Plant Molecular Biology

, Volume 56, Issue 4, pp 613–624 | Cite as

A geminivirus-induced gene silencing system for gene function validation in cassava

  • Ismael B. F. Fofana
  • Abdourahamane Sangaré
  • Ray Collier
  • Christopher Taylor
  • Claude M. FauquetEmail author


We have constructed an African cassava mosaic virus (ACMV) based gene-silencing vector as a reverse genetics tool for gene function analysis in cassava. The vector carrying a fragment from the Nicotiana tabacumsulfur gene (su), encoding one unit of the chloroplast enzyme magnesium chelatase, was used to induce the silencing of the cassava orthologous gene resulting in yellow–white spots characteristic of the inhibition of su expression. This result suggests that well developed sequence databases from model plants like Arabidopsis thaliana, Nicotiana benthamiana, N. tabacum, Lycopersicon esculentum and others could be used as a major source of information and sequences for functional genomics in cassava. Furthermore, a fragment of the cassava CYP79D2endogenous gene, sharing 89% homology with CYP79D1endogenous gene was inserted into the ACMV vector. The resultant vector was inducing the down regulation of the expression of these two genes which catalyze the first-dedicated step in the synthesis of linamarin, the major cyanogenic glycoside in cassava. At 21 days post-inoculation (dpi), a 76% reduction of linamarin content was observed in silenced leaves. Using transgenic plants expressing antisense RNA of CYP79D1and CYP79D2, Siritunga and Sayre (2003) obtained several lines with a reduction level varying from 60% to 94%. This result provides the first example of direct comparison of the efficiency of a virus-induced gene silencing (VIGS) system and the transgenic approach for suppression of a biosynthetic pathway. The ACMV VIGS system will certainly be a complement and in some cases an alternative to the transgenic approach, for gene discovery and gene function analysis in cassava.


cassava linamarin sulfur gene (suvirus induced gene silencing (VIGS) 



base pair


days post-inoculation








Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Akano, O., Dixon, O., Mba, C., Barrera, E., Fregene, M. 2002Genetic mapping of a dominant gene conferring resistance to cassava mosaic diseaseTheor. Appl. Genet.105521525CrossRefPubMedGoogle Scholar
  2. Baulcombe, D.C. 1999Fast forward genetics based on virus-induced gene silencingCurr. Opin. Plant Biol.2109113CrossRefPubMedGoogle Scholar
  3. Bellotti, A.C., Bohórquez, A., Arias, B., Vargas, J., Vargas, H.L., Mba, C., Duque, M.C. and Tohme, J. 2004. Recent advances in the identification of genes conveying whitefly (Aleurotrachelus socialis Bondar; Homoptera: Aleyrodidae) in cassava (Manihot esculenta Crantz). In: International Scientific Meeting of the Cassava Biotechnology Network. Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia, p. 119.Google Scholar
  4. Bradbury, M.G., Egan, S.V., Bradbury, J.H. 1999Picrate paper kits for determination of total cyanogens in cassava roots and all forms of cyanogens in cassava productsJ. Sci. Food Agric.79595601CrossRefGoogle Scholar
  5. Burton, R.A., Gibeaut, D.M., Bacic, A., Findlay, K., Roberts, K., Hamilton, A., Baulcombe, D.C., Fincher, G.B. 2000Virus-induced silencing of a plant cellulose synthase genePlant Cell12691706CrossRefPubMedGoogle Scholar
  6. Dellaporta, S.L., Wood, J., Hicks, J.B. 1983A plant DNA minipreparation: version IIPlant Mol. Biol. Rep.11921Google Scholar
  7. Egan, S.V., Yeoh, H.H., Bradbury, J.H. 1998Simple picrate paper kit for determination of the cyanogenic potential of cassava flourJ. Sci. Food Agric.763948CrossRefGoogle Scholar
  8. Fauquet, C., Fargette, D. 1990African cassava mosaic virus: etiology epidemiology, and controlPlant Dis.74404411Google Scholar
  9. Fauquet, C., Fargette, D., Thouvenel, J.C. 1988Some aspects of the epidemiology of African cassava mosaic virus in Ivory CoastTrop. Pest Manage.349296Google Scholar
  10. Fondong, V.N., Pita, J.S., Rey, M.E., de Kochko, A., Beachy, R.N., Fauquet, C.M. 2000Evidence of synergism between African cassava mosaic virus and a new double-recombinant geminivirus infecting cassava in CameroonJ. Gen. Virol.81287297PubMedGoogle Scholar
  11. Gosselé, V., Faché, I., Meulewaeter, F., Cornelissen, M., Metzlaff, M. 2002SVISS–a novel transient gene silencing system for gene function discovery and validation in tobacco plantsPlant J.32859866CrossRefPubMedGoogle Scholar
  12. Holzberg, S., Brosio, P., Gross, C., Pogue, G.P. 2002Barley stripe mosaic virus-induced gene silencing in a monocot plantPlant J. Cell Mol. Biol.30315327Google Scholar
  13. Jones, L., Hamilton, A.J., Voinnet, O., Thomas, C.L., Maule, A.J., Baulcombe, D.C. 1999RNA-DNA interactions and DNA methylation in post-transcriptional gene silencingPlant Cell1122912301CrossRefPubMedGoogle Scholar
  14. Kjemtrup, S., Sampson, K.S., Peele, C.G., Nguyen, L.V., Conkling, M.A., Thompson, W.F., Robertson, D. 1998Gene silencing from plant DNA carried by a geminivirusPlant J. Cell Mol. Biol.1491100Google Scholar
  15. Kumagai, M.H., Donson, J., Della-Cioppa, G., Harvey, D., Hanley, K., Grill, L.K. 1995Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNAProc. Natl. Acad. Sci. USA9216791683PubMedGoogle Scholar
  16. Liu, Y., Schiff, M., Dinesh-Kumar, S.P. 2002aVirus-induced gene silencing in tomatoPlant J.31777786Google Scholar
  17. Liu, Y.L., Schiff, M., Marathe, R., Dinesh-Kumar, S.P. 2002bTobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virusPlant J.30415429Google Scholar
  18. Llano, G.A., Alvarez, E., Fregene, M. and Muñoz, J.E. 2004. Identification of resistance-gene analogs in cassava (Manihot esculenta Crantz), and their relationship to three Phytophthora species. In: International Scientific Meeting of the Cassava Biotechnology Network. Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia, p. 121.Google Scholar
  19. Lopez, C., Jorge, V., Mba, C., Cortes, D., Soto, M., Restrepo, S., Piégu, B., Cooke, R., Delseny, M., Tohme, J. and Verdier, V. 2004. A catalogue of 6000 expressed genes in cassava: identification of genes implicated in cassava bacterial blight resistance and starch biosynthesis. In: International Scientific Meeting of the Cassava Biotechnology Network. Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia, p. 120Google Scholar
  20. Mak, Y.M., Ho, K.K. 1995Sequence of cassava ribulose-1,5-bisphosphate carboxylase small subunit precursor cDNADNA Seq5229232PubMedGoogle Scholar
  21. Monger, W.A., Seal, S., Cotton, S., Foster, G.D. 2001aIdentification of different isolates of cassava brown streak virus and development of a diagnostic testPlant Pathol.50768775Google Scholar
  22. Monger, W.A., Spence, N.J., Foster, G.D. 2001bMolecular evidence that the aphid-transmitted Tomato mild mottle virus belongs to the Potyviridae family but not the Potyvirus genusArch. Virol.14624352441Google Scholar
  23. Nweke, F., Dixon, A.G.O., Asiedu, R., Folayan, S.A. 1994Cassava varietal needs of farmers and the potential for production growth in Africa. COSCA working paper No. 10. Collaborative study of cassava in AfricaIn: International Institute of Tropical AgricultureIbadan, NigeriaGoogle Scholar
  24. Peele, C., Jordan, C.V., Muangsan, N., Turnage, M., Egelkrout, E., Eagle, P., Hanley-Bowdoin, L., Robertson, D. 2001Silencing of a meristematic gene using geminivirus-derived vectorsPlant J. Cell Mol. Biol.27357366Google Scholar
  25. Pita, J.S., Fondong, V.N., Sangaré, A., Kokora, R.N.N., Fauquet, C.M. 2001aGenomic and biological diversity of the African cassava geminivirusesEuphytica: Netherlands J. Plant Breed.120115125Google Scholar
  26. Pita, J.S., Fondong, V.N., Sangare, A., Otim-Nape, G.W., Ogwal, S., Fauquet, C.M. 2001bRecombination, pseudorecombination and synergism of geminiviruses are determinant keys to the epidemic of severe cassava mosaic disease in UgandaJ. Gen. Virol.82655665Google Scholar
  27. Ratcliff, F.G., MacFarlane, S.A., Baulcombe, D.C. 1999Gene silencing without DNA: RNA-mediated cross-protection between virusesPlant Cell1112071215PubMedGoogle Scholar
  28. Ratcliff, F., Martin-Hernandez, A.M., Baulcombe, D.C. 2001Tobacco rattle virus as a vector for analysis of gene function by silencingPlant J.25237245PubMedGoogle Scholar
  29. Ruiz, M.T., Voinnet, O., Baulcombe, D.C. 1998Initiation and maintenance of virus-induced gene silencingPlant Cell10937946PubMedGoogle Scholar
  30. Salcedo, A., Mancilla, L., Cortés, D., Chavariaga, P. and Tohme, J. 2004. Cloning carotene synthesis genes from cassava roots. In: International Scientific Meeting of the Cassava Biotechnology Network. Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia, p. 122Google Scholar
  31. Siritunga, D., Sayre, R.T. 2003Generation of cyanogen-free transgenic cassavaPlanta217367373PubMedGoogle Scholar
  32. Storey, H.H., Nichols, R.F.W. 1938Studies of the mosaic disease of cassavaAnn. Appl. Biol.25790806Google Scholar
  33. Thomas, C.L., Jones, L., Baulcombe, D.C., Maule, A.J. 2001Size constraints for targeting post-transcriptional gene silencing and for RNA-directed methylation in Nicotiana benthamiana using a potato virus X vectorPlant J.25417425PubMedGoogle Scholar
  34. Turnage, M.A., Muangsan, N., Peele, C.G., Robertson, D. 2002Geminivirus-based vectors for gene silencing in ArabidopsisPlant J. Cell Mol. Biol.30107114Google Scholar
  35. Vanitharani, R., Chellappan, P., Pita, J.S. and Fauquet, C.M. 2004. Differential roles of AC2 and AC4 of cassava geminiviruses in mediating synergism and posttranscriptional gene silencing suppression. J. Virol. In pressGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Ismael B. F. Fofana
    • 1
    • 2
    • 3
  • Abdourahamane Sangaré
    • 4
  • Ray Collier
    • 1
  • Christopher Taylor
    • 1
  • Claude M. Fauquet
    • 1
    • 2
    • 2
    Email author
  1. 1.Donald Danforth Plant Science Center (DDPSC)St LouisUSA
  2. 2.International Laboratory for Tropical Agricultural Biotechnology (ILTAB/DDPSC)St LouisUSA
  3. 3.UFR des Biosciences, Laboratoire de GénétiqueUniversité de CocodyAbidjan 22
  4. 4.Laboratoire Central de BiotechnologieCentre National de Recherche Agronomique (CNRA)Abidjan 01

Personalised recommendations