Identification Of Genes Involved In C-di-GMP Signaling Pathway And Its Regulation Of Virulence Expression In Xanthomonas Oryzae Pv. Oryzae

The bacterial leaf blight of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of theimportant bacterial diseases in rice production worldwide. Elucidation of its pathogenes is and theregulatory mechanism will contribute to effective control of the disease. Rice-Xoo interaction hasbecome a model to study the molecular interactions between plant and pathogens due to itseconomic importance and the convenience of genetic operation. The sequencing of three Xoo strainsKACC10331, MAFF311018, PXO99Ahave completed and provided the basis for further investigatingthe molecular mechanism of Xoo pathogenesis.C-di-GMP is a novel second messenger widely existing in bacteria, controlling the pathogenicity,biofilm formation, motility and other biological functions. The of diguanylate cyclases (DGC) harboringthe GGDEF domain control the synthesis of c-di-GMP, while phosphodiesterases (PDE) containingEAL or HD-GYP domain are responsible for its degradation. A large number of proteins containingthese domains have been found in bacterial genomes, and form a complex regulatory network. Throughbioinformatics analysis,26genes encoding GGDEF, EAL or HD-GYP domains were identified in thegenome of PXO99A. Previously, we and other research groups have demonstrated that several suchproteins including PdeR, VieA, and RpfG of Xoo are involved in the regulation of EPS production andpathogenicity. However, the mechanisms involved in the upstream and downstream of c-di-GMPsignaling pathway remain unknown. This study was designed to further elucidate the c-di-GMPsignaling pathway in Xoo, and provide a scientific basis to further understand the pathogenesis of Xoo.1. A two-component system PdeK/PdeR regulates c-di-GMP turnover and virulence of XooTwo-component systems (TCS) consisting of a histidine kinase (HK) and a cognate responseregulator (RR) play essential roles in bacteria to sense environmental signals and regulate cell functions.One type of response regulators are involved in metabolism of c-di-GMP.PdeR(PXO₀1019) is a response regulator with an GGDEF domain and an EAL domain. Deletionof pdeR in PXO99Aresulted in reduced virulence to rice and decreased EPS production. There are twohistidine kinase genes pdeK(PXO₀1018) and PXO₀1020flanking pdeR, which might form a TCSwith PdeR. To study the biochemical function of PdeR, we expressed and purified PdeR protein in vitro..By using colorimetric assays and and HPLC analysis, we showed the truncated protein containing onlyGGDEF-EAL domains had strong PDE activity. We constructed the pdeK and PXO₀1020mutantsthrough homologous recombination method. Deletion of pdeK in Xoo strain PXO99Ahad attenuated itsvirulence on rice. Compared with the wild type, pdeK produced lower amount of exopolysaccharide(EPS), but there was no difference observed in terms of motility or extracellular cellulase activity.Although PXO₀1020and pdeR are transcribed in the same operon, deletion of PXO₀1020did notaffect the virulence phenotypes. Further analysis of virulence gene transcripts in pdeR and pdeK byquantitative RT-PCR (qRT-PCR) indicated EPS synthesis-related gene gumMBKD, and type IIIsecretion system-related genes hrpG, hrpX and hpa1were significantly decreased, suggesting thesegenes are positively regulated by PdeK and PdeR. The Y2H experiments also showed that PdeR interacted with PdeK, but not PXO₀1020. Additionally, the PDE activity of full length PdeR needs tobe triggered by PdeK in the presence of ATP. All results suggested that PdeK and PdeR constituted atwo-component system to regulate the virulence of Xoo.2. Identification of PXO₀0403from Xoo as a c-di-GMP receptor to regulate the pathogenicityand induction of hypersensitive response in plantsC-di-GMP receptors are critical components in the downstream of c-di-GMP signaling pathway byvarious mechanisms. PliZ domain protein, transcription factor FleQ and Clp, and mRNA riboswitcheshave been identified as c-di-GMP receptors. And some degenerated GGDEF or EAL domains alsofunction as c-di-GMP binding proteins. Presence of different types of c-di-GMP receptors indicates thatdifferent levels of regulatory mechanism are used to regulated cellular functions, such as genetranscription, translation, and post-translation. PXO₀0403possesses both GGDEF and EAL domains.Mutation of PXO₀0403in PXO99Aattenuated the bacterial ability to multiply in susceptible rice, andcaused a delayed hypersensitive response (HR) on nonhost tobacco. qRT-PCR analysis showed typeIII secretion system (T3SS) genes hrpG, hrpX and hpa1were down-regulated in PXO₀0403. Both insilico analysis, enzyme assays and the ITC assays indicated that the GGDEF and EAL domains ofPXO₀0403were degenerate and inactive, and it binds c-di-GMP with high affinity as a stoichiometryof1:1in vitro. Therefore, we concluded that PXO₀0403acts as a c-di-GMP receptor in PXO99Atoregulate bacterial pathogenicity and induction of HR in plants.3. Functional analysis of GGDEF protein PXO₀0649and PXO₀2615PXO₀2615and PXO₀0649contains only GGDEF domain, which is related to c-di-GMPsynthesis. To study the fuction of PXO₀2615and PXO₀0649in Xoo, we constructed their genedeletion mutants through marker change. Compared with wild type, PXO₀2615showed increasedEPS production by20%, and enhanced virulence to rice. Deletion of PXO₀0649showed similarimpact on EPS production and bacterial virulence. However, they did not affect the motility, biofilmformation, or extracellular enzymes production. The results indicate that PXO₀2615and PXO₀0649are involved in the regulation of Xoo pathogenic ity and production of EPS. Whether they are functionalDGCs remain unclear and needs further work to find out.In summary, this study has revealed that the upstream c-di-GMP signaling pathway is regulated byphosphorylation signal (TCSs), and the downstream is regulated through c-di-GMP binding with thereceptor. Our work has provided scientific basis for further understanding of the c-di-GMP regulatorynetworks and the pathogenesis of Xoo.