Plant pathogen resistance genes and uses thereof

Details Plant pathogen resistance genes and uses thereof Plant pathogen resistance genes and uses thereof

1997-11-12

2001-05-01

Hutzell, Paula (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide confers pathogen or pest resistance

C435S069100, C435S468000, C435S410000, C435S411000, C435S419000, C435S220000, C536S023740, C536S023100, C800S278000, C800S288000, C800S290000, C800S295000, C800S317400

Reexamination Certificate

active

06225527

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-4 gene.
Plants are constantly challenged by potentially pathogenic microorganisms. Crop plants are particularly vulnerable, since they are usually grown as genetically uniform monocultures. However, plants have evolved an array of both preexisting and inducible defences which pathogens must circumvent, especially those 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 often (though not exclusively) specified by dominant R genes. When pathogens mutate to overcome R genes, the mutations are often recessive. For R genes to function, there must also be a corresponding gene in the pathogen termed an avirulence gene (Avr). To become virulent, pathogens (fungi, bacteria or viruses) must no longer produce a product that triggers R gene-dependent defence mechanisms (Flor, 1971). One model, often termed the elicitor/receptor model, is that R genes encode products which enable plants to detect the presence of pathogens, provided the pathogen carries the corresponding Avr gene (Gabriel and Rolfe, 1990). This recognition is subsequently transduced into the activation of a defence response.
The characterization of two fungal avirulence genes from the tomato leaf mould pathogen
Cladosporium fulvum
has been reported. The Avr9 and Avr4 genes encode small cysteine rich peptides (van Kan et al., 1991; Joosten et al., 1995) which, in their mature processed forms, are composed of 28 and 106 amino acid residues respectively. Avr9 and Avr4 confer avirulence to races of
C. fulvum
on tomato lines carrying the R genes Cf-9 and Cf-4 respectively. We have shown that these two R genes are genetically tightly linked, or possibly allelic (Jones et al., 1993; Balint-Kurti et al., 1994). We isolated the Cf-9 gene by transposon tagging (Jones et al., 1994; PCT/GB94/02812, published as WO 95/18230) and we report here the positional cloning of Cf-4 using more refined genetic analysis and Cf-9 DNA as a probe. The availability of cloned avirulence genes and their cognate resistance genes may ultimately be exploited to engineer broad-based and durable disease resistance in a wide range of crop plants (de Wit, 1992; Staskawicz et al., 1995).
In plants gene isolation has been achieved by two main approaches, positional cloning and transposon tagging. Several plant genes have been successfully isolated by positional cloning (reviewed in Tanksley et al., 1995) including several R genes i.e. Pto (Martin et al., 1993) and Cf-2 from tomato (Dixon et al., 1996) and RPS2 and RPM1 from Arabidopsis (Bent et al., 1994; Mindrinos et al., 1994; Grant et al., 1995). Most positional cloning strategies have relied heavily on the use of restriction fragment length polymorphism (RFLP) markers. Recently however, PCR-based strategies have been developed which are capable of detecting more subtle DNA sequence variation allowing a much greater number of DNA sequences to be inspected for polymorphism. These techniques include random amplified polymorphic DNA (RAPDs, Williams et al., 1990) and amplified restriction fragment polymorphism analysis (AFLP, Zabeau and Vos, 1992, EP-A-92402629.7; Vos et al. 1995; Thomas et al., 1995) and should expedite plant gene isolation by positional cloning strategies. Transposon tagging has been used to isolate the N gene from tobacco (Whitham et al., 1994), L
6
from flax (Lawrence et al., 1995) and Cf-9 from tomato (Jones et al., 1994; PCT/GB94/02812, published as WO 95/18230).
Amino acid sequence comparisons of plant R genes has shown they currently constitute two major classes. All except PTO appear to contain leucine rich repeat (LRR) motifs but the R genes L
6
, N, RPM1 and RPS2 appear to have additional domains not present in Cf-9 (Staskawicz et al., 1995) and Cf-2 (Dixon et al., 1996). Furthermore, L
6
, N and RPS2 are probably located intracellularly in contrast to Cf-9 and Cf-2 which are composed largely of LRRs and are predicted to be predominantly extra-cytoplasmic membrane-anchored proteins. Our analysis shows that the predicted Cf-4 protein is highly homologous to Cf-9.
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-4 (the subject of the present invention). Cf-9, Cf-4 and others (Cf-5, -2 etc.) are termed by those skilled in the art “pathogen resistance genes” or “disease resistance genes”. PGIP-encoding genes are not pathogen resistance genes. A pathogen resistance gene (R) enables a plant to detect the presence of a pathogen expressing a corresponding avirulence gene (Avr). When the pathogen is detected, a defence response such as the hypersensitive response (HR) is activated. By such means a plant may deprive the pathogen of living cells by localised cell death at sites of attempted pathogen ingress. On the other hand, the PGIP gene of WO93/11241 (for example) is a gene of the kind that is induced in the plant defence response resulting from detection of a pathogen by an R gene.
Thus, a pathogen resistance gene may be envisaged as encoding a receptor to a pathogen-derived and Avr dependent molecule. In this way it may be likened to the RADAR of a plant for detection of a pathogen, whereas PGIP is involved in the defence the plant mounts to the pathogen once detected and is not a pathogen resistance gene. Expression of a pathogen resistance gene in a plant causes activation of a defence response in the plant. This may be upon contact of the plant with a pathogen or a corresponding elicitor molecule, though the possibility of causing activation by over-expression of the resistance gene in the absence of elicitor has been reported. The defence response may be activated locally, e.g. at a site of contact of the plant with pathogen or elicitor molecule, or systemically. Activation of a defence response in a plant expressing a pathogen resistance gene may be caused upon contact of the plant with an appropriate, corresponding elicitor molecule, e.g. as produced by a
Cladosporium fulvum
avr gene as discussed. The elicitor may be contained in an extract of a pathogen such as
Cladosporium fulvum
, or may be wholly or partially purified and may be wholly or partially synthetic. An elicitor molecule may be said to “correspond” if it is a suitable ligand for the R gene product to elicit activation of a defence response.
The “Cf-x”/“Avrx” terminology is standard in the art. The Cf resistance genes and corresponding fungal avirulence genes (Avr) were originally defined genetically as interacting pairs of genes whose measurable activities fall into mutually exclusive interacting pairs. Avr9 elicits a necrotic response on Cf-9 containing tomatoes but no response on Cf-4 containing tomatoes, the moeity recognised by Cf-4 being different from that recognised by Cf-9.
Expression of Cf-4 function in a plant may be determined by investigating compatibility of various
C. fulvum
races.
A race of
C. fulvum
that carries functional copies of all known Avr genes (race 0) will grow (compatible) only on a tomato which lacks all the Cf genes. It will not grow (incompatible) on a plant carrying any functional Cf gene. If the
C. fulvum
race lacks a functional Avr4 gene (race 4) it will be able to grow not only on a plant lacking any Cf genes but also a plant carrying the Cf-4 gene. A race also lacking a functional Avr2 gene (race 2,4) will also be able to grow on a plant carrying the Cf-2 gene. A race only lacking a functional Avr2 gene (race 2) will not be able to grow on a plant carrying Cf-4. Similarly, a
C. fulvum
race 5 (lacking a functional Avr5 gene) will not be able to grow on a plant carrying a Cf-4 gene. Neither a race 4 nor a race 2,4 will be able to grow on a plant carrying any of th

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