# Patent

## Title
Method for increasing the efficacy of agricultural chemicals

## Abstract

The present invention is directed to increasing the efficacy of agricultural chemicals. This can be achieved by applying at least one agricultural chemical to a plant or plant seed under conditions effective for the agricultural chemical to perform its intended function and applying at least one hypersensitive response elicitor protein or polypeptide to the plant or plant seed under conditions effective to increase the efficacy of the agricultural chemical. Alternatively, the present invention relates to a method for increasing the efficacy of agricultural chemicals by applying an agricultural chemical to a transgenic plants or transgenic seeds transformed with nucleic acid molecule which encodes a hypersensitive response elicitor protein or polypeptide, wherein the agricultural chemical is applied under conditions effective for the agricultural chemical to perform its intended function but with increased efficacy.

## Background
This application is a national stage application under 35 U.S.C. §371 from PCT Application No. PCT/US03/40089, filed Dec. 15, 2003, which claims the priority benefit of U.S. Provisional Patent Application No. 60/433,893, filed Dec. 16, 2002.


FIELD OF INVENTION
The present invention relates to methods of increasing the efficacy of commonly used agricultural chemicals.
BACKGROUND
Modern agricultural practices rely heavily on the use of chemical inputs to maintain and increase productivity. Agricultural chemical inputs can be broadly categorized as pesticides, fertilizers, and plant growth regulators. Based on monetary expenditure, as well as physical volumes, the vast majority of chemical inputs are in the form of pesticides and fertilizers. In the common agricultural sense, pests are any organisms that contribute to a loss of value or productivity in a crop. Pesticides can be categorized into; insecticides, fungicides, herbicides, as well as minor categories such as acaricides, avicides, virucides, and nematicides. In 1996, U.S. farmers spent over $8.5 billion on pesticides. This translates to the use of over 355 million pounds of herbicides, 70 million pounds of insecticides, and 180 million pounds of fungicides and other pesticides in 1996 alone (Fernandez-Conejo and Jans, “Pest Management in the U.S. Agriculture.” Resource Economics Division, Economic Research Service, U.S. Department of agriculture. Agricultural Handbook No. 717.). With some exceptions, fertilizers are typically characterized as substances containing plant macronutrients or plant micronutrients, and are used in as proportionally as large of volumes as pesticides. In 1997, approximately 22 million tons of nutrients were applied in the United States alone (Data from the Economic Research Service, U.S. Department of Agriculture). Plant growth regulators are a class of agricultural chemical inputs whose use is minor compared to pesticides and fertilizers. Nonetheless, plant growth regulators have significant importance in specific agricultural sectors such as fruit production and ornamentals.
Though the increase in use of agricultural chemicals has directly contributed to an increase in productivity, the increased productivity has not come without a price. Most pesticides present inherent human and environmental health risks. Increasingly, municipalities are identifying hazardous agricultural chemicals, or their residues, in local water sources, streams, and lakes. In addition, the high volumes of pesticides being applied results in the development of pest resistance to the agricultural chemical being applied. Incidences of pest resistance have been documented in most classes of pesticide and a wide range of crop types. Resistance occurs after persistent use of a pesticide or closely related pesticides has decimated a local population of pests, but left a small sub-population of the same pest surviving. The sub-population, either through human pressure or natural divergence of ecotypes, has evolved to be less affected or resistant to the pesticide or closely related pesticides. After repeated cycles of heavy use of the pesticide, decimation of the local population, and survival of the resistant sub-populations, the resistant sub-population eventually multiplies to become the dominant population. The end result being, an entire pest population that is resistant to a given pesticide or closely related pesticides. A once effective and important pesticide is essentially rendered useless to the farmer or commercial grower. Prior to recognition of the actual existence of a resistant pest, the grower having recognized a decrease in efficacy of a pesticide will often intuitively increase the amount of pesticide being applied. Thus, compounding the situation by furthering the propagation of resistant pest through increased use of the pesticide, decreasing the profitability of the crop because of increased purchases of chemical inputs, and simultaneously increasing the human and environmental health risks.
Greater crop yields, resulting from an increased use of fertilizers, have not come without detrimental effects either. Fertilizers are applied to cropland to replenish or add nutrients that are needed by an existing or future crop. The vast majority of the nutrients applied are in the form of nitrogen, phosphorus, and potash (i.e. potassium). Depending on a combination of factor such as the soil's chemical structure, pH, and texture; fertilizer components can be highly susceptible to leaching. Leaching occurs when the amount of water present in the soil, either from irrigation of rainfall, is greater than the soil's water-holding capacity. When this occurs, solubilized fertilizer components are carried low into the soil and out of the plant root zone, thus effectively removing the nutrients for use by the plant. Nitrate-nitrogen (NO3−) is particularly prone to leaching, and can result in hazardous nitrate accumulation in groundwater. In the U.S. and abroad, cropland is commonly over-fertilized. Soil nutrient analysis is often viewed as timely and not economically feasible. Thus, fertilizers are often applied at regular intervals regardless of their need. As with pesticides, the over use of the fertilizers has potentially far reaching detrimental effect on agricultural profitability and risk to environmental health.
In recent years, farmers and agricultural researchers have begun to develop programs and techniques to aid in combating the cycles of increased chemical inputs and decreased profitability. These programs and techniques are commonly referred to as Integrated Pest Management (IPM), or more broadly, Integrated Crop Management (ICM). ICM programs and techniques are being advanced by a range of organizations including; the USDA, land-grant universities and the private sector. ICM Programs are specifically designed with respect to crop type, local environmental conditions, and local pest pressures. In contrast to previous agricultural practices, ICM practices draw on a broad range of techniques and tools including; early and persistent monitoring of pest populations, establishment of acceptable pest population thresholds, the development of chemical control programs that routinely rotate the chemicals being utilized, establishment of cultural control techniques (e.g. adjusting planting and harvesting dates, no-till systems, crop rotation, etc.), promotion of the use of specific plant varieties or transgenic plants, and the development of biological controls techniques (e.g. use of beneficial insects, use of pheromones traps, use of live micro-organisms such as Bacillius thuringensis, etc.). Although ICM practices show great promise for combating many of the problems associated with the high chemical input of modern agricultural practices, the ability to increase the efficacy of the commonly used agricultural chemicals would greatly aid in the over all effort. Increased efficacy would provide greater pest control and/or facilitate decreases in the volume of agricultural chemicals used.
As evident from the above discussion, modem agricultural practices dictate the need to apply several agricultural chemicals, often repeatedly, to a single crop over the course of a growing season. To facilitate this need to apply numerous chemicals to a single crop, it has become routine practice to make what is referred to as tank mixes. Tank mixes are a single application of one or more chemical at the same time. The agricultural chemicals that are desired to be applied are combined into one tank, mixed, soluablized if needed, and applied to the crop. There are limitations to this practice in that some agricultural chemicals are not compatible and may precipitate, become inactive, or decrease the efficacy of other chemicals when mixed together. Pesticide interactions are typically characterized as additive, synergistic, antagonistic, and enhancement. Additive effects occur when the combination of two pesticides produces the same amount of control as the combined effects of each of the chemicals applies independently. Synergistic effects occur when the combined effects of the chemicals is greater than the additive effects. It is assumed that in synergistic pesticide interactions the chemicals are not neutral to one another, and to some extent are chemically interacting with one another. Antagonistic effects are those resulting when the combination of chemicals is less than if the chemicals were used individually. Enhanced effects can occur when a pesticide is combined with an additive that is not a pesticide and the resulting control of the desired pest is greater than if the pesticide was used individually. Factor such as the quantity of water used, the order of mixing the chemicals, and the addition of ajuvants may also affect the utility of a tank mix (Petroff, “Pesticide Interaction and Compatibility,” Montana State University).
The present invention is directed towards improving the efficacy of agricultural chemicals.
SUMMARY OF THE INVENTION
The present invention relates to a method for increasing the efficacy of agricultural chemicals. In one embodiment of the present invention, the method is carried out by applying at least one agricultural chemical and at least one least one hypersensitive response elicitor protein or peptide to a plant or plant seed under conditions effective to increase the efficacy of the agricultural chemical.
In addition, the present invention relates to a method for increasing the efficacy of agricultural chemicals by applying one or more agricultural chemicals to a transgenic plants or transgenic seeds transformed with a nucleic acid molecule which encodes a hypersensitive response elicitor protein or polypeptide under conditions effective for the agricultural chemical to perform its intended function but with increased efficacy.
By the present invention, the efficacy of an agricultural chemical is increased. In achieving this objective, the present invention enables the grower to more effectively manage their crops with respect to fertilizers and plant growth regulators and to more effectively control pests such as insects, fungus, disease, and weeds. As a result of the increased efficacy in controlling common pest problems, growers can reduce yield losses resulting from pest problems. In addition, the present invention enables growers to utilize lower quantities of commonly utilized agricultural chemicals while maintaining or increasing yields. The reduction of agricultural chemical use will also result in profound health and ecological benefits.


DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method for increasing the efficacy of agricultural chemicals. In one embodiment of the present invention, the method is carried out by applying at least one agricultural chemical and at least one least one hypersensitive response elicitor protein or peptide to a plant or plant seed under conditions effective to increase the efficacy of the agricultural chemical.
Agricultural chemicals, according to the present invention, can be divided into several broad categories pesticides, fertilizers, and plant growth regulators. Pesticides, perhaps the most diverse category of agricultural chemicals, can be subdivided into categories based on the type of pest or organism which they are intended to control, such as; insecticides, intended for the control of insect; fungicides, intended for the control of fungi; herbicides, intended for the control of noxious weeds and plants; acaricides, intended for the control of arachnids or spiders; virucides intended for the control of viruses; and nematicides, intended for the control of nematodes.
For use in accordance with this method, suitable insecticides include but, are not limited to those listed in Table 1.







TABLE 1







Common Agricultural Insecticides











Common





Name of


Class of Active
Active

Example


Ingredient
Ingredient
Active ingredient
Product Name





carbamate
Aldricarb
2-methyl-2-(methylthio)propanal O-
Temik ® (Aventis



(ISO)
[(methylamino)carbonyl]oxime (CAS)
CropScience, Research





Triangle Park, NC)


organochlorine
Endosulfan
6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-
Thiodan ® (Aventis



(ISO)
hexahydro-6,9-methano-2,4,3-
CropScience, Research




benzodioxathiepin 3-oxide (CAS)
Triangle Park, NC)


nicotinoid
Imidacloprid
1-[(6-chloro-3-pyridinyl)methyl]-N-
Merit ® (Bayer



(ISO)
nitro-2-imidazolidinimine (CAS)
Ag, Leverkusen, Germany)


phosphoramidothioate
Acephate
O,S-dimethyl acetylphosphoramidothioate
Orthene ® (Valent



(ISO)
(CAS)
U.S.A. Corp.,





Walnut Creek, CA)


organothiophosphate
Dimethoate
O,O-dimethyl S-[2-(methylamino)-2-
Roxion ® (BASF



(ISO)
oxoethyl] phosphorodithioate (CAS)
Corp., Research





Triangle Park, NC)


pyrethroid
Permethrin
(3-phenoxyphenyl)methyl 3-(2,2-
Ambush ®



(ISO)
dichloroethenyl)-2,2-
(Syngenta, Greensboro NC)




dimethylcyclopropanecarboxylate (CAS)









Table 1 is intended as an example. Alternative example product names and classifications exist for the active ingredients cited and would fall within the scope of the present invention.
For use in accordance with this method, suitable fungicides include those listed in Table 2. In addition to Table 2, suitable fungicides include various forms of organic and inorganic copper. Examples of suitable copper compounds include, copper ammonium, copper hydroxide, copper oxychloride, and copper-zinc-chromate.







TABLE 2







Common Agricultural Fungicides











Common Name




Class of Active
of Active

Example Product


Ingredient
Ingredient
Active ingredient
Name





aromatic
Chlorothalonil
Tetrachloroisophthalonitrile (IUPAC)
Bravo ® (Syngenta,



(ISO)

Greensboro NC)


copper
copper hydroxide
copper hydroxide (Cu(OH)2) (CAS)
Kocide ® (Griffin





L.L.C., Valdosta GA)


sulfur
Flowers of Sulfur
sulfur
Kumulus ® (BASF





Corp., Research





Triangle Park, NC)


aliphatic nitrogen
Cymoxanil (ISO)
2-cyano-N-[(ethylamino)carbonyl]-2-
Curzate ® (DuPont




(methoxyimino)acetamide (CAS)
Crop Protection,





Wilmington, DE)


benzimidazole
Thiabendazole
2-(4-thiazolyl)-1H-benzimidazole
Thiabendazole ®



(ISO)
(CAS)
(Syngenta,





Greensboro NC)


dicarboximide
Captan (ISO)
3a,4,7,7a-tetrahydro-2-
Captan ® (Syngenta,




[(trichloromethyl)thio]-1H-isoindole-
Greensboro NC)




1,3(2H)-dione (CAS)


dicarboximide
Vinclozolin (ISO)
3-(3,5-dichlorophenyl)-5-ethenyl-5-
Ronilan ® (BASF




methyl-2,4-oxazolidinedione (CAS)
Corp., Research





Triangle Park, NC)


dithiocarbamate
Mancozeb (ISO)
[[1,2-ethanediylbis[carbamodithioato]]
Dithane ® (Rohm and




(2-)]manganese mixture with [[1,2-
Haas Co.,




ethanediylbis[carbamodithioato]](2-)]
Philadelphia, PA)




zinc (CAS)


dithiocarbamate
Maneb (ISO)
[[1,2-
Manex ® (Griffin




ethanediylbis[carbamodithioato]](2-)]
L.L.C., Valdosta GA)




manganese (CAS)


dithiocarbamate
Metiram (JMAFF)
zinc ammoniate
Polyram ® (BASF




ethylenebis(ditbiocarbamate) -
Corp., Research




poly(ethylenethiuram disulfide)
Triangle Park, NC)




(IUPAC)


dithiocarbamate
Thiram (ISO)
tetramethylthioperoxydicarbonic
Thiram ® (BASF




diamide ([[(CH3)2N]C(S)]2S2) (CAS)
Corp., Research





Triangle Park, NC)


dithiocarbamate
Ziram (ISO)
(T-4)-bis(dimethylcarbamoditbioato-
Ziram ® (UBC




S,S′)zinc
Agrochemicals,





Ghent, Belgium)


imidazole,
Iprodione (ISO)
3-(3,5-dichlorophenyl)-N-(1-
Rovral ® (Aventis


dicarboximide

methylethyl)-2,4-dioxo-1-
CropScience,




imidazolidinecarboxamide (CAS)
Research Triangle





Park, NC)


organophosphate
Fosetyl-aluminum
ethyl hydrogen phosphonate(CAS) as
Alientte ® (Aventis



(ISO)
an aluminum salt
CropScience,





Research Triangle





Park, NC)


strobin
Azoxystrobin
(αE)-methyl 2-[[6-(2-cyanophenoxy)-
Abound ® (Syngenta,



(ISO)
4-pyrimidinyl]oxy]-α-
Greensboro NC)




(methoxymethylene)benzeneacetate




(CAS)


anilide
Metalaxyl (ISO)
methyl N-(2,6-dimethylphenyl)-N-
Ridomil ® (Syngenta,




(methoxyacetyl)-DL-alaninate (CAS)
Greensboro NC)









Table 2 is intended as an example. Alternative example product names and classifications exist for the active ingredients cited and would fall within the scope of the present invention.
For use in accordance with this method, suitable herbicides include, but are not limited to those listed in Tables 3 and 4. Table 3 outlines a Site of Action Classification of Herbicides and is based on the classification system developed by the Weed Science Society of America (WSSA). The herbicide's site of action is defined as the specific biochemical process in the plant that the herbicide acts upon or disrupts. For example, an herbicide containing the active ingredient primisulfuron, has a site of action classification number 2. Table 3 indicates that a Classification Number 2 has as its site of action acetolactate synthase inhibition.







TABLE 3







Herbicide Site of Action and Classification Numbers.








Site of Action



Classification No.
Description of Site of Action











1
ACCase = acetyl-CoA carboxylase inhibitor


2
ALS = actolactate synthase inhibitor


3
MT = microtubule assembly inhibitor


4
GR = growth regulator


5
PSII(A) = photosynthesis II, binding



site A inhibitor


6
PSII(B) = photosynthesis II, binding



site B inhibitor


7
PSII(C) = photosynthesis II, binding



site C inhibitor


8
SHT = shoot inhibitor


9
EPSP = enolpyruvyl-shikimate-phosphate



synthase inhibitor


10
GS = glutamine synthase inhibitor


12
PDS = phytoene desaturase synthase inhibitor


13
DITERP = diterpene inhibitor


14
PPO = protoporphyrinogen oxidase inhibitor


15
SHT/RT = shoot and root inhibitor


22
ED = photosystem 1 electron diverter


28
HPPD = hydroxyphenlypyruvate dioxygenase



synthesis inhibitor
















TABLE 4







Common Agricultural Herbicides













Common






Name of


Site of
Class of Active
Active

Example


Action
Ingredient
Ingredient
Active ingredient
Product Name














1
Cyclohexene Oxime
Sethoxydim
2-[1-(ethoxyimino)butyl]-5-[2-
Poast ® (BASF




(ISO)
(ethylthio)propyl]-3-hydroxy-2-
Corp., Research





cyclohexen-1-one (CAS)
Triangle Park, NC)


1
Phenoxy
Quizalofop-P
(R)-2-[4-[(6-chloro-2-
Assure II ®




(ISO)
quinoxalinyl)oxy]phenoxy]propanoic
(DuPont Crop





acid (CAS)
Protection,






Wilmington, DE)


2
Sulfonylurea
Primisulfuron
2-[[[[[4,6-bis(difluoromethoxy)-2-
Beacon ®




(ISO)
pyrimidinyl]amino]carbonyl]amino]
(Syngenta,





sulfonyl]benzoic acid (CAS)
Greensboro NC)


2
Imidazolinone
Imazamox
2-[4,5-dihydro-4-methyl-4-(1-
Raptor ® (BASF




(ISO)
methylethyl)-5-oxo-1H-imidazol-2-
Corp., Research





yl]-5-(methoxymethyl)-3-
Triangle Park, NC)





pyridinecarboxylic acid (CAS)


3
Dinitroaniline
Trifluralin
2,6-dinitro-N,N-dipropyl-4-
Passport ®




(ISO)
(trifluoromethyl)benzenamine (CAS)
(BASF Corp.,






Research






Triangle Park, NC)


3
Dinitroaniline
Pendimethalin
N-(1-ethylpropyl)-3,4-dimethyl-2,6-
Prowl ® (BASF




(ISO)
dinitrobenzenamine (CAS)
Corp., Research






Triangle Park, NC)


4
Phenoxy
2,4-D (ISO)
(2,4-dichlorophenoxy)acetic acid
Amsol ®





(CAS)
(Aventis






CropScience,






Research






Triangle Park, NC)


4
Benzoic acid
Dicamba
3,6-dichloro-2-methoxybenzoic acid
Banvel ® (BASF




(ISO)
(CAS)
Corp., Research






Triangle Park, NC)


5
Triazine
Atrazine
6-chloro-N-ethyl-N′-(1-methylethyl)-
Atrazine ®




(ISO)
1,3,5-triazine-2,4-diamine (CAS)
(Syngenta,






Greensboro NC)


5
Triazine
Cyanazine
2-[[4-chloro-6-(ethylamino)-1,3,5-
Blandex ®




(ISO)
triazin-2-yl]amino]-2-
(BASF Corp.,





methylpropanenitrile (CAS)
Research






Triangle Park, NC)


6
Nitrite
Bromoxylin
3,5-dibromo-4-hydroxybenzonitrile
Buctril ®




(ISO)
(CAS)
(Aventis






CropScience,






Research






Triangle Park, NC)


7
Phenylurea
Diuron (ISO)
N′-(3,4-dichlorophenyl)-N,N-
Karmex ®





dimethylurea (CAS)
(Griffin L.L.C.,






Valdosta GA)


8
Thiocarbamate
EPTC (ISO)
S-ethyl dipropylcarbamothioate (CAS)
Eptam ®






(Syngenta,






Greensboro NC)


9
Organophosphorus
Glyphosate
N-(phosphonomethyl)glycine (CAS)
Roundup ®




(ISO)

(Monsanto Co.,






St Louis MO)


10
Organophosphorus
Glufosinate
2-amino-4-
Liberty ®




(ISO)
(hydroxymethylphosphinyl)butanoic
(Aventis





acid (CAS)
CropScience,






Research






Triangle Park, NC)


12
Pyridazinone
Norflurazon
4-chloro-5-(methylamino)-2-[3-
Zorial ®




(ISO)
(trifluoromethyl)phenyl]-3(2H)-
(Syngenta,





pyridazinone (CAS)
Greensboro NC)


13
unclassified
Clomazone
2-[(2-chlorophenyl)methyl]-4,4-
Command ®




(ISO)
dimethyl-3-isoxazolidinone (CAS)
(FMC Corp.,






Philadelphia, PA)


14
Diphenyl ether
Fomesafen
5-[2-chloro-4-
Reflex ®




(ISO)
(trifluoromethyl)phenoxy]-N-
(Syngenta,





(methylsulfonyl)-2-nitrobenzamide
Greensboro NC)





(CAS)


15
Chloroacetanilide
Alachlor
2-chloro-N-(2,6-diethylphenyl)-N-
Lasso ®




(ISO)
(methoxymethyl)acetamide (CAS)
(Monsanto Co.,






St. Louis MO)


15
Chloroacetanilide
Acetochlor
2-chloro-N-(ethoxymethyl)-N-(2-
Surpass ® (Dow




(ISO)
ethyl-6-methylphenyl)acetamide
AgroScience LLC,





(CAS)
Indianapolis, IN)


22
Quaternary
Diquat (ISO)
6,7-dihydrodipyrido[1,2-α:2′,1′-
Reglone ®



ammonium

c]pyrazinediium (CAS)
(Syngenta,






Greensboro NC)


28
Cyclopropylisoxazole
Isoxaflutole
(5-cyclopropyl-4-isoxazolyl)[2-
Balance ®




(ISO)
(methylsulfonyl)-4-
(Aventis





(trifluoromethyl)phenyl]methanone
CropScience,





(CAS)
Research






Triangle Park, NC)









Table 4 is intended as an example. Alternative example product names and classifications exist for the active ingredients cited and would fall within the scope of the present invention.
For use in accordance with this method, suitable fertilizers include, but are not limited to those containing plant micronutrients (molybdenum, copper, zinc, manganese, iron, boron, cobalt, and chlorine) and plant macronutrients (sulfur, phosphorus, magnesium, calcium, potassium, and nitrogen). Numerous combinations and forms of plant macro and micronutrients exist and are available in a wide range of formulations. The predominant fertilizers used in agriculture contain various combinations and concentrations of nitrogen, phosphorus, and potassium. Micronutrient specific fertilizers are also common and may contain a single micronutrient or a combination of several micronutrients.
For use in accordance with this method, suitable plant growth regulators include, but are not limited to those containing various form and combinations of auxins, cytokinins, defoliants, gibberellins, ethylene releaser, growth inhibitors, growth retardants, and growth stimulators. Specific plant growth regulators include but are not limited to those listed in Table 5.







TABLE 5







Common Plant Growth Regulators










Class of





Active
Common Name of

Example Product


Ingredient
Active Ingredient
Active ingredient
Name





Cytokinin
Zeatin
(E)-2-methyl-4-(1H-purin-6-ylamino)-2-





buten-1-ol


Defoliant
Thidiazuron (ISO)
N-phenyl-N′-1,2,3-thiadiazol-5-ylurea
Dropp ® (Aventis




(CAS)
CropScience,





Research Triangle





Park, NC)


Growth
Forchlorfenuron
N-(2-chloro-4-pyridinyl)-N′-phenylurea


stimulator

(CAS)


Growth
Mepiquat (ISO)
N,N-dimethylpiperdinum chloride (CAS)
Pix ® (BASF Corp.,


Inhibitor
chloride

Research Triangle





Park, NC)


Growth
Maleic Hydrazide
1,2-dihydro-3,6-pyridazinedione (CAS)
Sprout Stop ®


Inhibitor
(ISO-E)

(Drexel Chemical





Co., Memphis, TN)


Growth
Palclobutrazol (ISO)
(R*,R*)-β-[(4-chlorophenyl)methyl]-α-
Bonzi ® (Syngenta,


Retardant

(1,1-dimethylethyl)-1H-1,2,4-triazole-1-
Greensboro NC)




ethanol (CAS)


Difoliant,
Ethephon (ANSI)
(2-chloroethyl)phosphonic acid (CAS)
Prep ® (Aventis


ethylene


CropScience,


releaser


Research Triangle





Park, NC)


Gibberellin
Gibberellic acid
(1α,2β,4aα,4bβ,10β)-2,4a,7-trihydroxy-1-
RyzUp ® (Valent




methyl-8-methylenegibb-3-ene-1,10-
U.S.A. Corp.,




dicarboxylic acid 1,4a-lactone (CAS)
Walnut Creek, CA)


Auxin
α-naphthaleneacetic
1-naphthaleneacetic acid (CAS)
Tre-Hold ® (Amvac



acid (ISO)

Chemical Co., New





Port Beach, CA)


Auxin
IBA
Indole-3-butyric acid (CAS 8CI)
Seradix ® (Aventis





CropScience,





Research Triangle





Park, NC)


Gibberellin
BAP + Gibberellic
N-(phenylmethyl)-1H-purine-6-amine and
Accel ® (Agtrol



acid
gibberellic acid
International,





Huston, TX)




(S)-trans-2-Amino-4-(2-aminoethoxy)-3-
ReTain ® (Valent




butenoic acid hydrochloride
U.S.A. Corp.,





Walnut Creek, CA)









Table 5 is intended as an example. Alternative example product names and classifications exist for the active ingredients cited and would fall within the scope of the present invention.
For use in accordance with these methods, suitable hypersensitive response elicitor protein or polypeptide are from bacterial sources including, without limitation, 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).
The hypersensitive response elicitor protein or polypeptide is derived, preferably, from Erwinia chrysanthemi, Erwinia amylovora, Pseudomonas syringae, Pseudomonas solanacearum, or Xanthomonas campestris. 
A hypersensitive response elicitor protein or polypeptide from Erwinia chrysanthemi has an amino acid sequence corresponding to SEQ. ID. No. 1 as follows:








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






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:









cgattttacc cgggtgaacg tgctatgacc gacagcatca cggtattcga caccgttacg   60



 


gcgtttatgg ccgcgatgaa ccggcatcag gcggcgcgct ggtcgccgca atccggcgtc  120


 


gatctggtat ttcagtttgg ggacaccggg cgtgaactca tgatgcagat tcagccgggg  180


 


cagcaatatc ccggcatgtt gcgcacgctg ctcgctcgtc gttatcagca ggcggcagag  240


 


tgcgatggct gccatctgtg cctgaacggc agcgatgtat tgatcctctg gtggccgctg  300


 


ccgtcggatc ccggcagtta tccgcaggtg atcgaacgtt tgtttgaact ggcgggaatg  360


 


acgttgccgt cgctatccat agcaccgacg gcgcgtccgc agacagggaa cggacgcgcc  420


 


cgatcattaa gataaaggcg gcttttttta ttgcaaaacg gtaacggtga ggaaccgttt  480


 


caccgtcggc gtcactcagt aacaagtatc catcatgatg cctacatcgg gatcggcgtg  540


 


ggcatccgtt gcagatactt ttgcgaacac ctgacatgaa tgaggaaacg aaattatgca  600


 


aattacgatc aaagcgcaca tcggcggtga tttgggcgtc tccggtctgg ggctgggtgc  660


 


tcagggactg aaaggactga attccgcggc ttcatcgctg ggttccagcg tggataaact  720


 


gagcagcacc atcgataagt tgacctccgc gctgacttcg atgatgtttg gcggcgcgct  780


 


ggcgcagggg ctgggcgcca gctcgaaggg gctggggatg agcaatcaac tgggccagtc  840


 


tttcggcaat ggcgcgcagg gtgcgagcaa cctgctatcc gtaccgaaat ccggcggcga  900


 


tgcgttgtca aaaatgtttg ataaagcgct ggacgatctg ctgggtcatg acaccgtgac  960


 


caagctgact aaccagagca accaactggc taattcaatg ctgaacgcca gccagatgac 1020


 


ccagggtaat atgaatgcgt tcggcagcgg tgtgaacaac gcactgtcgt ccattctcgg 1080


 


caacggtctc ggccagtcga tgagtggctt ctctcagcct tctctggggg caggcggctt 1140


 


gcagggcctg agcggcgcgg gtgcattcaa ccagttgggt aatgccatcg gcatgggcgt 1200


 


ggggcagaat gctgcgctga gtgcgttgag taacgtcagc acccacgtag acggtaacaa 1260


 


ccgccacttt gtagataaag aagatcgcgg catggcgaaa gagatcggcc agtttatgga 1320


 


tcagtatccg gaaatattcg gtaaaccgga ataccagaaa gatggctgga gttcgccgaa 1380


 


gacggacgac aaatcctggg ctaaagcgct gagtaaaccg gatgatgacg gtatgaccgg 1440


 


cgccagcatg gacaaattcc gtcaggcgat gggtatgatc aaaagcgcgg tggcgggtga 1500


 


taccggcaat accaacctga acctgcgtgg cgcgggcggt gcatcgctgg gtatcgatgc 1560


 


ggctgtcgtc ggcgataaaa tagccaacat gtcgctgggt aagctggcca acgcctgata 1620


 


atctgtgctg gcctgataaa gcggaaacga aaaaagagac ggggaagcct gtctcttttc 1680


 


ttattatgcg gtttatgcgg ttacctggac cggttaatca tcgtcatcga tctggtacaa 1740


 


acgcacattt tcccgttcat tcgcgtcgtt acgcgccaca atcgcgatgg catcttcctc 1800


 


gtcgctcaga ttgcgcggct gatggggaac gccgggtgga atatagagaa actcgccggc 1860


 


cagatggaga cacgtctgcg ataaatctgt gccgtaacgt gtttctatcc gcccctttag 1920


 


cagatagatt gcggtttcgt aatcaacatg gtaatgcggt tccgcctgtg cgccggccgg 1980


 


gatcaccaca atattcatag aaagctgtct tgcacctacc gtatcgcggg agataccgac 2040


 


aaaatagggc agtttttgcg tggtatccgt ggggtgttcc ggcctgacaa tcttgagttg 2100


 


gttcgtcatc atctttctcc atctgggcga cctgatcggt t                     2141






The 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.

One particular hypersensitive response elicitor protein, known as harpinEa, is commercially available from Eden Bioscience Corporation (Bothell, Wash.) under the name of Messenger®. Messenger contains 3% by weight of harpinEa as the active ingredient and 97% by weight inert ingredients. HarpinEa is one type of hypersensitive response elicitor protein from Erwinia amylovora. HarpinEa has an amino acid sequence corresponding to SEQ. ID. No. 3 as follows:








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






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° 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:









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






The above nucleotide and amino acid sequences are disclosed and 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.

Another hypersensitive response elicitor protein or polypeptide derived from Erwinia amylovora has an amino acid sequence corresponding to SEQ. ID. No. 5 as follows:








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






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. 45 kDa. The DNA molecule encoding this hypersensitive response elicitor protein or polypeptide has a nucleotide sequence corresponding to SEQ. ID. No. 6 as follows:









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






The above nucleotide and amino acid sequences are disclosed and further described in PCT Application Publication No. WO 99/07208 to Kim et al., which is hereby incorporated by reference in its entirety.

A hypersensitive response elicitor protein or polypeptide derived from Pseudomonas syringae has an amino acid sequence corresponding to SEQ. ID. No. 7 as follows:








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






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 HarpinPss: 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:









atgcagagtc tcagtcttaa cagcagctcg ctgcaaaccc cggcaatggc ccttgtcctg   60



 


gtacgtcctg aagccgagac gactggcagt acgtcgagca aggcgcttca ggaagttgtc  120


 


gtgaagctgg ccgaggaact gatgcgcaat ggtcaactcg acgacagctc gccattggga  180


 


aaactgttgg ccaagtcgat ggccgcagat ggcaaggcgg gcggcggtat tgaggatgtc  240


 


atcgctgcgc tggacaagct gatccatgaa aagctcggtg acaacttcgg cgcgtctgcg  300


 


gacagcgcct cgggtaccgg acagcaggac ctgatgactc aggtgctcaa tggcctggcc  360


 


aagtcgatgc tcgatgatct tctgaccaag caggatggcg ggacaagctt ctccgaagac  420


 


gatatgccga tgctgaacaa gatcgcgcag ttcatggatg acaatcccgc acagtttccc  480


 


aagccggact cgggctcctg ggtgaacgaa ctcaaggaag acaacttcct tgatggcgac  540


 


gaaacggctg cgttccgttc ggcactcgac atcattggcc agcaactggg taatcagcag  600


 


agtgacgctg gcagtctggc agggacgggt ggaggtctgg gcactccgag cagtttttcc  660


 


aacaactcgt ccgtgatggg tgatccgctg atcgacgcca ataccggtcc cggtgacagc  720


 


ggcaataccc gtggtgaagc ggggcaactg atcggcgagc ttatcgaccg tggcctgcaa  780


 


tcggtattgg ccggtggtgg actgggcaca cccgtaaaca ccccgcagac cggtacgtcg  840


 


gcgaatggcg gacagtccgc tcaggatctt gatcagttgc tgggcggctt gctgctcaag  900


 


ggcctggagg caacgctcaa ggatgccggg caaacaggca ccgacgtgca gtcgagcgct  960


 


gcgcaaatcg ccaccttgct ggtcagtacg ctgctgcaag gcacccgcaa tcaggctgca 1020


 


gcctga                                                            1026






The 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.

Another hypersensitive response elicitor protein or polypeptide derived from Pseudomonas syringae has an amino acid sequence corresponding to SEQ. ID. No. 9 as follows:








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






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:









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






The above nucleotide and amino acid sequences are disclosed and further described in U.S. Pat. No. 6,172,184 to Collmer et al., which is hereby incorporated by reference in its entirety.

A hypersensitive response elicitor protein or polypeptide derived from Pseudomonas solanacearum has an amino acid sequence corresponding to SEQ. ID. No. 11 as follows:








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






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:









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






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.

A hypersensitive response elicitor polypeptide or protein derived from Xanthomonas campestris has an amino acid sequence corresponding to SEQ. ID. No. 13 as follows:








Met Asp Ser Ile Gly Asn Asn Phe Ser Asn Ile Gly Asn Leu Gln Thr



1               5                   10                  15


 


Met Gly Ile Gly Pro Gln Gln His Glu Asp Ser Ser Gln Gln Ser Pro


            20                  25                  30


 


Ser Ala Gly Ser Glu Gln Gln Leu Asp Gln Leu Leu Ala Met Phe Ile


        35                  40                  45


 


Met Met Met Leu Gln Gln Ser Gln Gly Ser Asp Ala Asn Gln Glu Cys


    50                  55                  60


 


Gly Asn Glu Gln Pro Gln Asn Gly Gln Gln Glu Gly Leu Ser Pro Leu


65                  70                  75                  80


 


Thr Gln Met Leu Met Gln Ile Val Met Gln Leu Met Gln Asn Gln Gly


                85                  90                  95


 


Gly Ala Gly Met Gly Gly Gly Gly Ser Val Asn Ser Ser Leu Gly Gly


            100                 105                 110


 


Asn Ala






This hypersensitive response elicitor polypeptide or protein has an estimated molecular weight of about 12 kDa based on the deduced amino acid sequence, which is consistent with a molecular weight of about 14 kDa as detected by SDS-PAGE. The above protein or polypeptide is encoded by a DNA molecule according to SEQ. ID. No. 14 as follows:









atggactcta tcggaaacaa cttttcgaat atcggcaacc tgcagacgat gggcatcggg  60



 


cctcagcaac acgaggactc cagccagcag tcgccttcgg ctggctccga gcagcagctg 120


 


gatcagttgc tcgccatgtt catcatgatg atgctgcaac agagccaggg cagcgatgca 180


 


aatcaggagt gtggcaacga acaaccgcag aacggtcaac aggaaggcct gagtccgttg 240


 


acgcagatgc tgatgcagat cgtgatgcag ctgatgcaga accagggcgg cgccggcatg 300


 


ggcggtggcg gttcggtcaa cagcagcctg ggcggcaacg cc                    342






The above nucleotide and amino acid sequences are disclosed and further described in U.S. patent application Ser. No. 09/829,124, which is hereby incorporated by reference in its entirety.

Other embodiments of the present invention include, but are not limited to, use of a hypersensitive response elicitor protein or polypeptide derived from Erwinia carotovora and Erwinia stewartii. Isolation of Erwinia carotovora hypersensitive response elicitor protein or polypeptide is described in Cui, et al., “The RsmA Mutants of Erwinia carotovora subsp. carotovora Strain Ecc7 Overexpress hrp NEcc 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. Cone. 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.
Other elicitors can be readily identified by isolating putative hypersensitive response elicitors 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.
The hypersensitive response elicitor protein or polypeptide can also be a fragment of the above referenced hypersensitive response elicitor proteins or polypeptides as well as fragments of full length elicitors from other pathogens.
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 subolones 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.
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.
Examples of suitable fragments of a hypersensitive response elicitor are described in WIPO International Publication Numbers: WO 98/54214 and WO 01/98501, which are hereby incorporated by reference in their entirety.
DNA molecules encoding a hypersensitive response elicitor protein or polypeptide can also include a DNA molecule that hybridizes under stringent conditions to the DNA molecule having a nucleotide sequences from one of the above identified hypersensitive response licitors. An example of suitable stringency conditions is when hybridization is carried out at a temperature of about 37° C. using a hybridization medium that includes 0.9M sodium citrate (“SSC”) buffer, followed by washing with 0.2×SSC buffer at 37° 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° C. to about 65° 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 μg/ml E. coli DNA, followed by washing carried out at between about 42° C. to about 65° C. in a 0.2×SSC buffer.
Variants of suitable hypersensitive response elicitor proteins or polypeptides can also be expressed. Variants may be made by, for example, the deletion, addition, or alteration of amino acids that have minimal influence on the properties, secondary structure and hydropathic nature of the polypeptide. For example, a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide.
The DNA molecule encoding the hypersensitive response elicitor polypeptide or protein can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
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 procaryotic organisms and eucaryotic cells grown in tissue culture.
Recombinant genes may also be introduced into viruses, such as vaccina virus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, 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.
A variety of host-vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria. The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.
Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (mRNA) translation).
Transcription of DNA is dependent upon the presence of a promotor which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eucaryotic promotors differ from those of procaryotic promotors. Furthermore, eucaryotic promotors and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promotors are not recognized and do not function in eucaryotic cells.
Similarly, translation of mRNA in 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.
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, Zac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) 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.
Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.
Specific initiation signals are also required for efficient gene transcription and translation in 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 a Shine-Dalgarno (“SD”) sequence about 7-9 bases 5′ to the initiation codon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include, but are not limited to, the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
Once the DNA molecule coding for a hypersensitive response elicitor protein or polypeptide has been ligated to its appropriate regulatory regions using well known molecular cloning techniques, it can then be introduced into a vector or otherwise introduced directly into a host cell (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). The recombinant molecule can be introduced into host cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like. Preferably the host cells are either a bacterial cell or a plant cell. The host cells, when grown in an appropriate medium, are capable of expressing the hypersensitive response elicitor protein or polypeptide, which can then be isolated therefrom and, if necessary, purified.
Alternatively, it is desirable for recombinant host cells to secrete the hypersensitive response elicitor protein or polypeptide into growth medium, thereby avoiding the need to lyse cells and remove cellular debris. To enable the host cell to secrete the hypersensitive response elicitor, the host cell can 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. After growing recombinant host cells which secrete the hypersensitive response elicitor into growth medium, isolation of the hypersensitive response elicitor protein or polypeptide from growth medium can be carried out substantially as described above.
The hypersensitive response elicitor of the present invention is preferably in isolated form (i.e. separated from its host organism) and more preferably produced in purified form (preferably at least about 60%,) by conventional techniques. Typically, the hypersensitive response elicitor of the present invention is produced but not secreted into the growth medium of recombinant host cells. Alternatively, the protein or polypeptide of the present invention is secreted into growth medium. In the case of unsecreted protein, to isolate the protein, the host cell (e.g., E. coli) carrying a recombinant plasmid is propagated, lysed by sonication, heat, or chemical treatment, and the homogenate is centrifuged to remove bacterial debris. The supernatant is then subjected to heat treatment and the hypersensitive response elicitor is separated by centrifugation. The supernatant fraction containing the hypersensitive response elicitor is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the fragment. If necessary, the protein fraction may be further purified by ion exchange or HPLC.
A composition suitable for treating plants or plant seeds with a hypersensitive response elicitor polypeptide or protein in an isolated form contains a hypersensitive response elicitor polypeptide or protein in a carrier. Suitable carriers include water, aqueous solutions, slurries, or dry powders. In this embodiment, the composition contains greater than 500 nM hypersensitive response elicitor polypeptide or protein.
Alternatively, application of the hypersensitive response elicitor protein or polypeptide can also be applied in a non-isolated but non-infectious form. When applied in non-isolated but non-infectious form, the hypersensitive response elicitor is applied indirectly to the plant via application of a bacteria which expresses and then secretes or injects the expressed hypersensitive response elicitor protein or polypeptide into plant cells or tissues. Such application can be carried out by applying the bacteria to all or part of a plant or a plant seed under conditions where the polypeptide or protein contacts all or part of the cells of the plant or plant seed. Alternatively, the hypersensitive response elicitor protein or polypeptide can be applied to plants such that seeds recovered from such plants themselves are able to achieve the effects of the present invention.
In the bacterial application mode of the present invention, the bacteria do not cause disease and have been transformed (e.g., recombinantly) with genes encoding a hypersensitive response elicitor polypeptide or protein. For example, E. coli, which does not elicit a hypersensitive response in plants, can be transformed with genes encoding a hypersensitive response elicitor polypeptide or protein and then applied to plants. Bacterial species other than E. coli can also be used in this embodiment of the present invention.
Alternatively, in the bacterial application mode of the present invention, a naturally occurring virulent bacteria that is capable of expressing and secreting a hypersensitive response elicitor is mutated or altered to be an aviralent pathogen while retaining its ability to express and secrete the hypersensitive response elicitoris. Examples of such naturally occurring virulent bacteria are noted above. In this embodiment, these bacteria are applied to plants or their seeds. For example, virulent Erwinia amylovora causes disease in apple. An avirulent Erwinia amylovora would not cause the disease in apples, but would retain its ability to express and secrete a hypersensitive response elicitor. Bacterial species other than Erwinia amylovora can also be used in this embodiment of the present invention.
The methods of the present invention which involve application of the agricultural chemicals and/or hypersensitive response elicitor polypeptides or proteins can be carried out through a variety of procedures in which all or part of the plant is treated, including leaves, stems, roots, etc. Application techniques may include but not limited to; foliar application, broadcast application, chemigation, high pressures injection, nesting, aerial spray, utilization of chemstations, root drench, and cutting drench. Application may, but need not, involve infiltration of the hypersensitive response elicitor polypeptide or protein into the plant. More than one application of the agricultural chemical and/or hypersensitive response elicitor protein or polypeptide may be desirable to realize maximal benefit over the course of a growing season.
Agricultural chemicals and/or hypersensitive response elicitor polypeptides or proteins can be applied to a plant or plant seed alone or mixed with additional components. Additional components can include one or more additional agricultural chemicals, carriers, adjuvants, buffering agents, coating agents, abrading agents, surfactants, preservatives, and color agents. These materials can be used to facilitate the process of the present invention. In addition, the agricultural chemicals and/or hypersensitive response elicitor polypeptides or proteins can be applied to plant seeds with other conventional seed formulation and treatment materials, including clays and polysaccharides.
When treating plant seeds in accordance with the application embodiment of the present invention, the agricultural chemicals and/or hypersensitive response elicitor polypeptides or proteins can be applied by low or high pressure spraying, seed dusting, seed soaking, and seed coating, or injection. Other suitable application procedures can be envisioned by those skilled in the art provided they are able to effect contact of the hypersensitive response elicitor polypeptide or protein with cells of the plant or plant seed.
Once treated with the agricultural chemical and/or hypersensitive response elicitor of the present invention, the seeds can be planted in natural or artificial soil and cultivated using conventional procedures to produce plants. After plants have been propagated from seeds treated in accordance with the present invention, the plants may also be treated with one or more applications of the agricultural chemicals and/or hypersensitive response elicitor polypeptides or proteins. Such propagated plants may, in turn, be useful in producing seeds or propagules (e.g., cuttings) suitable for carrying out the present invention.
Typically, the manufacturer or distributor's product label for specific agricultural chemicals and/or hypersensitive response elicitor polypeptides or proteins will provide suggested application rates, the crops on which use of the agricultural chemicals and/or hypersensitive response elicitor polypeptides or proteins has been approved, and preferred application techniques if they exist.
The present method, for increasing the efficacy of common agricultural chemicals, can be utilized while treating a wide variety of plants and plant seeds types. Suitable plants include dicots and monocots. More particularly, useful crop plants can include, but are not limited to: canola, alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum, and sugarcane. Examples of suitable ornamental plants are: Arabidopsis thaliana, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, and zinnia.
In another embodiment of the present invention, one or more agricultural chemicals are applied to a transgenic plants or transgenic seeds encoding a hypersensitive response elicitor protein or polypeptide. This technique involves the use of transgenic plants and transgenic seeds encoding a hypersensitive response elicitor protein or polypeptide, a hypersensitive response elicitor proteins or polypeptides need not be applied to the plant or seed. Instead, transgenic plants transformed with a gene encoding such a hypersensitive response elicitor protein or polypeptide are produced according to procedures well known in the art as described below.
The vector described above can be microinjected directly into plant cells by use of micropipettes to transfer mechanically the recombinant DNA. Crossway, Mol. Gen. Genetics, 202:179-85 (1985), which is hereby incorporated by reference in its entirety. The genetic material may also be transferred into the plant cell using polyethylene glycol. Krens, et al., Nature, 296:72-74 (1982), which is hereby incorporated by reference in its entirety.
Another approach to transforming plant cells with a gene is particle bombardment (also known as biolistic transformation) of the host cell. This can be accomplished in one of several ways. 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.
Yet another method of introduction is fusion of protoplasts with other entities, either minicells, cells, lysosomes, or other fusible lipid-surfaced bodies. Fraley, et al., Proc. Natl. Acad. Sci. USA, 79:1859-63 (1982), which is hereby incorporated by reference in its entirety.
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 expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and regenerate.
Another method of introducing the DNA molecule into plant cells is to infect a plant cell with Agrobacterium tumefaciens or A. rhizogenes previously transformed with the gene. Under appropriate conditions known in the art, the transformed plant cells are grown to form shoots or roots, and develop further into plants. Generally, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28° C.
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.
Heterologous genetic sequences can be introduced into appropriate plant cells, by means of the Ti plasmid of A. tumefaciens or the Ri plasmid of A. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome. J. Schell, Science, 237:1176-83 (1987), which is hereby incorporated by reference in its entirety.
After transformation, the transformed plant cells must be regenerated.
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. I, 1984, and Vol. III (1986), which are hereby incorporated by reference in their entirety.
It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugarcane, sugar beets, cotton, fruit trees, and legumes.
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.
After the expression cassette is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
Once transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure. Alternatively, transgenic seeds or propagules (e.g., cuttings) are recovered from the transgenic plants. The seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants. The transgenic plants are propagated from the planted transgenic seeds.
EXAMPLES
Example 1
Application of Messenger® with Roundup UltraMAX® to Improve Control of Various Weeds
The objective of this study was to determine if pre, post, or tank-mix application of Messenger (active ingredient harpinEa) affected Roundup UltraMAX's (active ingredient glyphosate, Monsanto, St. Louis, Mo.) ability to control weeds. In this experiment, control of two susceptible and two tolerant dicot weed species, as well as two susceptible and two tolerant monocot weed species was examined. Plots were constructed in the field and uniformilly planted with the respective weed seeds. Plots were maintained in ambient conditions. Messenger and Roundup UltraMAX applications were conducted at 2.25 oz. per acre and 16 oz. per acre, respectively. The various treatment groups were as follows; (1) Messenger application followed three days later by a Roundup UltraMAX application (Mess bf RU), (2) application of Messenger and Roundup UltraMAX at the same time via a tank-mix (MSS+RU), (3) application of Roundup UltraMAX followed one day (24 hours) later by a Messenger application (RU bf MSS), (4) Roundup UltraMAX application alone. Observations regarding the percent weed control of the specific weed species were made at seven and 14 days after treatments (DAT). Results are shown below in Tables 6 through 9.







TABLE 6







Effect of Messenger upon Roundup UltraMAX


Efficacy (susceptible dicots)










Common Lambsquarter
Common Cocklebur











Treatment
7 DAT
14 DAT
7 DAT
14 DAT





MSS bf RU
62 b
82 b
82 b
100


MSS + RU
73 a
94 a
91 a
100


RU bf MSS
72 a
91 a
92 a
100


RU
45 c
72 c
72 c
100





Same letters do not significantly differ (P = .05, Student-Newman-Keuls)













TABLE 7







Effect of Messenger upon Roundup UltraMAX


Efficacy (tolerant dicots)











Velvetleaf
Redroot Pigweed














Treatment
7 DAT
14 DAT
7 DAT
14 DAT







MSS bf RU
21 b
32 b
54 b
74 b



MSS + RU
32 a
44 a
81 a
96 a



RU bf MSS
33 a
46 a
77 a
94 a



RU
11 c
18 c
35 c
46 c







Same letters do not significantly differ (P = .05, Student-Newman-Keuls)













TABLE 8







Effect of Messenger upon Roundup UltraMAX


Efficacy (susceptible monocots)












Smooth Crabgrass

Giant Foxtail














Treatment
7 DAT
14 DAT
7 DAT
14 DAT







MSS bf RU
80 b
100
83 b
100



MSS + RU
92 a
100
93 a
100



RU bf MSS
91 a
100
92 a
100



RU
72 c
100
75 c
100







Same letters do not significantly differ (P = .05, Student-Newman-Keuls)













TABLE 9







Effect of Messenger upon Roundup UltraMAX


Efficacy (tolerant monocots)












Yellow Nutsedge

Shattercane














Treatment
7 DAT
14 DAT
7 DAT
14 DAT







MSS bf RU
 5 b
10 c
42 b
70 b



MSS + RU
14 a
29 a
75 a
97 a



RU bf MSS
13 a
24 d
72 a
93 a



RU
 2 c
 4 b
28 c
54 c







Same letters do not significantly differ (P = .05, Student-Newman-Keuls)






In each case where 100% control was not achieved, the inclusion of Messenger with Roundup UltraMAX significantly increased Roundup UltraMAX's control of the weed. Though Messenger treatment followed by Roundup UltraMAX treatment showed significantly increased weed control over that of Roundup Ultra Max alone, tank-mixing and application of Roundup UlItraMAX followed by Messenger application showed the greatest control of weeds.
Example 2
Application of Messenger® with Orthene® to Control Insects for Blue Mold in Tobacco Results in Lower Disease Incidence than Orthene Alone
Tobacco (Nicotiana tobacum), var. K-326, was planted in a small-plot, replicated (3 times) field trial. Application of Messenger (active ingredient harpinEa) Orthene (active ingredient acephate, Valent U.S.A. Corp., Walnut Creek, Calif.), and Messenger+Orthene were made beginning with the transplant water and were followed by 4 foliar sprays at approximately 14-d intervals. Orthene was used in this trial to control aphids, a common vector for blue mold disease (Peronospora tabacina) in tobacco.
The trial was not inoculated with insects or disease. Evaluation for blue mold was made approximately one week following the final (4th) foliar application of each treatment. Addition of Messenger to the Orthene treatment resulted in lower blue mold infestation than the Messenger alone treatment, while the combination of both products resulted in substantially lower disease incidence than the Orthene alone treatment (Table 10). These results indicate a positive trend for the inclusion of Messenger with Orthene to give a slightly greater disease control than either Messenger or Orthene alone (Table 10).







TABLE 10







Messenger, Orthene, and Messenger + Orthene treatments applied


to tobacco as transplant water drenches (TPW) and foliar sprays.












APPL. RATE
BLUE



APPL.
(FOLIAR
MOLD DISEASE


TREATMENT(S)
RATE (TPW)
SPRAY)
INCIDENCE (%)













Messenger
30 ppm
30 ppm
8.2


Orthene
12 oz/A
12 oz/A
27.8


Messenger +
30 ppm +
30 ppm +
7.0


Orthene
12 oz/A
12 oz/A









Messenger vs. Messenger+Orthene: 15% decrease in blue mold disease incidence.
Orthene vs. Messenger+Orthene: 75% decrease in blue mold disease incidence.
Example 3
Application of Messenger® with Temik® to Control Nematodes in Cotton Enhances Performance of Temik
Cotton, (Gossypium hirsutum), var. PM 1218, was planted to a small-plot, replicated (6 times) field trial. Plot size was 6-8 rows×50 feet with the center 4 rows treated and center 2 rows harvested. Ten-foot buffers were established between blocks. Temik (active ingredient aldricarb, Aventis CropScience, Research Triangle, N.C.) was applied in-furrow (at 5 lbs/A) at planting. Messenger (active ingredient harpinEa) foliar applications (at 2.23 oz/A) were made at various timing regimes on both Temik-treated and non-Temik treated cotton. Yield data in response to these treatments is shown in Table 11.







TABLE 11







Messenger, Temik, and Messenger + Temik


Treatments Effect on Cotton Seed Yield.










SEED COTTOT SEED
INCREASE OVER


TREATMENT
YIELD (LBS/A)
UNTREATED (%)












Messenger
2,2031
8.9


Messenger + Temik
2,3881
18.0


Temik
2,221 
9.8


Untreated
2,023 
—





1Seed cotton yield figures are averages from four treatment-timing combinations of Messenger and Messenger + Temik, respectively.






Results from this field trial indicated that both the individual Messenger and Temik treatments boosted seed cotton yield about 10% above the untreated. However, the Messenger+Temik treatment gave an 18% yield above the untreated suggesting that addition of Messenger to the Temik treatment enhanced Temik's ability to perform its intended function.
Example 4
Application of Messenger® with Equation Pro® to Control Late Blight in Tomatoes Enhances Performance of Equation Pro
Tomato seedlings were planted into greenhouse pots, 3 plants per pot replicated 4 pots per treatment. One week prior to artificial inoculation with Phytopthora infestans (Late blight), one set of plants received a single foliar spray of Messenger (active ingredient harpinEa) at approx. 20 ppm active ingredient (a.i.) followed by a second foliar spray approximately one week after inoculation. A second set of replicate pots received Messenger+Equation Pro (active ingredients famoxadone+cymoxanil, DuPont Crop Protection, Wilmington, Del.) while a third set of replicates received only the Equation Pro treatment. An untreated control treatment was included in the test. After the disease had spread to fully infect the untreated plants, treated plants were rated for disease symptoms; severity and index were both calculated for each treatment. Results are presented in Table 12.







TABLE 12







Messenger, Messenger + Equation Pro, and Equation


Pro Treatments Effect on Late Blight in Tomato.













DISEASE
SEVERITY
EFFICACY



TREATMENT
INDEX
(%)
(%)
















Messenger
0.891
17.9
71.0



Messenger +
0.301
6.0
90.2



Equation Pro



Equation Pro
0.59 
11.8
80.8



Untreated
3.07 
61.4
—







1Mean values of four replicate pots, three plants in each.






Results from this greenhouse trial indicated that both the individual Messenger and Equation Pro treatments provided substantial resistance to Late blight in tomato. However, the Messenger+Equation Pro treatment resulted in an even greater degree of disease control than either treatment alone, suggesting that the addition of Messenger to the Equation Pro treatment enhances Equation Pro's ability to perform its intended function.
Example 5
Inclusion of Messenger® in Aliette® Treatment Program Increases Control of Phytophthora cinnamomi Root Rot in Avocado
Five month old avocado seedlings (Topo Topa) were inoculated with Phytophthora cinnamomi. Treatment groups included; (1) Aliette (active ingredient fosetyl-aluminum ISO, Aventis CropScience, Research Triangle Park, N.C.) pre-treatment, applied seven days prior to inoculation, (2) Messenger (active ingredient harpinEa) treatments seven days prior to inoculation, 14 days post-inoculation and every 21 days there after, (3) the combination of treatments 1 and 2 described above, (4) inoculated untreated control, and (5) uninoculated untreated control. Each treatment group was replicated six times. Observations were recorded with respect to the percent of necrotic roots present in the total root mass. Avocado roots show a distinct blackening when infected with P. cinnamomi, whereas non-infected roots are brown-white in color. Table 13 summarizes the study details and resulting data.







TABLE 13







Messenger, Messenger + Aliette, and Aliette


Treatments Effect on Root Rot in Avocado.









Treatment
Application Technique
% Diseased Roots













Aliette
pre-treatment
60
bc


Messenger
foliar every 21 days
38.3
c


Aliette + Messenger
pre-treat + foliar 21 d
27.5
cd


UTC
none
96.5
a


UTC (no inoculation)
none
6.3
d





Same letters do not significantly differ.






Although the invention has been described in detail for the purpose of illustration, it is understood that such details are solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit of the scope of the invention which is defined by the following claims.

## Claims

1. A method of treating at least one plant or plant seed with at least one insecticide, fungicide, or herbicide where a local population of pests has resistance to the at least one insecticide, fungicide, or herbicide, said method comprising:
selecting the at least one plant or plant seed to be treated by the at least one insecticide, fungicide, or herbicide under conditions effective for the at least one insecticide, fungicide, or herbicide to perform its intended function, wherein the selected at least one plant or plant seed is planted where the local population of pests has resistance to the at least one insecticide, fungicide, or herbicide and
applying the at least one insecticide, fungicide, or herbicide and at least one hypersensitive response elicitor protein or polypeptide to said selected at least one plant or plant seed under conditions effective to treat the at least one plant or plant seed with the at least one insecticide, fungicide, or herbicide, wherein said at least one hypersensitive response elicitor is heat stable, glycine rich, and contains substantially no cysteine.

2. The method according to claim 1, wherein said selected plant is treated during said applying.

3. The method according to claim 1, wherein said selected plant seed is treated during said applying, said method further comprising:
planting said treated, selected plant seed in natural or artificial soil and
propagating a plant from said treated, selected plant seed planted in said natural or artificial soil.

4. The method according to claim 1, wherein said selected plants or plant seeds are selected from the group consisting of canola, alfalfa, rice, wheat, barley, rye, cotton, sunflower, peanut, corn, potato, sweet potato, bean, pea, chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic, eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber, apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple, soybean, tobacco, tomato, sorghum, avocado, sugarcane, Saintpaulia, petunia, pelargonium, poinsettia, chrysanthemum, carnation, and zinnia.

5. The method according to claim 1, wherein said applying the at least one insecticide, fungicide, or herbicide is conducted simultaneously or independently of said applying the at least one hypersensitive response elicitor protein or polypeptide.

6. The method according to claim 1, wherein the at least one insecticide is applied, said insecticide containing an active ingredient selected from the group consisting of carbamates, organochlorines, nicotinoids, phosphoramidothioates, organophosphates, and pyrethroids.

7. The method according to claim 1, wherein the at least one fungicide is applied, said fungicide containing an active ingredient selected from the group consisting of aliphatic nitrogens, benzimidazoles, dicarboximides, dithiocarbamates, imidazoles, strobins, anilides, aromatics, sulfur derivatives, and copper derivatives.

8. The method according to claim 1, wherein the at least one herbicide is applied, said herbicide is selected from the group consisting of acetyl-CoA carboxylase inhibitors (ACCase), actolactate synthase inhibitors (ALS), microtubule assembly inhibitors (MT), growth regulators (GR), photosynthesis II, binding site A inhibitors (PSII(A)), photosynthesis II, binding site B inhibitors (PSII(B)), photosynthesis II, binding site C inhibitors (PSII(C)), shoot inhibitors (SHT), enolpyruvyl-shikimate-phosphate synthase inhibitors (EPSP), glutamine synthase inhibitors (GS), phytoene desaturase synthase inhibitors (PDS), diterpene inhibitors (DITERP), protoporphyrinogen oxidase inhibitors (PPO), shoot and root inhibitors (SHT/RT), photosystem 1 electron diverters (ED), hydroxyphenlypyruvate dioxygenase synthesis inhibitors (HPPD), and combinations thereof.

9. The method according to claim 1, wherein the at least one hypersensitive response elicitor or polypeptide is derived from a species of pathogens selected from the group consisting of Erwinia, Pseudomonas, and Xanthomonas.

10. The method according to claim 9, wherein the at least one hypersensitive response elicitor protein or polypeptide is derived from an Erwinia species selected from the group consisting of Erwinia amylovora, Erwinia carotovora, Erwinia chrysanthemi, and Erwinia stewartii.

11. The method according to claim 9, wherein the at least one hypersensitive response elicitor protein or polypeptide is derived from a Pseudomonas species selected from the group consisting of Pseudomonas syringae and Pseudomonas solanacearum.

12. The method according to claim 9, wherein the at least one hypersensitive response elicitor or polypeptide is derived from Xanthomonas campestris.

13. The method according to claim 1, wherein the at least one insecticide is applied, said insecticide comprising nicotinoid.

14. The method according to claim 1, wherein the at least one fungicide is applied, said fungicide comprising strobin.

15. The method according to claim 1, wherein the at least one herbicide is applied, said herbicide comprising glyphosate.

16. The method according to claim 1, wherein the at least one herbicide and the at least one fungicide are applied, said herbicide comprising glyphosate and said fungicide comprising strobin.

17. The method according to claim 1, wherein the at least one herbicide and the at least one insecticide are applied, said herbicide comprising glyphosate and said insecticide comprising nicotinoid.

18. The method according to claim 1, wherein the at least one herbicide is applied, said herbicide comprising glyphosate and Dicamba.

19. The method according to claim 1, wherein the at least one herbicide and the at least one fungicide are applied, said herbicide comprising glyphosate, and Dicamba, and said fungicide comprising strobin.

20. The method according to claim 1, wherein the at least one herbicide and the at least one insecticide are applied, said herbicide comprising glyphosate, and Dicamba, and said insecticide comprising nicotinoid.

21. The method according to claim 8, wherein the at least one herbicide is applied, said herbicide comprising a enolpyruvyl-shikimate-phosphate synthase inhibitor (EPSP) glyphosate.

22. The method according to claim 6, wherein the at least one insecticide is applied, said insecticide comprising a pyrethroid.

23. The method according to claim 7, wherein the at least one fungicide is applied, said fungicide comprising a benzimidazole.

24. A method of treating at least one transgenic plant or transgenic seed with at least one insecticide, fungicide, or herbicide where a local population of pests has resistance to the at least one insecticide, fungicide, or herbicide, said method comprising:
selecting the at least one transgenic plant or transgenic seed, transformed with at least one nucleic acid molecule which encodes at least one hypersensitive response elicitor protein or polypeptide, to be treated by the at least one insecticide, fungicide, or herbicide under conditions effective for the at least one insecticide, fungicide, or herbicide to treat at least one pest, wherein the selected at least one transgenic plant or transgenic seed is planted where the local population of pests has resistance to the at least one insecticide, fungicide, or herbicide and
applying the at least one insecticide, fungicide, or herbicide to said selected transgenic plant or transgenic seed under conditions effective to treat the at least one transgenic plant or transgenic seed with the at least one insecticide, fungicide, or herbicide and for the at least one insecticide, fungicide, or herbicide to perform its intended functions, wherein said at least one hypersensitive response elicitor is heat stable, glycine rich, and contains substantially no cysteine.