Functional Analysis Of PXO₀1021Putatively-Encoding C-di-GMP Signaling Protein Of Xanthomonas Oryzae Pv. Oryzue

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial blight of rice, one of themost serious diseases of rice worldwide. There is a series of signaling transduction and regulation athorizons of molecular and cellular in Xoo during its interaction with rice,which facilitates its infectionto the host plant. Cyclic guanosine monophosphate (c-di-GMP) is a newly bacterial second messenger,which involved in the regulation of a lot of complex physiological process (virulence, pathogenicityetc.). The genomic sequence of Xoo PXO99Ahas revealed a number of signal proteins involved inmetabolism (synthesis and degradation) of c-di-GMP, a widely existing bacterial second messenger.This study focused on one of these proteins, PXO₀1021, a putative signaling protein containing PAS,GGDEF and EAL domains. However, its function,expression and regulation is not clear so far. Tounderstand the roles and functions of PXO₀1021and reveal the signals PXO₀1021might sense, thefollowing investigation was carried out.(1)A gene deletion mutant of PXO₀1021was generated by homologous recombination.Phenotypic analysis revealed that the mutant displayed increased virulence and reduced biofilmformation compared with wild type strain PXO99A. In contrast, no changes in motility and inductionof hypersensitive response (HR) on non-host tobacco were found. In the complemented strain, thechanged phenotypes were restored to certain degree(2)In vitro transcription of PXO₀1021gene in different conditions (simulation of ricevascular tissue:hypoxia, neutral and acid, copper ion distribution)was assayed by quantitative real-timePCR (RT-qPCR). The results demonstrated that the expression of PXO₀1021was significantlyinfluenced by the concentration of oxygen (O2),indicating the PAS domain may sense the density ofoxygen. Similar experiments showed different pH had no influence in the expression ofPXO₀10121.The expression of PXO₀10121was also regulated by Cu²⁺concentration. The analysisshowed that Cu²⁺concentration had a positive effect on the expression of PXO₀10121which isdosage-dependent.(3)To find out the external environmental signals PXO₀1021might sense,a plasmidcontaining the promoter of PXO₀1021gene was fused to the green fluorescence protein reporter gene(gfp), and was transferred into PXO99A. GFP was detected qualitatively under fluorescencemicroscopy, suggesting the promoter of PXO₀1021is functional. However, the fluorescence densityis not enough for quantitative detection, so that experiments need to be further optimized.(4) Transcriptional analysis of the gene cluster PXO₀1021/PXO₀1020/PXO₀1019/PXO₀1018showed that PXO₀1019and PXO₀1020share the same transcription unit and direction. WhilePXO₀1021and PXO₀1018show the same transcription direction, but are both single genetranscription.In summary, cloning of PXO₀1021gene and construction of ΔPXO₀1021and complementarystrain is reported in this study. PXO₀1021has a positive regulation of biofilm formation and plays anegative role in regulation of virulence. The concentration of oxygen and Cu²⁺has significantlyinfluenced the expression of PXO₀1021.The promoter of PXO₀1021is functional. PXO₀1020and PXO₀1019share a transcript unit in the gene cluster.In addition, to further elucidate the regulatory mechanisms, transcription leavels of type IIIsecretion system (T3SS)-encoded hrp genes were measured via RT-qPCR assays. Expression of hrpgenes was regulated by Rpf proteins. Therefore, it might be suggested that QS regulates T3SSexpression via the DSF-mediated signaling pathway(s).