Identification of protein secretion systems and type III effectors in wood-associated bacteria of the genus Xylophilus

To gain insights into the biology of bacteria of the genus Xylophilus , including the grapevine pathogen Xylophilus ampelinus , we scrutinized all available genome sequences of the genus for the presence of type III secretion and flagellar systems. We found three distinct flagellar systems within the genus, one of which was present in all 12 strains for which good-quality genome sequences were available. The other two flagellar systems were only detected in one or two strains. We also identified two sub-classes of type III secretion systems, likely under control of the AraC-type transcriptional activator HrpX. One system with resemblance to systems from plant-pathogenic bacteria was only found in the grapevine pathogen. The other system was found in three strains of Xylophilus , all isolated from plant material. We predicted which genes are co-regulated with the type III secretion systems, as supported by the presence of strongly conserved HrpX-binding promoter elements. We identified about 40 type III effectors in the grapevine pathogen with homologs in plant pathogenic bacteria. In contrast, a rhododendron flower isolate had only two type III effector gene candidates with conserved HrpX-binding promoter elements, but many genes without homologs beyond the species. Finally, we predicted and confirmed three novel effector candidates from X. ampelinus to contain a functional type III secretion signal using an AvrBs1 reporter approach. The presence of type III effectors suggests that effector-triggered immunity may exist in grapevine or non-host plants and that strategies targeting type III effectors for resistance engineering may contribute to suitable control measures. IMPORTANCE Xylophilus species include poorly characterized plant-associated bacteria, among them the grapevine pathogen Xylophilus ampelinus , yet their molecular mechanisms of host interaction have remained largely unknown. By curating public genomes and performing a genus-wide comparative analysis, this study clarifies Xylophilus taxonomy, reveals the distribution and evolutionary history of flagellar and type III secretion systems (T3SS), and identifies two distinct Hrp2/Hrc2-class T3SS types within the genus. Importantly, we define the predicted effector repertoires associated with these systems and experimentally validate three previously unrecognized type III effectors from X. ampelinus . These findings provide the first comprehensive framework for understanding virulence and host interaction strategies in Xylophilus , offering molecular targets and genomic markers relevant to plant pathology, disease surveillance, and the management of bacterial grapevine diseases.