Identification Of Novel T3Ss Effectors And Functional Analysis Of XopQ1-1in Two Pathovars Of Xanthomonas Oryzae In Rice

Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) cause bacterial blight (BB) and bacterial leaf streak (BLS) of rice (Oryza sativa), respectively. BB and BLS are two severe diseases in rice caused by phytopathogenic bacteria in China. As other Gram-negative plant pathogenic bacteria, Xoo and Xoc deliver effectors into host cells through type Ⅲ secretion system (T3SS) to cause pathogenicity or susceptibility. The genomic sequence of several Xanthomonas oryzae strain have been completed, however, how many effectors are secreted into rice cell by the T3SS of these two pathovars is unclear. Tsuge et al mentioned that many of the known T3S effectors share several characteristics:In xanthomonads, T3SS effectors, which are regulated by HrpX, an AraC-type transcriptional activator posses the conserved cis-regulatory element, a plant-inducible promoter (PIP) box (TTCGB-N15-TTCGB, B, C/G/T); N-terminal amino-acid patterns:a high percentage (more than20%) of Ser and Pro, a low percentage (less than6%) of Leu in the first50residues, no Asp and Glu in the first12residues, At least one of the third and fourth residues was Leu, Ile, Val, or Pro. In this study, we identified120potential T3SS effectors from the genomic sequence of Xanthomonas oryzae pv. oryzae strain of PXO99A following i) homologs of known T3S effectors in plant-pathogenic bacteria and ii) genes with expression regulated by HrpX as well as iii) proteins with N-terminal amino acid patterns exhibiting the T3SS singal.15candidates were chosen for further study. We found that one of these15candidate proteins, PXO-3819, which is T3SE, was secreted through T3SS by the AvrxalO reporter system. Specific PIP-BOX (TTCGC-N15-TTCGC) sequences were indentified in the promoter region of three genes PXO2065、PXO2774and PXO3819. We observed that the expression of these three genes was regulated by HrpX using fluorescence microscopy by gfp reporter system. These results were consistent with those acquired by RT-PCR. In conclusion, GFP and AvrXa10reporter systems are efficient to identify novel T3SEs of Xanthomonas oryzae.The genome bioinformatics of X. oryzae pv. oryzicola(Xoc), which is the causal agent of bacterial leaf streak in rice, reveals that there is a xopQ1-1gene in the pathogen homologous to hopQl-1in Pseudomonas syringae pv. tomato DC3000, causing bacterial speck diseases in tomato while HopQ1-1is secreted through type-III secretion system (T3S). However, it is unclear whether XopQ1-1is a T3S effector or not and what roles it takes in virulence of Xoc and Xo in rice. Here we reported that the mutant of Xoc in the xopQl-1gene was generated by knocking-out technique through marker-exchange. Pathogenicity assays demonstrated that the virulence of the mutant in rice was stronger than that caused by wild-type strain and the bacterial growth of the mutant in rice tissue was enhanced. Intriguingly, the xopQ1-1mutant at108-CFU/mL still induced hypersensitive response in tobacco, but caused necrosis lesions at104-CFU/mL after injected into tobacco leaves for8days. The complementation assay indicated that the xopQ1Xoc gene restored virulence and bacterial growth in rice to wild-type level as well as the ability not to cause necroses lesions in tobacco. The xopQlxoc gene from X. campestris pv. campestris, R. solanacearum and P. syringae pv. tomato DC3000) also recovered the phenotype of the mutant in rice and tobacco to wild type level. Reverse transcriptional polymerase chain reaction (RT-PCR) revealed that the expression of XopQl-1was not only induced by rice cells, but also controlled by hrpX. The yeast two-hybrid (Y2H) assay showed that XopQl-1was secreted through T3S apparatus and interacted with the translocon protein HrpF and other Hrp protein HpaA、Hpa4and HrpB5. A xopQ1-1-interacting gene was screened from the cDNA library of rice by Y2H using the xopQ1-1of Xoc as bait, designating OsXopQ1-1, which is inducible by the xopQ1-1of Xoc. These results strongly suggested that XopQ1-1is a conserved T3SE in the Gram-negative phytopathogenic bacteria and has similar function of interfering plant immunity system to benefit the growth of bacteria in host cell, providing a basis to understand molecular mechanisms in Xo-rice interactions.