Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-05-28
2003-06-24
Carlson, Karen Cochrane (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S012200, C530S350000, C530S300000, C536S023700, C536S023740, C435S411000, C435S069100, C800S298000
Reexamination Certificate
active
06583107
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to fragments of a hypersensitive response elicitor which fragments elicit a hypersensitive response and uses thereof.
BACKGROUND OF THE INVENTION
Interactions between bacterial pathogens and their plant hosts generally fall into two categories: (1) compatible (pathogen-host), leading to intercellular bacterial growth, symptom development, and disease development in the host plant; and (2) incompatible (pathogen-nonhost), resulting in the hypersensitive response, a particular type of incompatible interaction occurring, without progressive disease symptoms. During compatible interactions on host plants, bacterial populations increase dramatically and progressive symptoms occur. During incompatible interactions, bacterial populations do not increase, and progressive symptoms do not occur.
The hypersensitive response is a rapid, localized necrosis that is associated with the active defense of plants against many pathogens (Kiraly, Z., “Defenses Triggered by the Invader: Hypersensitivity,” pages 201-224 in:
Plant Disease: An Advanced Treatise
, Vol. 5, J. G. Horsfall and E. B. Cowling, ed. Academic Press New York (1980); Klement, Z., “Hypersensitivity,” pages 149-177 in:
Phytopathogenic Prokaryotes
, Vol. 2, M. S. Mount and G. H. Lacy, ed. Academic Press, New York (1982)). The hypersensitive response elicited by bacteria is readily observed as a tissue collapse if high concentrations (≧10
7
cells/ml) of a limited host-range pathogen like
Pseudonmonas syringae
or
Erwinia amylovora
are infiltrated into the leaves of nonhost plants (necrosis occurs only in isolated plant cells at lower levels of inoculum) (Klement, Z. “Rapid Detection of Pathogenicity of Phytopathogenic Pseudomonads,”
Nature
199:299-300; Klement, et al., “Hypersensitive Reaction Induced by Phytopathogenic Bacteria in the Tobacco Leaf,”
Phytopathology
54:474-477 (1963); Turner, et al., “The Quantitative Relation Between Plant and Bacterial Cells Involved in the Hypersensitive Reaction,”
Phytopathology
64:885-890 (1974); Klement, Z., “Hypersensitivity,” pages 149-177 in
Phytopathoenic Prokaryotes
, Vol. 2., M. S. Mount and G. H. Lacy, ed. Academic Press, New York (1982)). The capacities to elicit the hypersensitive response in a nonhost and be pathogenic in a host appear linked. As noted by Klement, Z., “Hypersensitivity,” pages 149-177 in
Phytopathogenic Prokaryotes
, Vol. 2., M. S. Mount and G. H. Lacy, ed. Academic Press, New York, these pathogens also cause physiologically similar, albeit delayed, necroses in their interactions with compatible hosts. Furthermore, the ability to produce the hypersensitive response or pathogenesis is dependent on a common set of genes, denoted hrp (Lindgren, P. B., et al., “Gene Cluster of
Pseudomonas syringae
pv. ‘phaseolicola’ Controls Pathogenicity of Bean Plants and Hypersensitivity on Nonhost Plants,”
J. Bacteriol.
168:512-22 (1986); Willis, D. K., et al., “hrp Genes of Phytopathogenic Bacteria,”
Mol. Plant
-
Microbe Interact.
4:132-138 (1991)). Consequently, the hypersensitive response may hold clues to both the nature of plant defense and the basis for bacterial pathogenicity.
The hrp genes are widespread in gram-negative plant pathogens, where they are clustered, conserved, and in some cases interchangeable (Willis, D. K., et al., “hrp Genes of Phytopathogenic Bacteria,”
Mol. Plant
-
Microbe Interact.
4:132-138 (1991); Bonas, U., “hrp Genes of Phytopathogenic Bacteria,” pages 79-98 in:
Current Topics in Microbiology and Immunology: Bacterial Pathogenesis of Plants and Animals—Molecular and Cellular Mechanisms
, J. L. Dangl, ed. Springer-Verlag, Berlin (1994)). Several hrp genes encode components of a protein secretion pathway similar to one used by Yersinia, Shigella, and Salmonella spp. to secrete proteins essential in animal diseases (Van Gijsegem, et al., “Evolutionary Conservation of Pathogenicity Determinants Among Plant and Animal Pathogenic Bacteria,”
Trends Microbiol.
1:175-180 (1993)). In
E. amylovora, P. syringae
, and
P. solanacearum
, hrp genes have been shown to control the production and secretion of glycine-rich, protein elicitors of the hypersensitive response (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), Wei, Z.-H., et al., “HrpI of
Erwinia amylovora
Functions in Secretion of Harpin and is a Member of a New Protein Family,”
J. Bacteriol.
175:7958-7967 (1993); Arlat, M. et al. “PopA1, a Protein Which Induces a Hypersensitive-like Response on Specific Petunia Genotypes, is Secreted via the Hrp Pathway of
Pseudomonas solanacearum,” EMBO J.
13:543-553 (1994)).
The first of these proteins was discovered in
E. amylovora
Ea321, a bacterium that causes fire blight of rosaceous plants, and was designated harpin (Wei, Z.-M., et al, “Harpin, Elicitor of the Hypersensitive Response Produced by the Plant Pathogen
Erwinia amylovora,” Science
257:85-88 (1992)). Mutations in the encoding hrpN gene revealed that harpin is required for
E. amylovora
to elicit a hypersensitive response in nonhost tobacco leaves and incite disease symptoms in highly susceptible pear fruit. The
P. solanacearum
GMI1000 PopA1 protein has similar physical properties and also elicits the hypersensitive response in leaves of tobacco, which is not a host of that strain (Arlat, et al. “PopA1, a Protein Which Induces a Hypersensitive-like Response on Specific Petunia Genotypes, is Secreted via the Hrp Pathway of
Pseudomnonas solanacearum,” EMBO J.
13:543-53 (1994)). However,
P. solanacearum
popA mutants still elicit the hypersensitive response in tobacco and incite disease in tomato. Thus, the role of these glycine-rich hypersensitive response elicitors can vary widely among gram-negative plant pathogens.
Other plant pathogenic hypersensitive response elicitors have been isolated, cloned, and sequenced. These include:
Erwinia chrysanthemi
(Bauer, et. al., “
Erwinia chrysanthemi
Harpin
Ech
: Soft-Rot Pathogenesis,”
MPMI
8(4): 484-91 (1995));
Erwinia carotovora
(Cui, et. al., “The RsmA
−
Mutants of
Erwinia carotovora
subsp.
carotovora
Strain Ecc71 Overexpress hrpN
Ecc
and Elicit a Hypersensitive Reaction-like Response in Tobacco Leaves,”
MPMI
9(7): 565-73 (1966));
Erwinia stewartii
(Ahmad, et. al., “Harpin is not Necessary for the Pathogenicity of
Erwinia stewartii
on Maize,” 8
th Int'l. Cong. Molec. Plant
-
Microb. Inter
. 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); and
Pseudomonas syringae
pv.
syringae
(WO 94/26782 to Cornell Research Foundation, Inc.).
The present invention seeks to identify fragments of hypersensitive response elicitor proteins or polypeptides, which fragments elicit a hypersensitive response, and uses of such fragments.
SUMMARY OF THE INVENTION
The present invention is directed to an isolated fragment of an Erwinia hypersensitive response elicitor protein or polypeptide where the fragment elicits a hypersensitive response in plants. Also disclosed are isolated DNA molecules which encode such fragments.
The fragments of hypersensitive response elicitors can be used to impart disease resistance to plants, to enhance plant growth, and/or to control insects. This involves applying the fragments in a non-infectious form to plants or plant seeds under conditions effective to impart disease resistance, to enhance plant growth, and/or to control insects on plants or plants grown from the plant seeds.
As an alternative to applying the fragments to plants or plant seeds in order to impart disease resistance, to enhance plant growth, and/or to control insects on plants, transgenic plants or plant seeds can be utilized. When utilizing transgenic plants, this involves providing a transgenic plant transformed with a DNA molecule encoding a fragment of a hypersensitive response elicitor protein or
Beer Steven V.
Laby Ron J.
Wei Zhong-Min
Carlson Karen Cochrane
Cornell Research Foundation Inc.
Kam Chih-Min
Nixon & Peabody LLP
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