Functional Analysis Of Hrcq Gene In Xanthomonas Oryzae Pv. Oryucola And Research On RepLACEment Of Hrp ReGULAtor HrpG Gene Between Xanthomonas Oryzae

Xanthomonas oryzae pv. oryzicola(Xoc) is a gram-negative plant pathogenic bacteria, which cause bacterial leaf streak (BLS) in rice(Oryza sativa). BLS is an important bacterial disease on rice, specially in Southeast Asian and China。hrcQ gene of Xoc is highly conserved in the animal and plant pathogens. It has been postulated that the differentiation at the N-terminal of HrcQ proteins in different plant pathogenic bacteria is related to the secretion of specific effectors through type III secretion system (T3SS). However, it is unclear whether HrcQ affects the formation of T3SS and secretion of T3SS effectors. Here we reported that the hrcQ mutant of Xoc by knocking-out mutagenesis through allelic exchange. Pathogenicity assays demonstrated that the hrcQ mutant lost the ability to trigger hypersensitive response (HR) in nonhost tobacco and pathogenicity in host rice. The expression of hrcQ was induced when the pathogen interacted with rice cells. The protein-protein interaction assay by yeast two-hybrid system showed that HrcQ could interact with Hpal, HrcN, HrpB5and HrpB2. Immunobloting assay confirmed that HrcQ was not secreted through T3SS, whereas the mutation of hrcQ led to no secretion of Hpal and HrpB2through T3SS. These results suggested that HrcQ is the core component of T3SS helping Hpal and HrpB2secret through T3SS to determine HR in tobacco and pathogenicity in rice. All the above also provides a basis for further understanding of the secretion mechanisms by T3SS of Xoc.hrpG and hrpX, two major hrp regulators in Xanthomonas oryzae pv. oryzae,(Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) control the expression of some genes which encode core component proteins of type III secretion system (T3SS) and type III effectors and is required for elicitation of the hypersensitive response (HR) in nonhost or resistant host plants and for pathogenesis in susceptible plants. In this study, we focused on the functional replacement of hrpG and hrpX between these two pathovar. Results showed that the hrpX genes from Xoo and Xoc can replace each other; the hrpGxoc gene can recover the ability of causing pathogenicity on rice and triggering HR on nonhost tobacco of the hrpGxoo mutant to wild type level; these symptom caused by the hrpGxoc mutant could not be complemented to wild type level when the hrpGxoo gene was transferred into, however, the expression of hrpG downstream genes hrpX, hrcT, hpa2, hrpD6and hpaR as well as the production of extracellular proteins secretion were not affected in the complementary strain. These evidences indicated that the hrpX genes of Xoo and Xoc have similar function; the hrpGxoc gene not only has the function of hrpGxoo, but also regulates expression of other unknown genes which are essential for pathogenicity of Xoc on host rice but not controlled by hrpGxoo, providing some clues for elucidating the difference of hrpG regulatory pathway in Xoo and Xoc.