Transcriptome profiling of type VI secretion system core gene tssM mutant of Xanthomonas perforans highlights regulators controlling diverse functions ranging from virulence to metabolism

Type VI secretion system (T6SS) is a versatile, contact dependent contractile nano-weapon in Gram-negative bacteria that fires proteinaceous effector molecules directly into prokaryotic and eukaryotic cells aiding in manipulation of the host and killing of competitors in complex niches. In plant pathogenic xanthomonads, T6SS has been demonstrated to play these diverse roles in individual pathosystems. However, the regulatory circuit involved in mediating biological functions carried out by T6SS are still elusive in Xanthomonas sp. To bridge this knowledge gap, we conducted an in vitro transcriptome screen using plant apoplast mimicking minimal medium, XVM2 medium, to decipher the effect of tssM deletion, a core gene belonging to T6SS-cluster i3*, on the regulation of gene expression in Xanthomonas perforans strain AL65. Transcriptomic data revealed that a total of 277 and 525 genes were upregulated, while 307 and 392 genes were downregulated in the mutant strain post 8 and 16 hours of growth in XVM2 medium. The transcript abundance of several genes associated with flagellum and pilus biogenesis as well as type III secretion system were downregulated in the mutant strain. Deletion of tssM of cluster-i3* resulted in upregulation of several T6SS genes belonging to cluster-i3*** and genes involved in biofilm and cell wall biogenesis. Similarly, transcription regulators like rpoN, Pho regulon, rpoE and csrA were identified to be upregulated in the mutant strain. Our results suggest that T6SS modulates the expression of global regulators like csrA, rpoN and pho regulons triggering a signaling cascade and co-ordinates the expression of suite of virulence factors, stress response genes and metabolic genes. ImportanceType VI secretion system (T6SS) has received attention due to its significance in mediating inter-organismal competition through contact-dependent release of effector molecules into prokaryotic and eukaryotic cells. Reverse-genetic studies targeting core genes of T6SS have indicated the role of T6SS in virulence in a variety of plant pathogenic bacteria, including Xanthomonas studied here. However, it is not clear whether such effect on virulence is merely because of a shift in the microbiome-mediated protection or if T6SS is involved in a complex regulatory network governing virulence in plant pathogens involving type III secretion system or c-di-GMP signaling pathways. In this study, we conducted in vitro transcriptome profiling in minimal medium to decipher the signaling pathways regulated by tssM-i3* in Xanthomonas perforans strain AL65. We show that TssM-i3* regulates the expression of a suite of genes associated with virulence and metabolism either directly or indirectly by altering the transcription of several regulators. These findings further expand our knowledge on the intricate molecular circuits regulated by T6SS in phytopathogenic bacteria.