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Xanthomonas oryzae pv. oryzae type III effector XopN targets OsVOZ2 and a putative thiamine synthase as a virulence factor in rice.

Cheong H, Kim CY, Jeon JS, Lee BM, Sun Moon J, Hwang I - PLoS ONE (2013)

Bottom Line: In contrast, the xopN KXO85 mutant exhibited significantly less virulence in flag leaves after flowering than the wild-type KXO85.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea.

ABSTRACT
Xanthomonasoryzae 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.

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Pathogenicity test for xop mutants of Xoo KXO85 in rice.A. Disease severity of each xop mutant in 3-month-old rice leaves. W, water; 85, wild-type KXO85; Q, KXO85 xopQKXO85::EZ-Tn5; X, KXO85 xopXKXO85::EZ-Tn5; P1, KXO85 xopP1KXO85::EZ-Tn5; P2, KXO85 xopP2KXO85::EZ-Tn5; N, KXO85 xopNKXO85::EZ-Tn5. B. Disease severity of the xopNKXO85 mutants in the flag leaves of rice grown in a paddy field. W, water; 85, KXO85; N, KXO85 xopNKXO85::EZ-Tn5; and NC, KXO85 xopNKXO85::EZ-Tn5 (pML122G2). Photographs were taken and lesion lengths were determined 21 days after inoculation. Vertical error bars indicate the standard deviations (SD). The data are the averages of 12–15 replicates for each treatment. Columns and lines not connected by the same letter are significantly different (P<0.05) as determined by a one-way ANOVA (P<0.001) followed by post hoc Tukey HSD analysis. C. Bacterial growth patterns of the KXO85, xopNKXO85 mutant, and complemented xopNKXO85 mutant strains in flag leaves of wild-type Dongjin. The data are shown as the average values for three replicates; vertical bars indicate the error ranges (±SD). The bacterial populations were assessed every 3 days after inoculation. Different letters at day 21 indicate significant differences (P<0.05) as determined by a one-way ANOVA (P<0.001) followed by post hoc Tukey HSD analysis.
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pone-0073346-g001: Pathogenicity test for xop mutants of Xoo KXO85 in rice.A. Disease severity of each xop mutant in 3-month-old rice leaves. W, water; 85, wild-type KXO85; Q, KXO85 xopQKXO85::EZ-Tn5; X, KXO85 xopXKXO85::EZ-Tn5; P1, KXO85 xopP1KXO85::EZ-Tn5; P2, KXO85 xopP2KXO85::EZ-Tn5; N, KXO85 xopNKXO85::EZ-Tn5. B. Disease severity of the xopNKXO85 mutants in the flag leaves of rice grown in a paddy field. W, water; 85, KXO85; N, KXO85 xopNKXO85::EZ-Tn5; and NC, KXO85 xopNKXO85::EZ-Tn5 (pML122G2). Photographs were taken and lesion lengths were determined 21 days after inoculation. Vertical error bars indicate the standard deviations (SD). The data are the averages of 12–15 replicates for each treatment. Columns and lines not connected by the same letter are significantly different (P<0.05) as determined by a one-way ANOVA (P<0.001) followed by post hoc Tukey HSD analysis. C. Bacterial growth patterns of the KXO85, xopNKXO85 mutant, and complemented xopNKXO85 mutant strains in flag leaves of wild-type Dongjin. The data are shown as the average values for three replicates; vertical bars indicate the error ranges (±SD). The bacterial populations were assessed every 3 days after inoculation. Different letters at day 21 indicate significant differences (P<0.05) as determined by a one-way ANOVA (P<0.001) followed by post hoc Tukey HSD analysis.

Mentions: Five xop genes, xopQKXO85 (XOO4466), xopXKXO85 (XOO4287), xopP1KXO85 (XOO3425), xopP2KXO85 (XOO3426), and xopNKXO85 (XOO0343) (Table S1), were characterized among 18 xop homologs in the strain KXO85 (www.xanthomonas.org/t3e.html), which showed significant homology with reported xop genes. EZ-Tn5 insertion mutants of xopQKXO85, xopXKXO85, xopP1KXO85, xopP2KXO85, and xopNKXO85 (Figure S1) were generated in the strain KXO85, and then the virulence of each xop gene knockout mutant was evaluated in 3-month-old leaves of the Japonica-type rice cultivar Dongjin. Mutations in the xopQKXO85, xopXKXO85, xopP1KXO85, xopP2KXO85, or xopNKXO85 gene did not significantly affect virulence (Figure 1A). When the xopNKXO85 mutant was inoculated into the flag leaves of Dongjin in the field, the mutant was significantly less virulent than the wild-type KXO85 (Figure 1B). Virulence the xopNKXO85 mutant carrying each wild-type xop gene in a multicopy plasmid was recovered to the wild-type level (Figure 1B). These observations indicate that xopNKXO85 exhibits important roles for virulence of Xoo. Therefore, we chose xopNKXO85 for further characterization. The bacterial population of the xopNKXO85 mutant was reduced up to 21 days after inoculation of flag leaves compared to the growth of wild-type strain KXO85 in Dongjin (Figure 1C).


Xanthomonas oryzae pv. oryzae type III effector XopN targets OsVOZ2 and a putative thiamine synthase as a virulence factor in rice.

Cheong H, Kim CY, Jeon JS, Lee BM, Sun Moon J, Hwang I - PLoS ONE (2013)

Pathogenicity test for xop mutants of Xoo KXO85 in rice.A. Disease severity of each xop mutant in 3-month-old rice leaves. W, water; 85, wild-type KXO85; Q, KXO85 xopQKXO85::EZ-Tn5; X, KXO85 xopXKXO85::EZ-Tn5; P1, KXO85 xopP1KXO85::EZ-Tn5; P2, KXO85 xopP2KXO85::EZ-Tn5; N, KXO85 xopNKXO85::EZ-Tn5. B. Disease severity of the xopNKXO85 mutants in the flag leaves of rice grown in a paddy field. W, water; 85, KXO85; N, KXO85 xopNKXO85::EZ-Tn5; and NC, KXO85 xopNKXO85::EZ-Tn5 (pML122G2). Photographs were taken and lesion lengths were determined 21 days after inoculation. Vertical error bars indicate the standard deviations (SD). The data are the averages of 12–15 replicates for each treatment. Columns and lines not connected by the same letter are significantly different (P<0.05) as determined by a one-way ANOVA (P<0.001) followed by post hoc Tukey HSD analysis. C. Bacterial growth patterns of the KXO85, xopNKXO85 mutant, and complemented xopNKXO85 mutant strains in flag leaves of wild-type Dongjin. The data are shown as the average values for three replicates; vertical bars indicate the error ranges (±SD). The bacterial populations were assessed every 3 days after inoculation. Different letters at day 21 indicate significant differences (P<0.05) as determined by a one-way ANOVA (P<0.001) followed by post hoc Tukey HSD analysis.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760903&req=5

pone-0073346-g001: Pathogenicity test for xop mutants of Xoo KXO85 in rice.A. Disease severity of each xop mutant in 3-month-old rice leaves. W, water; 85, wild-type KXO85; Q, KXO85 xopQKXO85::EZ-Tn5; X, KXO85 xopXKXO85::EZ-Tn5; P1, KXO85 xopP1KXO85::EZ-Tn5; P2, KXO85 xopP2KXO85::EZ-Tn5; N, KXO85 xopNKXO85::EZ-Tn5. B. Disease severity of the xopNKXO85 mutants in the flag leaves of rice grown in a paddy field. W, water; 85, KXO85; N, KXO85 xopNKXO85::EZ-Tn5; and NC, KXO85 xopNKXO85::EZ-Tn5 (pML122G2). Photographs were taken and lesion lengths were determined 21 days after inoculation. Vertical error bars indicate the standard deviations (SD). The data are the averages of 12–15 replicates for each treatment. Columns and lines not connected by the same letter are significantly different (P<0.05) as determined by a one-way ANOVA (P<0.001) followed by post hoc Tukey HSD analysis. C. Bacterial growth patterns of the KXO85, xopNKXO85 mutant, and complemented xopNKXO85 mutant strains in flag leaves of wild-type Dongjin. The data are shown as the average values for three replicates; vertical bars indicate the error ranges (±SD). The bacterial populations were assessed every 3 days after inoculation. Different letters at day 21 indicate significant differences (P<0.05) as determined by a one-way ANOVA (P<0.001) followed by post hoc Tukey HSD analysis.
Mentions: Five xop genes, xopQKXO85 (XOO4466), xopXKXO85 (XOO4287), xopP1KXO85 (XOO3425), xopP2KXO85 (XOO3426), and xopNKXO85 (XOO0343) (Table S1), were characterized among 18 xop homologs in the strain KXO85 (www.xanthomonas.org/t3e.html), which showed significant homology with reported xop genes. EZ-Tn5 insertion mutants of xopQKXO85, xopXKXO85, xopP1KXO85, xopP2KXO85, and xopNKXO85 (Figure S1) were generated in the strain KXO85, and then the virulence of each xop gene knockout mutant was evaluated in 3-month-old leaves of the Japonica-type rice cultivar Dongjin. Mutations in the xopQKXO85, xopXKXO85, xopP1KXO85, xopP2KXO85, or xopNKXO85 gene did not significantly affect virulence (Figure 1A). When the xopNKXO85 mutant was inoculated into the flag leaves of Dongjin in the field, the mutant was significantly less virulent than the wild-type KXO85 (Figure 1B). Virulence the xopNKXO85 mutant carrying each wild-type xop gene in a multicopy plasmid was recovered to the wild-type level (Figure 1B). These observations indicate that xopNKXO85 exhibits important roles for virulence of Xoo. Therefore, we chose xopNKXO85 for further characterization. The bacterial population of the xopNKXO85 mutant was reduced up to 21 days after inoculation of flag leaves compared to the growth of wild-type strain KXO85 in Dongjin (Figure 1C).

Bottom Line: In contrast, the xopN KXO85 mutant exhibited significantly less virulence in flag leaves after flowering than the wild-type KXO85.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea.

ABSTRACT
Xanthomonasoryzae 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.

Show MeSH
Related in: MedlinePlus