Abstract
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, xopX KXO85, 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 XopNKXO85 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 XopNKXO85 by yeast two-hybrid screening. Interactions between XopNKXO85 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 XopNKXO85 and OsVOZ2 play important roles both individually and together for Xoo virulence in rice.
Highlights
Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, which is one of the most serious diseases in rice (Oryza sativa L.)
Expression of xopNKXO85 is regulated by HrpXKXO85 As expression of hrp and xop genes in X. campestris pv. vesicatoria (Xcv) and other xanthomonads is controlled by two regulatory genes, hrpG and hrpX, we examined whether xopNKXO85 is regulated by HrpXKXO85 in Xoo KXO85
The population of xopNKXO85 mutant was smaller than that of wild-type KXO85 in the OsVOZ2 mutant line (Figure 4D). These results indicate that XopNKXO85 is a virulence factor and that its interactions with OsVOZ2 are critical for Xoo virulence in rice
Summary
Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, which is one of the most serious diseases in rice (Oryza sativa L.). The T3SS of plant pathogenic species of Pseudomonas, Xanthomonas, Erwinia, and Ralstonia is highly conserved and involved in translocation of T3SS-dependent effector proteins from bacterial cells into plant cells [7,8,9,10,11,12]. These effector proteins are categorized into two groups: transcription activator-like (TAL) effectors and non-TAL effectors [11,13,14,15]. In Xoo, T3SS that is essential for virulence is encoded by hypersensitive response and pathogenicity (hrp) genes, the expression of which is controlled by HrpX [13,16,17]
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