Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Sep 3;8(9):e73346.
doi: 10.1371/journal.pone.0073346. eCollection 2013.

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

Hoon Cheong  1 , Chi-Yeol Kim, Jong-Seong Jeon, Byoung-Moo Lee, Jae Sun Moon, Ingyu Hwang
Affiliations

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

Hoon Cheong et al. PLoS One. .

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.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. 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 xopQ KXO85::EZ-Tn5; X, KXO85 xopX KXO85::EZ-Tn5; P1, KXO85 xopP1 KXO85::EZ-Tn5; P2, KXO85 xopP2 KXO85::EZ-Tn5; N, KXO85 xopN KXO85::EZ-Tn5. B. Disease severity of the xopN KXO85 mutants in the flag leaves of rice grown in a paddy field. W, water; 85, KXO85; N, KXO85 xopN KXO85::EZ-Tn5; and NC, KXO85 xopN KXO85::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, xopN KXO85 mutant, and complemented xopN KXO85 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.
Figure 2
Figure 2. 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.
Figure 3
Figure 3. Localization of XopNKXO85, OsVOZ2, and OsXNP in plant cells.
Subcellular localization of the XopNKXO85-GFP, OsVOZ2-GFP, and OsXNP-GFP fusion proteins in maize mesophyll cells. OsABF1-RFP was used as a nuclear marker. GFP (green) fluorescence was merged with RFP (red) fluorescence. Bars = 10 µm.
Figure 4
Figure 4. 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 xopN KXO85 mutant in wild-type Dongjin rice and OsVOZ2 mutant rice. W, water; 85, KXO85; N, KXO85 xopN KXO85::EZ-Tn5; and NC, KXO85 xopN KXO85::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 xopN KXO85::EZ-Tn5; and NC, KXO85 xopN KXO85::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, xopN KXO85 mutant, and complemented xopN KXO85 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.
See this image and copyright information in PMC

References

    1. Lee B-M, Park Y-J, Park D-S, Kang H-W, Kim J-G et al. (2005) The genome sequence of Xanthomonas oryzae pathovar oryzae KACC10331, the bacterial blight pathogen of rice. Nucleic Acids Res 33: 577-586. doi:10.1093/nar/gki591. PubMed: 15673718. - DOI - PMC - PubMed
    1. Büttner D, He SY (2009) Type III protein secretion in plant pathogenic bacteria. Plant Physiol 150: 1656-1664. doi:10.1104/pp.109.139089. PubMed: 19458111. - DOI - PMC - PubMed
    1. Büttner D, Bonas U (2002) Port of entry-the type III secretion translocon. Trends Microbiol 10: 186-192. doi:10.1016/S0966-842X(02)02331-4. PubMed: 11912026. - DOI - PubMed
    1. Crabill E, Karpisek A, Alfano JR (2012) The Pseudomonas syringae HrpJ protein controls the secretion of type III translocator proteins and has a virulence role inside plant cells. Mol Microbiol 85: 225-238. doi:10.1111/j.1365-2958.2012.08097.x. PubMed: 22607547. - DOI - PubMed
    1. Galán JE, Collmer A (1999) Type III secretion machines: bacterial devices for protein delivery into host cells. Science 284: 1322-1328. doi:10.1126/science.284.5418.1322. PubMed: 10334981. - DOI - PubMed

Publication types

LinkOut - more resources