Xanthomonas oryzae pv. oryzae Type III Effector XopN Targets OsVOZ2 and a Putative Thiamine Synthase as a Virulence Factor in Rice

Xanthomonas oryzae pv. oryzae (Xoo) is spread systemically through the xylem tissue and causes bacterial blight in rice. We evaluated the roles of Xanthomonas outer proteins (Xop) in the Xoo strain KXO85 in a Japonica-type rice cultivar, Dongjin. Five xop gene knockout mutants (xopQ(KXO85), xop(XKXO85), xopP1(KXO85), xopP2(KXO85), and xopN(KXO85)) were generated by EZ-Tn5 mutagenesis, and their virulence was assessed in 3-month-old rice leaves. Among these mutants, the xopN(KXO85) mutant appeared to be less virulent than the wild-type KXO85; however, the difference was not statistically significant. In contrast, the xopN(KXO85) mutant exhibited significantly less virulence in flag leaves after flowering than the wild-type KXO85. These observations indicate that the roles of Xop in Xoo virulence are dependent on leaf stage. We chose the xopN gene for further characterization because the xopN(KXO85) mutant showed the greatest influence on virulence. We confirmed that XopN(KXO85) is translocated into rice cells, and its gene expression is positively regulated by HrpX. Two rice proteins, OsVOZ2 and a putative thiamine synthase (OsXNP), were identified as targets of XopN(KXO85) by yeast two-hybrid screening. Interactions between XopN(KXO85) and OsVOZ2 and OsXNP were further confirmed in planta by bimolecular fluorescence complementation and in vivo pull-down assays. To investigate the roles of OsVOZ2 in interactions between rice and Xoo, we evaluated the virulence of the wild-type KXO85 and xopN(KXO85) mutant in the OsVOZ2 mutant line PFG_3A-07565 of Dongjin. The wild-type KXO85 and xopN(KXO85) mutant were significantly less virulent in the mutant rice line. These results indicate that XopN(KXO85) and OsVOZ2 play important roles both individually and together for Xoo virulence in rice.