Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-07-15
2001-09-11
Myers, Carla J. (Department: 1655)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S252300, C435S320100, C435S071100, C435S069100, C435S471000, C435S419000, C435S440000, C536S023100, C536S023600, C536S024300
Reexamination Certificate
active
06287865
ABSTRACT:
The present invention relates to pathogen resistance in plants and more particularly the identification and use of pathogen resistance genes. It is based on cloning of the tomato Cf-2 gene.
Plants are constantly challenged by potentially pathogenic microorganisms. Crop plants are particularly vulnerable, because they are usually grown as genetically uniform monocultures; when disease strikes, losses can be severe. However, most plants are resistant to most plant pathogens. To defend themselves, plants have evolved an array of both preexisting and inducible defences. Pathogens must specialize to circumvent the defence mechanisms of the host, especially those biotrophic pathogens that derive their nutrition from an intimate association with living plant cells. If the pathogen can cause disease, the interaction is said to be compatible, but if the plant is resistant, the interaction is said to be incompatible. Race specific resistance is strongly correlated with the hypersensitive response (HR), an induced response by which (it is hypothesized) the plant deprives the pathogen of living host cells by localized cell death at sites of attempted pathogen ingress.
It has long been known that HR-associated disease resistance is often (though not exclusively) specified by dominant genes (R genes). Flor showed that when pathogens mutate to overcome such R genes, these mutations are recessive. Flor concluded that for R genes to function, there must also be corresponding genes in the pathogen, denoted avirulence genes (Avr genes). To become virulent, pathogens must thus stop making a product that activates R gene-dependent defence mechanisms (Flor, 1971). A broadly accepted working hypothesis, often termed the elicitor/receptor model, is that R genes encode products that enable plants to detect the presence of pathogens, provided said pathogens carry the corresponding Avr gene (Gabriel and Rolfe, 1990). This recognition is then transduced into the activation of a defence response.
Some interactions exhibit different genetic properties.
Helminthosporium carbonum
races that express a toxin (Hc toxin) infect maize lines that lack the Hm1 resistance gene. Mutations to loss of Hc toxin expression are recessive, and correlated with loss of virulence, in contrast to gene-for-gene interactions in which mutations to virulence are recessive. A major accomplishment was reported in 1992, with the isolation by tagging of the Hm1 gene (Johal and Briggs, 1992). Plausible arguments have been made for how gene-for-gene interactions could evolve from toxin-dependent virulence. For example, plant genes whose products were the target of the toxin might mutate to confer even greater sensitivity to the toxin, leading to HR, and the conversion of a sensitivity gene to a resistance gene. However, this does not seem to be the mode of action of Hm1, whose gene product inactivates Hc toxin.
Pathogen avirulence genes are still poorly understood. Several bacterial Avr genes encode hydrophilic proteins with no homology to other classes of protein, while others carry repeating units whose number can be modified to change the range of plants on which they exhibit avirulence (Keen, 1992; Long and Staskawicz, 1993). Additional bacterial genes (hzp genes) are required for bacterial Avr genes to induce
HR
, and also for pathogenicity (Keen, 1992; Long and Staskawicz, 1993). It is not clear why pathogens make products that enable the plant to detect them. It is widely believed that certain easily discarded Avr genes contribute to but are not required for pathogenicity, whereas other Avr genes are less dispensable (Keen, 1992; Long and Staskawicz, 1993). The characterization of one fungal avirulence gene has also been reported; the Avr9 gene of
Cladosporium fulvum
, which confers avirulence on
C. fulvum
races that attempt to attack tomato varieties that carry the Cf-9 gene, encodes a secreted cysteine-rich peptide with a final processed size of 28 amino acids but its role in compatible interactions is not clear (De Wit, 1992).
The technology for gene isolation based primarily on genetic criteria has improved dramatically in recent years, and many workers are currently attempting to clone a variety of R genes. Targets include (amongst others) rust resistance genes in maize, Antirrhinum and flax (by transposon tagging); downy mildew resistance genes in lettuce and Arabidopsis (by map based cloning and T-DNA tagging);
Cladosporium fulvum
(Cf) resistance genes in tomato (by tagging, map based cloning and affinity labelling with avirulence gene products); virus resistance genes in tomato and tobacco (by map based cloning and tagging); nematode resistance genes in tomato (by map based cloning); and genes for resistance to bacterial pathogens in Arabidopsis and tomato (by map based cloning).
The map based cloning of the tomato Pto gene that confers “gene-for-gene” resistance to the bacterial speck pathogen
Pseudomonas syringae
pv tomato (Pst) has been reported (Martin et al, 1993). A YAC (yeast artificial chromosome) clone was identified that carried restriction fragment length polymorphism (RFLP) markers that were very tightly linked to the gene. This YAC was used to isolate homologous cDNA clones. Two of these cDNAs were fused to a strong promoter, and after transformation of a disease sensitive tomato variety, one of these gene fusions was shown to confer resistance to Pst strains that carry the corresponding avirulence gene, AvrPto. These two cDNAs show homology to each other. Indeed, the Pto cDNA probe reveals a small gene family of at least six members, 5 of which can be found on the YAC from which Pto was isolated, and which thus comprise exactly the kind of local multigene family inferred from genetic analysis of other R gene loci.
The Pto gene CDNA sequence is puzzling for proponents of the simple elicitor/receptor model. It reveals unambiguous homology to serine/threonine kinases, consistent with a role in signal transduction Intriguingly, there is strong homology to the kinases associated with self incompatibility in Brassicas, which carry out an analogous role, in that they are required to prevent the growth of genotypically defined incompatible pollen tubes. However, in contrast to the Brassica SRK kinase (Stein et al 1991), the Pto gene appears to code for little more than the kinase catalytic domain and a potential N-terminal-myristoylation site that could promote association with membranes. It would be surprising if such a gene product could act alone to accomplish the specific recognition required to initiate the defence response only when the AvrPto gene is detected in invading microrganisms. The race-specific elicitor molecule made by Pst strains that carry AvrPto is still unknown and needs to be characterized before possible recognition of this molecule by the Pto gene product can be investigated.
Since the isolation of the Pto gene a number of other resistance genes have been isolated. The isolation of the tobacco mosaic resistance gene N from tobacco was reported by Whitham et al (1994). The isolation of the
Arabidopsis thaliana
gene for resistance to
Pseudomonas syringae
RPS2 was reported by Bent et al (1994) and by Mindrinos et al (1994). These genes probably encode cytoplasmic proteins that carry a P-loop and a leucine-rich repeat. The ligands with which they interact are uncharacterised and it is not known what other plant proteins they interact with to accomplish the defence response. Our own laboratory has reported the isolation of the tomato Cf-9 which confers resistance against the fungus
Cladosporium fulvum.
This is a subject of a previous patent application (PCT/GB94/02812 published as WO 95/18230) and has been reported in Jones et al (1994). Cf-9 and Avr9 sequences, and sequences of the encoded polypeptides are given in WO95/18320 and Jones et al (1994).
We have now cloned Cf-2 genes.
WO93/11241 reports the sequence of a gene encoding a polygalacturonase inhibitor protein (PGIP) that has some homology with Cf-9 and, as we have now discovered, Cf-2 (the subject of the present invention
Dixon Mark Stewart
Jones David A.
Jones Jonathan Dallas George
Myers Carla J.
Nixon & Vanderhye P.C.
Plant Bioscience Limited
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