Isolation and characterization of potato homologues of Arabidopsis thaliana genes operating in defense signal transduction

An increasing number of pathogen-defense related genes are being identified and characterized in Arabidopsis thaliana. So far, it is not known whether and which structural and functional homologues of these Arabidopsis genes have any role in natural variation of resistance to pathogens in crops. Using sequence database mining and PCR-based approaches, potato (Solanum tuberosum L.) gene fragments with high sequence similarity to 16 Arabidopsis defense signal transduction genes were obtained, sequenced and genetically positioned on potato molecular maps. Of 16 novel loci, five were positional candidates for known potato pathogen resistance QTL. One of the candidate loci, StAOS2 co-localizing with QTL for resistance to P. infestans and E. carotovora on linkage group XI, was further characterized in more detail. StAOS2 encodes a gene for allene oxide synthase, a cytochrome P450-enzyme, acting upstream in the jasmonic acid biosynthesis pathway. A metabolic block at the level of AOS completely abolishes JA production, which affects plant development (e.g. sterile pollen production) and various abiotic and biotic stress responses (e.g. P. infestans resistance in tomato, E. carotovora resistance in Arabidopsis). The chloroplastic localization of StAOS2-GFP was confirmed by confocal microscopy and functionality of the potato protein was proven by complementation of the male-sterile Arabidopsis aos mutant. StAOS2-RNAi transgenic lines in potato were generated in order to test role of StAOS2 in P. infestans resistance. The measurements of endogenous OPDA and JA in the silenced lines after wounding treatment revealed drastic decrease in the levels of above mentioned compounds (up to 25 folds less than in wild type plants). In addition, natural variation of StAOS2 locus was characterized. Sequencing of the locus across 38 potato chromosomes revealed high polymorphism. Thirteen distinct alleles were found, and four of them showed highly significant (P=0.000, R2=14%) linkage to P. infestans and E. carotovora QTL. Five alleles of StAOS2 were cloned. Sequence analyses revealed a substantial polymorphism on amino acid level, including non-conservative substitutions and an insertion/deletion within the cytochrome P450 domain. Currently, an ongoing quantitative complementation of the Ataos mutant with the five different StAOS2 alleles fused to the native AtAOS promoter, followed by OPDA and JA levels measurements in the transgenic lines, will possibly provide direct evidence for StAOS2 being the first plant resistance QTL identified