1.6.6S
A LARGE NATIVE PLASMID OF PSEUDOMONAS SYRINGAE PV. PHASEOLICOLA IS ASSOCIATED WITH PATHOGENICITY TOWARDS BEAN

R JACKSON1, J MANSFIELD2, M GIBBON1, D ARNOLD1, J MURILLO3 and A VIVIAN1

1Department of Biological and Biomedical Sciences, University of the West of England, Frenchay, Bristol BS16 1QY, UK; 2Department of Biological Sciences, Wye College, University of London, Wye, Ashford, Kent TN25 5AH, UK; 3Depto Produccion Agraria, Universidad Publica de Navarra, Pamplona 31006, Spain

Background and objectives
Pseudomonas syringae pv. phaseolicola (hereafter Pph) is the causal agent of halo blight of bean (Phaseolus spp.) and causes significant damage to crops worldwide. The host-pathogen interaction has been well studied and has led to the recognition of a race-cultivar level of interaction, arising from the interaction of matching avirulence and resistance genes [1]. However, host-specific pathogenicity determinants have not been identified, and it was the aim of our research to identify the genetic basis of pathogenesis.

Results and conclusions
Characterization of the nine races of Pph revealed eight of the races to harbour a highly stable plasmid of ca 150 kb. A plasmid origin of replication, previously cloned by Wood et al. [2], was introduced into races 4, 5 and 7 (strains 1302A, 1375A and 1449B, respectively) by electroporation. Some electroporants obtained were cured of the ca 150-kb plasmid and were tested in pods and leaves of bean. The cured strains were unable to cause disease and elicited a hypersensitive response (HR) in normally susceptible cultivars (cvs) and caused an altered HR in cv. Red Mexican; they remained avirulent towards the non-host, tobacco. The cured strains showed no change in the colony or culture appearance nor loss of secretion of phaseolotoxin or production of levan. Nine clones from a race 7 gene library restored a pathogenic phenotype to the cured race 7 strains, RW60 and RW63. Five of the clones restored the ability of the pathogen to cause watersoaking (disease) on bean cvs Tendergreen and Canadian Wonder and restored the HR in cv. Red Mexican. The remaining four clones restored watersoaking on cvs Tendergreen and Canadian Wonder, but also conferred partial watersoaking on cv. Red Mexican. The avirulence gene avrPphF.R1 [1] was present on clone pAV521, which accorded with the altered HR on cv. Red Mexican. Transposon insertions which abolished the pathogenicity phenotype conferred by the clones revealed a cluster of ca 10 kb in pAV518 and a smaller region associated with avrPphF in pAV521. The disposition of inactivating transposon insertions suggests the possibility that numerous genes are involved in the pathogenicity process. Variations in the efficiency of restoration of pathogenicity, in particular host-pathogen combinations, were observed for single restoring clones. We concluded that a large native plasmid of at least three races of Pph encoded genetic determinants required for pathogenicity towards the host plant bean.

References
1. Mansfield J, Tsiamis G, Pur, N et al., 1997. Pseudomonas syringae Pathovars and Related Pathogens, pp. 385-391.
2. Wood JR, Vivian A, Jenner C et al., 1994. Molecular Plant-Microbe Interactions 7, 534-537.