1.6.11
THE AVIRULENCE GENES avrPphE and avrPphB FROM PSEUDOMONAS SYRINGAE PV. PHASEOLICOLA ENCODE ELICITORS OF THE HYPERSENSITIVE REACTION IN BEAN

C STEVENS, E ATHANASSOPOULOS and JW MANSFIELD

Department of Biological Sciences, Wye College, University of London, Wye, Ashford, Kent TN25 5AH, UK

Background and objectives
The bean halo blight pathogen Pseudomonas syringae pv. phaseolicola (Psph) is differentiated into nine races based on the presence or absence of five avirulence (avr) genes in the bacterium which interact with corresponding resistance genes, R1-R5, in Phaseolus vulgaris [1]. The resistance gene R2 is matched by avrPphE which is located adjacent to the cluster of hrp genes, which are required for pathogenicity of Psph [2]. Although only races 2, 3, 5 and 7 are avirulent on cultivars with R2 (inducing the hypersensitive response; HR), homologues of avrPphE are present in all races of Psph. This work aimed to analyse the structure and function of alleles of avrPphE. Experiments were designed to answer the following questions. (i) Do changes in the avrPphE DNA sequence confer virulence on cultivars possessing R2? (ii) Do Avr proteins from Psph directly elicit the HR? (iii) Does avrPphE have a role in pathogenicity?

Results and conclusions
DNA sequencing of avrPphE alleles from races of Psph has demonstrated two routes to virulence: via single base-pair changes conferring amino-acid substitutions in races 1, 3, 6 and 9; and via an insertion of 104 bp in the allele in race 8. By generating transconjugants of a virulent race harbouring plasmids expressing the various alleles of avrPphE, we have demonstrated that these base changes are responsible for the difference between virulence and avirulence. The race 5 and race 7 alleles result in avirulence, whereas those from races 1 and 6 lead to a virulent phenotype on R2-containing beans. Transconjugants over-expressing the race 8 or 9 allele induce an intermediate response in R2-containing cultivars.

Agrobacterium tumefaciens-directed expression of avrPphE from race 4 in bean leaves induced the HR in a resistance gene-specific manner, suggesting that the AvrPphE protein is alone required for HR induction and is recognized within the plant cell. The allele from race 6, which is inactive if expressed in Psph, elicited a weak HR if expressed in planta whereas the allele from race 1 did not. Our results suggest that the affinity of interaction between AvrPphE homologues and an unknown plant receptor mediates the severity of the plant's response. The avirulence gene avrPphB which matches the R3 resistance gene also caused a gene-specific HR following expression in the plant after delivery by A. tumefaciens.

The role of avrPphE remains enigmatic and we have failed to demonstrate any affect on ability to colonize bean from a low or high level of inoculum after mutation of the gene. However, the location of avrPphE adjacent to the hrp cluster and its recognition within the plant cell would seem to suggest a role in pathogenicity. Using the in planta expression system we will continue to investigate the role of avrPphE, looking for any localization within the plant cell, in planta processing and any effect on plant physiology or interaction with pathogens.

References
1. Taylor JD, Teverson DM, Allen DJ, Pastor-Corrales MA, 1996. Plant Pathology 45, 469-478.
2. Mansfield JW, Jenner C, Hockenhull R et al., 1994. Molecular Plant-Microbe Interactions 7, 726-329.