5.4.5S
NEMATODE AND APHID RESISTANCE IN TOMATO

VM WILLIAMSON1, I KALOSHIAN2, J YAGHOOBI1, J BODEAU1, SB MILLIGAN1, DE ULLMAN3 and M ROSSI2

1Center for Engineering Plants for Resistance against Pathogens, 2Department of Nematology, and 3Department of Entomology, University of California, Davis, CA 95616, USA

Background and objectives
Many modern tomato varieties carry a gene, Mi, that confers resistance to several species of root-knot nematodes. Recently, a gene for resistance to the potato aphid was found to be tightly linked to Mi [1]. These traits were transferred into cultivated tomato from the wild tomato species Lycopersicon peruvianum and have remained linked through more than 50 years of introgression. The genetic and mechanistic relationship between the nematode and aphid resistance genes is a question of great interest. Identification of Mi by positional cloning has been a long-term goal of our research group. However, efforts to localize the gene genetically have been hampered by severe repression of recombination in the Mi region in crosses segregating for Mi. Recently we have circumvented this handicap by screening large populations of cultivated tomato and by screening for recombinants within L. peruvianum populations that differ in nematode resistance [2]. From this analysis, Mi was localized to a region of the genome of less than 65 kb. Identification of Mi within this region and characterization of the gene is described below.

Results and conclusions
Molecular markers flanking a 65-kb interval containing Mi were used to isolate tomato DNA spanning this gene in the form of bacterial artificial chromosome clones. 52 kb of contiguous DNA were sequenced and three open reading frames with similarity to cloned plant disease resistance genes were identified. A 4-kb mRNA hybridizing to these genes is present in roots of resistant and susceptible tomato. Susceptible tomato plants transformed with one of the genes, Mi-1.2, are resistant to root-knot nematodes, indicating that transfer of this gene is sufficient to confer resistance. Progeny of primary transformants segregate for resistance with complete correlation between resistance and the presence of the introduced gene. Sequence comparisons revealed Mi-1.2 to be most similar to Prf, a tomato gene required for resistance against Pseudomonas syringae. Prf and Mi-1.2 share structural motifs including a potential leucine zipper, a nucleotide binding site and a leucine-rich repeat region, features that are characteristic of a number of other plant resistance genes. Hybridization revealed approximately six Mi-related sequences in resistant tomato and eight copies in susceptible tomato, most or all clustered near Mi on chromosome 6. At least four copies are represented in the mRNA of roots and at least two different copies are expressed in leaf tissue. The cloned gene will allow us to explore the mechanism of resistance and, possibly, to transfer resistance to other crop species that are damaged by nematodes but for which no natural resistance is currently available. Plants with Mi and its homologues will be tested for aphid resistance to determine whether the leucine-rich repeat class of pathogen resistance genes can be extended to include resistance against insects. Manipulation of the cloned resistance genes should provide insights for designing resistance to other nematodes and insects.

References
1. Kaloshian I, Lange WH, Williamson VM, 1995. Proceedings of the National Academy of Sciences, USA 92, 622-625.
2. Kaloshian I, Yaghoobi J, Liharska T et al., 1997. Molecular and General Genetics, in press.