ROS accumulation and antiviral defence control by microRNA528 in rice

  • Nature Plants 3, Article number: 16203 (2017)
  • doi:10.1038/nplants.2016.203
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MicroRNAs (miRNAs) are key regulators of plant–pathogen interactions. Modulating miRNA function has emerged as a new strategy to produce virus resistance traits1,​2,​3,​4,​5. However, the miRNAs involved in antiviral defence and the underlying mechanisms remain largely elusive. We previously demonstrated that sequestration by Argonaute (AGO) proteins plays an important role in regulating miRNA function in antiviral defence pathways6. Here we reveal that cleavage-defective AGO18 complexes sequester microRNA528 (miR528) upon viral infection. We show that miR528 negatively regulates viral resistance in rice by cleaving L-ascorbate oxidase (AO) messenger RNA, thereby reducing AO-mediated accumulation of reactive oxygen species. Upon viral infection, miR528 becomes preferentially associated with AGO18, leading to elevated AO activity, higher basal reactive oxygen species accumulation and enhanced antiviral defence. Our findings reveal a mechanism in which antiviral defence is boosted through suppression of an miRNA that negatively regulates viral resistance. This mechanism could be manipulated to engineer virus-resistant crop plants.

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  1. 1.

    & Roles of plant small RNAs in biotic stress responses. Annu. Rev. Plant Biol. 60, 485–510 (2009).

  2. 2.

    & Role of small RNAs in host-microbe interactions. Annu. Rev. Phytopathol. 48, 225–246 (2010).

  3. 3.

    et al. Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature 449, 919–922 (2007).

  4. 4.

    & Antiviral immunity directed by small RNAs. Cell 130, 413–426 (2007).

  5. 5.

    Small RNAs and their roles in plant development. Annu. Rev. Cell Dev. Biol. 25, 21–44 (2009).

  6. 6.

    et al. Viral-inducible Argonaute18 confers broad-spectrum virus resistance in rice by sequestering a host microRNA. eLife 4, e05733 (2015).

  7. 7.

    microRNAs and the immune response. Trends Immunol. 29, 343–351 (2008).

  8. 8.

    , & MicroRNAs play critical roles during plant development and in response to abiotic stresses. Genet. Mol. Biol. 35, 1069–1077 (2012).

  9. 9.

    et al. Constitutive expression of rice MicroRNA528 alters plant development and enhances tolerance to salinity stress and nitrogen starvation in creeping bentgrass. Plant Physiol. 169, 576–593 (2015).

  10. 10.

    et al. T-DNA insertional mutagenesis for functional genomics in rice. Plant J. 22, 561–570 (2000).

  11. 11.

    et al. Degradome sequencing reveals endogenous small RNA targets in rice (Oryza sativa L. ssp. indica). Front. Biol. 5, 67–90 (2010).

  12. 12.

    et al. Rice MicroRNA effector complexes and targets. Plant Cell 21, 3421–3435 (2009).

  13. 13.

    et al. OsRFPH2-10, a ring-H2 finger E3 ubiquitin ligase, is involved in rice antiviral defense in the early stages of rice dwarf virus infection. Mol. Plant 7, 1057–1060 (2014).

  14. 14.

    , & A novel gene, OZONE-RESPONSIVE APOPLASTIC PROTEIN1, enhances cell death in ozone stress in rice. Plant Physiol. 169, 873–889 (2015).

  15. 15.

    , , , & The function of ascorbate oxidase in tobacco. Plant Physiol. 132, 1631–1641 (2003).

  16. 16.

    et al. Ascorbate oxidase-dependent changes in the redox state of the apoplast modulate gene transcript accumulation leading to modified hormone signaling and orchestration of defense processes in tobacco. Plant Physiol. 141, 423–435 (2006).

  17. 17.

    , & The impact of global change factors on redox signaling underpinning stress tolerance. Plant Physiol. 161, 5–19 (2013).

  18. 18.

    , , , & Potentiating antibacterial activity by predictably enhancing endogenous microbial ROS production. Nat. Biotechnol. 31, 160–165 (2013).

  19. 19.

    et al. ROS signaling: the new wave? Trends Plant Sci. 16, 300–309 (2011).

  20. 20.

    & Role of L-ascorbate in alleviating abiotic stresses in crop plants. Bot. Stud. 55, 1–19 (2014).

  21. 21.

    & Vitamins in plants: occurrence, biosynthesis and antioxidant function. Trends Plant Sci. 15, 582–592 (2010).

  22. 22.

    & Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373–399 (2004).

  23. 23.

    et al. Ascorbic acid deficiency in Arabidopsis induces constitutive priming that is dependent on hydrogen peroxide, salicylic acid, and the NPR1 gene. Mol. Plant Microbe Interact. 23, 340–351 (2010).

  24. 24.

    et al. Viral infection induces expression of novel phased microRNAs from conserved cellular microRNA precursors. PLoS Pathog. 7, e1002176 (2011).

  25. 25.

    et al. Arabidopsis Argonaute10 specifically sequesters miR166/165 to regulate shoot apical meristem development. Cell 145, 242–256 (2011).

  26. 26.

    et al. Reactive oxygen species are involved in brassinosteroid-induced stress tolerance in cucumber. Plant Physiol. 150, 801–814 (2009).

  27. 27.

    et al. Arabidopsis COP1/SPA1 complex and FHY1/FHY3 associate with distinct phosphorylated forms of phytochrome A in balancing light signaling. Mol. Cell 31, 607–613 (2008).

  28. 28.

    et al. Arabidopsis phytochrome B promotes SPA1 nuclear accumulation to repress photomorphogenesis under far-red light. Plant Cell 25, 115–133 (2013).

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We thank F. Qu (Ohio State University) and Y. Li (Tsinghua-Peking Center for Life Sciences) for critical reading of the manuscript, L. Li (Peking University) for technical assistance, G. Liu (Tsinghua University) for assistance with the AO and AsA assay and the Integrated R & D Services – WuXi AppTec for generating the antisera used in this study. This work was supported by grants from the National Basic Research Program 973 (2014CB138400), Natural Science Foundation of China (91540203, 31530062, 31420103904, 31123007 and 31272018), the National Key Research and Development Program of China (2016YFD0100904), the Transgenic Research Program (2016ZX08010-001 and 2016ZX08009001-005) and state key laboratories of protein and plant gene research, plant genomics. J.W. was supported in part by the Postdoctoral Fellowship of Peking-Tsinghua Center for Life Sciences.

Author information

Author notes

    • Jianguo Wu
    • , Rongxin Yang
    •  & Zhirui Yang

    These authors contributed equally to this work.


  1. The State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China

    • Jianguo Wu
    • , Zhirui Yang
    • , Shengze Yao
    • , Shanshan Zhao
    • , Yu Wang
    • , Lian Jin
    •  & Yi Li
  2. State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou 350002, China

    • Jianguo Wu
    •  & Lianhui Xie
  3. State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

    • Rongxin Yang
    • , Xianwei Song
    • , Chengcai Chu
    •  & Xiaofeng Cao
  4. Agriculture and Agri-Food Canada, Morden, Manitoba R6M 1Y5, Canada

    • Pingchuan Li
  5. Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China

    • Tong Zhou
    •  & Ying Lan
  6. State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China

    • Xueping Zhou
  7. Center for Plant Biology, Tsinghua-Peking Center for Life Sciences, College of Life Sciences, Tsinghua University, Beijing 100084, China

    • Yijun Qi
  8. CAS Center for Excellence in Molecular Plant Sciences, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

    • Xiaofeng Cao


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J.W., Z.Y., X.C. and Y. Li designed the experiments; J.W., Z.Y., R.Y., Z.Y., S.Z., Y.W., L.J., P.L., X.S., T.Z. and Y. Lan performed the experiments; J.W., Z.Y., R.Y., L.X., X.Z., C.C., Y.Q., X.C. and Y. Li analysed the data; J.W., Z.Y. and Y. Li wrote the paper. All the authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Xiaofeng Cao or Yi Li.

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    Supplementary Information

    Supplementary Figures 1–10, Supplementary Tables 1–3.