Functional Identification Of Six Hypothetical Secreted Proteins In Xanthomonas Oryzae Pv. Oryzicola

Xanthomonas oryzae consists of two pathogenic variants, X. oryzae pv. oryzicola （Xoc）and X. oryzae pv. oryzae（Xoo）, which cause bacterial steak and bacterial blight in rice respectively. Xoc invades rice leaves through stomatal while Xoo through hydathodes. Both diseases are very serious in rice production in China. Currently, studies on Xoc pathogenesis have focused on the fuction of important genes, such as LuxR gene, avr gene, genes encoding quorum sensing receptor protein, genes related to type â…¢ secretion system, two-component regulation system or DSF-based quorum sensing regulation system. But researches on hypothetical proteins were fewer reported.Through bioinformatic analysis, we made a survey in the genome of X. oryzae pv. oryzicola strain BLS256, and found another six hypothetical secreted proteins （Xoryp₄540, Xoryp₁1980, Xoryp₁5235, Xoryp₁8640, Xoryp₁0851and Xoryp₁2495）, which were predicted to be absent in the genome of X. oryzae pv. oryzae by comparative genomics. Interestingly, these proteins are absent in the whole genome of X. oryzae pv. oryzae, which is a xylem-invading pathogen （colonies in the vascular tissues and propagates in the xylem of rice leaves） and causes bacterial blight of rice. This result showed the novel roles of unique putative hypothetical secreted proteins in X. oryzae pv. oryzicola compared to X. oryzae pv. oryzae.1. These six genes were cloned from X. oryzae pv. oryzicola strain Rs105, with degenerated primers by PCR. Using leaf-piercing inoculation to test the pathogenicity of six genes mutants, the results showed that only Xoryp₁5235mutant reduced pathogenicity. But compared to the wild-type strain, the six mutant strains had the same cell morphology and could also stimulate HR in nonhost tobacco. In the present study, mutagenesis study revealed that one of them, Xoryp₁5235, is required for the optimal virulence. Then, the genetics and biological roles of Xoryp₁5235in the virulence and virulence-associated functions are presented.2. The Xoryp₁5235gene was amplified by PCR. We constructed AXoryp₁5235, the deletion mutants by using double crossover method, and deter mined cell morphology, motility, pathogenicity in host rice and hypersensitive response （HR） in nonhost tobacco, bacterial growth in rice and MMX medium （Peptone, Yeast extract, Asparagine, Aspartic acid, Gluta mine, Glutamate, Ammonium sulfate, Urea, Potassium nitrate or Alanine）. In-frame deletion of Xoryp₁5235in Xoc dramatically impaired pathogen virulence, and extracellular polysaccharide （EPS） production, one of the important known virulence-associated functions in Xanthomonas. According to our experimental results, we defined Xoryp₁5235as epv （extracellular polysaccharide and virulence related gene） inX. oryzae pv. oryzicola.Quantitative Real-Time Reverse Transcription PCR （qRT-PCR） showed that both gumB （one gene of the gum operon encoding the key enzymes required for the final step of EPS biosynthesis） and a known virulence-related gene pgk （encodes phosphoglycerate kinase） were obviously reduced in the epv-deletion mutant compared to the wild-type strain Rs105. In addition, we observed that epv was positively regulated both by diffusible signal factor （DSF） and global regulator Clp in X. oryzae pv. oryzicola. Taken together, we showed for the first time that the novel roles and genetics of epv of X. oryzae pv. oryzicola in the EPS production and virulence. To our knowledge, this was also the first report of genetics and roles of epv and its homolog in the genus of Xanthomonas.