Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-07-09
2003-07-22
Myers, Carla J. (Department: 1634)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S006120, C435S070100, C435S071100, C435S252300, C435S252330, C435S320100, C435S325000, C536S023100, C536S023400, C536S023700, C536S024100, C530S412000
Reexamination Certificate
active
06596509
ABSTRACT:
DESCRIPTION OF DEPOSITED BIOLOGICAL MATERIALS
The biological material listed below has been deposited with the American Type Culture Center (10801 University Blvd., Manassas, Va.):
ATCC
Deposit No.
Description
Date Deposited
PTA-3287
Escherichia coli
containing the cloned hrp
Apr. 13, 2001
gene cluster of
Erwinia chrysanthemi
:
DH5&agr;(pCPP2156)
PTA-3288
Escherichia coli
containing the cloned hrp
Apr. 13, 2001
gene cluster of
Erwinia amylovora
:
DH5(pCPP430)
BACKGROUND OF THE INVENTION
The most common bacterial pathogens of plants colonize the apoplast, and from that location outside of the walls of living cells they incite a variety of diseases in most cultivated plants (Alfano et al., “Bacterial Pathogens in Plants: Life Up Against the Wall,”
Plant Cell
8:1683-1698 (1996)). The majority of these are Gram-negative bacteria in the genera Erwinia, Pseudomonas, Xanthomonas, and Ralstonia. Most are host specific and will elicit the hypersensitive response (“HR”) in nonhosts. The HR is a rapid, programmed death of plant cells in contact with the pathogen. Some of the defense responses associated with the HR are localized at the periphery of plant cells at the site of bacterial contact, but what actually stops bacterial growth is not known (Brown et al., “hrp genes in
Xanthomonas campestris
pv.
vesicatoria
Determine Ability to Suppress Papilla Deposition in Pepper Mesophyll Cells,”
MPMI
8:825-836 (1995); Young et al., “Changes in the Plasma Membrane Distribution of Rice Phospholipase D During Resistant Interactions With
Xanthomonas oryzae
pv.
oryzae,” Plant Cell
8:1079-1090 (1996); Bestwick et al., “Localization of Hydrogen Peroxide Accumulation During the Hypersensitive Reaction of Lettuce Cells to
Pseudomonas syringae
pv.
phaseolicola,” Plant Cell
9:209-221 (1997)). Pathogenesis in host plants, in contrast, involves prolonged bacterial multiplication, spread to surrounding tissues, and the eventual production of macroscopic symptoms characteristic of the disease. Although these bacteria are diverse in their taxonomy and pathology, they all possess hrp genes which direct their ability to elicit the HR in nonhosts or to be pathogenic (and parasitic) in hosts (Lindgren, “The Role of hrp Genes During Plant-Bacterial Interactions,”
Annu. Rev. Phytopathol.
35:129-152 (1997)). The hrp genes encode a type III protein secretion system that appears to be capable of delivering Avr (avirulence) proteins across the walls and plasma membranes of living plant cells (Alfano et al., “The Type III (Hrp) Secretion Pathway of Plant Pathogenic Bacteria: Trafficking Harpins, Avr Proteins, and Death,”
J. Bacteriol.
179:5655-5662 (1997), which is hereby incorporated by reference). The Avr proteins are so named because they can betray the parasite to the R gene-encoded surveillance system of plants, thereby triggering the HR (Vivian et al., “Avirulence Genes in Plant-Pathogenic Bacteria: Signals or Weapons?,”
Microbiology
143:693-704 (1997); Leach et al., “Bacterial Avirulence Genes,”
Annul. Rev. Phytopathol.
34:153-179 (1996)). But Avr-like proteins also appear to be key to parasitism in compatible host plants, where the parasite proteins are undetected and the HR is not triggered. Thus, bacterial avirulence and pathogenicity are interrelated phenomena and explorations of HR elicitation are furthering our understanding of parasitic mechanisms.
Despite the emerging importance of Avr proteins, there is no direct evidence that they travel the Hrp pathway, there is no knowledge of their function in virulence, it appears likely that only a subset of those that are produced by typical host-specific pathogens have been identified, and there is no evidence that they are produced at all by host-promiscuous pathogens. The evidence that Avr proteins are transferred by the Hrp pathway into plants is most complete, although still indirect, with
Pseudomonas syringae
AvrB and AvrPto proteins. Nonpathogenic
Escherichia coli
and
Pseudomonas fluorescens
cells that harbor the functional cluster of
Pseudomonas syringae
hrp genes carried on cosmid pHIR11 can elicit an HR that is dependent on both the type III secretion system and either AvrB or AvrPto (Gopalan et al., “Expression of the
Pseudomonas Syringae
Avirulence Protein AvrB in Plant Cells Alleviates its Dependence on the Hypersensitive Response and pathogenicity (Hrp) Secretion System in Eliciting Genotype-specific Hypersensitive Cell Death,”
Plant Cell
8:1095-1105 (1996); Pirhonen et al., “Phenotypic Expression of
Pseudomonas Syringae
avr Genes in
E. coli
is Linked to the Activities of the hrp-encoded Secretion System,”
MPMI
9:252-260 (1996)). Both Avr proteins trigger an R gene-dependent HR when transiently expressed inside plant cells (Gopalan et al., “Expression of the
Pseudomonas Syringae
Avirulence Protein AvrB in Plant Cells Alleviates its Dependence on the Hypersensitive Response and pathogenicity (Hrp) Secretion System in Eliciting Genotype-specific Hypersensitive Cell Death,”
Plant Cell
8:1095-1105 (1996)) and the interaction of AvrPto and Pto in the yeast two-hybrid system correlates with biological activity (Tang et al.,
Science
274:2060 (1996); Scofield et al.,
Science
274:2063-2065 (1996)). However, neither
Pseudomonas syringae, Escherichia coli
(pHIR11), nor
Pseudomonas fluorescens
(pHIR11) secrete AvrB or AvrPto in culture, presumably because these proteins travel the type III pathway directly into host cells and only upon host cell contact, as with the Yop virulence proteins of Yersinia spp. (Gopalan et al., “Expression of the
Pseudomonas syringae
Avirulence Protein AvrB in Plant Cells Alleviates its Dependence on the Hypersensitive Response and Pathogenicity (Hrp) Secretion System in Eliciting Genotype-specific Hypersensitive Cell Death,”
Plant Cell
8:1095-1105 (1996); Cornelis et al., “The Yersinia Yop Regulon: A Bacterial System for Subverting Eukaryotic Cells,”
Mol. Microbiol.
23:861-867 (1997)). Other known Avr proteins have been observed only in the bacterial cytoplasm (Leach et al., “Bacterial Avirulence Genes,”
Annu. Rev. Phytopathol.
34:153-179 (1996); Knoop et al., “Expression of the Avirulence Gene avrBs3 from
Xanthomonas campestris
pv.
vesicatoria
is not Under the Control of hrp Genes and is Independent of Plant Factors,”
J. Bacteriol.
173:7142-7150 (1991); Puri et al., “Expression of avrPphB, an Avirulence Gene from
Pseudomonas Syringae
pv. Phaseolicola, and the Delivery of Signals Causing the Hypersensitive Reaction in Bean,”
MPMI
10:247-256 (1997)).
Many proteins and polypeptides, including hormones and enzymes, are in high demand for pharmacological and industrial use. Once the gene encoding a desired protein or polypeptide has been isolated, the protein can be produced readily through fermentation in rapidly growing bacteria.
Escherichia coli
is used most commonly for large-scale protein production. Current technology enables the production of relatively large intracellular concentrations of the desired proteins or polypeptides. Extraction of the desired protein or polypeptide from the bacterial cells requires lysing of the cell membrane. After lysing the cell membrane, the desired protein or polypeptide is contaminated with other proteins and, therefore, subject to degradation. The resulting contamination requires significant purification to obtain the isolated protein or polypeptide and degradation of the desired protein or polypeptide limits the obtainable yield.
In addition to fermentation technologies for production of proteins or polypeptides, gene therapy involving transgenic plants is emerging as an important tool for enhancing agricultural productivity and reducing disease losses. For example, transgenic plants expressing bacterial and viral proteins are now used for herbicide tolerance and resistance to viral diseases, respectively. Because of the ease with which foreign proteins can be expressed in most major crops, it is feasible to bioprospect for proteins that will alter plant metabolism to enhance productivity and prevent losses due t
Bauer David W.
Beer Steven V.
Bogdanove Adam J.
Collmer Alan
Ham Jong Hyun
Cornell Research Foundation Inc.
Johannsen Diana
Myers Carla J.
Nixon & Peabody LLP
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