BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATION OF THE BACTERIAL PsaR1 SENSOR IN THE PSEUDOMONAS SYRINGAE PV. ACTINIDIAE-KIWIFRUIT INTERACTION

Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is responsible for important economic losses in all major areas of kiwifruit production worldwide, including Italy. As for many other bacterial diseases, current plant defense strategies against Psa are mainly based on the use of copper-containing products, which raise eco-toxicological problems. Revision and restriction processes regarding the use of high amounts of copper in agriculture impose an urgent study of new solutions, efficient and eco-compatible, avoiding at the same time the occurrence of new resistance to active molecules. Innovative strategies are based for instance on the application of targeted treatments for “weakening” the pathogen, i.e. to reduce its virulence within its host. However, this requires improving our knowledge regarding molecular mechanisms controlling bacterial virulence induction. A key regulator of bacterial virulence is the so-called ‘quorum-sensing’ (QS), that links bacterial density to gene expression. This mechanism allows bacteria to communicate within the bacterial community and with their environment, via small diffusible molecules. The prototypical QS system of Gram-negative bacteria consists of a LuxI-type synthase that produces the signal molecules acyl homoserine lactones (AHLs) and a cognate LuxR-type receptor/regulator that senses signal specific threshold concentration. An interesting subgroup of LuxR receptors lacks a genetically linked LuxI and has been termed “solos”. These “solos” are assumed to sense AHLs from neighboring bacteria, bacterial molecules other than AHLs or still unknown plant-produced compounds in the case of phytopathogenic bacteria. Interestingly, Psa does not produce AHLs but possesses three LuxR solos, which likely contribute to Psa virulence. As a first candidate for a targeted inhibition strategy against Psa, we are currently investigating the biochemical properties of the sensor PsaR1. To that purpose, several tentative have been made to obtain the soluble recombinant sensor in a heterologous system. Once achieved, we demonstrated that it does not bind AHLs, thus excluding the possibility to sense AHLs from neighboring bacteria, and we are currently setting a chemical screening, based on thermal shift assay, to identify the class(es) of molecules able to bind to the sensor. On the other hand, we aim to identify the pathway(s) regulated by PsaR1 during Psa interaction with kiwifruit, during different phases of the infection. Thus, a microarray analysis is being performed to compare the transcriptomic profiles of wild-type and psaR1 knockout Psa strains at both exponential and stationary growth phase, in conditions mimicking the interaction with the host plant, i.e. minimal medium supplemented with kiwifruit extract.