Exposure to host resistance drives evolution of bacterial virulence in plants; investigating the excision and mobility of genomic island PPHGI-1

The ability of bacteria to infect animal and plant hosts is determined by proteins known as pathogenicity factors. These factors are encoded by genes, which are frequently found on mobile pieces of DNA known as genomic islands (GI). Transmission of GIs between bacteria is predicted to disseminate pathogenicity determinants amongst bacteria causing the spread of virulence. In plant pathogenic bacteria the acquisition or loss of pathogenicity determinants can result in a change of virulence. We have previously found that the pathogenicity determinant avrPphB resides on a GI designated PPHGI-1, which if deleted from the chromosome of the plant pathogen Pseudomonas syringae pathovar phaseolicola strain 1302A, causes a change in pathogenicity on differential cultivars of bean. Located on the GI are genes predicted to mediate the mobilisation of the GI as well as genes thought to be involved in conjugation of mobile elements from one bacterium to another. This proposal will investigate, using molecular techniques, the function of the genes predicted to mediate movement and conjugation of the PPHGI-1 carrying avrPphB. We hypothesise that these mobility determinants will be involved in movement and transmission of avrPphB. The research will help to elucidate ubiquitous mechanisms involved in the transfer of pathogenicity in plant and animal pathogens, a knowledge of which could impact on medicinal and agricultural treatments against pathogens.

The avirulence gene avrPphB of Pseudomonas syringae pv. phaseolicola strain 1302A causes the induction of a rapid hypersensitive response (HR) in certain bean cultivars. Exposure to the HR led to the selection of strains lacking avrPphB through the deletion of a 106-kb genomic island (PPHGI-1) that contains an integrase required for excision. The island excised from chromosomal att loci to form an episome that was rapidly lost from bacteria in leaves undergoing the HR. PPHGI-1 shares features with integrative and conjugative elements and also pathogenicity islands (PAIs) in diverse bacteria. This proposal will investigate the conditions of PPHGI-1 excision and the role of several genes in mobility and transmission of the island. Mobility of PAIs is thought to be tightly regulated and so the expression of genes predicted to mediate mobility of PPHGI-1 will be screened using real-time RT-PCR. Expression of genes responsible for excision in the plant will be investigated using a series of fluorescent protein tagged strains and a multiphoton microscope. A variety of plant extracts will also be used be define the conditions which activate excision. Experimental systems will be established to investigate the ability of the episome to transfer and recombine into other bacteria. Loss of PPHGI-1 does not compromise the ability of the bacteria to grow and cause disease within the bean plant after artificial inoculation. However, PPHGI-1 encodes a type IV pilus related to pili involved in motility of bacterial cells as well as the presence of a series of photosensory and chemotaxis signalling genes which may be important fitness determinants under different environmental conditions such as the high light intensities. Fitness under different light regimes will therefore be investigated. These experiments will provide a more complete molecular explanation of how exposure to resistance mechanisms in plants drives the evolution of new virulent forms of plant pathogens.