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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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Virulence assay in wild-type Dongjin rice and the OsVOZ2 mutant line PFG_3A-07565.A. Schematic representation of the T-DNA insertion in OsVOZ2 T7 transgenic rice. OsVOZ2 consists of four exons (orange boxes) and three introns (line between the orange boxes). The T-DNA was located in the second intron from the translational start site. F and R are the primers used for RT-PCR analysis, which showed the expected size of OsVOZ2 in wild-type Dongjin but not in the OsVOZ2 mutant rice PFG_3A-07565. Actin1 was used for normalization of the cDNA quantity. B. Virulence assay of the xopNKXO85 mutant in wild-type Dongjin rice and OsVOZ2 mutant rice. W, water; 85, KXO85; N, KXO85 xopNKXO85::EZ-Tn5; and NC, KXO85 xopNKXO85::EZ-Tn5 (pML122G2). Photographs were taken 21 days after inoculation. C. Measurement of disease severity in flag leaves of wild-type Dongjin rice (□) and OsVOZ2 mutant rice (▨). W, water; 85, KXO85; N, KXO85 xopNKXO85::EZ-Tn5; and NC, KXO85 xopNKXO85::EZ-Tn5 (pML122G2). Lesion lengths were determined 21 days after inoculation. Vertical error bars indicate the standard deviation (SD). The statistical significance was determined using a two-way ANOVA as compared to wild-type Dongjin rice with the post hoc Tukey HSD test (***, P<0.001). D. Growth patterns of the KXO85, xopNKXO85 mutant, and complemented xopNKXO85 mutant in the flag leaves of OsVOZ2 mutant rice (PFG_3A-07565). The data are the average values of 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-g004: Virulence assay in wild-type Dongjin rice and the OsVOZ2 mutant line PFG_3A-07565.A. Schematic representation of the T-DNA insertion in OsVOZ2 T7 transgenic rice. OsVOZ2 consists of four exons (orange boxes) and three introns (line between the orange boxes). The T-DNA was located in the second intron from the translational start site. F and R are the primers used for RT-PCR analysis, which showed the expected size of OsVOZ2 in wild-type Dongjin but not in the OsVOZ2 mutant rice PFG_3A-07565. Actin1 was used for normalization of the cDNA quantity. B. Virulence assay of the xopNKXO85 mutant in wild-type Dongjin rice and OsVOZ2 mutant rice. W, water; 85, KXO85; N, KXO85 xopNKXO85::EZ-Tn5; and NC, KXO85 xopNKXO85::EZ-Tn5 (pML122G2). Photographs were taken 21 days after inoculation. C. Measurement of disease severity in flag leaves of wild-type Dongjin rice (□) and OsVOZ2 mutant rice (▨). W, water; 85, KXO85; N, KXO85 xopNKXO85::EZ-Tn5; and NC, KXO85 xopNKXO85::EZ-Tn5 (pML122G2). Lesion lengths were determined 21 days after inoculation. Vertical error bars indicate the standard deviation (SD). The statistical significance was determined using a two-way ANOVA as compared to wild-type Dongjin rice with the post hoc Tukey HSD test (***, P<0.001). D. Growth patterns of the KXO85, xopNKXO85 mutant, and complemented xopNKXO85 mutant in the flag leaves of OsVOZ2 mutant rice (PFG_3A-07565). The data are the average values of 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: To determine whether OsVOZ2 and its interactions with XopNKXO85 are critical for Xoo virulence, the OsVOZ2 knockout mutant line PFG_3A-07565 from the rice T-DNA Insertion Sequence Database (http://signal.salk.edu/cgi-bin/RiceGE) [41] was inoculated with wild-type KXO85. In the mutant line PFG_3A-07565, T-DNA is inserted 929 nucleotides downstream from the translational start site of OsVOZ2. RT-PCR analysis detected OsVOZ2 transcript in wild-type Dongjin but not in the OsVOZ2 mutant line PFG_3A-07565 (Figure 4A), which confirmed knockout mutation in OsVOZ2. Wild-type KXO85 and xopNKXO85 mutant strains were inoculated into wild-type Dongjin and the OsVOZ2 mutant line, and the xopNKXO85 mutant was shown to exhibit reduced virulence in the wild-type Dongjin. However, both strains showed significantly reduced disease severity in the OsVOZ2 mutant line compared to the wild-type Dongjin (Figure 4B). The xopNKXO85 mutant was less virulent in the OsVOZ2 mutant line than the wild-type KXO85 (Figure 4B and 4C). 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.


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)

Virulence assay in wild-type Dongjin rice and the OsVOZ2 mutant line PFG_3A-07565.A. Schematic representation of the T-DNA insertion in OsVOZ2 T7 transgenic rice. OsVOZ2 consists of four exons (orange boxes) and three introns (line between the orange boxes). The T-DNA was located in the second intron from the translational start site. F and R are the primers used for RT-PCR analysis, which showed the expected size of OsVOZ2 in wild-type Dongjin but not in the OsVOZ2 mutant rice PFG_3A-07565. Actin1 was used for normalization of the cDNA quantity. B. Virulence assay of the xopNKXO85 mutant in wild-type Dongjin rice and OsVOZ2 mutant rice. W, water; 85, KXO85; N, KXO85 xopNKXO85::EZ-Tn5; and NC, KXO85 xopNKXO85::EZ-Tn5 (pML122G2). Photographs were taken 21 days after inoculation. C. Measurement of disease severity in flag leaves of wild-type Dongjin rice (□) and OsVOZ2 mutant rice (▨). W, water; 85, KXO85; N, KXO85 xopNKXO85::EZ-Tn5; and NC, KXO85 xopNKXO85::EZ-Tn5 (pML122G2). Lesion lengths were determined 21 days after inoculation. Vertical error bars indicate the standard deviation (SD). The statistical significance was determined using a two-way ANOVA as compared to wild-type Dongjin rice with the post hoc Tukey HSD test (***, P<0.001). D. Growth patterns of the KXO85, xopNKXO85 mutant, and complemented xopNKXO85 mutant in the flag leaves of OsVOZ2 mutant rice (PFG_3A-07565). The data are the average values of 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

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getmorefigures.php?uid=PMC3760903&req=5

pone-0073346-g004: Virulence assay in wild-type Dongjin rice and the OsVOZ2 mutant line PFG_3A-07565.A. Schematic representation of the T-DNA insertion in OsVOZ2 T7 transgenic rice. OsVOZ2 consists of four exons (orange boxes) and three introns (line between the orange boxes). The T-DNA was located in the second intron from the translational start site. F and R are the primers used for RT-PCR analysis, which showed the expected size of OsVOZ2 in wild-type Dongjin but not in the OsVOZ2 mutant rice PFG_3A-07565. Actin1 was used for normalization of the cDNA quantity. B. Virulence assay of the xopNKXO85 mutant in wild-type Dongjin rice and OsVOZ2 mutant rice. W, water; 85, KXO85; N, KXO85 xopNKXO85::EZ-Tn5; and NC, KXO85 xopNKXO85::EZ-Tn5 (pML122G2). Photographs were taken 21 days after inoculation. C. Measurement of disease severity in flag leaves of wild-type Dongjin rice (□) and OsVOZ2 mutant rice (▨). W, water; 85, KXO85; N, KXO85 xopNKXO85::EZ-Tn5; and NC, KXO85 xopNKXO85::EZ-Tn5 (pML122G2). Lesion lengths were determined 21 days after inoculation. Vertical error bars indicate the standard deviation (SD). The statistical significance was determined using a two-way ANOVA as compared to wild-type Dongjin rice with the post hoc Tukey HSD test (***, P<0.001). D. Growth patterns of the KXO85, xopNKXO85 mutant, and complemented xopNKXO85 mutant in the flag leaves of OsVOZ2 mutant rice (PFG_3A-07565). The data are the average values of 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: To determine whether OsVOZ2 and its interactions with XopNKXO85 are critical for Xoo virulence, the OsVOZ2 knockout mutant line PFG_3A-07565 from the rice T-DNA Insertion Sequence Database (http://signal.salk.edu/cgi-bin/RiceGE) [41] was inoculated with wild-type KXO85. In the mutant line PFG_3A-07565, T-DNA is inserted 929 nucleotides downstream from the translational start site of OsVOZ2. RT-PCR analysis detected OsVOZ2 transcript in wild-type Dongjin but not in the OsVOZ2 mutant line PFG_3A-07565 (Figure 4A), which confirmed knockout mutation in OsVOZ2. Wild-type KXO85 and xopNKXO85 mutant strains were inoculated into wild-type Dongjin and the OsVOZ2 mutant line, and the xopNKXO85 mutant was shown to exhibit reduced virulence in the wild-type Dongjin. However, both strains showed significantly reduced disease severity in the OsVOZ2 mutant line compared to the wild-type Dongjin (Figure 4B). The xopNKXO85 mutant was less virulent in the OsVOZ2 mutant line than the wild-type KXO85 (Figure 4B and 4C). 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.

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