Functional Characterization Of The Transcriptional Regulator FleQxoo And The Alternative Sigma Factor σ⁵⁴ RpoNxoo In Xanthomonas Oryzae Pv. Oryzae

Flagellum is one of the major bacterial organelles for motility by using which bacteria swim in a liquid environment and swarm over a semisolid surface. In addition to these well-established functions, it has been demonstrated in many pathogenic bacteria that flagella play multiple roles in adhesion, biofilm formation, chemotaxis, colonization and invasion in the pathogenesis. The biosynthesis of flagella is tightly and hierarchically regulated. In some of the important animal and human pathogenic bacteria including Pseudomonas aeruginosa, Vibrio spp. and Helicobater pylori, there is a four-tiered transcription regulation hierarchy mainly controlled by the transcriptional regulator FleQ and the alternative sigma factorσ⁵⁴.Bacterial blight of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important diseases that constrain production of this staple crop in many parts of the world. The disease system becomes one of the models to study the plant-pathogen interactions. Although it has been shown that single polar flagellum of Xoo is one of the most important factors in virulence, the regulation of bacterial flagellation is still unknown. Genomic analysis of Xoo revealed that about 60 flagellar genes are clustered, and fleQ homologue (fleQxoo) and rpoN homologue (rpoNxoo) was found. There seems to be a N-tiered transcriptional regulatory circuit with FleQxoo and RpoNxoo (σ⁵⁴), both of which function as the master regulators in Xoo.Molecular identification and characterization of fleQxoo and rpoNxoo was performed through gene cloning, sequencing, deletion and expression analysis. The goal is to better understand the role of FleQxoo and RpoNxoo in bacterial behavier (such as motility and virulence/pathogenicity) and to help develop the more innovative, effective and durable strategies for the disease control.FleQxoo and rpoNxoo was cloned from the genomic DNA of the wild-type PXO99^A. FleQxoo, an NtrC family activator protein, was structurally featured in theσ⁵⁴-interacting domain and DNA-binding domain with a helix-turn-helix motif, which serves as a cognate activator ofσ⁵⁴ in transcription from severalσ⁵⁴-dependent promoters of flagellar genes. RpoNxoo had three conserved domains including the activator interacting domain (AID), core binding domain (CBD) and DNA binding domain (DBD). FleQ and RpoN were found highly conserved in plant-pathogenic Xanthomonas spp. with 81.06-98.08% of amino acid homolegous, respectively.â–³fleQxoo andâ–³rpoNxoo, the gene deletion mutants were constructed after a double crossover recombination event between Gentamycin resistance gene (GmR) and fleQxoo or rpoNxoo through the marker exchange. Both mutants were non-flagellated and attenuated in flagellar motility on the semi-solid medium, which can be restored by complementation with fleQxoo and rpoNxoo, respectively. Moreover, no significant changes in production of extracellular cellulase and xylanase in vitro, in pathogenicity on rice and induction of hypersensitive response (HR) on non-host tobacco were observed in both mutants compared to PXO99^A.Putative sequences with consensus to GG-(N10)-GC and CC-(N4)-C-(N3)-T of RpoNxoo- and FleQxoo-binding sites were identified from the promoters of flagellar genes. Putative sequence with consensus to TAAA-(N15)-GCCCGTT ofσ²⁸-binding sites was identified from the promoters of 3 genes (fliAxoo, cheAxoo and cheWxoo). Real-time quantitative PCR analysis of the transcription of 6 flagellar genes (fleQxoo, rpoNxoo, fliAxoo, fliCxoo, fliDxoo and fliLxoo) inâ–³fleQxoo andâ–³rpoNxoo indicates that both FleQxoo and RpoNxoo not only up-regulate the transcription of fliAxoo, fliDxoo and fliLxoo directly and up-regulate fliCxoo through the control of fliAxoo indirectly, but also down-regulate theirself.In addition, 3 genes involved in TTSS (hrpG, hrpXo and hpaI) are up-regulated remarkably inâ–³fleQxoo andâ–³rpoNxoo compared to PXO99^A, suggesting both FleQxoo and RpoNxoo repress hrpG, hrpXo and hpaI genes.In summary, both FleQxoo and RpoNxoo as the major regulators play important roles of flagellation and motility in Xoo. It is suggested that there is likely a transcription regulation hierarchy controlled by the transcriptional regulator FleQxoo and the sigma factorsσ⁵⁴ andσ²⁸ in Xoo.