Secreening Of Harpin-like Genes And Identification Of Extracellular Polysaccharide-Associated Genes In Xanthomonas Oryzae Pv. Oryzicola

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak (BLS) in rice. BLS causes serious loses of rice production.The harpin gene identified in Xanthomonas oryzae pv. oryzicola(Xoc) so far, that is hpa1,which locates in the core hrp gene cluster. Hpal protein is dependent on an exit control protein HpaB through typeⅢsecretion system (T3SS). Previously, we found that the hpa1 single mutant and the hpal hpaB double mutant of Xoc still could trigger HR in nonhost, which suggested that there are (is) harpin-like protein(s) in Xoc not Hpa1. In this study, we constructed a mutant library containing ten thousand transformants in the background of hpa1 hpaB double mutant of Xoc by Tn5 transposon. We observed the hypersensitive response (HR) in two nonhost plants tomato and tobacco, individually. At bacterial concentrations of OD600=0.5, Four mutants could induce HR in tomato and tobacco simultaneously, and forty-two mutants could induced HR only in tobacco not tomato, as well as two mutants could induced delayed HR in tobacco. When at OD600=0.3, nine mutants could induce HR in tomato and tobacco simultaneously, and thirty-six mutants could induced HR only in tobacco not tomato, as well as only one mutant could induced delayed HR in tobacco. These results provided a strong foundation for further identification of harpin gene.The same to the other plant pathogenic Xanthomonas, Xoc also products different extracellular enzymes and abundant exocellular polysaccharide (EPS). The EPS of plant pathogenic Xanthomonas mainly controled by three gene cluster gum,xan, wxoc, but their genetic compositions are very different. xanthan gum is the most clearly identified EPS in Xanthomonas campestris pv. campestris, the proteins related to xanthan biosynthesis are encoded by the gum gene cluster consisted of a tandem array of 12 ORFs (from gumB to gumM). xan gene cluster is highly conservative in Xanthomonas. wxocA and wxocB gene only exist in Xoc, Does there exist other unkown gene or gene cluster which controls EPS production? Previously, seventeen extracellular polysaccharide-associated mutants of Xanthomonas oryzae pv. oryzicola(Xoc) had been acquired from our randomly Tn5-inserted mutant library. Our results showed that the Tn5 transposon was inserted in genes of the gum, xan and wxoc clusters in the majority of EPS-reduced mutants. Of the EPS-reduced mutants, three were due to the Tn5 insertion in Xotyp₄217, Xoryp₂488 and Xoryp₀918 genes, respectively, which were first reported to be involved in the EPS production. In six EPS-increased mutants, three were in mutagenesis in fimO, pilY and xopQ genes, respectively, resulting in higher EPS production than the wild-type strain RS105, while other three were because of the mutation in Xoryp2392, Xoryp₄221 and Xoryp₃511 genes. Interestingly, XocORF-3511 only exists in X. oryzae but not in other Xanthomomas species. Our findings provide fundamental clues to further understand not only the metabolic pathways for EPS synthesis in Xoc but also the specific roles of EPS-associated genes in Xoc-rice interactions.