Journal of General Plant Pathology

, Volume 82, Issue 1, pp 12–17 | Cite as

Analysis of rice RNA-dependent RNA polymerase 6 (OsRDR6) gene in response to viral, bacterial and fungal pathogens

  • S. G. Wagh
  • M. M. Alam
  • K. Kobayashi
  • T. Yaeno
  • N. Yamaoka
  • T. Toriba
  • H.-Y. Hirano
  • M. NishiguchiEmail author
Host Responses


RNA-dependent RNA polymerases (RDRs) play key roles in gene silencing. The rice RDR6 gene was analyzed in response to viral, bacterial and fungal pathogens, after inoculation of a rice mutant line of OsRDR6, shl2-rol, with Cucumber mosaic virus, Rice necrosis mosaic virus, Xanthomonas oryzae pv. oryzae or Magnaporthe oryzae. Compared with the wild type, the mutant line accumulated more viral RNA after inoculation with the viruses and developed more severe symptoms after inoculation with the bacterium or fungus. Thus, the OsRDR6-mediated RNA silencing pathway seems to participate in defense against not only viruses, but also bacterial and fungal pathogens.


OsRDR6 shl2-rol Rice Cucumber mosaic virus Rice necrosis mosaic virus Xanthomonas oryzae pv. oryzae Magnaporthe oryzae 



We are grateful to D. Murphy for checking the English in the manuscript and Md. Panna Ali for the RT-qPCR analysis. We also thank Drs. M. Mori and H. Ochiai for seeds of Sensyou and Asominori, respectively. This work was supported partly by the Program for Promotion of Basic and Applied Researches in Bio-oriented Industry and Science, the Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry, the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for Scientific Research for Scientific Research (C), no. 24580065 to MN and (B), no. 26292026 to KK) and by grants from the Ministry of Agriculture, Forestry and Fisheries of Japan (Rice Genome Project to MN).

Supplementary material

10327_2015_630_MOESM1_ESM.pptx (2.8 mb)
Fig. S1 Expression of OsRDR6 in response to mock inoculation with various pathogens as determined by RT-PCR. (a) Cucmber mosaic virus/Rice necrosis mosaic virus. Lane numbers represent days post inoculation. (b) Xanthomonas oryzae pv. oryzae/Magnaporthe oryzae. Lane numbers represent hours after inoculation. Total RNA was extracted from the leaves of treated plants at theh indicated times. Primers were OsRDR6-F and OsRDR6-R. The actin gene was used as a standard to show normalization of the amount of PCR templates. Fig. S2 Expression of OsRDR6 after inoculation with Xanthomonas oryzae pv. oryzae and Magnaporthe oryzae as determined by RT-PCR. Total RNA was extracted from inoculated leaves at 36 h after inoculation. RT-PCR primers were OsRDR6-F and OsRDR6-R. The actin gene was used as a standard to show normalization of the amount of PCR templates. M, mock; I, inoculated. Fig. S3 Accumulation of Cucmber mosaic virus (CMV) and Rice necrosis mosaic virus (RNMV) in WT (+/+), homozygous (-/-)/heterozygous (±) shl2-rol. Young leaves were inoculated with CMV and total RNA extracted at 7 dpi. Total RNA was extracted from the leaves of plants grown in RNMV-infested soil for 30 days. RT-PCR primers were RNMV-R1-F-5′ and RNMV-R1-R-3′ for RNMV, CMV-R3-cDNA-F-5′ and CMV-R3-cDNA-F-3′ for CMV. The actin gene was used as standard control to show the normalization of the amount of PCR templates. (PPTX 2845 kb)
10327_2015_630_MOESM2_ESM.docx (21 kb)
Supplementary material 2 (DOCX 22 kb)


  1. Alam MM, Nakamura H, Ichikawa H, Miyao A, Hirochika H, Kobayashi K, Yamaoka N, Nishiguchi M (2014) Response of an aspartic protease gene OsAP77 to fungal, bacterial and viral infections in rice. Rice 7:9PubMedCrossRefGoogle Scholar
  2. Alam MM, Nakamura H, Ichikawa H, Kobayashi K, Yaeno T, Yamaoka N, Nishiguchi M (2015) Overexpression of OsHAP2E for a CCAAT-binding factor confers resistance to Cucumber mosaic virus and Rice necrosis mosaic virus. J Gen Plant Pathol 81:32–41CrossRefGoogle Scholar
  3. Baulcombe D (2004) RNA silencing in plants. Nature 431:356–363PubMedCrossRefGoogle Scholar
  4. Chen J, Xia X, Yin W (2009) Expression profiling and functional characterization of a DREB2-type gene from Populus euphratica. Biochem Biophys Res Commun 378:483–487PubMedCrossRefGoogle Scholar
  5. Chen H, Tamai A, Mori M, Ugaki M, Tanaka Y, Samadder P, Miyao A, Hirochika H, Yamaoka N, Nishiguchi M (2010) Analysis of rice RNA-dependent RNA polymerase 1 (OsRDR1) in virus-mediated RNA silencing after particle bombardment. J Gen Plant Pathol 76:152–160CrossRefGoogle Scholar
  6. Chen H, Kobayashi K, Miyao A, Hirochika H, Yamaoka N, Nishiguchi M (2013) Both OsRecQ1 and OsRDR1 are required for the production of small RNA in response to DNA-damage in rice. PLoS One 8:e55252PubMedCentralPubMedCrossRefGoogle Scholar
  7. Csorba T, Pantaleo V, Burgyán J (2009) RNA silencing: an antiviral mechanism. Adv Virus Res 75:35–71PubMedCrossRefGoogle Scholar
  8. Ding SW (2010) RNA-based antiviral immunity. Nat Rev Immunol 10:632–644PubMedCrossRefGoogle Scholar
  9. Gao Q, Liu Y, Wang M, Zhang J, Gai Y, Zhu C, Guo X (2009) Molecular cloning and characterization of an inducible RNA-dependent RNA polymerase gene, GhRdRP, from cotton (Gossypium hirsutum L.). Mol Biol Rep 36:47–56PubMedCrossRefGoogle Scholar
  10. Guo H, Song X, Xie C, Huo Y, Zhang F, Chen X, Geng Y, Fang R (2013) Rice yellow stunt rhabdovirus protein 6 suppresses systemic RNA silencing by blocking RDR6-mediated secondary siRNA synthesis. Mol Plant Microbe Interact 26:927–936PubMedCrossRefGoogle Scholar
  11. He J, Dong Z, Jia Z, Wang J, Wang G (2010) Isolation, expression and functional analysis of a putative RNA-dependent RNA polymerase gene from maize (Zea mays L.). Mol Biol Rep 37:865–874PubMedCrossRefGoogle Scholar
  12. Hunter LJR, Westwood JH, Heath G, Macaulay K, Smith AG, Stuart A, MacFarlane SA, Palukaitis P, Carr JP (2013) Regulation of RNA-dependent RNA polymerase 1 and isochorismate synthase gene expression in Arabidopsis. PLoS One 8:e66530PubMedCentralPubMedCrossRefGoogle Scholar
  13. Jiang L, Qian D, Zheng H, Meng LY, Chen J, Le WJ, Zhou T, Zhoub YJ, Weia CH, Li Y (2012) RNA-dependent RNA polymerase 6 of rice (Oryza sativa) plays role in host defense against negative-strand RNA virus, Rice stripe virus. Virus Res 163:512–519PubMedCrossRefGoogle Scholar
  14. Kaku H, Kimura T (1987) Difference in resistance expression of rice to Xanthomonas campestris pv. oryzae as controlled by resistance genes 1. Resistance expression controlled by resistance gene Xa-1. Ann Phytopathol Soc Jpn 53:14–20CrossRefGoogle Scholar
  15. Kawano Y, Akamatsu A, Hayashi K, Housen Y, Okuda J, Yao A, Nakashima A, Takahashi H, Yoshida H, Wong HL, Kawasaki T, Shimamoto K (2010) Activation of a Rac GTPase by the NLR family disease resistance protein Pit plays a critical role in rice innate immunity. Cell Host Microbe 7:362–375PubMedCrossRefGoogle Scholar
  16. Lee WS, Fu SF, Verchot-Lubicz J, Carr JP (2011) Genetic modification of alternative respiration in Nicotiana benthamiana affects basal and salicylic acid-induced resistance to potato virus X. BMC Plant Biol 11:41PubMedCentralPubMedCrossRefGoogle Scholar
  17. Li S, Yu F, Wang M, Guo X, Li H (2012) Molecular characterization of a Nicotiana tabacum NtRDR6 gene. Plant Mol Biol Rep 30:1375–1384CrossRefGoogle Scholar
  18. Liao YWK, Sun ZH, Zhou YH, Shi K, Li X, Zhang GQ, Xia XJ, Chen ZX, Yu JQ (2013) The role of hydrogen peroxide and nitric oxide in the induction of plant-encoded RNA-dependent RNA polymerase 1 in the basal defense against Tobacco mosaic virus. PLoS One 8:e76090PubMedCentralPubMedCrossRefGoogle Scholar
  19. Liu Y, Gao Q, Wu B, Ai T, Guo X (2009) NgRDR1, an RNA dependent RNA polymerase isolated from Nicotiana glutinosa, was involved in biotic and abiotic stresses. Plant Physiol Biochem 47:359–368PubMedCrossRefGoogle Scholar
  20. Mourrain P, Béclin C, Elmayan T, Feuerbach F, Godon C, Morel JB, Jouette D, Lacombe AM, Nikic S, Picault N, Rémoué K, Sanial M, Vo TA, Vaucheret H (2000) Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell 101:533–542PubMedCrossRefGoogle Scholar
  21. Nagasaki H, Itoh J, Hayashi K, Hibara K, Satoh-Nagasawa N, Nosaka M, Mukouhata M, Ashikari M, Kitano H, Matsuoka M, Nagato Y, Sato Y (2007) The small interfering RNA production pathway is required for shoot meristem initiation in rice. Proc Natl Acad Sci USA 104:14867–14871PubMedCentralPubMedCrossRefGoogle Scholar
  22. Navarro L, Jay F, Nomura K, He SY, Voinnet O (2008) Suppression of the microRNA pathway by bacterial effector proteins. Science 321:964–967PubMedCentralPubMedCrossRefGoogle Scholar
  23. Pandey SP, Baldwin IT (2007) RNA-directed RNA polymerase 1 (RdR1) mediates the resistance of Nicotiana attenuata to herbivore attack in nature. Plant J 50:40–53PubMedCrossRefGoogle Scholar
  24. Pumplin N, Voinnet O (2013) RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence. Nat Rev Microbiol 11:745–760PubMedCrossRefGoogle Scholar
  25. Qi X, Bao FS, Xie Z (2009) Small RNA deep sequencing reveals role for Arabidopsis thaliana RNA-dependent RNA polymerases in viral siRNA biogenesis. PLoS One 4:e4971PubMedCentralPubMedCrossRefGoogle Scholar
  26. Qiao Y, Liu L, Xiong Q, Flores C, Wong J, Shi J, Wang X, Liu X, Xiang Q, Jiang S, Zhang F, Wang Y, Judelson HS, Chen X, Ma W (2013) Oomycete pathogens encode RNA silencing suppressors. Nat Genet 45:330–333PubMedCentralPubMedCrossRefGoogle Scholar
  27. Qiao Y, Shi J, Yi Z, Hou Y, Ma W (2015) Phytophthora effector targets a novel component of small RNA pathway in plants to promote infection. Proc Natl Acad Sci USA 112:5850–5855PubMedCentralPubMedCrossRefGoogle Scholar
  28. Song X, Wang D, Ma L, Chen Z, Li P, Cui X, Liu C, Cao S, Chu C, Tao Y, Cao X (2012) Rice RNA-dependent RNA polymerase 6 acts in small RNA biogenesis and spikelet development. Plant J 71:378–389PubMedGoogle Scholar
  29. Toriba T, Suzaki T, Yamaguchi T, Ohmori Y, Tsukaya H, Hirano HY (2010) Distinct regulation of adaxial–abaxial polarity in anther patterning in rice. Plant Cell 22:1452–1462PubMedCentralPubMedCrossRefGoogle Scholar
  30. Voinnet O (2009) Origin, biogenesis, and activity of plant micro RNAs. Cell 136:669–687PubMedCrossRefGoogle Scholar
  31. Wagh SG, Kobayashi K, Yaeno T, Yamaoka N, Masuta C, Nishiguchi M (2015) Rice necrosis mosaic virus, a fungal transmitted Bymovirus: complete nucleotide sequence of the genomic RNAs and subgrouping of bymoviruses. J Gen Plant Pathol. doi: 10.1007/s10327-015-0618-7 Google Scholar
  32. Wang XB, Wu Q, Ito T, Cillo F, Li WX, Chen X, Yu JL, Ding SW (2009) RNAi-mediated viral immunity requires amplification of virus-derived siRNAs in Arabidopsis thaliana. Proc Natl Acad Sci USA 107:484–489PubMedCentralPubMedCrossRefGoogle Scholar
  33. Wang M, Li S, Yang H, GAO Z, Wu C, Guo X (2012) Characterization and functional analysis of GhRDR6, a novel RDR6 gene from cotton (Gossypium hirsutum L.). Biosci Rep 32:139–151PubMedCrossRefGoogle Scholar
  34. Wassenegger M, Krczal G (2006) Nomenclature and functions of RNA-directed RNA polymerases. Trends Plant Sci 11:142–151PubMedCrossRefGoogle Scholar
  35. Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, Ziberman D, Jacobsen SE, Carrington JC (2004) Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2:e104PubMedCentralPubMedCrossRefGoogle Scholar
  36. Yang JH, Seo HH, Han SJ, Yoon EK, Yang MS, Lee WS (2008) Phytohormone abscisic acid control RNA-dependent RNA polymerase 6 gene expression and post-transcriptional gene silencing in rice cells. Nucl Acids Res 36:1220–1226PubMedCentralPubMedCrossRefGoogle Scholar
  37. Yang H, Wang M, Gao Z, Zhu C, Guo X (2011) Isolation of a novel RNA dependent RNA polymerase 6 from Nicotiana glutinosa, NgRDR6, and analysis of its response to biotic and abiotic stresses. Mol Biol Rep 38:929–937PubMedCrossRefGoogle Scholar
  38. Yu D, Fan B, MacFarlane SA, Chen Z (2003) Analysis of the involvement of an inducible Arabidopsis RNA-dependent RNA polymerase in antiviral defense. Mol Plant Microbe Interact 16:206–216PubMedCrossRefGoogle Scholar

Copyright information

© The Phytopathological Society of Japan and Springer Japan 2015

Authors and Affiliations

  • S. G. Wagh
    • 1
  • M. M. Alam
    • 2
  • K. Kobayashi
    • 2
  • T. Yaeno
    • 2
  • N. Yamaoka
    • 2
  • T. Toriba
    • 3
    • 4
  • H.-Y. Hirano
    • 3
  • M. Nishiguchi
    • 2
    Email author
  1. 1.The United Graduate School of Agricultural SciencesEhime UniversityMatsuyamaJapan
  2. 2.Faculty of AgricultureEhime UniversityMatsuyamaJapan
  3. 3.Department of Biological Sciences, Graduate School of ScienceUniversity of TokyoTokyoJapan
  4. 4.National Institute for Basic BiologyOkazakiJapan

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