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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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Interactions between XopNKXO85 and OsVOZ2 and OsXNP.A. Screening for interactors of XopNKXO85 in rice using a yeast two-hybrid system. S (strong: pEXP TM32/Krev1 + pEXP TM22/RalGDS-wt), W (weak: pEXP TM32/Krev1 + pEXP TM22/RalGDS-m1), and A (absent: pEXP TM32/Krev1 + pEXP TM22/RalGDS-m2) indicate the strength of each interaction. Three independent and representative colonies are shown for each bait–prey combination. B. In vivo pull-down analysis of XopNKXO85 and OsVOZ2 (left panel) and XopNKXO85 and OsXNP (right panel). Total proteins from N. benthamiana leaves co-expressing XopNKXO85-6× His and Flag-OsVOZ2 or XopNKXO85-6× His and OsXNP-Flag protein were purified by Ni+ affinity chromatography followed by Western blotting using anti-His and anti-Flag antibodies. The expected molecular weights were as follows: XopNKXO85-6× His = 78.7 kDa; Flag-OsVOZ2 = 74.6 kDa; OsXNP-Flag = 40.1 kDa; +, protein expressed; and -, vector control. C. BiFC analysis of XopNKXO85 -OsVOZ2, XopNKXO85 -OsXNP, and XopNKXO85 -OsVOZ1 interactions in N. benthamiana leaves. Negative, pDEST-SCYNE(R)GW + pDEST-SCYCE(R)GW; positive, pEXP-SCYNE(R)-Cnx7 + pEXP-SCYCE(R)-Cnx6. Bars = 50 µm.
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pone-0073346-g002: Interactions between XopNKXO85 and OsVOZ2 and OsXNP.A. Screening for interactors of XopNKXO85 in rice using a yeast two-hybrid system. S (strong: pEXP TM32/Krev1 + pEXP TM22/RalGDS-wt), W (weak: pEXP TM32/Krev1 + pEXP TM22/RalGDS-m1), and A (absent: pEXP TM32/Krev1 + pEXP TM22/RalGDS-m2) indicate the strength of each interaction. Three independent and representative colonies are shown for each bait–prey combination. B. In vivo pull-down analysis of XopNKXO85 and OsVOZ2 (left panel) and XopNKXO85 and OsXNP (right panel). Total proteins from N. benthamiana leaves co-expressing XopNKXO85-6× His and Flag-OsVOZ2 or XopNKXO85-6× His and OsXNP-Flag protein were purified by Ni+ affinity chromatography followed by Western blotting using anti-His and anti-Flag antibodies. The expected molecular weights were as follows: XopNKXO85-6× His = 78.7 kDa; Flag-OsVOZ2 = 74.6 kDa; OsXNP-Flag = 40.1 kDa; +, protein expressed; and -, vector control. C. BiFC analysis of XopNKXO85 -OsVOZ2, XopNKXO85 -OsXNP, and XopNKXO85 -OsVOZ1 interactions in N. benthamiana leaves. Negative, pDEST-SCYNE(R)GW + pDEST-SCYCE(R)GW; positive, pEXP-SCYNE(R)-Cnx7 + pEXP-SCYCE(R)-Cnx6. Bars = 50 µm.

Mentions: To identify XopNKXO85 target proteins in rice, we carried out yeast two-hybrid screening using GAL4-XopN as a bait protein and a rice cDNA library constructed in the prey vector in the Saccharomyces cerevisiae strain MaV203. We found two possible candidates: Oryza sativa vascular plant one zinc finger protein 2 (OsVOZ2: NP_001056041, Os05g0515700) and O. sativa XopNKXO85 binding protein (OsXNP: NP_001059841, Os07g0529600) (Figure 2A and Table S2). The OsVOZ2 gene is 3,630 bp in length consisting of four exons and three introns and encodes a protein of 69,901 Da. OsVOZ2 is a homolog of Arabidopsis thaliana vascular plant one zinc finger protein 2 (AtVOZ2; At2g42400) that has a conserved zinc finger domain (Figure S5 and Figure S6). The OsXNP gene is 1,489 bp in length with two exons and one intron and possibly encodes a putative protein of 37,224 Da that has significant homology with thiamine biosynthetic enzyme in Saccharum hybrid cultivar GT28 (Table S2). XopNKXO85, OsVOZ2, and OsXNP were expressed in yeast as confirmed by immunoblot using anti-GAL4BD and anti-GAL4AD antibodies (Figure S4).


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)

Interactions between XopNKXO85 and OsVOZ2 and OsXNP.A. Screening for interactors of XopNKXO85 in rice using a yeast two-hybrid system. S (strong: pEXP TM32/Krev1 + pEXP TM22/RalGDS-wt), W (weak: pEXP TM32/Krev1 + pEXP TM22/RalGDS-m1), and A (absent: pEXP TM32/Krev1 + pEXP TM22/RalGDS-m2) indicate the strength of each interaction. Three independent and representative colonies are shown for each bait–prey combination. B. In vivo pull-down analysis of XopNKXO85 and OsVOZ2 (left panel) and XopNKXO85 and OsXNP (right panel). Total proteins from N. benthamiana leaves co-expressing XopNKXO85-6× His and Flag-OsVOZ2 or XopNKXO85-6× His and OsXNP-Flag protein were purified by Ni+ affinity chromatography followed by Western blotting using anti-His and anti-Flag antibodies. The expected molecular weights were as follows: XopNKXO85-6× His = 78.7 kDa; Flag-OsVOZ2 = 74.6 kDa; OsXNP-Flag = 40.1 kDa; +, protein expressed; and -, vector control. C. BiFC analysis of XopNKXO85 -OsVOZ2, XopNKXO85 -OsXNP, and XopNKXO85 -OsVOZ1 interactions in N. benthamiana leaves. Negative, pDEST-SCYNE(R)GW + pDEST-SCYCE(R)GW; positive, pEXP-SCYNE(R)-Cnx7 + pEXP-SCYCE(R)-Cnx6. Bars = 50 µm.
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Related In: Results  -  Collection

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

pone-0073346-g002: Interactions between XopNKXO85 and OsVOZ2 and OsXNP.A. Screening for interactors of XopNKXO85 in rice using a yeast two-hybrid system. S (strong: pEXP TM32/Krev1 + pEXP TM22/RalGDS-wt), W (weak: pEXP TM32/Krev1 + pEXP TM22/RalGDS-m1), and A (absent: pEXP TM32/Krev1 + pEXP TM22/RalGDS-m2) indicate the strength of each interaction. Three independent and representative colonies are shown for each bait–prey combination. B. In vivo pull-down analysis of XopNKXO85 and OsVOZ2 (left panel) and XopNKXO85 and OsXNP (right panel). Total proteins from N. benthamiana leaves co-expressing XopNKXO85-6× His and Flag-OsVOZ2 or XopNKXO85-6× His and OsXNP-Flag protein were purified by Ni+ affinity chromatography followed by Western blotting using anti-His and anti-Flag antibodies. The expected molecular weights were as follows: XopNKXO85-6× His = 78.7 kDa; Flag-OsVOZ2 = 74.6 kDa; OsXNP-Flag = 40.1 kDa; +, protein expressed; and -, vector control. C. BiFC analysis of XopNKXO85 -OsVOZ2, XopNKXO85 -OsXNP, and XopNKXO85 -OsVOZ1 interactions in N. benthamiana leaves. Negative, pDEST-SCYNE(R)GW + pDEST-SCYCE(R)GW; positive, pEXP-SCYNE(R)-Cnx7 + pEXP-SCYCE(R)-Cnx6. Bars = 50 µm.
Mentions: To identify XopNKXO85 target proteins in rice, we carried out yeast two-hybrid screening using GAL4-XopN as a bait protein and a rice cDNA library constructed in the prey vector in the Saccharomyces cerevisiae strain MaV203. We found two possible candidates: Oryza sativa vascular plant one zinc finger protein 2 (OsVOZ2: NP_001056041, Os05g0515700) and O. sativa XopNKXO85 binding protein (OsXNP: NP_001059841, Os07g0529600) (Figure 2A and Table S2). The OsVOZ2 gene is 3,630 bp in length consisting of four exons and three introns and encodes a protein of 69,901 Da. OsVOZ2 is a homolog of Arabidopsis thaliana vascular plant one zinc finger protein 2 (AtVOZ2; At2g42400) that has a conserved zinc finger domain (Figure S5 and Figure S6). The OsXNP gene is 1,489 bp in length with two exons and one intron and possibly encodes a putative protein of 37,224 Da that has significant homology with thiamine biosynthetic enzyme in Saccharum hybrid cultivar GT28 (Table S2). XopNKXO85, OsVOZ2, and OsXNP were expressed in yeast as confirmed by immunoblot using anti-GAL4BD and anti-GAL4AD antibodies (Figure S4).

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