DECIPHERING Pseudomonas syringae pv. actinidiae VIRULENCE AND COMMUNICATION WITH HOST PLANT

The gram-negative bacterium Pseudomonas syringae pv. actinidiae (Psa) is the causal agent of the bacterial canker of kiwifruits leading to important economic losses in New Zealand, Italy, Korea and Japan the main producers worldwide. To identify virulence targets and develop new effective targeted control strategies, molecular mechanisms involved in host plant recognition by Psa and related infection processes was currently studied. To this aim, the Psa CRAFRU 8.43 strain was transformed with constructs carrying the green fluorescent protein (GFP)-encoding reporter gene under the control of the promoter of several Psa genes, selected for their putative role in pathogenicity based on literature. Our results showed that hrpA1 gene promoter, involved in the early steps of bacterial infection, is induced in a minimal medium (mimicking apoplast conditions), with an earlier and at higher levels in presence of Actinidia deliciosa leaf extract, indicating a role downstream of host recognition by bacteria. The characterization of hrpA1-inducing kiwi extract showed that such signal(s) are kiwi-specific and smaller than 10KDa in size. Moreover, to elucidate the signalling pathway(s) involved in host-mediated hrpA1 induction, a chemical library was screened to identify molecules able to block such activation. Nineteen candidate molecules were obtained, displaying different inhibition levels. According to the role of HrpA1 in plant hypersensitive response (HR) induction, HR in model plants wasquantifiedto obtain a visible phenotype correlated with hrpA1 promoter induction and thus demonstrate the inhibitory effect of selected molecule, dicoumarol, in a plant system. Moreover, the molecules responsible for Psa quorum sensing activation are still unknown, since Psa possess three LuxR-solos proteins (PsaR1, R2, R3) lacking LuxI enzymes responsible for AHL synthesis. Thus, to identify putative PsaR1 ligands, its recombinant autoinducer-binding domain was produced in E. coli and chemical libraries were screened using a high throughput fluorescence-based thermal shift assay. Four molecules inducing a significant thermal shift were identified as putative PsaR1 ligands; moreover, the presence of the same or other putative PsaR1 ligands in kiwi plants was confirmed by a similar thermal shift effect in presence of kiwi plant extracts. Quercetin and luteolin, PsaR1 putative ligands, tested on Psa virulence traits (motility and biofilm) showed an increase of swarming motility and reduction biofilm formation.