cggtatcggt atgaaagcgg
gcattcaggc gctgaatgat 900 atcggtacgc acaggcacag ttcaacccgt
tctttcgtca ataaaggcga tcgggcgatg 960 gcgaaggaaa tcggtcagtt
catggaccag tatcctgagg tgtttggcaa gccgcagtac 1020 cagaaaggcc
cgggtcagga ggtgaaaacc gatgacaaat catgggcaaa agcactgagc 1080
aagccagatg acgacggaat gacaccagcc agtatggagc agttcaacaa agccaagggc
1140 atgatcaaaa ggcccatggc gggtgatacc ggcaacggca acctgcaggc
acgcggtgcc 1200 ggtggttctt cgctgggtat tgatgccatg atggccggtg
atgccattaa caatatggca 1260 cttggcaagc tgggcgcggc ttaagctt 1288
[0029] The above nucleotide and amino acid sequences are disclosed
are further described in U.S. Pat. No. 5,849,868 to Beer et al. and
U.S. Pat. No. 5,776,889 to Wei et al., which are hereby
incorporated by reference in their entirety.
[0030] Another hypersensitive response elicitor protein or
polypeptide derived from Erwinia amylovora has an amino acid
sequence corresponding to SEQ. ID. No. 5 as follows:
5 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
[0031] 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. The DNA molecule encoding this
hypersensitive response elicitor protein or polypeptide has a
nucleotide sequence corresponding to SEQ. ID. No. 6 as follows:
6 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
[0032] The above nucleotide and amino acid sequences are disclosed
and further described in U.S. patent application Ser. No.
09/120,927 to Beer et al., which is hereby incorporated by
reference in its entirety.
[0033] A hypersensitive response elicitor protein or polypeptide
derived from Pseudomonas syringae has an amino acid sequence
corresponding to SEQ. ID. No. 7 as follows:
7 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
[0034] This hypersensitive response elicitor protein or polypeptide
has a molecular weight of 34-35 kDa. It is rich in glycine (about
13.5%) and lacks cysteine and tyrosine. Further information about
the hypersensitive response elicitor derived from Pseudomonas
syringae is found in He, S. Y., et al., "Pseudomonas syringae pv.
syringae Harpin.sub.Pss: a Protein that is Secreted via the Hrp
Pathway and Elicits the Hypersensitive Response in Plants," Cell
73:1255-1266 (1993), which is hereby incorporated by reference in
its entirety. The DNA molecule encoding this hypersensitive
response elicitor from Pseudomonas syringae has a nucleotide
sequence corresponding to SEQ. ID. No. 8 as follows:
8 atgcagagtc tcagtcttaa cagcagctcg ctgcaaaccc cggcaatggc ccttgtcctg
60 gtacgtcctg aagccgagac gactggcagt acgtcgagca aggcgcttca
ggaagttgtc 120 gtgaagctgg ccgaggaact gatgcgcaat ggtcaactcg
acgacagctc gccattggga 180 aaactgttgg ccaagtcgat ggccgcagat
ggcaaggcgg gcggcggtat tgaggatgtc 240 atcgctgcgc tggacaagct
gatccatgaa aagctcggtg acaacttcgg cgcgtctgcg 300 gacagcgcct
cgggtaccgg acagcaggac ctgatgactc aggtgctcaa tggcctggcc 360
aagtcgatgc tcgatgatct tctgaccaag caggatggcg ggacaagctt ctccgaagac
420 gatatgccga tgctgaacaa gatcgcgcag ttcatggatg acaatcccgc
acagtttccc 480 aagccggact cgggctcctg ggtgaacgaa ctcaaggaag
acaacttcct tgatggcgac 540 gaaacggctg cgttccgttc ggcactcgac
atcattggcc agcaactggg taatcagcag 600 agtgacgctg gcagtctggc
agggacgggt ggaggtctgg gcactccgag cagtttttcc 660 aacaactcgt
ccgtgatggg tgatccgctg atcgacgcca ataccggtcc cggtgacagc 720
ggcaataccc gtggtgaagc ggggcaactg atcggcgagc ttatcgaccg tggcctgcaa
780 tcggtattgg ccggtggtgg actgggcaca cccgtaaaca ccccgcagac
cggtacgtcg 840 gcgaatggcg gacagtccgc tcaggatctt gatcagttgc
tgggcggctt gctgctcaag 900 ggcctggagg caacgctcaa ggatgccggg
caaacaggca ccgacgtgca gtcgagcgct 960 gcgcaaatcg ccaccttgct
ggtcagtacg ctgctgcaag gcacccgcaa tcaggctgca 1020 gcctga 1026
[0035] The above nucleotide and amino acid sequences are disclosed
and further described in U.S. Pat. No. 5,708,139 to Collmer et al.
and U.S. Pat. No. 5,776,889 to Wei et al., which are hereby
incorporated by reference in their entirety.
[0036] Another hypersensitive response elicitor protein or
polypeptide derived from Pseudomonas syringae has an amino acid
sequence corresponding to SEQ. ID. No. 9 as follows:
9 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
[0037] This protein or polypeptide is acidic, glycine-rich, lacks
cysteine, and is deficient in aromatic amino acids. The DNA
molecule encoding this hypersensitive response elicitor from
Pseudomonas syringae has a nucleotide sequence corresponding to
SEQ. ID. No. 10 as follows:
10 tccacttcgc tgattttgaa attggcagat tcatagaaac gttcaggtgt
ggaaatcagg 10 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
[0038] The above nucleotide and amino acid sequences are disclosed
and further described in U.S. patent application Ser. No.
09/120,817 to Collmer et al., which is hereby incorporated by
reference in its entirety.
[0039] A hypersensitive response elicitor protein or polypeptide
derived from Pseudomonas solanacearum has an amino acid sequence
corresponding to SEQ. ID. No. 11 as follows:
11 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
[0040] Further information regarding this hypersensitive response
elicitor protein or polypeptide derived from Pseudomonas
solanacearum is set forth in Arlat, M., et al., "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 in its entirety. It is encoded by a DNA
molecule from Pseudomonas solanacearum having a nucleotide sequence
corresponding SEQ. ID. No. 12 as follows:
12 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
[0041] The above nucleotide and amino acid sequences are disclosed
and further described in U.S. Pat. No. 5,776,889 to Wei et al.,
which is hereby incorporated by reference in its entirety.
[0042] Other embodiments of the present invention include, but are
not limited to, use of hypersensitive response elicitor proteins or
polypeptides derived from Erwinia carotovora and Erwinia stewartii.
Isolation of an Erwinia carotovora hypersensitive response elicitor
protein or polypeptide is described in Cui, et al., "The RsmA
Mutants of Erwinia carotovora subsp. carotovora Strain Ecc71
Overexpress hrpN.sub.Ecc and Elicit a Hypersensitive Reaction-like
Response in Tobacco Leaves," MPMI, 9(7):565-73 (1996), which is
hereby incorporated by reference in its entirety. A 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 in their entirety.
[0043] Hypersensitive response elicitor proteins or polypeptides
from various Phytophthora species 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 Defense Genes, Production of
Salicylic Acid, and Induction of Systemic Acquired Resistance,"
Plant J., 8(4):551-60 (1995), and Bonnet, et al., "Acquired
Resistance Triggered by Elicitors in Tobacco and Other Plants,"
Eur. J. Plant Path., 102:181-92 (1996), which are hereby
incorporated by reference in their entirety.
[0044] Another hypersensitive response elicitor protein or
polypeptide which can be used in accordance with the present
invention is derived from Clavibacter michiganensis subsp.
sepedonicus and is described in U.S. patent application Ser. No.
09/136,625, which is hereby incorporated by reference in its
entirety.
[0045] Fragments of the above hypersensitive response elicitor
proteins or polypeptides as well as fragments of full length
elicitors from other pathogens can also be used according to the
present invention.
[0046] Suitable fragments can be produced by several means.
Subclones of the gene encoding a known elicitor protein can be
produced using conventional molecular genetic manipulation for
subcloning gene fragments, such as described by Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory,
Cold Springs Harbor, N.Y. (1989), and Ausubel et al. (ed.), Current
Protocols in Molecular Biology, John Wiley & Sons (New York,
N.Y.) (1999 and preceding editions), which are hereby incorporated
by reference in their entirety. The subclones then are expressed in
vitro or in vivo in bacterial cells to yield a smaller protein or
polypeptide that can be tested for elicitor activity, e.g., using
procedures set forth in Wei, Z -M., et al., Science 257: 85-88
(1992), which is hereby incorporated by reference in its
entirety.
[0047] In another approach, based on knowledge of the primary
structure of the protein, fragments of the elicitor protein gene
may be synthesized using the PCR technique together with specific
sets of primers chosen to represent particular portions of the
protein. Erlich, H. A., et al., "Recent Advances in the Polymerase
Chain Reaction," Science 252:1643-51 (1991), which is hereby
incorporated by reference in its entirety. These can then be cloned
into an appropriate vector for expression of a truncated protein or
polypeptide from bacterial cells as described above.
[0048] 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.
[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 elicit a hypersensitive response are
fragments of the Erwinia amylovora hypersensitive response elicitor
protein or polypeptide of SEQ. ID. No. 3. The fragments can be 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. DNA molecules encoding these fragments can also be utilized in
the chimeric gene of the present invention.
[0051] Variants may also (or alternatively) be modified by, for
example, the deletion or addition of amino acids that have minimal
influence on the properties, secondary structure and hydropathic
nature of the polypeptide. For example, a polypeptide may be
conjugated to a signal (or leader) sequence at the N-terminal end
of the protein which co-translationally or post-translationally
directs transfer of the protein. The polypeptide may also be
conjugated to a linker or other sequence for ease of synthesis,
purification, or identification of the polypeptide.
[0052] The hypersensitive response elicitor proteins or
polypeptides used in accordance with the present invention are
preferably produced in purified form (preferably at least about
80%, more preferably 90%, pure) by conventional techniques.
Typically, the protein or polypeptide of the present invention is
secreted into the growth medium of recombinant host cells
(discussed infra). Alternatively, the protein or polypeptide of the
present invention is produced but not secreted into growth medium.
In such cases, 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 sequential ammonium sulfate precipitation. The
fraction containing the hypersensitive response elicitor protein or
polypeptide of interest is subjected to gel filtration in an
appropriately sized dextran or polyacrylamide column to separate
the proteins. If necessary, the protein fraction may be further
purified by HPLC.
[0053] Other hypersensitive response elicitors can be readily
identified by isolating putative protein or polypeptide candidates
and testing them for elicitor activity as described, for example,
in Wei, Z-M., et al., "Harpin, Elicitor of the Hypersensitive
Response Produced by the Plant Pathogen Erwinia amylovora," Science
257:85-88 (1992), which is hereby incorporated by reference in its
entirety. Cell-free preparations from culture supernatants can be
tested for elicitor activity (i.e., local necrosis) by using them
to infiltrate appropriate plant tissues. Once identified, DNA
molecules encoding a hypersensitive response elicitor can be
isolated using standard techniques known to those skilled in the
art.
[0054] DNA molecules encoding other hypersensitive response
elicitor proteins or polypeptides can also be identified by
determining whether such DNA molecules hybridizes under stringent
conditions to a DNA molecule having the nucleotide sequence of SEQ.
ID. Nos. 2, 4, 6, 8, 10, or 12. An example of suitable stringency
conditions is when hybridization is carried out at a temperature of
about 37.degree. C. using a hybridization medium that includes 0.9M
sodium citrate ("SSC") buffer, followed by washing with
0.2.times.SSC buffer at 37.degree. C. Higher stringency can readily
be attained by increasing the temperature for either hybridization
or washing conditions or increasing the sodium concentration of the
hybridization or wash medium. Nonspecific binding may also be
controlled using any one of a number of known techniques such as,
for example, blocking the membrane with protein-containing
solutions, addition of heterologous RNA, DNA, and SDS to the
hybridization buffer, and treatment with RNase. Wash conditions are
typically performed at or below stringency. Exemplary high
stringency conditions include carrying out hybridization at a
temperature of about 42.degree. C. to about 65.degree. C. for up to
about 20 hours in a hybridization medium containing 1M NaCl, 50 mM
Tris-HCl, pH 7.4, 10 mM EDTA, 0.1 % sodium dodecyl sulfate (SDS),
0.2% ficoll, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin,
and 50 .mu.g/ml E. coli DNA, followed by washing carried out at
between about 42.degree. C. to about 65.degree. C. in a
0.2.times.SSC buffer.
[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 proper 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 in its entirety, 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 prokaryotic
organisms and eukaryotic 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 in its
entirety), 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 in its entirety), 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 in its entirety.
[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
promoter which is a DNA sequence that directs the binding of RNA
polymerase and thereby promotes mRNA synthesis. The DNA sequences
of eukaryotic promoters differ from those of prokaryotic promoters.
Furthermore, eukaryotic promoters and accompanying genetic signals
may not be recognized in or may not function in a prokaryotic
system, and, further, prokaryotic promoters are not recognized and
do not function in eukaryotic cells.
[0062] Similarly, translation of mRNA in prokaryotes depends upon
the presence of the proper prokaryotic signals which differ from
those of eukaryotes. Efficient translation of mRNA in prokaryotes
requires a ribosome binding site called the Shine-Dalgarno ("SD")
sequence on the mRNA. This sequence is a short nucleotide sequence
of mRNA that is located before the start codon, usually AUG, which
encodes the amino-terminal methionine of the protein. The SD
sequences are complementary to the 3'-end of the 16S rRNA
(ribosomal RNA) and probably promote binding of mRNA to ribosomes
by duplexing with the rRNA to allow correct positioning of the
ribosome. For a review on maximizing gene expression, see Roberts
and Lauer, Methods in Enzymology, 68:473 (1979), which is hereby
incorporated by reference in its entirety.
[0063] Promoters 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, promoters such as the T7
phage promoter, lac promoter, trp promoter, recA promoter,
ribosomal RNA promoter, the P.sub.R and P.sub.L 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) promoter or other E. coli promoters produced by
recombinant DNA or other synthetic DNA techniques may be used to
provide for transcription of the inserted gene.
[0064] Bacterial host cell strains and expression vectors may be
chosen which inhibit the action of the promoter 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 prokaryotic 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 promoter, 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] Because it is desirable for recombinant host cells to
secrete the hypersensitive response elicitor protein or
polypeptide, it is preferable that the host cell also be
transformed with a type III secretion system in accordance with Ham
et al., "A Cloned Erwinia chrysanthemi Hrp (Type III Protein
Secretion) System Functions in Escherichia coli to Deliver
Pseudomonas syringae Avr Signals to Plant Cells and Secrete Avr
Proteins in Culture," Microbiol. 95:10206-10211 (1998), which is
hereby incorporated by reference in its entirety.
[0068] Isolation of the hypersensitive response elicitor protein or
polypeptide from the host cell or growth medium can be carried out
as described above.
[0069] The methods of the present invention can be performed by
treating the fruit or vegetable either prior to or after harvest of
the fruit or vegetable.
[0070] Suitable preharvest application methods include, without
limitation, high or low pressure spraying of the entire plant and
fruits. Suitable postharvest application methods include, without
limitation, low or high pressure spraying, coating, or immersion.
Other suitable application procedures (both preharvest and
postharvest) can be envisioned by those skilled in the art provided
they are able to effect contact of the hypersensitive response
elicitor polypeptide or protein with the fruit or vegetable. Once
treated, the fruits or vegetables can be handled, packed, shipped,
and processed using conventional procedures to deliver the produce
to processing plants or end-consumers.
[0071] The hypersensitive response elicitor polypeptide or protein
can be applied to fruits or vegetables in accordance with the
present invention alone or in a mixture with other materials.
Alternatively, the hypersensitive response elicitor polypeptide or
protein can be applied separately to fruits or vegetables with
other materials being applied at different times.
[0072] A composition suitable for treating fruits or vegetables in
accordance with the application embodiment of the present invention
contains an isolated hypersensitive response elicitor polypeptide
or protein in a carrier. Suitable carriers include water, aqueous
solutions, slurries, or dry powders. The composition preferably
contains greater than about 500 nM hypersensitive response elicitor
polypeptide or protein, although greater or lesser amounts of the
hypersensitive response elicitor polypeptide or protein depending
on the rate of composition application and efficacy of different
hypersensitive response elicitor proteins or polypeptides.
[0073] 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.
[0074] Other suitable additives include buffering agents, wetting
agents, coating agents, and ripening agents. These materials can be
used either to facilitate the process of the present invention or
to provide additive benefits to inhibit postharvest disease and
desiccation.
[0075] As indicated above, one embodiment of the present invention
involves treating fruits or vegetables with an isolated
hypersensitive response elicitor protein or polypeptide. The
hypersensitive response elicitor protein or polypeptide can be
isolated from its natural source (e.g., Erwinia amylovora,
Pseudomonas syringae, etc.) or from recombinant source transformed
with a DNA molecule encoding the protein or polypeptide.
[0076] Another aspect of the present invention relates to a DNA
construct as well as host cells, expression systems, and transgenic
plants which contain the heterologous DNA construct.
[0077] The DNA construct includes a DNA molecule encoding a
hypersensitive response elicitor protein or polypeptide, a
plant-expressible promoter operably coupled 5' to the DNA molecule
and which is effective to transcribe the DNA molecule in fruit or
vegetable tissue, and a 3' regulatory region operably coupled to
the DNA molecule. Expression of the DNA molecule in fruit or
vegetable tissue imparts to a fruit or vegetable resistance against
postharvest disease or desiccation.
[0078] Expression of such heterologous DNA molecules requires a
suitable promoter which is operable in plant tissues. In some
embodiments of the present invention, it may be desirable for the
heterologous DNA molecule to be expressed in many, if not all,
tissues. Such promoters yield constitutive expression of coding
sequences under their regulatory control. Exemplary constitutive
promoters include, without limitation, the nopaline synthase
promoter (Fraley et al., Proc. Natl. Acad. Sci. USA 80:4803-4807
(1983), which is hereby incorporated by reference in its entirety)
and the cauliflower mosaic virus 35S promoter (O'Dell et al.,
"Identification of DNA Sequences Required for Activity of the
Cauliflower Mosaic Virus 35S Promoter," Nature, 313(6005):810-812
(1985), which is hereby incorporated by reference in its
entirety).
[0079] While constitutive expression is generally suitable for
expression of the DNA molecule, it should be apparent to those of
skill in the art that temporally or tissue regulated expression may
also be desirable, in which case any regulated promoter can be
selected to achieve the desired expression. Typically, the
temporally or tissue regulated promoters will be used in connection
with the DNA molecule that are expressed at only certain stages of
development or only in certain tissues.
[0080] In another embodiment of the present invention, expression
of the heterologous DNA molecule is directed in a tissue-specific
manner or environmentally-regulated manner (i.e., inducible
promoters). Tissue-specific promoters under developmental control
include promoters that initiate transcription only in certain
tissues.
[0081] For example, the E4 and E8 promoters of tomato have been
used to direct fruit-specific expression of a heterologous DNA
sequence in transgenic tomato plants (Cordes et al., Plant Cell
1:1025-1034 (1989); Deikman et al., EMBO J. 7:3315-3320 (1988); and
Della Penna et al., Proc. Natl. Acad. Sci. USA 83:6420-6424 (1986),
which are hereby incorporated by reference in their entirety).
Another fruit-specific promoter is the PG promoter (Bird et al.,
Plant Molec. Biol. 11:651-662 (1988), which is hereby incorporated
by reference in its entirety). Another tissue-specific promoter is
the AP2 promoter from the ovule-specific BEL1 gene promoter
described in Reiser et al., Cell 83:735-742 (1995), which is hereby
incorporated by reference in its entirety.
[0082] Promoters useful for expression in seed tissues include,
without limitation, the promoters from genes encoding seed storage
proteins, such as napin, cruciferin, phaseolin, and the like (see
U.S. Pat. No. 5,420,034 to Kridl et al., which is hereby
incorporated by reference in its entirety). Other suitable
promoters include those from genes encoding embryonic storage
proteins.
[0083] Promoters useful for expression in leaf tissue include the
Rubisco small subunit promoter.
[0084] Promoters useful for expression in tubers, particularly
potato tubers, include the patatin promoter.
[0085] Examples of environmental conditions that may affect
transcription by inducible promoters include anaerobic conditions,
elevated temperature, or the presence of light. In some plants, it
may also be desirable to use promoters which are responsive to
pathogen infiltration or stress. For example, it may be desirable
to limit expression of the protein or polypeptide in response to
infection by a particular pathogen of the plant. One example of a
pathogen-inducible promoter is the gstl promoter from potato, which
is described in U.S. Pat. Nos. 5,750,874 and 5,723,760 to
Strittmayer et al., which are hereby incorporated by reference in
their entirety.
[0086] Expression of the DNA molecule in isolated plant cells or
tissue or whole plants also utilizes appropriate transcription
termination and polyadenylation of mRNA. Any 3' regulatory region
suitable for use in plant cells or tissue can be operably linked to
the first and second DNA molecules. A number of 3' regulatory
regions are known to be operable in plants. Exemplary 3' regulatory
regions include, without limitation, the nopaline synthase 3'
regulatory region (Fraley, et al., "Expression of Bacterial Genes
in Plant Cells," Proc. Nat'l. Acad. Sci. USA, 80:4803-4807 (1983),
which is hereby incorporated by reference in its entirety) and the
cauliflower mosaic virus 3' regulatory region (Odell, et al.,
"Identification of DNA Sequences Required for Activity of the
Cauliflower Mosaic Virus 35S Promoter," Nature, 313(6005):810-812
(1985), which is hereby incorporated by reference in its
entirety).
[0087] The promoter and a 3' regulatory region can readily be
ligated to the DNA molecule using well known molecular cloning
techniques described in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y.
(1989), which is hereby incorporated by reference in its
entirety.
[0088] One approach to transforming plant cells with a DNA molecule
of the present invention 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
in their entirety. 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. Other variations of particle
bombardment, now known or hereafter developed, can also be
used.
[0089] Another method of introducing the DNA molecule into plant
cells is fusion of protoplasts with other entities, either
minicells, cells, lysosomes, or other fusible lipid-surfaced bodies
that contain the DNA molecule. Fraley, et al., Proc. Natl. Acad.
Sci. USA, 79:1859-63 (1982), which is hereby incorporated by
reference in its entirety.
[0090] 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 its
entirety. In this technique, plant protoplasts are electroporated
in the presence of plasmids containing the DNA molecule. 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.
[0091] Another method of introducing the DNA molecule into plant
cells is to infect a plant cell with Agrobacterium tumefaciens or
Agrobacterium rhizogenes previously transformed with the DNA
molecule. 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.
[0092] 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.
[0093] Heterologous genetic sequences such as a DNA molecule a
hypersensitive response elicitor protein or polypeptide 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. Schell, J.,
Science, 237:1176-83 (1987), which is hereby incorporated by
reference in its entirety.
[0094] Plant tissue suitable for transformation include leaf
tissue, root tissue, meristems, zygotic and somatic embryos, and
anthers.
[0095] After transformation, the transformed plant cells can be
selected and regenerated.
[0096] Preferably, transformed cells are first identified using,
e.g., a selection marker simultaneously introduced into the host
cells along with the DNA molecule of the present invention.
Suitable selection markers include, without limitation, markers
coding for antibiotic resistance, such as kanamycin resistance
(Fraley, et al., Proc. Natl. Acad. Sci. USA, 80:4803-4807 (1983),
which is hereby incorporated by reference in its entirety). A
number of antibiotic-resistance markers are known in the art and
other are continually being identified. Any known
antibiotic-resistance marker can be used to transform and select
transformed host cells in accordance with the present invention.
Cells or tissues are grown on a selection media containing an
antibiotic, whereby generally only those transformants expressing
the antibiotic resistance marker continue to grow.
[0097] Once a recombinant plant cell or tissue has been obtained,
it is possible to regenerate a full-grown plant therefrom. Thus,
another aspect of the present invention relates to a transgenic
plant that includes a heterologous DNA molecule encoding a
hypersensitive response elicitor protein or polypeptide, wherein
the heterologous DNA molecule is under control or a promoter that
induces transcription of the DNA molecule fruit or vegetable
tissues. Preferably, the DNA molecule is stably inserted into the
genome of the transgenic plant of the present invention.
[0098] 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 in their entirety.
[0099] It is known that practically all plants can be regenerated
from cultured cells or tissues, including both monocots and
dicots.
[0100] 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.
[0101] After the DNA molecule encoding the hypersensitive response
elicitor protein or polypeptide is stably incorporated in
transgenic plants, it can be transferred to other plants by sexual
crossing or by preparing cultivars. With respect to sexual
crossing, any of a number of standard breeding techniques can be
used depending upon the species to be crossed. Cultivars can be
propagated in accord with common agricultural procedures known to
those in the field.
[0102] With regard to the use of the hypersensitive response
elicitor protein or polypeptide in imparting postharvest disease
resistance, absolute immunity against infection may not be
conferred, but the severity of the disease can be reduced and
symptom development can be delayed. Lesion number, lesion size, and
extent of sporulation of fungal pathogens are all decreased. This
method of controlling postharvest disease has the potential for
controlling previously untreatable diseases and avoiding the use of
infectious agents or environmentally harmful materials.
[0103] With respect to desiccation, complete protection against
desiccation may not be conferred, but the severity of desiccation
can be reduced. Desiccation protection inevitably will depend, at
least to some extent, on other conditions such as storage
temperatures, light exposure, etc. However, this method of
controlling desiccation has the potential for eliminating some
other treatments (i.e., use of coating waxes) which may contribute
to reduced costs or, at least, substantially no increase in
costs.
[0104] The methods of the present invention can be used to control
a number of postharvest diseases caused by a variety of pathogens.
These postharvest diseases and the causative agents which can be
treated according to the present invention include, without
limitation, the following: Penicillium (e.g., Penicillium
digitatum), Botrytis (e.g., Botrytis cinereaon), Phytophthora
(e.g., Phytophthora citrophthora), and Erwinia (e.g. Erwinia
carotovora).
[0105] A further aspect of the present invention relates to a
method of enhancing the longevity of fruit or vegetable
ripeness.
[0106] According to one embodiment, this aspect of the present
invention is carried out by treating a fruit or vegetable with a
hypersensitive response elicitor protein or polypeptide under
conditions effective to enhance the longevity of fruit or vegetable
ripeness. Preferably, as noted above, the hypersensitive response
elicitor protein or polypeptide is in isolated form. Treating of
the fruit or vegetable can be performed either prior to harvest
after harvest of the fruit or vegetable, using the techniques
described above.
[0107] According to another embodiment, this aspect of the present
invention is carried out by providing a transgenic plant or plant
seed transformed with a DNA molecule encoding a hypersensitive
response elicitor polypeptide or protein and then growing the
transgenic plant or transgenic plant produced from the transgenic
plant seed under conditions effective to enhance the longevity of
fruit or vegetable ripeness in a fruit or vegetable harvested from
the transgenic plant. This aspect of the present invention may
further include applying the hypersensitive response elicitor
polypeptide or protein to the fruit or vegetable to enhance the
longevity of fruit or vegetable ripeness. Treating of the fruit or
vegetable can be performed either prior to harvest or after harvest
of the fruit or vegetable, using the techniques described
above.
[0108] The methods of the present invention can be utilized to
treat a wide variety of fruits and vegetables to control
postharvest disease or desiccation as well as enhance the longevity
of fruit or vegetable ripeness. Fruits and vegetables which can be
treated include any edible plant product, particularly those from
traditional crop plants, such as seed, root, tuber, stem, leaf,
flower, and fruit. Exemplary transgenic fruit plants and fruits
that can be treated include, without limitation, apple, pear,
peach, nectarine, apricot, plum, cherry, olive, melon, citrus,
grape, strawberry, raspberry, blueberry, currant, pineapple,
papaya, guava, banana, and kiwi. Exemplary transgenic vegetable
plants and vegetables that can be treated include, without
limitation, asparagus, potato, sweet potato, bean, pea, chicory,
lettuce, parsley, basil, endive, cabbage, brussel sprout, beet,
parsnip, turnip, cauliflower, broccoli, spinach, onion, garlic,
eggplant, pepper, celery, leek, radish, carrot, squash, pumpkin,
zucchini, cucumber, soybean, tobacco, tomato, sorghum, rhubarb, and
sugarcane. Exemplary transgenic grain plants and grain products
which can be treated include, without limitation, alfalfa, rice,
wheat, barley, corn, and rye.
EXAMPLES
[0109] The following examples are provided to illustrate
embodiments of the present invention but are by no means intended
to limit its scope.
[0110] As used in the following Examples, Messenger.RTM. refers to
a product available from Eden Bioscience Corporation (Bothell,
Wash.), which contains 3% by weight of harpin.sub.Ea as the active
ingredient and 97% by weight inert ingredients. Harpin.sub.Ea is
one type of hypersensitive response elicitor protein from Erwinia
amylovora, identified herein by SEQ. ID. No. 3.
Example 1--Effect of Treating Orange Fruits with Messenger.RTM. on
Postharvest Orange Storage
[0111] On day 0, Fall-GLO orange fruits were treated by spraying
Messenger.RTM. solution (ca. 15 ug/ml) or buffer solution (5 mM
KPO.sub.4, pH 6.8) on the surface of fruits in a 22.degree. C.
greenhouse. The Messenger.RTM. or buffer solutions on oranges were
then dried by air, and the treated oranges were marked, mixed
together, and put into a plastic container (Clear View 66 Qt/63L
made by Sterilite Corporation). The container with treated oranges
was then put into a 18.degree. C. growth chamber for storage. On
day 7, orange fruits were inoculated with Penicillium digitatum and
Botrytis cinereaon by spraying a 10.sup.5 cfu/ml suspension on the
surface of orange fruit. The above procedure was performed on 40
orange fruits per treatment.
[0112] Measurements of disease were conducted on days 20, 24, and
26 following treatment with Messenger.RTM. or buffer solution.
Grades 0-5 indicate different disease scales--Grade 0: No symptoms;
Grade 1: 1/5 an individual fruit has disease symptoms; Grade 2: 2/5
an individual fruit has disease symptoms; Grade 3: 3/5 an
individual fruit has disease symptoms; Grade 4: 4/5 an individual
fruit has disease symptoms; Grade 5: whole fruit has disease
symptoms. The results of these treatments are set forth in Table 1
below.
13TABLE 1 Reduction of Disease Index in Oranges Days After Grade
T-test Sample Treatment 0 1 2 3 4 5 Index Efficacy p < 0.05 p
< 0.01 Messenger .RTM. 20 33 3 1 0 2 1 0.09 58.14% yes yes
Buffer 20 23 8 0 2 6 1 0.22 n/a -- -- Messenger .RTM. 24 25 2 6 4 1
2 0.20 45.21% yes yes Buffer 24 16 7 3 3 4 7 0.37 n/a -- --
Messenger .RTM. 26 19 4 6 4 5 2 0.29 36.96% yes yes Buffer 26 16 3
3 0 7 11 0.46 n/a -- --
[0113] The data listed in Table 1 above shows that the
Messenger.RTM. was more effective than buffer as a fruit spray
treatment in reducing disease index for Penicillium digitatum and
Botrytis cinereaon and providing longer storage life.
Messenger.RTM. treatment can reduce orange disease about 58.14% at
21 days, about 45.21% at 25 days, and 36.97% at 27 days after
spraying treatment and 18.degree. C. storage conditions. T-test
shows that there are statistically significant differences at both
95% and 99% confidence levels for the results obtained from
Messenger treatment.RTM. and buffer treatment.
Example 2--Effect of Treating Tomato (Hot House) Fruits with
Messenger.RTM. on Postharvest Tomato Storage
[0114] On day 0, Hot House tomato fruits were treated by spraying
Messenger.RTM. solution (ca. 15 ug/ml) or buffer solution (5mM
KPO.sub.4, pH 6.8) on the surface of fruits in a 22.degree. C.
greenhouse. The Messenger.RTM. or buffer solutions on tomatoes were
then dried by air, and the treated tomatoes were marked, mixed
together, and put into a plastic container (Clear View 66 Qt/63L
made by Sterilite Corporation). The container with treated tomatoes
was then put into 18.degree. C. growth chamber for storage. On day
7, tomatoes were inoculated with Penicillium digitatum and Botrytis
cinereaon by spraying a 10.sup.5 cfu/ml suspension on the surface
of tomato fruit. The above procedure was performed on 15 tomatoes
fruits per treatment.
[0115] Measurements of disease were conducted on days 21 and 27
following treatment with Messenger.RTM. or buffer solution. Grades
are indicated according to the criteria set forth in Example 1. The
results of these treatments are set forth in Table 2 below.
14TABLE 2 Reduction of Disease Index in Tomatoes Days After Grade
T-test Sample Treatment 0 1 2 3 4 5 Index Efficacy p < 0.05 p
< 0.0l Messenger .RTM. 21 7 2 2 3 1 0 0.25 58.70% yes yes Buffer
21 3 1 2 1 2 6 0.61 n/a -- -- Messenger .RTM. 27 2 2 4 3 2 2 0.49
30.19% yes yes Buffer 27 1 1 2 2 3 6 0.71 n/a -- --
[0116] The data listed in Table 2 above shows that the
Messenger.RTM. was more effective than buffer as a fruit spray
treatment in reducing disease index for Penicillium digitatum and
Botrytis cinereaon and providing longer storage life.
Messenger.RTM. treatment can reduce tomato disease about 58.70% at
21 days and about 30.19% at 27 days after spraying treatment and
18.degree. C. storage conditions. T-test shows that there are
statistically significant differences at both 95% and 99%
confidence levels for the results obtained from Messenger
treatment.RTM. and buffer treatment.
Example 3--Effect of Treating Grape Fruits with Messenger.RTM. on
Postharvest Grape Storage
[0117] On day 0, Red G. Grape fruits were treated by spraying
Messenger.RTM. solution (ca. 15 ug/ml) or buffer solution (5 mM
KPO.sub.4, pH 6.8) on the surface of fruits in a 22.degree. C.
greenhouse. The Messenger.RTM. or buffer solutions on grapes were
then dried by air, and the treated grapes were marked, mixed
together, and put into a plastic container (Clear View 66 Qt/63L
made by Sterilite Corporation). The container with treated grapes
was then put into a 18.degree. C. growth chamber for storage. On
day 7, grapes were inoculated with Penicillium digitatum and
Botrytis cinereaon by spraying a 10.sup.5 cfu/ml suspension on the
surface of grape fruit. The above procedure was performed on about
3700 g of grape fruits per treatment.
[0118] Measurements of disease were conducted on days 14 and 21
following treatment with Messenger.RTM. or buffer solution. Grades
are indicated according to the criteria set forth in Example 1. The
results of these treatments are set forth in Table 3 below.
15TABLE 3 Reduction of Disease Index in Grapes Days After Grade
T-test Sample Treatment 0 1 2 3 4 5 Index Efficacy p < 0.05 p
< 0.01 Messenger .RTM. 14 225 99 42 39 21 13 0.20 45.65% yes yes
Buffer 14 98 130 91 52 38 48 0.38 n/a -- -- Messenger .RTM. 21 66
83 126 98 39 27 0.42 39.35% yes yes Buffer 21 18 36 64 72 119 137
0.69 n/a -- --
[0119] The data listed in Table 3 above shows that the
Messenger.RTM. was more effective than buffer as a fruit spray
treatment in reducing disease index for Penicillium digitatum and
Botrytis cinereaon and providing longer storage life.
Messenger.RTM. treatment can reduce grape disease by about 45.65%
at 14 days and about 39.35% at 21 days after spraying treatment and
18.degree. C. storage conditions. T-test shows that there are
statistically significant differences at both 95% and 99%
confidence levels for the results obtained from Messenger
treatments and buffer treatment.
Example 4--Effect of Treating Grapefruit Fruits with Messenger.RTM.
on Postharvest Grapefruit Storage
[0120] On day 0, FL 33935 grapefruit fruits were treated by
spraying Messenger.RTM. solution (ca. 15 ug/ml) or buffer solution
(5 mM KPO.sub.4, pH 6.8) on the surface of fruits in a 22.degree.
C. greenhouse. The Messenger.RTM. or buffer solutions on
grapefruits were then dried by air, and the treated grapefruits
were marked, mixed together, and put into a plastic container
(Clear View 66 Qt/63L made by Sterilite Corporation). The container
with treated grapefruit fruits was then put into a 18.degree. C.
growth chamber for storage. On day 7, grapefruit fruits were
inoculated with Phytophthora citrophthora by spraying a 10.sup.5
cfu/ml suspension on the surface of grapefruit fruit. The above
procedure was performed on 6 grapefruit fruits per treatment.
[0121] Measurements of disease were conducted on days 87, 97, 103,
and 111 following treatment with Messenger.RTM. or buffer solution.
Grades are indicated according to the criteria set forth in Example
1. The results of these treatments are set forth in Table 4
below.
16TABLE 4 Reduction of Disease Index in Grapefruits Days After
Grade T-test Sample Treatment 0 1 2 3 4 5 Index Efficacy p <
0.05 p < 0.01 Messenger .RTM. 87 5 1 0 0 0 0 0.03 75.00% yes yes
Buffer 87 4 1 0 1 0 0 0.13 n/a -- -- Messenger .RTM. 97 5 0 0 1 0 0
0.10 50.00% yes yes Buffer 97 4 0 1 0 1 0 0.20 n/a -- -- Messenger
.RTM. 103 4 1 0 0 1 0 0.17 28.57% yes yes Buffer 103 3 2 0 0 0 1
0.23 n/a -- -- Messenger .RTM. 111 4 1 0 0 0 1 0.20 33.33% yes yes
Buffer 111 3 1 0 1 0 1 0.30 n/a -- --
[0122] The data listed in Table 4 above shows that the
Messenger.RTM. was more effective than buffer as a fruit spray
treatment in reducing disease index for Phytophthora citrophthora
and providing longer storage life. Messenger.RTM. treatment can
reduce grapefruit disease by about 75.00% at 87 days, about 50.00%
at 97 days, about 28.57% at 103 days, and about 33.33% at 111 days
after spraying treatment and 18.degree. C. storage conditions.
T-test shows that there are statistically significant differences
at both 95% and 99% confidence levels for the results obtained from
Messenger treatment.RTM. and buffer treatment.
Example 5--Effect of Treating Apple (Fuji) Fruits with
Messenger.RTM. on Postharvest Apple Storage
[0123] On day 0, Fuji apple fruits were treated by spraying
Messenger.RTM. solution (ca. 15 ug/ml) or buffer solution (5 mM
KPO.sub.4, pH 6.8) on the surface of fruits in a 22.degree. C.
greenhouse. The Messenger.RTM. or buffer solutions on apples were
then dried by air, and the treated apples were marked, mixed
together, and put into a plastic container (Clear View 66 Qt/63L
made by Sterilite Corporation). The container with treated apples
was then put into a 18.degree. C. growth chamber for storage. On
day 7, apples were inoculated with Penicillium digitatum and
Phytophthora citrophora by spraying a 10.sup.5 cfu/ml suspension on
the surface of apples. The above procedure was performed on 20
apples per treatment.
[0124] Measurements of disease were conducted on days 50, 61, 70,
78, and 85 following treatment with Messenger.RTM. or buffer
solution. Grades are indicated according to the criteria set forth
in Example 1. The results of these treatments are set forth in
Table 5 below.
17TABLE 5 Reduction of Disease Index in Apples Days After Grade
T-test Sample Treatment 0 1 2 3 4 5 Index Efficacy p < 0.05 p
< 0.01 Messenger .RTM. 50 20 0 0 0 0 0 0.00 100.00% yes yes
Buffer 50 18 1 1 0 0 0 0.03 n/a -- -- Messenger .RTM. 61 19 1 0 0 0
0 0.01 88.89% yes yes Buffer 61 16 2 1 0 0 1 0.09 n/a -- --
Messenger .RTM. 70 18 0 2 0 0 0 0.04 71.43% yes yes Buffer 70 14 2
2 1 0 1 0.14 n/a -- -- Messenger .RTM. 78 15 2 3 0 0 0 0.08 57.89%
yes yes Buffer 78 13 2 2 1 0 2 0.19 n/a -- -- Messenger .RTM. 85 13
3 1 1 2 0 0.16 40.74% yes yes Buffer 85 10 5 0 0 3 2 0.27 n/a --
--
[0125] The data listed in Table 5 above shows that the
Messenger.RTM. was more effective than buffer as a fruit spray
treatment in reducing disease index for Penicillium digitatum and
Phytophthora citrophora and providing longer storage life.
Messenger.RTM. treatment can reduce apple disease by about 100.00%
at 51 days, 88.89% at 61 days, 71.43% at 70 days, 57.89% at 78
days, and 40.74% at 85 days after spraying treatment and 1
8.degree. C. storage conditions. T-test shows that there are
statistically significant differences at both 95% and 99%
confidence levels for the results obtained from Messenger
treatment.RTM. and buffer treatment.
Example 6--Effect of Treating Apple (Granny Smith) Fruits with
Messenger.RTM. on Postharvest Apple Storage
[0126] On day 0, Granny Smith apple fruits were treated by spraying
Messenger.RTM. solution (ca. 15 ug/ml) or buffer solution (5 mM
KPO.sub.4, pH 6.8) on the surface of fruits in a 22.degree. C.
greenhouse. The Messenger.RTM. or buffer solutions on apples were
then dried by air, and the treated apples were marked, mixed
together, and put into a plastic container (Clear View 66 Qt/63L
made by Sterilite Corporation). The container with treated apples
was then put into a 18.degree. C. growth chamber for storage. On
day 7, apples were inoculated with Penicillium digitatum and
Phytophthora citrophora by spraying a 10.sup.5 cfu/ml suspension on
the surface of apples. The above procedure was performed on 20
apples per treatment.
[0127] Measurements of disease were conducted on days 50, 61, 70,
78, and 85 following treatment with Messenger.RTM. or buffer
solution. Grades are indicated according to the criteria set forth
in Example 1. The results of these treatments are set forth in
Table 6 below.
18TABLE 6 Reduction of Disease Index in Apples Days After Grade
T-test Sample Treatment 0 1 2 3 4 5 Index Efficacy p < 0.05 p
< 0.01 Messenger .RTM. 50 20 0 0 0 0 0 00.00 100.00% yes yes
Buffer 50 19 1 0 0 0 0 0.01 n/a -- -- Messenger .RTM. 61 13 5 2 0 0
0 0.09 50.00% yes yes Buffer 61 7 9 3 1 0 0 0.18 n/a -- --
Messenger .RTM. 70 7 10 3 0 0 0 0.16 36.00% yes yes Buffer 70 2 12
5 1 0 0 0.25 n/a -- -- Messenger .RTM. 78 6 10 3 1 0 0 0.19 32.14%
yes yes Buffer 78 2 11 5 1 1 0 0.28 n/a -- -- Messenger .RTM. 85 7
9 2 1 1 0 0.20 23.08 yes yes Buffer 85 4 10 4 1 0 1 n/a -- --
[0128] The data listed in Table 6 above shows that the
Messenger.RTM. was more effective than buffer as a fruit spray
treatment in reducing disease index for Penicillium digitatum and
Phytophthora citrophora and providing longer storage life.
Messenger.RTM. treatment can reduce apple disease by about 100.00%
at 51 days, 50.00% at 61 days, 36.00% at 70 days, 32.14% at 78
days, and 23.08% at 85 days after spraying treatment and 18.degree.
C. storage conditions. T-test shows that there are statistically
significant differences at both 95% and 99% confidence levels for
the results obtained from Messenger treatment.RTM. and buffer
treatment.
Example 7--Effect of Treating Tomato Fruits with Messenger.RTM. on
Postharvest Tomato Storage
[0129] On day 0, tomato fruits were treated by spraying
Messenger.RTM. solution (ca. 15 ug/ml) or buffer solution (5mM
KPO.sub.4, pH 6.8) on the surface of fruits in a 22.degree. C.
greenhouse. After the Messenger.RTM. or buffer solutions on
tomatoes were dried by air, the treated tomatoes were marked, mixed
together, and put into a plastic container (Clear View 66 Qt/63L
made by Sterilite Corporation). The container with treated tomatoes
was then put into a 18.degree. C. growth chamber for storage. On
day 7, tomatoes were inoculated with Penicillium digitatum and
Botrytis cinereaon by spraying a 10.sup.5 cfu/ml suspension on the
surface of tomatoes. The above procedure was performed on 44
tomatoes per treatment.
[0130] Measurements of disease were conducted on days 18, 27, 35,
and 42 following treatment with Messenger.RTM. or buffer solution.
Grades are indicated according to the criteria set forth in Example
1. The results of these treatments are set forth in Table 7
below.
19TABLE 7 Reduction of Disease Index in Tomatoes Days After Grade
T-test Sample Treatment 0 1 2 3 4 5 Index Efficacy p < 0.05 p
< 0.01 Messenger .RTM. 18 21 18 5 0 0 0 0.13 37.78% yes yes
Buffer 18 11 21 12 0 0 0 0.20 n/a -- -- Messenger .RTM. 27 16 18 9
1 0 0 0.18 25.00% yes yes Buffer 27 8 24 8 4 0 0 0.24 n/a -- --
Messenger .RTM. 35 7 14 13 10 0 0 0.32 16.67% yes yes Buffer 35 1
16 15 10 2 0 0.38 n/a -- -- Messenger .RTM. 42 1 10 9 12 9 3 0.52
12.88% yes yes Buffer 42 0 3 15 10 11 5 0.60 n/a -- --
[0131] The data listed in Table 7 above shows that the
Messenger.RTM. was more effective than buffer as a fruit spray
treatment in reducing disease index for Penicillium digitatum and
Botrytis cinereaon and providing longer storage life.
Messenger.RTM. treatment can reduce tomato disease by about 37.78%
at 18 days, 25.00% at 27 days, 16.67% at 35 days, and 12.88% at 42
days after spraying treatment and 18.degree. C. storage conditions.
T-test shows that there are statistically significant differences
at both 95% and 99% confidence levels for the results obtained from
Messenger.RTM. treatment and buffer treatment.
Example 8--Effect of Preharvest and Postharvest Messenger.RTM.
Treatments on Tomato (Sanibel) Fruit Postharvest Storage
[0132] Plots of red and green Sanibel variety tomatoes were grown
under either standard conditions or full Messenger.RTM. treatment
over the course of the growing season. The standard conditions,
also known as grower's standard, included fungicide treatment
sprayed every seven days after transplanting using primarily
fungicides containing copper-based active ingredients. The
Messenger.RTM. treatment included six sprays at rate of 2.2 oz of
the product per acre.
[0133] Red and green fruits were harvested from both the
Messenger.RTM. treated and grower standard plots. It was noted that
green tomatoes from the grower standard treatment plots were
smaller (i.e. less mature) then green tomatoes from the messenger
treated plants.
[0134] Harvested fruits were treated as follows:
[0135] (1) Fruits from Messenger.RTM. treated plots were further
treated with Messenger.RTM. after harvest;
[0136] (2) Fruits from standard plots were treated with
Messenger.RTM. after harvest;
[0137] (3) Fruits from Messenger.RTM. treated plots received no
additional treatment following harvest; and
[0138] (4) Fruits from standard plots received no additional
treatment following harvest.
[0139] Postharvest treatment of fruits from groups (1) and (2) was
carried out by spraying with Messenger.RTM. at a rate of 20 ppm
harpin.sub.Ea concentration using a backpack-sprayer at about 30
p.s.i. The fruit were rolled during application to assure full
coverage of the spray. The postharvest treated tomatoes were
allowed to air dry and then tomatoes from groups (1)-(4) were
marked and mixed together in storage in a single layer. Storage
temperatures ranged from about 18 to 32.degree. C. and light
intervals were approximately 12 hours of light and darkness.
Tomatoes were checked daily for rot and desiccation for a total of
31 days after harvest. The results are shown in Table 8 below.
20TABLE 8 Affect of Preharvest and Postharvest Treatment on Rot and
Desiccation Ripe- No. Days After Harvest No. % Group ness Fruit 14
19 21 22 23 25 31 Desiccated Marketable (1) Pre/Postharvest Red 5 0
0 0 1 1 1 2 0 60% Messenger .RTM. Green 4 0 0 0 0 0 0 0 0 100% (2)
Postharvest Red 5 0 0 0 0 0 0 0 4 20% Messenger .RTM. Only Green 4
0 0 0 0 0 0 0 1 75% (3) Preharvest Red 5 0 0 0 0 0 0 2 0 60%
Messenger .RTM. Only Green 5 0 0 0 0 0 0 0 0 100% (4) No Messenger
.RTM. Red 5 1 3 1 5 5 5 5 0 0% Green 5 0 0 0 0 0 0 0 1 80%
[0140] The red tomatoes from group (4) all rotted by day 21. In
contrast, all red tomatoes which received some form of
Messenger.RTM. treatment showed reduced rate of decay and rot. Near
the end of the trial a number of tomatoes were observed to have
desiccated, exhibiting shriveled skins but no rot. These were
included as non-marketable. These results are suggestive that both
preharvest and postharvest Messenger.RTM. treatments can reduce the
level of rotting and desiccation, thereby extending fresh storage
time.
Example 9--Effect of Messenger on Post Harvested Maturity and Fruit
Decay on Tomato During Ambient Storage
[0141] The tomatoes were grown under either standard conditions
(identified in Example 8) or full Messenger.RTM. treatment over the
course of the growing season (identified in Example 8) and then
hand picked at the time of commercial harvest. Mature green fruit
of uniform size (5/6) were collected throughout the field in four
replicate samples of 25 fruit per sample, placed directly into
fruit bags and transported to a laboratory facility for postharvest
treatment and/or analysis. Three different treatment regimen were
examined as follows:
[0142] (1) Fruits from Messenger.RTM. treated plots received no
additional treatment following harvest;
[0143] (2) Fruits from standard plots were treated with
Messenger.RTM. after harvest;
[0144] (3) Fruits from standard plots received no additional
treatment following harvest.
[0145] Postharvest treatment of fruits from group (2) was carried
out by dipping the fruit in a Messenger.RTM. solution (20 ppm
harpin.sub.Ea). The postharvest treated tomatoes were allowed to
air dry and then tomatoes from groups (1)-(3) were marked and mixed
together in tomato crates for storage. Storage temperatures ranged
from about 23 to 26.degree. C. (75-80.degree. F.). The tomatoes
were then rated for color development and decay over time using the
rating scale below.
Grade Description
[0146] 1 Mature Green: When fruit cut in half, no seeds cut; fruit
entirely green with no color break;
[0147] 2 Pink: Initial sign of color break noticed on some areas of
fruit; these areas are usually pink;
[0148] 3 Pink/Red: Intermediate ripening: Fruit is not total red;
some pink still remains;
[0149] 4 Red: Fruit totally red in color;
[0150] 5 Decay: Some areas of the fruit beginning to break down
from decay.
[0151] The results of this test are summarized in Table 9
below.
21TABLE 9 Affect of Preharvest and Postharvest Treatment on
Maturity and Decay Days After Grade T-test Group Treatment 1 2 3 4
5 Index Efficacy p < 0.05 p < 0.01 1 10 11 6 8 75 0 0.69
7.28% yes yes 2 10 5 7 11 77 0 0.72 3.81% yes yes 3 10 5 3 6 86 1
0.75 N/A N/A N/A 1 14 4 5 5 86 0 0.75 2.61% yes yes 2 14 2 6 5 87 0
0.75 1.57% yes yes 3 14 2 4 4 89 1 0.77 N/A N/A N/A 1 17 0 0 3 92 5
0.80 3.37% yes yes 2 17 0 1 4 82 13 0.81 2.16% yes yes 3 17 0 0 1
82 17 0.83 N/A N/A N/A 1 20 0 0 0 89 11 0.82 2.61% yes yes 2 20 0 0
0 80 20 0.84 0.47% yes yes 3 20 0 0 1 76 23 0.84 N/A N/A N/A
[0152] The data generated in this trial indicate that treatment of
tomatoes with Messenger.RTM., either through field sprays or as a
post harvest dip, resulted in earlier fruit red ripening compared
to grower's standard. In addition, although early ripening was
observed, the Messenger.RTM. treatments maintained the red ripe
condition longer than the grower's standard with delay of breakdown
and decay.
Example 10--Effect on Messenger on Post Harvested Maturity and
Fruit Decay of Tomato Under Cold Storage Conditions
[0153] The tomatoes were grown under either standard conditions
(identified in Example 8) or full Messenger.RTM. treatment over the
course of the growing season (identified in Example 8) and then
hand picked at the time of commercial harvest. Mature green fruit
of uniform size (5/6) were collected throughout the field in four
replicate samples of 25 fruit per sample, placed directly into
fruit bags and transported to a laboratory facility for postharvest
treatment and/or analysis. Four different treatment regimen were
examined as follows:
[0154] (1) Fruits from Messenger.RTM. treated plots received no
additional treatment following harvest;
[0155] (2) Fruits from Messenger.RTM. treated plots were further
treated with Messenger.RTM. after harvest;
[0156] (3) Fruits from standard plots were treated with
Messenger.RTM. after harvest; and
[0157] (4) Fruits from standard plots received no additional
treatment following harvest.
[0158] Postharvest treatment of fruits from groups (2) and (3) were
carried out by dipping the fruit in a Messenger.RTM. solution (20
ppm harpin.sub.Ea). The postharvest treated tomatoes were allowed
to air dry and then tomatoes from groups (1)-(4) were marked and
mixed together in tomato crates for storage in a Custom Packing
House cooler at 11.degree. C. (52.degree. F.). The tomatoes were
then rated for color development and decay over time using the
rating scale described in Example 8. The results of this study
appear in Table 10 below.
22TABLE 10 Affect of Preharvest and Postharvest Treatment on
Maturity and Decay Days After Grade T-test Group Treatment 1 2 3 4
5 Index Efficacy p < 0.05 p < 0.01 1 7 66 34 0 0 0 0.27 0.00%
yes yes 2 7 67 33 0 0 0 0.27 0.75% yes yes 3 7 76 24 0 0 0 0.27
7.46% yes yes 4 7 68 30 2 0 0 0.27 N/A yes yes 1 10 59 31 8 0 0
0.30 7.53% yes yes 2 10 60 28 12 0 0 0.30 5.00% yes yes 3 10 65 35
0 0 0 0.27 15.63% yes yes 4 10 49 42 9 0 0 0.32 N/A N/A N/A 1 17 19
35 28 18 0 0.49 7.20% yes yes 2 17 20 38 28 14 0 0.47 10.61% yes
yes 3 17 19 28 39 14 0 0.50 6.06% yes yes 4 17 17 27 31 25 0 0.53
N/A N/A N/A 1 21 11 28 29 32 0 0.56 6.62% N/A N/A 2 21 15 26 37 22
0 0.53 11.92% yes yes 3 21 10 33 35 22 0 0.54 10.93% yes yes 4 21
10 18 32 40 0 0.60 N/A N/A N/A 1 26 3 15 23 59 0 0.68 -2.26% yes
yes 2 26 9 19 25 41 6 0.63 4.39% yes yes 3 26 3 23 31 43 0 0.63
5.00% yes yes 4 26 2 19 23 50 1 0.66 N/A N/A N/A 1 32 3 15 23 59 0
0.68 -2.26% yes yes 2 32 9 19 25 41 6 0.63 4.39% yes yes 3 32 3 23
31 43 0 0.63 5.00% yes yes 4 32 2 19 23 50 1 0.66 N/A N/A N/A 1 38
0 4 10 84 2 0.77 0.26% yes yes 2 38 1 10 15 65 9 0.74 3.64% yes yes
3 38 1 5 14 78 2 0.75 2.60% yes yes 4 38 0 3 13 80 4 0.77 N/A N/A
N/A 1 45 0 3 11 74 12 0.79 2.95% yes yes 2 45 1 4 12 69 14 0.78
3.93% yes yes 3 45 0 1 11 81 7 0.79 3.19% yes yes 4 45 0 0 10 73 17
0.81 N/A N/A N/A 1 50 0 3 10 63 23 0.82 3.55% yes yes 2 50 0 4 11
58 27 0.82 3.55% yes yes 3 50 0 0 8 78 14 0.81 4.02% yes yes 4 50 0
0 3 71 26 0.85 N/A N/A N/A 1 55 0 0 0 73 27 0.85 1.84% yes yes 2 55
0 0 0 68 32 0.86 0.69% yes yes 3 55 0 0 2 80 18 0.83 4.37% yes yes
4 55 0 0 0 65 35 0.87 N/A N/A N/A 1 60 0 0 0 65 35 0.87 2.47% yes
yes 2 60 0 0 0 63 37 0.87 2.02% yes yes 3 60 0 0 0 74 26 0.85 4.48%
yes yes 4 60 0 0 0 54 46 0.89 N/A N/A N/A 1 65 0 0 0 53 47 0.89
1.76% yes yes 2 65 0 0 0 58 42 0.88 2.86% yes yes 3 65 0 0 0 65 35
0.87 4.40% yes yes 4 65 0 0 0 45 55 0.91 N/A N/A N/A
[0159] In previous trials when tomatoes were treated with
Messenger.RTM. in the field and/or with a post harvest dip, the
fruit appeared to develop to red ripe more quickly than the
grower's standard, when held at ambient temperatures (75-80.degree.
F.). Although this early ripening was observed, these red fruit did
not begin to decay earlier than the grower's standard. In this
study, the fruit were held at a constant 52.degree. F. in a
commercial cold storage room at a tomato packinghouse facility. It
appears that this lower temperature slows the ripening process, as
would be expected, and Messenger.RTM. treatments did not increase
the rate of the red ripening for the first 30 days, as observed in
previous tests. The Messenger.RTM. treatments did, however, seem to
maintain the red ripe condition longer than the grower's standard
without breakdown and decay.
Example 11--Effect of Messenger on Post Harvested Maturity and
Fruit Decay on Tomato
[0160] The tomatoes were grown under either standard conditions
(identified in Example 8) or full Messenger.RTM. treatment over the
course of the growing season (identified in Example 8) and then
hand picked at the time of commercial harvest. Mature green fruit
of uniform size (5/6) were collected throughout the field in four
replicate samples of 25 fruit per sample, placed directly into
fruit bags and transported to a laboratory facility for postharvest
treatment and/or analysis. Four different treatment regimen were
examined as follows:
[0161] (1) Fruits from Messenger.RTM. treated plots received no
additional treatment following harvest;
[0162] (2) Fruits from Messenger.RTM. treated plots were further
treated with Messenger.RTM. after harvest;
[0163] (3) Fruits from standard plots were treated with
Messenger.RTM. after harvest; and
[0164] (4) Fruits from standard plots received no additional
treatment following harvest.
[0165] Postharvest treatment of fruits from groups (2) and (3) were
carried out by dipping the fruit in a Messenger.RTM. solution (20
ppm harpin.sub.Ea). The postharvest treated tomatoes were allowed
to air dry and then tomatoes from groups (1)-(4) were marked and
mixed together in tomato crates for storage. Storage temperatures
ranged from about 23 to 26.degree. C. (75-80.degree. F.). The
tomatoes were then rated for color development and decay over time
using the commercial rating scale from the Florida Tomato Committee
color guide as follows:
Grade Description
[0166] 1 Green: When fruit cut in half, no seeds cut; fruit
entirely green with no color break;
[0167] 2 Breakers: Initial sign of color break on 10% or less of
the area of fruit; these areas are usually pink;
[0168] 3 Turning: Pink or red on 10 to 30% of the fruit
surface;
[0169] 4 Pink: Pink or red on 30 to 60% of the fruit surface;
[0170] 5 Light Red: Pink on over 60% of fruit surface and red color
no more than 90% of fruit surface;
[0171] 6 Red: Fruit totally red in color; and
[0172] 7 Decay: Some areas of the fruit beginning to break down
from decay.
[0173] The results of this treatment are set forth in Table 11
below.
23TABLE 11 Affect of Preharvest and Postharvest Treatment on
Maturity and Decay Data Days After Grade T-test Group Treatment 1 2
3 4 5 6 7 Index Efficacy p < 0.05 p < 0.01 1 3 80 18 2 0 0 0
0 0.17 0.00% no no 2 3 73 17 9 1 0 0 0 0.20 -13.11% yes yes 3 3 78
19 3 0 0 0 0 0.18 -2.46% yes yes 4 3 80 18 2 0 0 0 0 0.17 N/A no no
1 7 36 23 22 12 5 2 0 0.33 3.72% yes no 2 7 37 23 17 19 4 0 0 0.33
4.96% yes no 3 7 40 17 15 18 9 1 0 0.35 0.00% yes no 4 7 35 22 19
15 8 1 0 0.35 N/A no no 1 14 2 5 8 8 13 65 0 0.74 8.02% yes yes 2
14 2 3 5 9 8 72 1 0.77 4.44% yes yes 3 14 4 4 7 8 17 60 0 0.73
9.41% yes yes 4 14 0 0 6 5 13 72 4 0.80 N/A no no 1 17 0 0 2 3 6 89
0 0.83 2.51% yes yes 2 17 1 1 1 0 7 88 2 0.83 2.35% yes yes 3 17 1
2 0 0 9 88 0 0.83 3.18% yes yes 4 17 0 0 0 0 7 89 4 0.85 N/A no no
1 21 0 0 0 0 0 97 3 0.86 1.31% yes yes 2 21 0 0 0 0 0 97 3 10.86
1.31% yes yes 3 21 0 0 0 0 3 95 2 0.86 1.96% yes yes 4 21 0 0 0 0 1
87 12 0.87 N/A no no 1 28 0 0 0 0 0 85 15 0.88 2.84% yes yes 2 28 0
0 0 0 0 91 9 0.87 3.79% yes yes 3 28 0 0 0 0 0 81 19 0.88 2.21% yes
yes 4 28 0 0 0 0 0 67 33 0.90 N/A no no 1 32 0 0 0 0 0 22 78 0.97
2.16% yes yes 2 32 0 0 0 0 0 16 84 0.98 1.30% yes yes 3 32 0 0 0 0
0 55 45 0.92 6.93% yes yes 4 32 0 0 0 0 0 7 93 0.99 N/A no no 1 37
0 0 0 0 0 14 86 0.98 1.15% yes yes 2 37 0 0 0 0 0 7 93 0.00 0.14%
yes yes 3 37 0 0 0 0 0 9 91 0.99 0.43% yes yes 4 37 0 0 0 0 0 6 94
0.99 N/A no no 1 42 0 0 0 0 0 12 88 0.98 1.01% yes yes 2 42 0 0 0 0
0 7 93 0.99 0.29% yes yes 3 42 0 0 0 0 0 8 92 0.99 0.43% yes yes 4
42 0 0 0 0 0 5 95 0.99 N/A no no 1 45 0 0 0 0 0 8 92 0.99 0.57% no
no 2 45 0 0 0 0 0 4 96 0.99 0.00% no no 3 45 0 0 0 0 0 4 96 0.99
0.00% no no 4 45 0 0 0 0 0 4 96 0.99 N/A no no 1 50 0 0 0 0 0 7 93
0.99 0.43% no no 2 50 0 0 0 0 0 4 96 0.99 0.00% no no 3 50 0 0 0 0
0 4 96 0.99 0.00% no no 4 50 0 0 0 0 0 4 96 0.99 N/A no no
[0174] In previous trials tomatoes treated with Messenger.RTM. in
the field and/or with a post harvest dip appeared to develop to red
ripe more quickly, but decayed slower than the grower's standard.
The data generated from this trial support these observations. By
twenty-one days post harvest, 97% of the Messenger.RTM. treated
tomatoes were full red ripe, compared to 87% of the grower's
standard. Although it may be assumed that fruit which reach
maturity more quickly will also start to break down more quickly,
the results of the present Examples surprisingly demonstrate that
these earlier-maturing tomatoes were actually 15% slower to decay
than the grower's standard tomatoes. This phenomenon should be of
great interest of several segments of the tomato market. The
growers may be able to reduce ethylene gashouse timings, and the
retail market should be able to significantly reduce inventory
shrinkage.
[0175] 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
12 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 5 447 PRT
Erwinia amylovora 5 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 6 1344 DNA
Erwinia amylovora 6 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 7 341 PRT Pseudomonas syringae 7
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
8 1026 DNA Pseudomonas syringae 8 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 9 424 PRT Pseudomonas
syringae 9 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
10 1729 DNA Pseudomonas syringae 10 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 11 344 PRT Pseudomonas solanacearum 11 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
12 1035 DNA Pseudomonas solanacearum 12 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
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