Plant Molecular Biology

, Volume 70, Issue 3, pp 265–272 | Cite as

Dose-dependent RNAi-mediated geminivirus resistance in the tropical root crop cassava

  • Hervé VanderschurenEmail author
  • Adrian Alder
  • Peng Zhang
  • Wilhelm GruissemEmail author


Cassava mosaic disease is a major constraint for cassava production in Africa, resulting in significant economic losses. We have engineered transgenic cassava with resistance to African cassava mosaic virus (ACMV), by expressing ACMV AC1-homologous hairpin double-strand RNAs. Transgenic cassava lines with high levels of AC1-homologous small RNAs have ACMV immunity with increasing viral load and different inoculation methods. We report a correlation between the expression of the AC1-homologous small RNAs and the ACMV resistance of the transgenic cassava lines. Characterization of the small RNAs revealed that only some of the hairpin-derived small RNAs fall into currently known small interfering RNA classes in plants. The method is scalable to stacking by targeting multiple virus isolates with additional hairpins.


Virus resistance Cassava RNA interference Hairpin RNA processing 



We thank Dr. John Stanley (John Innes Centre) for the ACMV clones, and Drs. Johannes Fütterer (ETH Zurich), Daniel Schöner (ETH Zurich) and Thomas Hohn (University of Basel) for helpful discussions. This work was supported by grants from the Eiselen-Foundation-Ulm and the Bill & Melinda Gates Foundation Grand Challenges in Global Health Initiative.

Supplementary material

11103_2009_9472_MOESM1_ESM.doc (131 kb)
Table S1 Sequences and functions of primers and oligonucleotides used in the experiments. (DOC 131 kb)
11103_2009_9472_MOESM2_ESM.tif (3.1 mb)
Fig. S1 Supplementary material 2 (TIFF 3175 kb)
11103_2009_9472_MOESM3_ESM.tif (3.1 mb)
Fig. S2 Supplementary material 3 (TIFF 3175 kb)


  1. Akbergenov R, Si-Ammour A, Blevins T, Amin I, Kutter C, Vanderschuren H, Zhang P, Gruissem W, Meins F Jr, Hohn T, Pooggin MM (2006) Molecular characterization of geminivirus-derived small RNAs in different plant species. Nucleic Acids Res 34(2):462–471. doi: 10.1093/nar/gkj447 PubMedCrossRefGoogle Scholar
  2. Belostotsky DA, Rose AB (2005) Plant gene expression in the age of systems biology: integrating transcriptional and post-transcriptional events. Trends Plant Sci 10(7):347–353. doi: 10.1016/j.tplants.2005.05.004 PubMedCrossRefGoogle Scholar
  3. Blevins T, Rajeswaran R, Shivaprasad PV, Beknazariants D, Si-Ammour A, Park HS, Vazquez F, Robertson D, Meins F Jr, Hohn T, Pooggin MM (2006) Four plant dicers mediate viral small RNA biogenesis and DNA virus induced silencing. Nucleic Acids Res 34(21):6233–6246. doi: 10.1093/nar/gkl886 PubMedCrossRefGoogle Scholar
  4. Bonfim K, Faria JC, Nogueira EOPL, Mendes EA, Aragao FJL (2007) RNAi-mediated resistance to Bean golden mosaic virus in genetically engineered common bean (Phaseolus vulgaris). Mol Plant Microbe Interact 20(6):717–726. doi: 10.1094/MPMI-20-6-0717 PubMedCrossRefGoogle Scholar
  5. Boulton MI (2003) Geminiviruses: major threats to world agriculture. Ann Appl Biol 142(2):143. doi: 10.1111/j.1744-7348.2003.tb00239.x CrossRefGoogle Scholar
  6. Chellappan P, Masona MV, Vanitharani R, Taylor NJ, Fauquet CM (2004) Broad spectrum resistance to ssDNA viruses associated with transgene-induced gene silencing in cassava. Plant Mol Biol 56(4):601–611. doi: 10.1007/s11103-004-0147-9 PubMedCrossRefGoogle Scholar
  7. Fauquet C, Fargette D (1990) African cassava mosaic-virus—etiology, epidemiology, and control. Plant Dis 74(6):404–411. doi: 10.1094/PD-74-0404 CrossRefGoogle Scholar
  8. Frischmuth T, Stanley J (1998) Recombination between viral DNA and the transgenic coat protein gene of African cassava mosaic geminivirus. J Gen Virol 79:1265–1271PubMedGoogle Scholar
  9. Fuentes A, Ramos PL, Fiallo E, Callard D, Sanchez Y, Peral R, Rodriguez R, Pujol M (2006) Intron-hairpin RNA derived from replication associated protein C1 gene confers immunity to Tomato Yellow Leaf Curl Virus infection in transgenic tomato plants. Transgenic Res 15(3):291–304. doi: 10.1007/s11248-005-5238-0 PubMedCrossRefGoogle Scholar
  10. Hanley-Bowdoin L, Settlage SB, Orozco BM, Nagar S, Robertson D (2000) Geminiviruses: models for plant DNA replication, transcription, and cell cycle regulation. Crit Rev Biochem Mol Biol 35(2):105–140PubMedGoogle Scholar
  11. Hily J-M, Scorza R, Webb K, Ravelonandro M (2005) Accumulation of the long class of siRNAs is associated with resistance to plum pox virus in a transgenic woody perennial plum tree. Mol Plant Microbe Interact 18(8):794–799. doi: 10.1094/MPMI-18-0794 PubMedCrossRefGoogle Scholar
  12. Hong Y, Stanley J (1996) Virus resistance in Nicotiana benthamiana conferred by African cassava mosaic virus replication-associated protein (AC1) transgene. Mol Plant Microbe Interact 9(4):219–225Google Scholar
  13. Kalantidis K, Psaradakis S, Tabler M, Tsagris M (2002) The occurrence of CMV-specific short RNAs in transgenic tobacco expressing virus-derived double-stranded RNA is indicative of resistance to the virus. Mol Plant Microbe Interact 15(8):826–833. doi: 10.1094/MPMI.2002.15.8.826 PubMedCrossRefGoogle Scholar
  14. Katiyar-Agarwal S, Gao S, Vivian-Smith A, Jin H (2007) A novel class of bacteria-induced small RNAs in Arabidopsis. Genes Dev 21(23):3123–3134. doi: 10.1101/gad.1595107 PubMedCrossRefGoogle Scholar
  15. Kunik T, Salomon R, Zamir D, Navot N, Zeidan M, Michelson I, Gafni Y, Czosnek H (1994) Transgenic tomato plants expressing the tomato yellow leaf curl virus capsid protein are resistant to the virus. Biotechnology (NY) 12(5):500–504. doi: 10.1038/nbt0594-500 CrossRefGoogle Scholar
  16. Lee SR, Collins K (2006) Two classes of endogenous small RNAs in Tetrahymena thermophila. Genes Dev 20(1):28–33. doi: 10.1101/gad.1377006 PubMedCrossRefGoogle Scholar
  17. Legg JP, Fauquet CM (2004) Cassava mosaic geminiviruses in Africa. Plant Mol Biol 56(4):585–599. doi: 10.1007/s11103-004-1651-7 PubMedCrossRefGoogle Scholar
  18. Legg JP, Owor B, Sseruwagi P, Ndunguru J (2006) Cassava mosaic virus disease in East and central Africa: epidemiology and management of a regional pandemic. Adv Virus Res 67:355–418. doi: 10.1016/S0065-3527(06)67010-3 PubMedCrossRefGoogle Scholar
  19. Mansoor S, Zafar Y, Briddon RW (2006) Geminivirus disease complexes: the threat is spreading. Trends Plant Sci 11(5):209–212. doi: 10.1016/j.tplants.2006.03.003 PubMedCrossRefGoogle Scholar
  20. Noris E, Accotto GP, Tavazza R, Brunetti A, Crespi S, Tavazza M (1996) Resistance to tomato yellow leaf curl geminivirus in Nicotiana benthamiana plants transformed with a truncated viral C1 gene. Virology 224(1):130–138. doi: 10.1006/viro.1996.0514 PubMedCrossRefGoogle Scholar
  21. Pooggin M, Shivaprasad PV, Veluthambi K, Hohn T (2003) RNAi targeting of DNA virus in plants. Nat Biotechnol 21(2):131–132. doi: 10.1038/nbt0203-131b PubMedCrossRefGoogle Scholar
  22. Ribeiro SG, Lohuis H, Goldbach R, Prins M (2007) Tomato chlorotic mottle virus is a target of RNA silencing but the presence of specific short interfering RNAs does not guarantee resistance in transgenic plants. J Virol 81(4):1563–1573. doi: 10.1128/JVI.01238-06 PubMedCrossRefGoogle Scholar
  23. Rose AB (2004) The effect of intron location on intron-mediated enhancement of gene expression in Arabidopsis. Plant J 40(5):744–751. doi: 10.1111/j.1365-313X.2004.02247.x PubMedCrossRefGoogle Scholar
  24. Shepherd DN, Mangwende T, Martin DP, Bezuidenhout M, Kloppers FJ, Carolissen CH, Monjane AL, Rybicki EP, Thomson JA (2007) Maize streak virus-resistant transgenic maize: a first for Africa. Plant Biotechnol J 5(6):759–767. doi: 10.1111/j.1467-7652.2007.00279.x PubMedCrossRefGoogle Scholar
  25. Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Gene expression—total silencing by intron-spliced hairpin RNAs. Nature 407(6802):319–320. doi: 10.1038/35030305 PubMedCrossRefGoogle Scholar
  26. Soni R, Murray JA (1994) Isolation of intact DNA and RNA from plant tissues. Anal Biochem 218(2):474–476. doi: 10.1006/abio.1994.1214 PubMedCrossRefGoogle Scholar
  27. Swiezewski S, Crevillen P, Liu F, Ecker JR, Jerzmanowski A, Dean C (2007) Small RNA-mediated chromatin silencing directed to the 3′ region of the Arabidopsis gene encoding the developmental regulator, FLC. Proc Natl Acad Sci USA 104(9):3633–3638. doi: 10.1073/pnas.0611459104 PubMedCrossRefGoogle Scholar
  28. Vanderschuren H, Stupak M, Futterer J, Gruissem W, Zhang P (2007a) Engineering resistance to geminiviruses—review and perspectives. Plant Biotechnol J 5(2):207–220. doi: 10.1111/j.1467-7652.2006.00217.x PubMedCrossRefGoogle Scholar
  29. Vanderschuren H, Akbergenov R, Pooggin MM, Hohn T, Gruissem W, Zhang P (2007b) Transgenic cassava resistance to African cassava mosaic virus is enhanced by viral DNA-A bidirectional promoter-derived siRNAs. Plant Mol Biol 64(5):549–557. doi: 10.1007/s11103-007-9175-6 PubMedCrossRefGoogle Scholar
  30. Vanitharani R, Chellappan P, Fauquet CM (2003) Short interfering RNA-mediated interference of gene expression and viral DNA accumulation in cultured plant cells. Proc Natl Acad Sci USA 100(16):9632–9636. doi: 10.1073/pnas.1733874100 PubMedCrossRefGoogle Scholar
  31. Wang M-B, Abbott DC, Waterhouse PM (2000) A single copy of a virus-derived transgene encoding hairpin RNA gives immunity to barley yellow dwarf virus. Mol Plant Pathol 1:347–356. doi: 10.1046/j.1364-3703.2000.00038.x CrossRefGoogle Scholar
  32. Wang MB, Helliwell CA, Wu LM, Waterhouse PM, Peacock WJ, Dennis ES (2008) Hairpin RNAs derived from RNA polymerase II and polymerase III promoter-directed transgenes are processed differently in plants. RNA 14(5):1–11Google Scholar
  33. Waterhouse PM, Fusaro AF (2006) Viruses face a double defense by plant small RNAs. Science 313(5783):54–55. doi: 10.1126/science.1130818 PubMedCrossRefGoogle Scholar
  34. Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27(6):581–590. doi: 10.1046/j.1365-313X.2001.01105.x PubMedCrossRefGoogle Scholar
  35. Zhang P, Potrykus I, Puonti-Kaerlas J (2000) Efficient production of transgenic cassava using negative and positive selection. Transgenic Res 9:405–415. doi: 10.1023/A:1026509017142 PubMedCrossRefGoogle Scholar
  36. Zhang P, Vanderschuren H, Futterer J, Gruissem W (2005) Resistance to cassava mosaic disease in transgenic cassava expressing antisense RNAs targeting virus replication genes. Plant Biotechnol J 3(4):385–397. doi: 10.1111/j.1467-7652.2005.00132.x PubMedCrossRefGoogle Scholar
  37. Zrachya A, Kumar PP, Ramakrishnan U, Levy Y, Loyter A, Arazi T, Lapidot M, Gafni Y (2007) Production of siRNA targeted against TYLCV coat protein transcripts leads to silencing of its expression and resistance to the virus. Transgenic Res 16(3):385–398. doi: 10.1007/s11248-006-9042-2 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  1. 1.Department of Biology, Institute of Plant SciencesETH ZürichZurichSwitzerland
  2. 2.Shanghai Center for Cassava Biotechnology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological SciencesChinese Academy of SciencesShanghaiChina

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