Abstract
Traditionally, developmental studies in plant biology have suffered from the lack of a convenient means to study gene function in non-model plant species. Here we show that virus-induced gene silencing (VIGS) is an effective new tool to study the function of orthologs of floral homeotic genes such as DEFICIENS(DEF) in non-model systems. We used a tobacco rattle virus (TRV)-based VIGS approach to study the function of the Nicotiana benthamiana DEFortholog (NbDEF). Silencing of NbDEFin N. benthamianausing TRV-VIGS was similar to that of Antirrhinum defand Arabidopsis ap3mutants and caused transformation of petals into sepals and stamens into carpels. Molecular analysis of the NbDEF-silenced plants revealed a dramatic reduction of the levels of NbDEFmRNA and protein in flowers. NbDEFsilencing was specific and has no effect on the mRNA levels of NbTM6, the closest paralog of NbDEF. A dramatic reduction of the levels of N. benthamiana GLOBOSA(NbGLO) mRNA and protein was also observed in flowers of NbDEF-silenced plants, suggesting that cross-regulation of this GLO-like gene by NbDEF. Taken together, our results suggest that NbDEF is a functional homolog of Antirrhinum DEF. Our results are significant in that they show that TRV efficiently induces gene silencing in young and differentiating flowers and that VIGS is a promising new tool for analyses of developmental gene function in non-model organisms.
Similar content being viewed by others
References
Angenent, G.C., Busscher, M., Franken, J., Dons, H.J. and van Tunen, A.J. 1995. Functional interaction between the homeotic genes fbp1 and pMADS1 during petunia floral organogenesis. Plant Cell 7: 507-516.
Angenent, G.C., Franken, J., Busscher, M., Colombo, L. and van Tunen, A.J. 1993. Petal and stamen formation in petunia is regulated by the homeotic gene fbp1. Plant J. 4: 101-112.
Baulcombe, D.C. 1999. Fast forward genetics based on virus-induced gene silencing. Curr. Op Plant Biol. 2: 109-113.
Bowman, J.L., Smyth, D.R. and Meyerowitz, E.M. 1989. Genes directing flower development in Arabidopsis. Plant Cell 1: 37-52.
Canas, L.A., Busscher, M., Angenent, G.C., Beltran, J.P. and Tunen, A.J. 1994. Nuclear localization of the petunia MADS box protein FBP1. Plant J. 6: 597-604.
Carpenter, R., Copsey, L., Vincent, C., Doyle, S., Magrath, R. and Coen, E. 1995. Control of flower development and phyllotaxy by meristem identity genes in antirrhinum. Plant Cell 7: 2001-2011.
Coen, E.S. and Meyerowitz, E.M. 1991. The war of the whorls: genetic interactions controlling flower development. Nature 353: 31-37.
Davies, B., Di Rosa, A., Eneva, T., Saedler, H. and Sommer, H. 1996a. Alteration of tobacco floral organ identity by expression of combinations of Antirrhinum MADS-box genes. Plant J. 10: 663-677.
Davies, B., Egea-Cortines, M., de Andrade Silva, E., Saedler, H. and Sommer, H. 1996b. Multiple interactions amongst floral homeotic MADS box proteins. EMBO J. 15: 4330-4343.
Garcia-Maroto, F., Salamini, F. and Rohde, W. 1993. Molecular cloning and expression patterns of three alleles of the Deficiens-homologous gene St-Deficiens from Solanum tuberosum. Plant J. 4: 771-780.
Gomez, P., Jamilena, M., Capel, J., Zurita, S., Angosto, T. and Lozano, R. 1999. Stamenless, a tomato mutant with homeotic conversions in petals and stamens. Planta 209: 172-179.
Goto, K. and Meyerowitz, E.M. 1994. Function and regulation of the Arabidopsis floral homeotic gene PISTILLATA. Genes Dev. 8: 1548-1560.
Huijser, P., Klein, J., Lonning, W.E. Meijer, H., Saedler, H. and Sommer, H. 1992. Bracteomania, an inflorescence anomaly, is caused by the loss of function of the MADSbox gene squamosa in Antirrhinum majus. EMBO J. 11: 1239-1249.
Hull, R. 2002. Matthews' Plant Virology. Fourth edition, Academic Press, NY.
Jack, T., Brockman, L.L. and Meyerowitz, E.M. 1992. The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens. Cell 68: 683-697.
Jenik, P.D. and Irish, V.F. 2000. Regulation of cell proliferation patterns by homeotic genes during Arabidopsis floral development. Development 127: 1267-1276.
Jenik, P.D. and Irish, V.F. 2001. The Arabidopsis floral homeotic gene APETALA3 differentially regulates intercellular signaling required for petal and stamen development. Development 128: 13-23.
Jofuku, K.D., den Boer, B.G.W., Montagu, M.V. and Okamuro, J.K. 1994. Control of Arabidopsis flower and seed development by the homeotic gene APETALA2. Plant Cell 6: 1211-1225.
Kramer, E.M., Dorit, R.L. and Irish, V.F. 1998. Molecular evolution of genes controlling petal and stamen development: duplication and divergence within the APETALA3 and PISTILLATA MADS-box gene lineages. Genetics 149: 765-783.
Kramer, E.M. and Irish, V.F. 1999. Evolution of genetic mechanisms controlling petal development. Nature 399: 144-148.
Liu, Y., Schi., M. and Dinesh-Kumar, S.P. 2002a. Virus-induced gene silencing in tomato. Plant J. 31: 777-786.
Liu, Y., Schi., M., Marathe, R. and Dinesh-Kumar, S.P. 2002b. Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J. 30: 415-429.
Lohmann, J.U. and Weigel, D. 2002. Building beauty: the genetic control of floral patterning. Dev. Cell 2: 135-142.
McGonigle, B., Bouhidel, K. and Irish, V.F. 1996. Nuclear localization of the Arabidopsis APETALA3 and PISTILLATA homeotic gene products depends on their simultaneous expression. Genes Dev. 10: 1812-1821.
Olmstead, R.G., Kim, K.J., Jansen, R.K. and Wagsta., S.J. 2000. The phylogeny of the Asteridae sensu lato based on chloroplast ndhF gene sequences. Mol. Phylogenet. Evol. 16: 96-112.
Olmstead, R.G. and Palmer, J.D. 1992. A chloroplast DNA phylogeny of the Solanaceae: subfamilial relationships and character evolution. Annu Missouri Bot. Garden. 79: 346-360.
Ratcli., F., Martin-Hernandez, A.M. and Baulcombe, D.C. 2001. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J. 25: 237-245.
Riechmann, J.L., Krizek, B.A. and Meyerowitz, E.M. 1996. Dimerization specificity of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA and AGAMOUS. Proc Natl. Acad. Sci. 93: 4793-4798.
Schwarz-Sommer, Z., Davies, B. and Hudson, A. 2003. An everlasting pioneer: the story of Antirrhinum research. Nat. Rev. Genet. 4: 657-66.
Schwarz-Sommer, Z., Hue, I., Huijser, P., Flor, P.J., Hansen, R., Tetens, F., Lonnig, W.E., Saedler, H. and Sommer, H. 1992. Characterization of the Antirrhinum floral homeotic MADS-box gene deficiens: evidence for DNA binding and autoregulation of its persistent expression throughout flower development. EMBO J. 11: 251-263.
Smith, H.H. 1979. The Nicotiana genus as a genetic resource. USDA Tech. Bull. No. 1586: 1-16.
Trobner, W., Ramirez, L., Motte, P., Hue, I., Huijser, P., Lonnig, W.E., Saedler, H., Sommer, H. and Schwarz-Sommer, Z. 1992. GLOBOSA: a homeotic gene which interacts with DEFICIENS in the control of Antirrhinum floral organogenesis. EMBO J. 11: 4693-4704.
Tsuchimoto, S., Mayama, T., van der Krol, A. and Ohtsubo, E. 2000. The whorl-specific action of a petunia class B floral homeotic gene. Genes Cells 5: 89-99.
Vander Krol, A.R., Brunelle, A., Tsuchimoto, S. and Chua, N.H. 1993. Functional analysis of petunia floral homeotic MADS box gene pMADS1. Genes Dev. 7: 1214-1228.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Liu, Y., Nakayama, N., Schiff, M. et al. Virus Induced Gene Silencing of a DEFICIENS Ortholog in Nicotiana Benthamiana . Plant Mol Biol 54, 701–711 (2004). https://doi.org/10.1023/B:PLAN.0000040899.53378.83
Issue Date:
DOI: https://doi.org/10.1023/B:PLAN.0000040899.53378.83