Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-09-13
2002-11-26
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C435S456000, C435S069100, C435S069700, C435S325000, C536S023100, C536S023400, C536S024100
Reexamination Certificate
active
06485977
ABSTRACT:
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 (“hypersensitive response and pathogenicity”) 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 proteins, known as effector proteins, across the walls and plasma membranes of living plant cells. Such effector proteins are variously known as hypersensitive response elicitors, Avr (Avirulence) proteins, Hop (hypersensitive response and pathogenicity-dependent outer proteins), Vir (virulence) proteins, or Pth (pathogenicity) proteins, depending on the phenotype by which they were discovered (see, e.g., 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.
A current model for plant-bacterium interaction and co-evolution based on Hrp delivery of Avr proteins into plant cells proposes that (i) Avr-like proteins are the primary effectors of parasitism, (ii) conserved Hrp systems are capable of delivering many, diverse Avr-like proteins into plant cells, and (iii) genetic changes in host populations that reduce the parasitic benefit of an effector protein or allow its recognition by the R-gene surveillance system will lead to a proliferation of complex arsenals of avr-like genes in co-evolving bacteria (Alfano et al., “Bacterial Pathogens in Plants: Life Up Against the Wall,”
Plant Cell,
8:1683-1698 (1996)). There are still many gaps in this model. For example, the physical transfer of Avr proteins into plant cells has never been observed, the virulence functions of Avr proteins are unknown, and it is likely that previous searches for Avr genes in various bacteria have yielded incomplete inventories of the genes in various bacteria and, thus, incomplete inventories of the genes encoding effector proteins.
Until recently, Avr proteins had not been reported outside of the cytoplasm of living
Pseudomonas syringae
and Xanthomonas spp. cells (Leach et al., “Bacterial Avirulence Genes,”
Annul. Rev. Phytopathol,
34:153-179 (1996); 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)), but it now appears that the Hrp systems of Erwinia spp. can secrete Avr proteins in culture. A homolog of the
Pseudomonas syringae
pv.
tomato
avrE gene has been found in
Erwinia amylovora
and designated dspA in strain CFBP1430 and dspE in strain Ea321 (Gaudriault et al., “DspA, an Essential Pathogenicity Factor of
Erwinia amylovora
Showing Homology with AvrE of
Pseudomonas syringae,
is Secreted via the Hrp Secretion Pathway in a DspB-dependent Way,”
Mol. Microbiol.,
26:1057-1069 (1997); Bogdanove et al., “Homology and Functional Similarity of a hrp-linked Pathogenicity Operon, dspEF, of
Erwinia amylovora
and the avrE locus of
Pseudomonas syringae
Pathovar Tomato,”
Proc. Natl. Acad. Sci. USA,
95:1325-1330 (1998)). dsp genes are required for the pathogenicity of
Erwinia amylovora,
but not for HR elicitation. A protein of the expected size of DspA is secreted in a Hrp- and DspB-dependent manner by CFBP1430 (DspB is a potential chaperone) (Gaudriault et al., “DspA, an Essential Pathogenicity Factor of
Erwinia amylovora
Showing Homology with AvrE of
Pseudomonas syringae,
is Secreted via the Hrp Secretion Pathway in a DspB-dependent Way,”
Mol. Microbiol.,
26:1057-1069 (1997)). Specific antibodies were used to demonstrate unambiguously that DspE is efficiently secreted in a Hrp-dependent manner by strain Ea321 (Bogdanove et al., “
Erwinia amylovora
Secretes DspE, a Pathogenicity Factor and Functional AvrE Homolog, Through the Hrp (Type III Secretion) Pathway,”
J. Bacteriol.,
180(8):2244-2247 (1998)).
Furthermore, the
Erwinia chrysanthemi
Hrp system enables
E. coli
to secrete effector proteins of
P. syringae
and Yersinia spp. (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 to Secrete Avr Proteins in Culture,”
Proc. Natl. Acad. Sci. USA
95:10206-10211 (1998); Anderson et al., “Reciprocal Secretion of Proteins by the Bacterial Type III Machines of Plant and Animal Pathogens Suggests Universal Recognition of mRNA Targeting Signals,”
Proc. Natl. Acad. Sci. USA
96:12839-12843 (1999); Mudgett and Staskawicz, “Characterization of the
Pseudomonas syringae
pv.
tomato
AvrRpt2 Protein: Demonstration of Secretion and Processing During Bacterial Pathogenesis,”
Mol. Microbiol.
32:927-941 (1999)). Also, conditions have now been defined that permit detection of Hrp-dependent secretion of effector proteins by
P. syringae
and
X. campestris.
Rossier et al., “The Xanthomonas Hrp Type III System Secretes Proteins from Plant and Mammalian Bacterial Pathogens,”
Proc. Natl. Acad. Sci. USA
96:9368-9373 (1999); van Dijk et al., “The Avr (Effector) Proteins HrmA (HopPsyA) and AvrPto are Secreted in Culture from
Pseudomonas syringae
Pathovars via the Hrp (Type III) Protein Secretion System in a Temperature and pH-Sensitive Manner,”
J. Bacteriol.
181:4790-4797 (1999)).
The biochemical activities or parasite-promoting functions of effector proteins remain unclear, although several of those known make measurable contributions to virulence (Leach et al., “Bacterial Avirulence Genes,”
Annul. Rev. Phytopathol,
34:153-179 (1996)). Members of the AvrBs3 family in Xanthomonas spp. are targeted to the plant nucl
Beer Steven V.
Collmer Alan
Cornell Research Foundation Inc.
Guzo David
Nixon & Peabody LLP
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