3.4.24
CHARACTERISATION AND CLONING OF TOMATO GENES REQUIRED FOR CF-2-MEDIATED RESISTANCE AGAINST CLADOSPORIUM FULVUM

C GOLSTEIN, MS DIXON, L MULDER, CM THOMAS and JDG JONES

Sainsbury Laboratory, John lnnes Centre, Coiney Lane, Norwich, NR4 7UH, UK

Background and objectives
The understanding of plant disease resistance was recently advanced by the cloning of numerous resistance (R) genes. The next objective is to unravel the molecular events which eventually trigger the defence response. The Cladosporium fulvum/tomato interaction has been exploited to identify additional genes involved in the resistance pathway. The first rcr genes (required for C. fulvum resistance) were identified by mutagenesis of L. esculentum Cf9 seed (containing the Cf-9 R gene), as two mutations which diminish Cf-9-mediated resistance [1]. Here, we report the identification and the progress in the cloning and characterisation of new Rcr genes required for Cf2-mediated resistance.

Results and conclusions
By screening 4000 M2 families of chemically mutagenised Cf2 seed, we identified 4 mutants which abolished Cf-2-mediated resistance: rcr3, rcr4, rcr6 and rcr7. There are two functional Cf-2 genes per haploid genome, therefore mutations in sensitive plants are likely to map at other loci. lntercrossing the mutants revealed that rcr-3 and rcr-4 are allelic. A backcross progeny test showed that Rcr-3 is not required for Cf-9-mediated resistance. The level of fungal growth on rcr3 and rcr6 plants looked never as great as on a CfO plant which lacks any functional Cf gene. Microscopic analysis and quantitative data will be discussed. Rcr-3 and Rcr-6 are being cloned by an AFLP-based positional cloning [2]. Mapping populations were established by crossing rcr3 and rcr6 to the wild tomato accession L. pennellii. Both F1 progeny were resistant, therefore L. pennellii has both functional Rcr genes. L. pennellii markers linked to Rcr-3 and Rcr-6 were identified by AFLP analysis of S (sensitive) and R (resistant) pools of backcross plants. Using a stock of near isogenic lines (NIL) of L. esculentum containing fragments of L. pennellii, these linked markers were mapped to the long arm of chromosome 2. RFLP probes known to map to this region were subsequently used to further define both Rcr-3 and windows to the same 10 cM interval and to select for plants which contain closely linked recombination events. AFLPs carried out on S and R pools of selected recombinants allowed us to detect cosegregating markers A for rcr-3, B and C for rcr-6. We are currently screening an L. pennellii BIBAC library with these markers.

References
1. Hammond-Kosack KE, Jones DA, Jones JDG, 1994. Plant Cell 6, 361-374.
2. Vos P, Hogers R, Bleeker M., Reijans M, Van de Lee T, Homes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M, 1995. Nucleic Acids Research 23, 4407-4414.