Molecular characterization of avrPphD, a widely-distributed gene from Pseudomonas syringae pv. phaseolicola involved in non-host recognition by pea (Pisum sativum)

A genomic library clone pPPY40, from Pseudomonas syringae pv. phaseolicola was previously shown to consist of two regions: region I potentially encode d an avirulence gene involved in non-host recognition in pea and region II contained a replicase gene. In this study, region I was sequenced and shown to contain a single open reading frame (ORF) of 2130 nt with a 55 % G + C ratio encoding a deduced peptide product of 710 amino acids with a predicte d molecular mass of 75.4 kDa. Both the entire ORF and an ORF truncated by f ive residues were isolated by PCR amplification. The full length ORF was su ccessfully cloned in the broad host range vector pBBR1MCS-2, but not in the RK2-based broad host range vector pLAFR3. Truncated ORFs were successfully cloned in both vectors. The constructs obtained were transferred to Pseudo monas syringae pv. pisi strain PT10 and the transconjugants tested for path ogenicity on pea plants. Only the full length ORF conferred avirulence and the gene was designated avrPphD. Inactivation of avrPphD by marker-exchange did not affect the ability of P. syringae pv. phaseolicola to cause a non- host hypersensitive reaction. PCR amplification using avrPphD-specific prim ers and hybridization analysis indicated that homologues of avrPphD occurre d in a wide range of P. syringae pathovars. (C) 2001 Academic Press.