Comparative transcriptomics reveal a highly polymorphic Xanthomonas HrpG virulence regulon

Bacteria of the genus Xanthomonas cause economically significant diseases in various crops. Their virulence is dependent on the translocation of type III effectors (T3Es) into plant cells by the type III secretion system (T3SS), a process regulated by the master response regulator HrpG. Although HrpG has been studied for over two decades, its regulon across diverse Xanthomonas species, particularly beyond type III secretion, remains understudied. In this study, we conducted transcriptome sequencing to explore the HrpG regulons of 17 Xanthomonas strains, encompassing six species and nine pathovars, each exhibiting distinct host and tissue specificities. We employed constitutive expression of plasmid-borne hrpG*, which encodes a constitutively active form of HrpG, to induce the regulon. Our findings reveal substantial inter- and intra-specific diversity in the HrpG* regulons across the strains. Besides 21 genes directly involved in the biosynthesis of the T3SS, the core HrpG* regulon is limited to only five additional genes encoding the transcriptional activator HrpX, the two T3E proteins XopR and XopL, a major facility superfamily (MFS) transporter, and the phosphatase PhoC. Interestingly, genes involved in chemotaxis and genes encoding enzymes with carbohydrate-active and proteolytic activities are variably regulated by HrpG*. The diversity in the HrpG* regulon suggests that HrpG-dependent virulence in Xanthomonas might be achieved through several distinct strain-specific strategies, potentially reflecting adaptation to diverse ecological niches. These findings enhance our understanding of the complex role of HrpG in regulating various virulence and adaptive pathways, extending beyond T3Es and the T3SS. IMPORTANCEIn the decades since its discovery, HrpG and its role in the regulation of the type III secretion system (T3SS) and its associated type III effectors (T3Es) in Xanthomonas has been the subject of extensive research. Despite notable progress in understanding its molecular regulatory mechanisms, the full spectrum of processes under control of HrpG, particularly beyond the T3SS and T3Es, and the degree of regulatory conservation across plant-pathogenic Xanthomonas species, remained unclear. To address this knowledge gap, we systematically compared the transcriptomes of 17 Xanthomonas strains, expressing a constitutively active form of HrpG, called HrpG*. We showed that HrpG* regulates different physiological processes other than the T3SS and T3Es and that this regulation shows substantial variation across the different strains. Taken together, our results provide new insights into Xanthomonas-plant interactions through the regulation of different metabolic and virulence pathways by the master response regulator HrpG.