The Transcriptional Regulator FleQxoo Binds To The Promoters Of Flagellar Genes Of Xanthomonas Oryzae Pv.oryzae

Xanthomonas oryzae pv. oryzae (Xoo) is a gram-negative bacterium that causes bacterial blight, a destructive disease of rice responsible for severe crop losses all over the world. Recent studies on the molecular mechanisms of pathogenicity of Xoo have shown the single polar flagellum not only is an important organ for motility, but also a potential pathogenic factor. Previous work in our laboratory on flagellar biosynthesis in Xoo has found that flagellar genes are located in a gene cluster harboring 60 open reading frames (ORFs). The cluster also includes genes for associated motility. Expression of the gene cluster is regulated by the transcriptional regulator FleQxoo. However, the mechanism by which FleQxoo regulates gene expression and biosynthesis of flagellum is unknown.The objective of this study was to understand how FleQxoo regulates flagellar motility and gene expression in Xoo. This knowledge will helpful for the development of effective and sustainable disease management. Analysis of gene regulation was made by prokaryotic expression and purification of FleQxoo, observation of FleQxoo binding with the promoters of flagellar genes, and tests using a deletion mutant of fliExoo.1. Expression, purification and binding of FleQxoo protein. Primers for the fleQxoo gene were designed based on the genomic sequence of KACC10331, which were cloned from the wild type strain PXO99A using the PCR method. The expression plasmid pET21-fleQxoo was constructed, and transformed into the expression host strain BL21(DE3)pLysS. FleQxoo was then expressed under IPTG induction. The resulting FleQxoo protein (molecular weight 56 kDa) was then purified through Ni-NTA affinity chromatography. Western blotting was used to detect the FleQxoo protein with anti-His antibody. Partial promoters of flagellar genes in Xoo were then analyzed and cloned. Observations were made of binding between the promoter fragments and the FleQxoo protein using Electrophoretic Mobility Shift Assay (EMSA). In vitro binding of FleQxoo to potential promoters of flagellar genes, fliExoo,flgFxoo and flhFxoo, was observed. This suggested that flagellar gene transcription might be regulated through direct binding of FleQxoo to promoter regions of flagellar genes.2. Analysis of deletion mutation of fliExoo encoding a flagellar basal body protein of Xoo. A gene deletion mutant△fliExoo was generated by the GmR marker exchange method from PXO99A. Non-flagellum, cell precipitation in culture and significantly attenuated motility on 0.3% semi-solid medium were observed in△fliExoo compared to those of PXO99A. While there were no apparent alterations in bacterial growth and activity of cellulase in vitro, significant decreases in extracellular polysaccharide (EPS) production and biofilm formation were found in△fliExoo. Both pathogenicity on rice (Oryza sativa L cv. Nipponbare) and induction of hypersensitive response (HR) on tobacco (Nicotiana tabacum L.) of△fliExoo were attenuated. The complementary strain,△fliExoo-C, restored the phenotype of the mutant partially or completely. The results indicate that the fliExoo mutation not only influences flagellar motility, but also virulence-related phenotypes in Xoo. 3. Detection of biofilm formation of flagellar gene mutants of Xoo. To demonstrate the biofilm formation of Xoo, the effects of culture conditions (plastic and glass surfaces, culture time, temperature and medium) on biofilm formation were evaluated, and the biofilms of wild-type, gene deletion mutants and complementation strains of Xoo were assayed by the crystal violet staining method. The results indicated the optimizing culture conditions for biofilm formation of PXO99A, whereas the bacterium were grown overnight in M210 medium, sub-cultured into M210 medium in 96-well polystyrene microplates, and incubated statically at 23°C for 24 h. The effects of deletion mutations of flagellar genes (fleQxoo, fliExoo and rbfCxoo) on biofilm formation were found. While the biofilm formation of wild-type strains collected from south and north China were observed, there were somewhat differences in capability of biofilm formation between the strains, and the difference in biofilm formation was not co-related to the origins of the strains. Results suggest the biofilm formation was influenced by the mutations of genes involved in flagellar motility in Xoo.In conclusion, these studies provide us with the evidence that FleQxoo might regulate the flagellar gene expression by direct binding to the promoters in Xoo.