Towards Positional Cloning of the Pto Bacterial Resistance Locus from Tomato

Abstract

Resistance to Pseudomonas syringae pv. tomato (Pst) in tomato is conferred by a single dominant locus, Pto. We are attempting to isolate the Pto locus using a multi-step positional cloning strategy. First, using near-isogenic lines (NILs) we identified RFLP and RAPD markers that are tightly linked to the locus (4). Second, we developed and screened a large F₂ population (251 plants) that segregated for Pst resistance. This population was used to create a high resolution (0.2 cM) linkage map that allowed the precise placement of Pto in relation to molecular markers (6). One marker, TG538, was found to cosegregate with Pto. Third, we screened a tomato YAC library (5) with TG538 and identified five YACs ranging from 400 to 600 kb that carry sequences derived from the Pto region. Mapping the ends of the cloned tomato DNA from the smallest YAC (PTY538-1) showed that this YAC encompasses the Pto locus. Fourth, PTY538-1 was used to identify cDNA clones from a library made from Pst-induced leaf tissue. Two cDNA clones CD106 and CD127-1, were isolated by this method. Linkage mapping of these clones showed that CD106 maps 0.4 cM from the Pto locus, whereas CD127 cosegregates with Pto. Once we have identified a full-length clone of CD127 we will introduce it (and the corresponding genomic sequence) into tomato plants in an effort to transform a Pst-susceptible line to resistance. The eventual isolation of Pto should open up many avenues of research into the molecular mechanism of plant disease resistance.