A universal primer for isolation of fragments of a gene encoding phytoene desaturase for use in virus-induced gene silencing (VIGS) studies
Yin-Chih, L., Imsabai, W. , Reid, M. S., Cai-Zhong, J. and Wagstaff, C. (2010) A universal primer for isolation of fragments of a gene encoding phytoene desaturase for use in virus-induced gene silencing (VIGS) studies. Acta Horticulturae, 877. pp. 1697-1704. ISSN 0567-7572
Official URL: http://www.actahort.org/books/877/877_233.htm
We have been using Virus-Induced Gene Silencing (VIGS) to test the function of genes that are candidates for involvement in floral senescence. Although VIGS is a powerful tool for assaying the effects of gene silencing in plants, relatively few taxa have been studied using this approach, and most that have are in the Solanaceae. We typically use silencing of phytoene desaturase (PDS) in preliminary tests of the feasibility of using VIGS. Silencing this gene, whose product is involved in carotene biosynthesis, results in a characteristic photobleaching phenotype in the leaves. We have found that efficient silencing requires the use of fragments that are more than 90% homologous to the target gene. To simplify testing the effectiveness of VIGS in a range of species, we designed a set of universal primers to a region of the PDS gene that is highly conserved among species, and that therefore allows an investigator to isolate a fragment of the homologous PDS gene from the species of interest. We report the sequences of these primers and the results of VIGS experiments in horticultural species from the Asteraceae, Leguminosae, Balsaminaceae and Solanaceae.
Baulcombe, D.C. 1999. Fast forward genetics based on virus-induced gene silencing. Current Opinion in Plant Biology 2:109-123. Burch-Smith, T.M., Anderson, J.C., Martin, G.B. and Dinesh-Kumar, S.P. 2004. Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant Journal 39(5):734-746. Chen, J.C., Jiang, C.Z., Gookin, T.E., Hunter, D.A., Clark, D.G. and Reid, M.S. 2004. Chalcone synthase as a reporter in virus-induced gene silencing studies of flower senescence. Plant Mol. Biol. 55:521-530. Chen, J.C., Johnson, F., Clark, D.G., Gookin, T. and Reid, M.S. 2003. Potential application of virus-induced gene silencing (VIGS) in flower senescence studies. Acta Hort. 669:147-152. Chicas, A. and Macino, G. 2001. Characteristics of post-transcriptional gene silencing. EMBO Reports 2:992-996. Gould, B. and Kramer, E.M. 2007. Virus-induced gene silencing as a tool for functional analyses in the emerging model plant Auilegia (columbine, Ranunculaceae). Plant Methods 3:6. Liu, Y., Schiff, M., Marathe, R. and Dinesh-Kumar, S.P. 2002a. Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J. 30:415-429. Liu, Y., Schiff, M. and Dinesh-Kumar, S.P. 2002b. Virus-induced gene silencing in tomato. Plant J. 31:777-786. Wege, S., Scholz, A., Gleissberg, S. and Becke, A. 2007. Highly efficient virus-induced gene silencing (VIGS) in California poppy (Eschscholzia californica): An evaluation of VIGS as a strategy to obtain functional data from non-model plants. Annals of Botany 100:641-649.