| Bacterial blight of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious diseases of rice in the world. The disease system has become an important model to study the plant-pathogen interactions. In recent years, availability of complete genome sequence of three Xoo strains: MAFF311008, KACC10331 and PXO99A has provided a substantial foundation for deeper and more profound research on Xoo biology.The single polar flagellum of Xoo is the major organelles for motility, previously studies in our lab have found that bothσ54 factor(RpoNxoo) and FleQxoo play important roles in flagellation and motility in Xoo. In order to elucidate the transcriptional hierarchy of Xoo, this research examed the genome sequence of PXO99A, identified the function of another important transcription factorσ28 (FliAxoo) which is located in flagellar cluster and confirmed the flagellar transcription mechanism by real-time quantitative PCR method, furthermore, this research also revleaved the function of a response regulator FlgRRxoo and flagellin protein Flicxoo. This project aimed to definite the flagellar biogenesis and identify the flagellin functions in the rice-Xoo interaction mechanisms.Alterative sigma factorσ28 is required in the regulation of flagellar gene transcription has been verified in different bacteria species, but its function in Xoo is still unknown. The fliAxoo gene deletion mutant was generated from wide type strain PXO99A by marker exchange method. Compared to PXO99A ,△fliAxoo was non-flagellated and greatly attenuated in motility, which can be restored by the complementation. Whereas, no significant changes in production of extracellular polysaccharide (EPS), extracellular cellulase and xylanase in vitro, and the virulence on rice were observed in the mutant compared to PXO99A. These results demonstrated thatσ28 is involved in flagellar biogenesis in Xoo, but not in production of virulence factors and pathogenicity on rice.The complete genome sequence of PXO99A revealed two non-contiguous chromosomal loci with clusters of flagellar genes (2591336-2656886, 2803423-2868973), spanning from PXO-01016 (flgM) to PXO-00954 (cheA) and PXO-06150 (flgM) to PXO-06212 (cheA). The organizations of the two clusters are almost the same, except that 63 genes in one cluster but 61 genes in the other, besides that, each gene in one cluster shares 100% identity and locations with the counterpart. These flagellar genes mainly appear to be organized into 15 transcriptional units, based on the close or overlapping coding sequences and lack of transcriptional terminators. Real-time quantitative PCR analysis the transcription of rpoN,fleQ,fliA,fliC,fliE,fliD,fliL,fliQ,flhF,fliS,flgB,flgG,flgM,flhBand rbfC genes in△rpoNxoo,△fleQxoo and△fliAxoo respectively. The results indicate that there is a three-tiered transcriptional regulation hierarchy in Xoo mainly controlled by the transcriptional regulator FleQ and two alternative sigma factorsσ54,σ28:σ54 and FleQ are in class I which are the master regulators in the cascade;σ28 coding gene fliA together with fliE,fliD,fliL,fliQ,flhF,flgB,flgG,flgM,flhB,rbfC are class II genes which are regulated by FleQ andσ54 ; fliC,fliS,flgM are in class III regulated byσ28.Response regulators play important roles in signal transduction in bacteria, response regulator with the REC signal domain could be act as a stand-alone module. Response regulator FlgRRxoo is located betweenσ54 and FleQxoo and structurally featured with REC signal domain, in order to clarify its function, the flgRRxoo mutant was constructed through the marker exchange.△flgRRxoo demonstrated greatly deficiency in EPS production and obviously low virulence compared with PXO99A, however, no significant changes in flagellar motility and production of extracellular cellulase and xylanase in vitro were observed in△flgRRxoo compared to PXO99A. Real-time quantitative PCR analysis showed that the expression of EPS biosynthesis-related genes gumBDGKM was down-regulated whereas rpoNxoo,fleQxoo,fliAxoo genes were up-regulated in△flgRRxoo. Therefore, FlgRRxoo may act as a regulator involved in regulation of EPS production and virulence of Xoo, but might negatively regulated the flagellar transcriptional regulators.Flagellin is the subunit of filament, which is essential for flagellar assembling and motility, meanwhile, flagellin could also act as PAMP to induce plant defense responses, fliCxoo is class III flagellar genes belongs toσ28 regulons in the three-tiered transcriptional regulation hierarchy of Xoo. The fliCxoo gene deletion mutant was generated through the marker exchange.△fliCxoo was non-flagellated and greatly attenuated in motility compared to the wide type, whereas, no significant changes in production of EPS, extracellular cellulase and xylanase in vitro, and the virulence on rice were observed in the mutant. The results indicated that FliCxoo is an important component of flagellum. In addition, Xoo Flagellin could not induce alkalinization in rice suspension cells as well as H2O2 generation and high cell death, which means Xoo Flagellin could not induce HR response in rice suspension cells.In summary, this work elucidates the three transcriptional hierarchy of Xoo, identified regulation mechanisms of FlgRRxoo in EPS biosynthesis and virulence. Furthermore, initially demonstrated the function of FliCxoo in motility and rice-flagellin interaction, provided new prospective for further studies in rice-Xoo interaction.
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