Kirsten Lahre

The Pto from tomato is capable of functioning in Nicotiana benthamiana but does not appear to be functional in lettuce, a non-Solanaceous species. However, several lettuce varieties elicit the HR in response to Agrobacterium-mediated transient expression of the AvrPto gene. I am interested in comparing the reaction to AvrPto in lettuce to the reaction in tomato. Previous work in Dr. Michelmore's lab has generated a large database of lettuce EST's. Using this data I have identified EST's with amino acid sequences similar to Pto. I am now cloning full-length cDNAs of these genes using RACE PCR. These clones will then be tested using the yeast two-hybrid system to see if they will physically interact with AvrPto. Transient in planta assays will be used to determine the gene's ability to elicit HR when challenged with AvrPto in N. benthamiana and tomato. If I identify a Pto homolog from lettuce that recognizes avrPto in yeast but is incapable of doing so in N. benthamiana and tomato, this will be the template for DNA shuffling experiments with the tomato Pto to identify the molecular basis for the taxonomic specificity. If I do not identify such a homolog, I will generate a cDNA library from lettuce to screen for interactors with avrPto using the yeast two-hybrid system. A second line of investigation stems from another graduate student's DNA shuffling library of Pto with Pto paralogs from tomato that do not interact with avrPto. She has used yeast two hybrid and in planta transient assays to analyze which regions of Pto are required for interaction with avrPto and for the generation of HR.