Cloning and validation of novel miRNA from basmati rice indicates cross talk between abiotic and biotic stresses

  • Neeti Sanan-Mishra
  • Vikash Kumar
  • Sudhir K. Sopory
  • Sunil K. Mukherjee
Original Paper


Most of the physiological processes are controlled by the small RNAs in several organisms including plants. A huge database exists on one type of small RNA, i.e., microRNAs (miRs) identified from diverse species. However, the processes of data-mining of miRs in most of the species are still incomplete. Rice feeds the hungry trillions and hence understanding its developmental processes as well as its stress biology, which might be largely controlled by the small RNA pathways, is certainly a worthwhile task. Here, we report the cloning and identification of ~40 new putative miRs from local basmati rice variety in accordance to the annotation suggested by Meyers et al. (Plant Cell 20:3186–3190, 2008). About 23 sequences were derived from rice exposed to salt stress while 18 were derived from rice infected with tungro virus. A few of these putative miRs were common to both. Our data showed that at least two of these miRs were up-regulated in response to both abiotic and biotic stresses. The miR target predictions indicate that most of the putative miRs target specific metabolic processes. The up-regulation of similar miRs in response to two entirely different types of stresses suggests a converging functional role of miRs in managing various stresses. Our findings suggest that more rice miRs need to be identified and a thorough understanding of the function of such miRs will help unravel the mysteries of rice stress biology.


miR Cloning Salt stress Tungro virus Indica rice 



This research was supported by grants from Department of Science and Technology and Department of Biotechnology, New Delhi, India.

Supplementary material

438_2009_478_MOESM1_ESM.xls (32 kb)
Table 1 List of the cloned sequences that are homologous with the already known miRs from rice (XLS 32 kb)
438_2009_478_MOESM2_ESM.xls (56 kb)
Supplemental material Table 2 Homology of the cloned small RNA sequences with the rice genome sequence available in the GenBank accessions with NCBI. The table lists the small RNA sequence, physiology of the tissue from which it was isolated along with the details of accession number, clone name, nucleotide positions, polarity of the homologous rice genomic or cDNA regions. The 5’-3’ strand is designated as the + strand while the complementary 3’-5’ strand is designated as the – strand (XLS 56 kb)
438_2009_478_MOESM3_ESM.xls (78 kb)
Supplemental Table 3 Homology of the cloned small RNA sequences with the mature miR sequences in the Sanger’s database. The table lists the small RNA sequence, tissue and physiology from which it was isolated along with the details of homologous miR including expect (E) value, accession number and genomic coordinates, number of predicted targets and polarity of the strand. The 5’-3’ strand is designated as the + strand while the complementary 3’-5’ strand is designated as the – strand (XLS 78 kb)
438_2009_478_MOESM4_ESM.xls (58 kb)
Supplemental Table 4 Predicted precursors of the 44 small RNA sequences having 0-3 mismatches with the available rice genome. The table lists the cultivar of rice and accession number from which the precursor was predicted. The length, sequence and free energy (dG) of the putative precursor is mentioned (XLS 57 kb)
438_2009_478_MOESM5_ESM.xls (58 kb)
Supplemental Table 5 Predicted targets of the putative miR sequences determined by using miRU software. The table lists accession number, sequence and function of the predicted target region. The maximum score and prediction parameters are mentioned in the table (XLS 58 kb)
438_2009_478_MOESM6_ESM.xls (47 kb)
Supplemental Table 6 Homologs of putative miR sequences identified in plant species other than rice. The summarized presentation of homologs of putative miR sequences identified in plant species other than rice using nucleotide BLAST search. The sequences specific to rice genome have been separately highlighted (XLS 47 kb)
438_2009_478_MOESM7_ESM.ppt (3.1 mb)
Supplemental Figure 1 Foldback structures. Foldback structures of the putative precursors of the 44 small RNA sequences having 0-3 mismatches with the available rice genome. The sequences are numbered starting from the 5’end, so the top strand represents the 5’ arm while the bottom strand represents the 3’ arm. The putative miR sequence is highlighted in green colour. dG indicates the free energy of the folded precursort (PPT 3205 kb)


  1. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297CrossRefPubMedGoogle Scholar
  2. Bartel B, Bartel DP (2003) MicroRNAs: at the root of plant development? Plant Physiol 132:709–717CrossRefPubMedGoogle Scholar
  3. Bonnet E, Wuyts J, Rouzé P, Van de Peer Y (2004) Detection of 91 potential conserved plant microRNAs in Arabidopsis thaliana and Oryza sativa identifies important target genes. Proc Natl Acad Sci USA 101:11511–11516CrossRefPubMedGoogle Scholar
  4. Chapman EJ, Carrington JC (2007) Specialization and evolution of endogenous small RNA pathways. Nat Rev Genet 8:884–896CrossRefPubMedGoogle Scholar
  5. Chapman EJ, Prokhnevsky AI, Gopinath K, Dolja VV, Carrington JC (2004) Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes Dev 18:1179–1186CrossRefPubMedGoogle Scholar
  6. Chekanova JA, Belostotsky DA (2006) MicroRNAs and messenger RNA turnover. Methods Mol Biol 342:73–85PubMedGoogle Scholar
  7. Chen X (2008) MicroRNA metabolism in plants. Curr Top Microbiol Immunol 320:117–136CrossRefPubMedGoogle Scholar
  8. Chen PY, Meister G (2005) MicroRNA-guided posttranscriptional gene regulation. Biol Chem 386:1205–1218CrossRefPubMedGoogle Scholar
  9. Chinnusamy V, Gong Z, Zhu JK (2008) Nuclear RNA export and its importance in abiotic stress responses of plants. Curr Top Microbiol Immunol 326:235–255CrossRefPubMedGoogle Scholar
  10. Christensen AB, Thordal-Christensen H, Zimmermann G, Gjetting T, Lyngkjaer MF, Dudler R, Schweizer P (2004) The germin-like protein GLP4 exhibits superoxide dismutase activity and is an important component of quantitative resistance in wheat and barley. Mol Plant Microbe Interact 17:109–117CrossRefPubMedGoogle Scholar
  11. Clouse SD, Langford M, McMorris TC (1996) A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development. Plant Physiol 111:671–678CrossRefPubMedGoogle Scholar
  12. Dezulian T, Remmert M, Palatnik JF, Weigel D, Huson DH (2006) Identification of plant microRNA homologs. Bioinformatics 22:359–360CrossRefPubMedGoogle Scholar
  13. Ebhardt HA, Thi EP, Wang MB, Unrau PJ (2005) Extensive 3′ modification of plant small RNAs is modulated by helper component-proteinase expression. Proc Natl Acad Sci USA 102:13398–13403CrossRefPubMedGoogle Scholar
  14. Eckardt NA (2004) Two genomes are better than one: widespread paleopolyploidy in plants and evolutionary effects. Plant Cell 16:1647–1649CrossRefPubMedGoogle Scholar
  15. Fahlgren N, Howell MD, Kasschau KD, Chapman EJ, Sullivan CM, Cumbie JS, Givan SA, Law TF, Grant SR, Dangl JL, Carrington JC (2007) High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes. PLoS ONE 2:e219CrossRefPubMedGoogle Scholar
  16. Fritz JH, Girardin SE, Philpott DJ (2006) Innate immune defense through RNA interference. Sci STKE pe27Google Scholar
  17. Fujita M, Fujita Y, Noutoshi Y, Takahashi F, Narusaka Y, Yamaguchi-Shinozaki K, Shinozaki K (2006) Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks. Curr Opin Plant Biol 9:436–442CrossRefPubMedGoogle Scholar
  18. Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ (2006) miRBase: microRNA sequences, targets and gene nomenclature. Nucl Acid Res 34(Database Issue):D140–D144CrossRefGoogle Scholar
  19. He L, Hannon GJ (2004) MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5:522–531CrossRefPubMedGoogle Scholar
  20. He XF, Fang YY, Feng L, Guo HS (2008) Characterization of conserved and novel microRNAs and their targets, including a TuMV-induced TIR-NBS-LRR class R gene-derived novel miRNA in Brassica. FEBS Lett 582:2445–2452CrossRefPubMedGoogle Scholar
  21. Jaillon O, Aury JM, Noel B, Policriti A, Clepet C, Casagrande A, Choisne N, Aubourg S, Vitulo N, Jubin C et al (2007) The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 449:463–467CrossRefPubMedGoogle Scholar
  22. Jin H (2008) Endogenous small RNAs and antibacterial immunity in plants. FEBS Lett 582:2679–2684CrossRefPubMedGoogle Scholar
  23. Jones-Rhoades MW, Bartel DP (2004) Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Mol Cell 14:787–799CrossRefPubMedGoogle Scholar
  24. Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAS and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53CrossRefPubMedGoogle Scholar
  25. Kasschau KD, Xie Z, Allen E, Llave C, Chapman EJ, Krizan KA, Carrington JC (2003) P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function. Dev Cell 4:205–217CrossRefPubMedGoogle Scholar
  26. Khan-Barozai MY, Irfan M, Yousaf R, Ali I, Qaisar U, Maqbool A, Zahoor M, Rashid B, Hussnain T, Riazuddin S (2008) Identification of micro-RNAs in cotton. Plant Physiol Biochem 46:739–751CrossRefPubMedGoogle Scholar
  27. Kidner CA, Martienssen RA (2005) The developmental role of microRNA in plants. Curr Opin Plant Biol 8:38–44CrossRefPubMedGoogle Scholar
  28. Kotchoni SO, Gachomo EW (2006) The reactive oxygen species network pathways: an essential prerequisite for perception of pathogen attack and the acquired disease resistance in plants. J Biosci 31:389–404CrossRefPubMedGoogle Scholar
  29. Kunii M, Kanda M, Nagano H, Uyeda I, Kishima Y, Sano Y (2004) Reconstruction of DNA virus from endogenous rice tungro bacilliform virus-like sequences in the rice genome: implications for integration and evolution. BMC Genomics 5:80CrossRefPubMedGoogle Scholar
  30. Kunz BA, Cahill DM, Mohr PG, Osmond MJ, Vonarx EJ (2006) Plant responses to UV radiation and links to pathogen resistance. Int Rev Cytol 255:1–40CrossRefPubMedGoogle Scholar
  31. Kurihara Y, Watanabe Y (2004) Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc Natl Acad Sci USA 101:12753–12758CrossRefPubMedGoogle Scholar
  32. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T (2001) Identification of novel genes coding for small expressed RNAs. Science 294:853–858CrossRefPubMedGoogle Scholar
  33. Laporte P, Merchan F, Amor BB, Wirth S, Crespi M (2007) Riboregulators in plant development. Biochem Soc Trans 35:1638–1642CrossRefPubMedGoogle Scholar
  34. Lau NC, Lim LP, Weinstein EG, Bartel DP (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294:858–862CrossRefPubMedGoogle Scholar
  35. Lee RC, Ambros V (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294:862–864CrossRefPubMedGoogle Scholar
  36. Li J, Yang Z, Yu B, Liu J, Chen X (2005) Methylation protects miRs and siRNAs from a 3′-end uridylation activity in Arabidopsis. Curr Biol 15:1501–1507CrossRefPubMedGoogle Scholar
  37. Lindow M, Jacobsen A, Nygaard S, Mang Y, Krogh A (2007) Intragenomic matching reveals a huge potential for miRNA-mediated regulation in plants. PLoS Comput Biol 3:e238CrossRefPubMedGoogle Scholar
  38. Liu PP, Montgomery TA, Fahlgren N, Kasschau KD, Nonogaki H, Carrington JC (2007) Repression of AUXIN RESPONSE FACTOR10 by microRNA160 is critical for seed germination and post-germination stages. Plant J 52:133–146CrossRefPubMedGoogle Scholar
  39. Llave C, Kasschau KD, Rector MA, Carrington JC (2002) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14:1605–1619CrossRefPubMedGoogle Scholar
  40. Lu XY, Huang XL (2008) Plant miRNAs and abiotic stress responses. Biochem Biophys Res Commun 368:458–462CrossRefPubMedGoogle Scholar
  41. Lu S, Sun YH, Shi R, Clark C, Li L, Chiang VL (2005) Novel and mechanical stress-responsive MicroRNAs in Populus trichocarpa that are absent from Arabidopsis. Plant Cell 17:2186–2203CrossRefPubMedGoogle Scholar
  42. Luo YC, Zhou H, Li Y, Chen JY, Yang JH, Chen YQ, Qu LH (2006) Rice embryogenic calli express a unique set of microRNAs, suggesting regulatory roles of microRNAs in plant post-embryogenic development. FEBS Lett 580:5111–5116CrossRefPubMedGoogle Scholar
  43. Manavella PA, Arce AL, Dezar CA, Bitton F, Renou JP, Crespi M, Chan RL (2006) Cross-talk between ethylene and drought signalling pathways is mediated by the sunflower Hahb-4 transcription factor. Plant J 48:125–137CrossRefPubMedGoogle Scholar
  44. Meyers BC, Axtell MJ, Bartel B, Bartel DP, Baulcombe D, Bowman JL, Cao X, Carrington JC, Chen X, Green PJ, Griffiths-Jones S, Jacobsen SE, Mallory AC, Martienssen RA, Poethig RS, Qi Y, Vaucheret H, Voinnet O, Watanabe Y, Weigel D, Zhu JK (2008) Criteria for annotation of plant microRNAs. Plant Cell 20:3186–3190CrossRefPubMedGoogle Scholar
  45. Moxon S, Jing R, Szittya G, Schwach F, Rusholme-Pilcher RL, Moulton V, Dalmay T (2008) Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening. Genome Res 18:1602–1609CrossRefPubMedGoogle Scholar
  46. Nagano H, Oka A, Kishima Y, Sano Y (2000) DNA sequences homologous to rice tungro bacilliform virus (RTBV) present in the rice genome. Rice Genetic Newsl 17:103–105Google Scholar
  47. Navarro L, Jay F, Nomura K, He SY, Voinnet O (2008) Suppression of the microRNA pathway by bacterial effector proteins. Science 321:964–967CrossRefPubMedGoogle Scholar
  48. Nobuta K, Venu RC, Lu C, Belo A, Vemaraju K, Kulkarni K, Wang W, Pillay M, Green PJ, Wang GL et al (2007) An expression atlas of rice mRNAs and small RNAs. Nat Biotechnol 25:473–477CrossRefPubMedGoogle Scholar
  49. Nogueira FT, Sarkar AK, Chitwood DH, Timmermans MC (2006) Organ polarity in plants is specified through the opposing activity of two distinct small regulatory RNAs. Cold Spring Harb Symp Quant Biol 71:157–164CrossRefPubMedGoogle Scholar
  50. Phillips JR, Dalmay T, Bartels D (2007) The role of small RNAs in abiotic stress. FEBS Lett 581:3592–3597CrossRefPubMedGoogle Scholar
  51. Pontes O, Pikaard CS (2008) siRNA and miRNA processing: new functions for Cajal bodies. Curr Opin Genet Dev 18:197–203CrossRefPubMedGoogle Scholar
  52. Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002) MicroRNAs in plants. Genes Dev 16:1616–1626CrossRefPubMedGoogle Scholar
  53. Reyes JL, Chua NH (2007) ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination. Plant J 49:592–606CrossRefPubMedGoogle Scholar
  54. Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP (2002) Prediction of plant microRNA targets. Cell 110:513–520CrossRefPubMedGoogle Scholar
  55. Sanan-Mishra N, Mukherjee SK (2007) A peep into the plant miRNA world. Open Plant Sci J 1:1–9Google Scholar
  56. Sunkar R, Zhu JK (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001–2019CrossRefPubMedGoogle Scholar
  57. Sunkar R, Girke T, Zhu JK (2005) Identification and characterization of endogenous small interfering RNAs from rice. Nucleic Acids Res 33:4443–4454CrossRefPubMedGoogle Scholar
  58. Sunkar R, Chinnusamy V, Zhu J, Zhu JK (2007) Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci 12:301–309CrossRefPubMedGoogle Scholar
  59. Sunkar R, Zhou X, Zheng Y, Zhang W, Zhu JK (2008) Identification of novel and candidate miRNAs in rice by high throughput sequencing. BMC Plant Biol 8:25CrossRefPubMedGoogle Scholar
  60. Tagami Y, Inaba N, Kutsuna N, Kurihara Y, Watanabe Y (2007) Specific enrichment of miRNAs in Arabidopsis thaliana infected with Tobacco mosaic virus. DNA Res 14:227–233CrossRefPubMedGoogle Scholar
  61. Terzi LC, Simpson GG (2008) Regulation of flowering time by RNA processing. Curr Top Microbiol Immunol 326:201–218CrossRefPubMedGoogle Scholar
  62. Tuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, Putnam N, Ralph S, Rombauts S, Salamov A et al (2006) The genome of black cottonwood Populus trichocarpa (Torr & Gray). Science 313:1596–1604CrossRefPubMedGoogle Scholar
  63. Wang QL, Li ZH (2007) The functions of microRNAs in plants. Front Biosci 12:3975–3982PubMedGoogle Scholar
  64. Wang XJ, Reyes JL, Chua NH, Gaasterland T (2004) Prediction and identification of Arabidopsis thaliana microRNAs and their mRNA targets. Genome Biol 5:R65CrossRefPubMedGoogle Scholar
  65. Wang L, Wang MB, Tu JX, Helliwell CA, Waterhouse PM, Dennis ES, Fu TD, Fan YL (2007) Cloning and characterization of microRNAs from Brassica napus. FEBS Lett 581:3848–3856CrossRefPubMedGoogle Scholar
  66. Willmann MR, Poethig RS (2007) Conservation and evolution of miRNA regulatory programs in plant development. Curr Opin Plant Biol 10:503–511CrossRefPubMedGoogle Scholar
  67. Wu CY, Trieu A, Radhakrishnan P, Kwok SF, Harris S, Zhang K, Wang J, Wan J, Zhai H, Takatsuto S, Matsumoto S, Fujioka S, Feldmann KA, Pennell RI (2008) Brassinosteroids regulate grain filling in rice. Plant Cell 20:2130–2145CrossRefPubMedGoogle Scholar
  68. Xie Z, Qi X (2008) Diverse small RNA-directed silencing pathways in plants. Biochem Biophys Acta 1779:720–724PubMedGoogle Scholar
  69. Yao C, Zhao B, Li W, Li Y, Qin W, Huang B, Jin Y (2007) Cloning of novel repeat-associated small RNAs derived from hairpin precursors in Oryza sativa. Acta Biochim Biophys Sin (Shanghai) 39:829–834CrossRefGoogle Scholar
  70. Zhang Y (2005) miRU: an automated plant miRNA target prediction server. Nucleic Acids Res 33:W701–W704CrossRefPubMedGoogle Scholar
  71. Zhao B, Liang R, Ge L, Li W, Xiao H, Lin H, Ruan K, Jin Y (2007) Identification of drought-induced microRNAs in rice. Biochem Biophys Res Commun 354:585–590CrossRefPubMedGoogle Scholar
  72. Zhao B, Ge L, Liang R, Li W, Ruan K, Lin H, Jin Y (2009) Members of miR-169 family are induced by high salinity and transiently inhibit the NF-YA transcription factor. BMC Mol Biol 10:29CrossRefPubMedGoogle Scholar
  73. Zhou X, Wang G, Zhang W (2007) UV-B responsive microRNA genes in Arabidopsis thaliana. Mol Syst Biol 3:103CrossRefPubMedGoogle Scholar
  74. Zhu JK (2008) Reconstituting plant miRNA biogenesis. Proc Natl Acad Sci USA 105:9851–9852CrossRefPubMedGoogle Scholar
  75. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Neeti Sanan-Mishra
    • 1
  • Vikash Kumar
    • 1
  • Sudhir K. Sopory
    • 1
  • Sunil K. Mukherjee
    • 1
  1. 1.International Center for Genetic Engineering and BiotechnologyNew DelhiIndia

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