Highly conserved sequences flank avirulence genes: isolation of novel avirulence genes from Pseudomonas syringae pv. pisi

Abstract

DNA sequences flanking two avr genes (avrPpiA1 and avrPpiB1) from Pseudomonas syringae pv. pisi show a high degree of similarity. Specific primers designed from the conserved regions were used in PCR amplifications with all P. syringae pv. pisi races. As well as amplifying the expected avrPpiA- and avrPpiB-containing fragments, two additional fragments were amplified: one contained a single open reading frame (ORF1) and was found in races of genomic group II (2, 3A, 4A and 6); the second fragment contained two open reading frames (ORF2 and ORF3), separated by 658 nt, and was detected in all races. All three ORFs had G+C ratios (46.9-48 mol%) that were significantly less than that for P. syringae and each was preceded by a potential hrp box promoter. In P. syringae pv. phaseolicola, ORF1 and ORF2 each elicited a strong non-host hypersensitive reaction on bean leaves; ORF1 was designated avrPpiG, the product of which had strong similarity to AvrRxv, AvrBsT and YopP. ORF2 was identical to a gene, designated avrPpiC, previously isolated from P. syringae pv. pisi race 5. ORF3 was always found in association with avrPpiC and both were detected in a wide range of P. syringae pathovars. In contrast, avrPpiG was only detected in strains of P. syringae pv. pisi genomic group II and P. syringae pv. coronafaciens (ICMP 3113). In P. syringae pv. pisi, avrPpiG was plasmid-borne and avrPpiC and ORF3 were chromosomal. This conservation of flanking sequences has implications for the horizontal transfer of avirulence and virulence genes, suggesting that specific regions of the bacterial genome act as sites for their integration/excision.