Plant Molecular Biology

, Volume 92, Issue 1–2, pp 39–55 | Cite as

Tight regulation of the interaction between Brassica napus and Sclerotinia sclerotiorum at the microRNA level

  • Jia-Yi Cao
  • You-Ping Xu
  • Li Zhao
  • Shuang-Sheng Li
  • Xin-Zhong Cai


MicroRNAs (miRNAs) are multifunctional non-coding short nucleotide molecules. Nevertheless, the role of miRNAs in the interactions between plants and necrotrophic pathogens is largely unknown. Here, we report the identification of the miRNA repertoire of the economically important oil crop oilseed rape (Brassica napus) and those involved in interacting with its most devastating necrotrophic pathogen Sclerotinia sclerotiorum. We identified 280 B. napus miRNA candidates, including 53 novel candidates and 227 canonical members or variants of known miRNA families, by high-throughput deep sequencing of small RNAs from both normal and S. sclerotiorum-inoculated leaves. Target genes of 15 novel candidates and 222 known miRNAs were further identified by sequencing of degradomes from the two types of samples. MiRNA microarray analysis revealed that 68 miRNAs were differentially expressed between S. sclerotiorum-inoculated and uninoculated leaves. A set of these miRNAs target genes involved in plant defense to S. sclerotiorum and/or other pathogens such as nucleotide binding site-leucine-rich repeat (NBS-LRR) R genes and nitric oxygen and reactive oxygen species related genes. Additionally, three miRNAs target AGO1 and AGO2, key components of post-transcriptional gene silencing (PTGS). Expression of several viral PTGS suppressors reduced resistance to S. sclerotiorum. Arabidopsis mutants of AGO1 and AGO2 exhibited reduced resistance while transgenic lines over-expressing AGO1 displayed increased resistance to S. sclerotiorum in an AGO1 expression level-dependent manner. Moreover, transient over-expression of miRNAs targeting AGO1 and AGO2 decreased resistance to S. sclerotiorum in oilseed rape. Our results demonstrate that the interactions between B. napus and S. sclerotiorum are tightly regulated at miRNA level and probably involve PTGS.


AGO Brassica napus MiRNA Post-transcriptional gene silencing Resistance Sclerotinia sclerotiorum 





Cucumber mosaic virus




Minimal folding free energies index


Effector-triggered immunity


K-Means Clustering




Nucleotide binding site-leucine-rich repeat




Potato dextrose agar


Pseudomonas syringae pv. tomato


Post-transcriptional gene silencing


Potato virus X


Quantitative real-time PCR


Reactive oxygen species


Tomato bushy stunt virus


Turnip crinkle virus



We are grateful to Prof. Shou-Wei Ding (Department of Plant Pathology and Microbiology, University of California, Riverside, USA) for providing the Arabidopsis thaliana mutant ago1-27 and Prof. Yi-Jun Qi (Tsinghua-Peking Center for Life Sciences, and School of Life Sciences, Tsinghua University, China) for providing the A. thaliana mutants ago1-33 and ago2-1. We acknowledge Professor David Baulcombe (Sainsbury Laboratory, John Innes Centre) and Plant Bioscience Limited (Colney Lane, Norwich NR4 7UH, England) for providing the plasmids expressing silencing suppressors. This work was supported by the National Natural Science Foundation of China (31371892), the Special Fund for Agro-scientific Research in the Public Interest (201103016), the SRFDP (20110101110092) and the Fundamental Research Funds for the Central Universities (2016FZA6014).

Author contribution

Jia-Yi Cao performed miRNA idenfication and verification analyses. Li Zhao constructed AGO1-OE transgenic plants and evaluated their resistance. Jia-Yi Cao and Shuang-Sheng Li carried out the resistance evaluation analyses for ago mutants. Jia-Yi Cao and You-Ping Xu conducted gene expression and statistical analyses. Xin-Zhong Cai and You-Ping Xu conceived of the study and participated in its design and coordination. Xin-Zhong Cai and Jia-Yi Cao prepared the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11103_2016_494_MOESM1_ESM.pdf (17 kb)
Supplementary material 1 (PDF 16 KB)
11103_2016_494_MOESM2_ESM.pdf (3 mb)
Supplementary material 2 (PDF 3054 KB)
11103_2016_494_MOESM3_ESM.pdf (28 mb)
Supplementary material 3 (PDF 28703 KB)
11103_2016_494_MOESM4_ESM.pdf (192 kb)
Supplementary material 4 (PDF 191 KB)
11103_2016_494_MOESM5_ESM.pdf (215 kb)
Supplementary material 5 (PDF 214 KB)
11103_2016_494_MOESM6_ESM.pdf (242 kb)
Supplementary material 6 (PDF 241 KB)
11103_2016_494_MOESM7_ESM.xls (26 kb)
Supplementary material 7 (XLS 26 KB)
11103_2016_494_MOESM8_ESM.xls (192 kb)
Supplementary material 8 (XLS 192 KB)
11103_2016_494_MOESM9_ESM.xls (96 kb)
Supplementary material 9 (XLS 96 KB)
11103_2016_494_MOESM10_ESM.xls (50 kb)
Supplementary material 10 (XLS 50 KB)
11103_2016_494_MOESM11_ESM.xls (26 kb)
Supplementary material 11 (XLS 25 KB)
11103_2016_494_MOESM12_ESM.xls (6.7 mb)
Supplementary material 12 (XLS 6890 KB)
11103_2016_494_MOESM13_ESM.xls (101 kb)
Supplementary material 13 (XLS 101 KB)
11103_2016_494_MOESM14_ESM.xls (44 kb)
Supplementary material 14 (XLS 44 KB)
11103_2016_494_MOESM15_ESM.xls (38 kb)
Supplementary material 15 (XLS 37 KB)


  1. Addo-Quaye C, Eshoo TW, Bartel DP, Axtell MJ (2008) Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome. Curr Biol 18:758–762CrossRefPubMedPubMedCentralGoogle Scholar
  2. Addo-Quaye C, Miller W, Axtell MJ (2009) CleaveLand: a pipeline for using degradome data to find cleaved small RNA targets. Bioinformatics 25:130–131CrossRefPubMedGoogle Scholar
  3. Allen E, Xie Z, Gustafson AM, Carrington JC (2005) MicroRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121:207–221CrossRefPubMedGoogle Scholar
  4. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297CrossRefPubMedGoogle Scholar
  5. Bolton MD, Thomma B, Nelson BD (2006) Sclerotinia sclerotiorum (Lib.) de Bary: biology and molecular traits of a cosmopolitan pathogen. Mol Plant Pathol 7:1–16CrossRefPubMedGoogle Scholar
  6. Buhtz A, Springer F, Chappell L, Baulcombe DC, Kehr J (2008) Identification and characterization of small RNAs from the phloem of Brassica napus. Plant J 53:739–749CrossRefPubMedGoogle Scholar
  7. Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH et al (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 60:591–602Google Scholar
  8. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743CrossRefPubMedGoogle Scholar
  9. Ding SW, Voinnet O (2007) Antiviral immunity directed by small RNAs. Cell 130:413–426CrossRefPubMedPubMedCentralGoogle Scholar
  10. Doukhanina EV, Chen S, van der Zalm E, Godzik A, Reed J et al (2006) Identification and functional characterization of the BAG protein family in Arabidopsis thaliana. J Biol Chem 281:18793–18801CrossRefPubMedGoogle Scholar
  11. Guo N, Ye WW, Wu XL, Shen DY, Wang YC et al (2011) Microarray profiling reveals microRNAs involving soybean resistance to Phytophthora sojae. Genome 54:954–958CrossRefPubMedGoogle Scholar
  12. Harvey JJW, Lewsey MG, Patel K, Westwood J, Heimstaedt S et al (2011) An antiviral defense role of AGO2 in plants. PLoS One 6:e14639CrossRefPubMedPubMedCentralGoogle Scholar
  13. Huang DQ, Koh C, Feurtado JA, Tsang EWT, Cutler AJ (2013) MicroRNAs and their putative targets in Brassica napus seed maturation. BMC Genomics 14:140CrossRefPubMedGoogle Scholar
  14. Jaubert M, Bhattacharjee S, Mello AFS, Perry KL, Moffett P (2011) ARGONAUTE2 mediates RNA-silencing antiviral defenses against Potato virus X in Arabidopsis. Plant Physiol 156:1556–1564CrossRefPubMedPubMedCentralGoogle Scholar
  15. Jin W, Wu F (2015) Characterization of miRNAs associated with Botrytis cinerea infection of tomato leaves. BMC Plant Biol 15:1CrossRefPubMedPubMedCentralGoogle Scholar
  16. Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAs and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53CrossRefPubMedGoogle Scholar
  17. Kabbage M, Dickman MB (2008) The BAG proteins: a ubiquitous family of chaperone regulators. Cell Mol Life Sci 65:1390–1402CrossRefPubMedGoogle Scholar
  18. Korbes AP, Machado RD, Guzman F, Almerao MP, Valter de Oliveira LF et al (2012) Identifying conserved and novel microRNAs in developing seeds of Brassica napus using deep sequencing. PLoS One 7:e50663CrossRefPubMedPubMedCentralGoogle Scholar
  19. Li Y, Zhang QQ, Zhang J, Wu L, Qi Y et al (2010) Identification of microRNAs involved in pathogen-associated molecular pattern-triggered plant innate immunity. Plant Physiol 152:2222–2231CrossRefPubMedPubMedCentralGoogle Scholar
  20. Li F, Pignatta D, Bendix C, Brunkard JO, Cohn MM et al (2012) MicroRNA regulation of plant innate immune receptors. Proc Natl Acad Sci USA 109:1790–1795CrossRefPubMedPubMedCentralGoogle Scholar
  21. Li Y, Lu YG, Shi Y, Wu L, X YJ et al (2014) Multiple rice microRNAs are involved in immunity against the blast fungus Magnaporthe oryzae. Plant Physiol 164:1077–1092CrossRefPubMedGoogle Scholar
  22. Lu SF, Sun YH, Amerson H, Chiang VL (2007) MicroRNAs in loblolly pine (Pinus taeda L.) and their association with fusiform rust gall development. Plant J 51:1077–1098CrossRefPubMedGoogle Scholar
  23. Ma Z, Coruh C, Axtell MJ (2010) Arabidopsis lyrata small RNAs: transient MIRNA and small interfering RNA loci within the Arabidopsis genus. Plant Cell 22:1090–1103CrossRefPubMedPubMedCentralGoogle Scholar
  24. Meyers BC, Axtell MJ, Bartel B, Bartel DP, Baulcombe D et al (2008) Criteria for annotation of plant microRNAs. Plant Cell 20:3186–3190CrossRefPubMedPubMedCentralGoogle Scholar
  25. Morel JB, Godon C, Mourrain P, Beclin C, Boutet S et al (2002) Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance. Plant Cell 14:629–639CrossRefPubMedPubMedCentralGoogle Scholar
  26. Nakahara KS, Masuta C (2014) Interaction between viral RNA silencing suppressors and host factors in plant immunity. Curr Opin Plant Biol 20:88–95CrossRefPubMedGoogle Scholar
  27. Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N et al (2006) A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science 312:436–439CrossRefPubMedGoogle Scholar
  28. Navarro L, Jay F, Nomura K, He SY, Voinnet O (2008) Suppression of the microRNA pathway by bacterial effector proteins. Science 321:964–967CrossRefPubMedPubMedCentralGoogle Scholar
  29. Perchepied L, Balague C, Riou C, Claudel-Renard C, Riviere N et al (2010) Nitric oxide participates in the complex interplay of defense-related signaling pathways controlling disease resistance to Sclerotinia sclerotiorum in Arabidopsis thaliana. Mol Plant Microb Interact 23:846–860CrossRefGoogle Scholar
  30. Radwan O, Liu Y, Clough SJ (2011) Transcriptional analysis of soybean root response to Fusarium virguliforme, the causal agent of sudden death syndrome. Mol Plant Microb Interact 24:958–972CrossRefGoogle Scholar
  31. Robert-Seilaniantz A, MacLean D, Jikumaru Y, Hill L, Yamaguchi S et al (2011) The microRNA miR393 re-directs secondary metabolite biosynthesis away from camalexin and towards glucosinolates. Plant J 67:218–231CrossRefPubMedGoogle Scholar
  32. Ruiz-Ferrer V, Voinnet O (2009) Roles of plant small RNAs in biotic stress responses. Annu Rev Plant Biol 60:485–510CrossRefPubMedGoogle Scholar
  33. Scholthof HB, Alvarad VY, Vega-Arreguin JC, Ciomperlik J, Odokonyero D et al (2011) Identification of an ARGONAUTE for antiviral RNA silencing in Nicotiana benthamiana. Plant Physiol 156:1548–1555CrossRefPubMedPubMedCentralGoogle Scholar
  34. Shen D, Suhrkamp I, Wang Y, Liu S, Menkhaus J et al (2014) Identification and characterization of microRNAs in oilseed rape (Brassica napus) responsive to infection with the pathogenic fungus Verticillium longisporum using Brassica AA (Brassica rapa) and CC (Brassica oleracea) as reference genomes. New Phytol 204:577–594CrossRefPubMedGoogle Scholar
  35. Shen EH, Zou J, Behrens FH, Chen L, Ye CY et al (2015) Identification, evolution, and expression partitioning of miRNAs in allopolyploid Brassica napus. J Exp Bot. doi: 10.1093/jxb/erv420 Google Scholar
  36. Shivaprasad PV, Chen HM, Patel K, Bond DM, Santos, BACM et al (2012) A microRNA superfamily regulates nucleotide binding site-leucine-rich repeats and other mRNAs. Plant Cell 24:859–874CrossRefPubMedPubMedCentralGoogle Scholar
  37. Sunkar R, Li YF, Jagadeeswaran G (2012) Functions of microRNAs in plant stress responses. Trends Plant Sci 17:196–203CrossRefPubMedGoogle Scholar
  38. Wang L, Wang MB, Tu JX, Helliwell CA, Waterhouse PM et al (2007) Cloning and characterization of microRNAs from Brassica napus. FEBS Lett 581:3848–3856CrossRefPubMedGoogle Scholar
  39. Weiberg A, Wang M, Lin FM, Zhao H, Zhang Z et al (2013) Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways. Science 342:118–123CrossRefPubMedPubMedCentralGoogle Scholar
  40. Williams B, Kabbage M, Kim HJ, Britt R, Dickman MB (2011) Tipping the balance: Sclerotinia sclerotiorum secreted oxalic acid suppresses host defenses by manipulating the host redox environment. PLoS Pathog 7:e1002107CrossRefPubMedPubMedCentralGoogle Scholar
  41. Xie FL, Huang SQ, Guo K, Xiang AL, Zhu YY et al (2007) Computational identification of novel microRNAs and targets in Brassica napus. FEBS Lett 581:1464–1474CrossRefPubMedGoogle Scholar
  42. Xin MM, Wang Y, Yao YY, Xie CJ, Peng HR et al (2010) Diverse set of microRNAs are responsive to powdery mildew infection and heat stress in wheat (Triticum aestivum L.) BMC Plant Biol 10:123CrossRefPubMedPubMedCentralGoogle Scholar
  43. Xu MY, Dong Y, Zhang QX, Zhang L, Luo YZ et al (2012a) Identification of miRNAs and their targets from Brassica napus by high-throughput sequencing and degradome analysis. BMC Genomics 13:421CrossRefPubMedPubMedCentralGoogle Scholar
  44. Xu QF, Cheng WS, Li SS, Li W, Zhang ZX et al (2012b) Identification of genes required for Cf-dependent hypersensitive cell death by combined proteomics and RNA interfering analyses. J Exp Bot 63:2421–2435CrossRefPubMedPubMedCentralGoogle Scholar
  45. Yang L, Jue D, Li W, Zhang R, Chen M et al (2013) Identification of miRNA from eggplant (Solanum melongena L.) by small RNA deep sequencing and their response to Verticillium dahliae infection. PLoS One 8:e72840CrossRefPubMedPubMedCentralGoogle Scholar
  46. Yin Z, Li Y, Han X, Shen F (2012) Genome-wide profiling of miRNAs and other small non-coding RNAs in the Verticillium dahliae-inoculated cotton roots. PLoS One 7:e35765CrossRefPubMedPubMedCentralGoogle Scholar
  47. Zhai J, Jeong DH, De Paoli E, Park S, Rosen BD et al (2011) MicroRNAs as master regulators of the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs. Genes Dev 25:2540–2553CrossRefPubMedPubMedCentralGoogle Scholar
  48. Zhang BH, Pan XP, Cannon CH, Cobb GP, Anderson TA (2006) Conservation and divergence of plant microRNA genes. Plant J 46:243–259CrossRefPubMedGoogle Scholar
  49. Zhang XM, Zhao HW, Gao S, Wang WC, Katiyar-Agarwal S et al (2011) Arabidopsis Argonaute 2 regulates innate immunity via miRNA393*-mediated silencing of a Golgi-localized SNARE gene, MEMB12. Mole. Cell 42:356–366Google Scholar
  50. Zhao YT, Wang M, Fu SX, Yang WC, Qi CK et al (2012) Small RNA profiling in two Brassica napus cultivars identifies microRNAs with oil production- and development-correlated expression and new small RNA classes. Plant Physiol 158:813–823CrossRefPubMedGoogle Scholar
  51. Zhao Y, Liu W, Xu YP, Cao JY, Braam J et al (2013) Genome-wide identification and functional analyses of calmodulin genes in Solanaceous species. BMC Plant Biol 13:70CrossRefPubMedPubMedCentralGoogle Scholar
  52. Zhou ZS, Song JB, Yang ZM (2012) Genome-wide identification of Brassica napus microRNAs and their targets in response to cadmium. J Exp Bot 63:4597–4461CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Jia-Yi Cao
    • 1
  • You-Ping Xu
    • 2
  • Li Zhao
    • 1
  • Shuang-Sheng Li
    • 1
  • Xin-Zhong Cai
    • 1
  1. 1.Institute of Biotechnology, College of Agriculture and BiotechnologyZhejiang UniversityHangzhouChina
  2. 2.Centre of Analysis and MeasurementZhejiang UniversityHangzhouChina

Personalised recommendations