1.1.32
CHARACTERIZATION OF avrPphF.R1, A GENE FOR CULTIVAR-SPECIFIC AVIRULENCE FROM PSEUDOMONAS SYRINGAE PV. PHASEOLICOLA

G TSIAMIS1, J MANSFIELD1, J TAYLOR2 and D TEVERSON2

1Department of Biological Sciences, Wye College, University of London, Ashford, Kent TN25 5AH, UK; 2Horticulture Research International, Wellesboume, Warwickshire CV35 9EP, UK

Background and objectives
The ability of Pseudomonas syringae pv. phaseolicola to cause halo-blight disease in cultivars of Phaseolus vulgaris is determined primarily by three sets of genes which control (i) basic pathogenicity (hrp genes), (ii) toxin production and (iii) cultivar-specific avirulence (avr genes) [1]. Recent characterization of strains of P.s. pv. phaseolicola, based on their virulence towards Phaseolus genotypes, has demonstrated the existence of gene-for-gene interactions based, in theory, on the presence of five genes for avirulence in P.s. pv. phaseolicola which match five genes for resistance (R1-5) in bean. Incompatibility is expressed by the hypersensitive reaction (HR) at inoculation sites in leaves and pods [2]. We have characterized the avr gene matching Rl .

Results and conclusions

A genomic clone pPPY503 isolated from a library of race 5, strain 1375, was found to confer avirulence to the differential cvs Red Mexican and Guatemala, thought to possess the Rl gene for avirulence. The location of avrPphF was determined by saturating the genomic clone pPPY503 with transposons (Tn3-gus) and by subcloning. Twelve transposon insertions altered the phenotype conferred by pPPY503 from avirulent to virulent, allowing avrPphF to be located to a 1.3-kb region flanked by BamHI and EcoRI restriction sites.

Co-segregation of reactions to race 6 (pPPY503) and race 1 (Al ) was demonstrated in F2 plant progeny of a cross between Red Mexican (Rl, R4) and Tendergreen (R3) and also Red Mexican and Canadian Wonder (no resistance genes). The Al/R1 gene-for-gene interaction was therefore confirmed. Phytoalexin accumulation was examined within infected tissues.

The nucleotide sequence of avrPphF revealed an 'hrp box' preceding two open reading frames (ORFs), which encoded proteins of 21.9 and 15.6 kDa, respectively. No homology to any known DNA or protein sequence has been found. Subcloning into vectors allowing overexpression showed that both ORFs are required for full avirulence activity. Transconjugants of E. coli harbouring avrPphF were able to deliver signals causing the HR in Red Mexican only if they also expressed the hrp cluster. The requirement for two proteins to confer avirulence suggests that they may be involved in some coordinated enzyme activity rather than themselves acting as elicitors.

References
1. Jenner C, Hitchin E, Mansfield J, et al., 1991. Molecular Plant-Microbe Interactions 4, 553-562.
2. Mansfield J, Jenner C, Hockenhull R et al., 1994. Molecular Plant-Microbe Interactions 7, 726-739.