Decoding the functions of LuxR solos and environmental factors in shaping Pseudomonas syringae virulence

The virulence of Pseudomonas syringae results from the joint action of multilevel regulatory processes that coordinate and integrate intracellular processes and environmental signals that the bacterium perceives during plant infection. LuxR proteins, especially LuxR solos, act as key sensors in bacterial quorum sensing. They provide a versatile communication system that allows bacteria to detect different signals, coordinate group responses, and precisely regulate adaptati on and virulence. In the highly virulent biovar 3 of P. syringae pv. actinidiae (Psa3), the LuxR solo PsaR3, encoded by a gene located in plasmid genome, is a peculiar and biovar-specific regulator with potential roles in modulating the virulence of the pathogen. However, the absence of a cognate synthase, the lack of a known ligand, and limited knowledge about the complex regulatory role have so far hindered a clear understanding of the function and mechanisms by which this protein becomes functionally active. In plant pathogens, and in P. syringae, one of the systems adopted to overcome host defences is undoubtedly the type III secretion system (T3SS), a fundamental determinant whose induction is regulated by both genetic factors and environmental stimuli. At the same time, the literature on how environmental factors govern T3SS induction, particularly the effects of pH and plant signals, remains limited; strain-specific responses complicate the modelling of host-pathogen interactions. Integrating these two levels of analysis may help identify the molecular and ecological determinants underlying the virulence of P. syringae. This thesis, therefore, aims to provide a new perspective on the multilevel regulation of P. syringae by (i) studying the functions of the PsaR3 LuxR solo and (ii) characterizing the influence of environmental pH and plant signals on T3SS activation in strains belonging to different phylogroups. The overall objective is to provide an integrated picture of intracellular regulatory circuits, extracellular signals, and how they converge to modulate virulence. To do this, a combined experimental and in silico analysis approach was adopted. Bioinformatic analyses combined with transcriptomic data obtained with a strain expressing an arabinose-inducible PsaR3 enabled a functional and genomic characterization of the protein. With the aim to control PsaR3 activation through post-translational regulation, a chimeric receptor was further designed, supported by in silico structural modelling, by fusing the DNA-binding domain of PsaR3 with the AHL-responsive sensory domain of CviR (Chromobacterium violaceum). Functional analyses evaluated the impact of the chimeric protein, as well as the constitutively overexpressed native PsaR3 for comparison, on T3SS induction, biofilm formation, c-di-GMP levels, and siderophore production, revealing an unexpected phenotype providing hypotheses regarding possible self-regulatory effects. In parallel, the use of a reporter system highlighted that T3SS activation was strongly influenced by environmental pH, with phylogroup- and strain-specific sensitivity ranges. The presence of plant signals often broadened or modified these ranges, indicating possible combinatorial effects between acidity and host-derived signal molecules. Significantly, pH-dependent modulation was also observed in rich media, where T3SS is typically considered repressed, revealing a broader and more complex regulatory landscape than expected. Overall, the results outline the virulence of P. syringae as a multi-layered process that is highly dependent on the dynamic environmental context and can be further shaped by interactions among the pathogen’s intracellular regulators. PsaR3 is a unique, potentially self-regulated transcriptional element whose influence intersects virulence pathways. Environmental pH, in turn, emerges as a key, hitherto underestimated determinant, whose effects can be modulated by plant-derived signals, with variable outcomes across strains. This integrated perspective helps elucidate aspects of the molecular logic underlying the regulation of P. syringae virulence. It offers new conceptual foundations for understanding and managing the pathogen’s behaviour in the presence of the plant and in dynamic ecological contexts.