TRANSCRIPTOMIC AND BIOCHEMICAL APPROACHES TO UNRAVEL THE BASIS OF BACTERIAL VIRULENCE IN Pseudomonas syringae pv. actinidiae

Pseudomonas syringae pv. actinidiae (Psa), is a Gram-negative bacterium, aerobic, motile, able to grow epiphytically and endophytically on plants, and it is the causative agent of kiwifruit bacterial canker. Cool temperature and high humidity promote the multiplication of this pathogen and increase the severity of plant disease. Psa can enter into its host plant through natural openings such as stomata and hydatodes or following mechanical wounding, and then spreads systematically into the whole plant. Psa strains are divided into 5 different biovars, based on the country of origin, the outbreak period, genomic characteristics, toxin production and the virulence. In particular, the biovar 1 was identified in Japan in 1984; the biovar 2 spread in Korea in 1997; the biovar 3, identified both in New Zealand and Italy in 2008-2010, is the causative agent of the last worldwide outbreaks and is recognized as the most virulent biovar; the biovar 4 identified in New Zealand, firstly classified as a low virulence Psa biovar, was re-assigned to another pathovar; finally, the biovar 5 and 6 were identified more recently in Japan but is not yet characterized. In the frame of the PhD project, we performed a microarray analysis in order to elucidate the differences in the transcriptomic profile among the Psa stains (biovar 1, 2 and 3), which could account for their different aggressiveness. Then, we focused on a class of proteins that play a role in signal recognition and transcription regulation called LuxR solos. Psa is characterize by three LuxR solos proteins, namely PsaR1, PsaR2 and PsaR3 which could be involved in the interaction between kiwifruit and Psa but whose role is not elucidate yet. Interestingly a bioinformatics analysis performed in our laboratory revealed that PsaR3-encoding gene is specific of biovar 3 and is localized on a plasmid, within a conserved cluster of genes. Furthermore, the cluster is characterized by the presence of an intergenic region that could play a role as bidirectional promoter and could be regulated by PsaR3 itself according to the predicted presence of cis-acting elements putatively regulated by LuxR proteins. Finally, an RNA-seq analysis revealed the Psa pathways regulated by PsaR3 and studies of promoter activities of the intergenic region which could be putatively regulated by PsaR3 revealed that this LuxR solo could recognize a signal molecule produced by kiwifruit, thus mediating an inter-kingdom communication and likely playing a crucial role in the host recognition and in the mediation of the high virulence of the biovar 3.