Identification And Cloning Of A Gene Involved In Pathogenicity Of Xanthomonas Oryzae Pv.oryzae From Guangxi Strain

Xanthomonas oryzae pv. oryzae (hereafter Xoo) is one of the most serious bacterial diseases in the world’s rice production, which could cause bacterial leaf blight (BLB), resulting noteworthy losses in our country. Recently, the whole genome of Xoo strains PXO99A (Philippines), MAFF311018 (Japan) and KACC10331 (Korea) have been completely sequenced and published, it becomes much better for the researchers to study the pathogenic mechanism. However, constructing a mutant library is an indispensable component of functional genomics research.In order to explore pathogenicity-related genes of Xoo, The strategy of transposon mutagensis was used. The EZ::Tn5 which carried in <R6kγori/KAN-2> Tnp TransposomeTM was introduced into Guangxi strain Xoo K74 genomics by electrical transformation, and 10,873 mutants of K74 were obtained by several transformation. Southern blot analysis, which containing 20 randomly Tn5-inserted mutants, indicated that transposon Tn5 was randomly inserted and most mutants were inserted by Tn5 only once. According to the leaf clip inoculation on the susceptible host Nipponbare,12 mutants which virulence decrease significantly were identified. Result of localized transposon Tn5 showed that mutagenesis genes included the familiar gumD, rfbD, hrpB,, and type Ⅱ secretion system genes. Moreover six mutants were obtained, which mutagensis genes were both in lipopolysaccharide cluster.LPS is an extracellular component of Gram-negative bacteria that is involved in the outer membrane. As a member of the lipopolysaccharide cluster, the function of wxoC gene is not clear. In this study, two mutant strains M355 and spl, which both have a single copy Tn5 and Tn5-inserted wxoC gene, performed the complementary experiment. The result showed that the virulence of the two mutants could recover to the wide-type strain Xoo K74, it suggests that wxoC gene is related to pathogenicity. However, the colonial morphology does just partial recovery, it still needs further study. It’s posible that the transcription of the downstream gene wxoD is affected by Tn5-inserted wxoC gene, but the wxoD gene is not essential for the virulence of Xoo K74, and it’s only relates to the colonial morphology of bacterial.Therefore, this study laid a solid foundation for clarifying the pathogenic molecular mechanism, developing environmental-friendly drugs to control Xoo, exploring resistance gene, and improving rice varieties.