A High Throughput Barley Stripe Mosaic Virus Vector for Virus Induced Gene Silencing in Monocots and Dicots

Abstract

Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.

Long noncoding miRNA gene represses wheat β-diketone waxes

“…Fragments of W1-COE were integrated into a VIGS system using the barley stripe mosaic virus (35) and were applied to the leaves of glaucous AG2 plants at the tillering stage, before visible glaucousness was apparent. Then the development of glaucousness was monitored for 4-6 wk.…”

Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants

“…FoMV-based VIGS was performed by using N. benthamiana as an intermediate host as described (Yu et al, 2003;Yuan et al, 2011). FoMVbased constructs were introduced into Agrobacterium tumefaciens strain GV3101.…”

“…By contrast, to date only four RNA viruses and one DNA virus have been modified as vectors for VIGS in monocot species, of which, Barley stripe mosaic virus (BSMV)-based VIGS has been applied for functional genomics in barley (Hordeum vulgare L.) and wheat (Triticum aestivum; Holzberg et al, 2002;Tai et al, 2005;Meng et al, 2009;Pacak et al, 2010;Yuan et al, 2011); Brome mosaic virus (BMV) in rice (Oryza sativa L.), barley, and maize (Zea mays L.; Ding et al, 2006); Bamboo mosaic virus and its satellite RNA in N. benthamiana and Brachypodium distachyon (Liou et al, 2014); and Rice tungro bacilliform virus in rice (Purkayastha et al, 2010). Very recently, Cucumber mosaic virus-based VIGS in maize is reported (Wang et al, 2016) BMV and BMV-based vectors are frequently used for VIGS in some monocot plants.…”

<i>Peanut stunt virus</i>-induced gene silencing in white lupin (<i>Lupinus albus</i>)

“…The results indicate that there are size constraints of insert fragments in the PSV VIGS vector and that 50-190 bp is the optimal length needed to induce silencing. The size constraints of insert fragments have also been reported in other viral vector systems, where vectors with larger inserts are more susceptible to losing the inserts within the plants (Hsieh et al 2013;Huang et al 2009;Igarashi et al 2009;Liu and Page 2008;Peele et al 2001;SenthilKumar and Mysore 2011b;Yuan et al 2011).…”