Virus-induced gene silencing (VIGS) of genes expressed in root, leaf, and meiotic tissues of wheat
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Barley stripe mosaic virus (BSMV)-based virus-induced gene silencing (VIGS) is an effective strategy for rapid functional analysis of genes in wheat leaves, but its utility to transiently express genes, and silencing in other tissues including root, flower, and developing grains, has not been demonstrated in monocots. We monitored green fluorescent protein (GFP) expression to demonstrate the utility of BSMV as a transient expression vector and silenced genes in various wheat tissues to expand VIGS utility to characterize tissue-specific genes. An antisense construct designed for coronatine insensitive1 (COI1) showed an 85% decrease in COI1 transcript level in roots accompanied by a 26% reduction in root length. Similarly, silencing of seed-specific granule-bound starch synthase by antisense and hairpin constructs resulted in up to 82% reduction in amylose content of the developing grains. VIGS of meiosis-specific genes demonstrated by silencing wheat homologue of disrupted meiosis cDNA1 (DMC1) by an antisense construct resulted in a 75–80% reduction in DMC1 transcript level accompanied by an average of 37.2 univalents at metaphase I. The virus-based transient GFP expression was observed in the leaf, phloem, and root cortex at 10–17 days post-inoculation. A novel observation was made that 8–11% of the first selfed generation progeny showed VIGS inheritance and that this proportion increased to 53–72% in the second and to 90–100% in the third generations. No viral symptoms were observed in the progeny, making it possible to study agronomic traits by VIGS. VIGS inheritance is particularly useful to study genes expressing during seed germination or other stages of early plant growth.
KeywordsVIGS Wheat BSMV Gene silencing VIGS inheritance Transient gene expression
The authors thank Dr. Gregory Pogue (Large Scale Biology Corporation, CA, USA) for providing VIGS vectors and Dr. Simon Chuong (Franchesci Electron Microscopy Center, Washington State University) for expert assistance with confocal microscopy. This work was supported by Washington Wheat Commission, The Vogel Endowment funds, and USDA ARS Project no. 5248-22000-012-00D (P.O.).
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