1.1.29
ANALYSIS OF DISEASE RESISTANCE GENE-LIKE SEQUENCES IN TOMATO

T OHMORI, M MURATA and F MOTOYOSHI

Research Institute for Bioresources, Okayama University, Kurashiki 710, Japan

Background and objectives
The genes Tm-l and Tm-2, conferring resistance to tomato mosaic virus (TOMV) in tomato, Lycopersicon esculentum, originated from the wild relatives of tomato, L. hirusutum and L. peruvianum, respectively. Using near-isogenic lines (NILs) of tomato, we have mapped these two gene loci with a number of random amplified polymorphic DNA (RAPD) markers and sequence-characterized amplified-region (SCAR) markers converted from the RAPDs [1-4]. We have been examining whether the NILs with Tm-1 or Tm-2 are related to the disease resistance genes already isolated and characterized.

Recently, genes for resistance to pathogens have been cloned from several plant species. Proteins encoded by these genes are categorized into four distinct classes: those encoding cytoplasmic receptor-like proteins that contain a leucine-rich repeat (LRR) domain and nucleotide binding site (NBS) (class 1); those encoding a serine-threonine kinase (class 2); those encoding putative transmembrane receptors with large extracytoplasmic LRR domains (class 3); and those encoding putative transmembrane receptors with an extracellular LRR domain and an intracellular serine-threonine kinase domain (class 4). In this study, consensus amino acid sequences in certain domains in proteins of classes 1, 2 and 3 were chosen to design PCR primers for amplifying genomic DNA fragments of the NILs. Some amplified DNA fragments showed high or moderate similarities to some of the known resistance genes. Here, we present the structural properties of these PCR products, and also discuss their relationship to the Tm-1 and Tm-2 genes.

Results and conclusions
DNA fragments were amplified from the genomic DNA of the NILs with Tm-1 or Tm-2. Twelve PCR-amplified products cloned were shown to have sequence homology to disease resistance genes of classes 1, 2 or 3. Out of 12 clones, three have significant sequence similarities to the class 1 genes including tobacco N, Arabidopsis thaliana RPS2 and RPM1, flax L6 and M and tomato Prf and I2C-1. These three clones contained NBS motifs such as the kinase-2 and/or kinase-3a motifs, conserved among these resistance genes. Seven clones were identified to have homology to the class 2 genes. Out of seven clones, two have 100% identity in the nucleotide sequences to pto and fen, the alleles of Pto and Fen in L. esculentum. The other five clones also have significant sequence similarities to Pto and Fen. Four of five clones contained the subdomains and the invariant residues that are characteristic of functional protein kinases. The other clone may be a pseudogene because it contains multiple stop codons and frame-shift mutations. Moreover, two other clones were homologous to the class 3 resistance genes such as tomato Cf9 and Cf2. These two clones contained the LRRs conserved in both Cf9 and Cf2. Most of the clones hybridized to multiple bands in Southern hybridization to the genomic DNAs of the NILs carrying either Tm-1 or Tm-2 or none of these resistance genes. This suggests that the genomes of the NILs contain multiple copies of sequences homologous to some of the known disease resistance genes. No evidence was obtained to show that the Tm-1 and/or Tm-2 loci encode proteins corresponding to the three classes, since no polymorphic banding patterns between the NILs were detected by Southern hybridization.

References
1. Ohmori T, Murata M, Motoyoshi F, 1995. Theoretical and Applied Genetics 90, 307-311.
2. Ohmori T, Murata M, Motoyoshi F, 1995. Japanese Journal of Genetics 70, 179-184.
3. Ohmori T, Murata M, Motoyoshi F, 1996. Theoretical and Applied Genetics 92, 151-156.
4. Moytoyoshi F, Ohmori T, Murata M, 1996. Symposia of the Society for Experimental Biology 50, 65-70.