Analysis of HrpG regulons and HrpG-interacting proteins by ChIP-seq and affinity proteomics in Xanthomonas campestris

Gamma-proteobacteria Xanthomonas spp. cause at least 350 different plant diseases among important agricultural crops, which result in serious yield losses. Xanthomonas spp. rely mainly on the type III secretion system (T3SS) to infect their hosts and induce a hypersensitive response in nonhosts. HrpG, the master regulator of the T3SS, plays the dominant role in bacterial virulence. In this study, we used chromatin immunoprecipitation followed by sequencing (ChIP-seq) and tandem affinity purification (TAP) to systematically characterize the HrpG regulon and HrpG interacting proteins in vivo. We obtained 186 candidate HrpG downstream genes from the ChIP-seq analysis, which represented the genomic-wide regulon spectrum. A consensus HrpG-binding motif was obtained and three T3SS genes, hpa2, hrcU, and hrpE, were confirmed to be directly transcriptionally activated by HrpG in the inducing medium. A total of 273 putative HrpG interacting proteins were identified from the TAP data and the DNA-binding histone-like HU protein of Xanthomonas campestris pv. campestris (HU_xcc ) was proved to be involved in bacterial virulence by increasing the complexity and intelligence of the bacterial signalling pathways in the T3SS.