Abstract
Virus-Induced Gene Silencing (VIGS) is a useful method for transient downregulation of gene expression in crop plants. The geminivirus Cotton leaf crumple virus (CLCrV) has been modified to serve as a VIGS vector for persistent gene silencing in cotton. Here the use of Green Fluorescent Protein (GFP) is described as a marker for identifying silenced tissues in reproductive tissues, a procedure that requires the use of transgenic plants. Suggestions are given for isolating and cloning combinations of target and marker sequences so that the total length of inserted foreign DNA is between 500 and 750 bp. Using this strategy, extensive silencing is achieved with only 200–400 bp of sequence homologous to an endogenous gene, reducing the possibility of off-target silencing. Cotyledons can be inoculated using either the gene gun or Agrobacterium and will continue to show silencing throughout fruit and fiber development. CLCrV is not transmitted through seed, and VIGS is limited to genes expressed in the maternally derived seed coat and fiber in the developing seed. This complicates the use of GFP as a marker for VIGS because cotton fibers must be separated from unsilenced tissue in the seed to determine if they are silenced. Nevertheless, fibers from a large number of seeds can be rapidly screened following placement into 96-well plates. Methods for quantifying the extent of silencing using semiquantitative RT-PCR are given.
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References
Senthil-Kumar M, Mysore KS (2011) New dimensions for VIGS in plant functional genomics. Trends Plant Sci 16:656–665
Zhu X, Pattathil S, Mazumder K et al (2010) Virus-induced gene silencing offers a functional genomics platform for studying plant cell wall formation. Mol Plant 3:818–833
Tuttle JR, Idris AM, Brown JK et al (2008) Geminivirus-mediated gene silencing from cotton leaf crumple virus is enhanced by low temperature in Gossypium hirsutum. Plant Physiol 148:41–50
Qu J, Ye J, Geng YF et al (2012) Dissecting functions of KATANIN and WRINKLED1 in cotton fiber development by virus-induced gene silencing. Plant Physiol 160:738–748
Gao X, Shan L (2013) Functional genomic analysis of cotton genes with agrobacterium-mediated virus-induced gene silencing. Methods Mol Biol 975:157–165
Idris AM, Tuttle JR, Robertson D et al (2010) Differential cotton leaf crumple virus-VIGS-mediated gene silencing and viral genome localization in different Gossypium hirsutum genetic backgrounds. Physiol Mol Plant Pathol 75:13–22
Pang J, Zhu Y, Li Q et al (2013) Development of Agrobacterium-mediated virus-induced gene silencing and performance evaluation of four marker genes in Gossypium barbadense. PLoS One 8:e73211
Ascencio-Ibanez JT, Sozzani R, Lee TJ et al (2008) Global analysis of Arabidopsis gene expression uncovers a complex array of changes impacting pathogen response and cell cycle during geminivirus infection. Plant Physiol 148:436–454
Nagar S, Hanley-Bowdoin L, Robertson D (2002) Host DNA replication is induced by geminivirus infection of differentiated plant cells. Plant Cell 14:2995–3007
Peele C, Jordan CV, Muangsan N et al (2001) Silencing of a meristematic gene using geminivirus-derived vectors. Plant J 27:357–366
Tuttle JR (2011) Development of a virus-induced gene silencing system for cotton and its application for functional genomics in fiber. PhD thesis, North Carolina State University
Himber C, Dunoyer P, Moissiard G et al (2003) Transitivity-dependent and -independent cell-to-cell movement of RNA silencing. EMBO J 22:4523–4533
Pang SZ, Jan FJ, Gonsalves D (1997) Nontarget DNA sequences reduce the transgene length necessary for RNA-mediated tospovirus resistance in transgenic plants. Proc Natl Acad Sci U S A 94:8261–8266
Tuttle JR, Haigler CH, Robertson D (2012) Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system. Plant Methods 8:27
Sunilkumar G, Mohr L, Lopata-Finch E et al (2002) Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP. Plant Mol Biol 50:463–474
Haigler CH, Singh B, Wang G et al (2009) Genomics of cotton fiber secondary wall deposition and cellulose biogenesis. In: Paterson AH (ed) Genetics and genomics of cotton, vol 3, Plant genetics and genomics: crops and models. Springer, New York, NY, pp 385–417
Wang H, Qi M, Cutler AJ (1993) A simple method of preparing plant samples for PCR. Nucleic Acids Res 21:4153–4154
Acknowledgements
We thank Cotton Incorporated, Cary NC, and the National Science Foundation (Grant # IOS1025947) for support of research related to this technology.
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Tuttle, J.R., Haigler, C.H., Robertson, D.(. (2015). Virus-Induced Gene Silencing of Fiber-Related Genes in Cotton. In: Mysore, K., Senthil-Kumar, M. (eds) Plant Gene Silencing. Methods in Molecular Biology, vol 1287. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2453-0_16
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DOI: https://doi.org/10.1007/978-1-4939-2453-0_16
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