Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat

Abstract

BackgroundGene silencing vectors based on Barley stripe mosaic virus (BSMV) are used extensively in cereals to study gene function, but nearly all studies have been limited to genes expressed in leaves of barley and wheat. However since many important aspects of plant biology are based on root-expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created a need for tools to study gene function in these species.ResultsHere we demonstrate the successful BSMV-mediated virus induced gene silencing (VIGS) of three different genes in barley roots, i.e. the barley homologues of the IPS1, PHR1, and PHO2 genes known to participate in Pi uptake and reallocation in Arabidopsis. Attempts to silence two other genes, the Pi transporter gene HvPht1;1 and the endo-β-1,4-glucanase gene HvCel1, in barley roots were unsuccessful, probably due to instability of the plant gene inserts in the viral vector. In B. distachyon leaves, significant silencing of the PHYTOENE DESATURASE (BdPDS) gene was obtained as shown by photobleaching as well as quantitative RT-PCR analysis. On the other hand, only very limited silencing of the oat AsPDS gene was observed in both hexaploid (A. sativa) and diploid (A. strigosa) oat. Finally, two modifications of the BSMV vector are presented, allowing ligation-free cloning of DNA fragments into the BSMV-γ component.ConclusionsOur results show that BSMV can be used as a vector for gene silencing in barley roots and in B. distachyon leaves and possibly roots, opening up possibilities for using VIGS to study cereal root biology and to exploit the wealth of genome information in the new cereal model plant B. distachyon. On the other hand, the silencing induced by BSMV in oat seemed too weak to be of practical use. The new BSMV vectors modified for ligation-free cloning will allow rapid insertion of plant gene fragments for future experiments.

Brachypodium as a Model for the Grasses: Today and the Future

“…Both approaches have been used with Brachypodium (Olsen et al, 2006;Demircan and Akkaya, 2009;Pacak et al, 2010), and recently, a T-DNA mutation in Brachypodium has been complemented with an Arabidopsis ortholog, bridging dicotyledonous and monocotyledonous models (Vain et al, 2011). In addition, biotechnological approaches for crop improvement can be tested by introducing and expressing heterologous genes (e.g.…”

Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants

“…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. However, both virus vectors do not work in many other monocot species and not all cultivars within a particular host species (Ding et al, 2006;Pacak et al, 2010).…”

“…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.…”

SILENCING OF G-PROTEIN -SUBUNIT IN BARLEY (Hordeum vulgare L.) BY VIGS AND ITS EFFECT ON THE PLANT GROWTH

“…Notably, we found complete silencing of pSL038-1/G two weeks after inoculation in barley plants. A similar study has shown that BSMV-VIGS can be used for down regulation of important genes in B. distachyon (Pacak et al, 2010). VIGS is a fast and reliable method for stable knock-down of a desired gene which would produce a phenotype; therefore it could be employed as a quick method for cereal species where stable genetic transformation is required but a very laborious choice (Wege et al, 2007;Ma et al, 2012).…”