Barley stripe mosaic virus‐induced gene silencing in a monocot plant

Abstract

SummaryRNA silencing of endogenous plant genes can be achieved by virus-mediated, transient expression of homologous gene fragments. This powerful, reverse genetic approach, known as virus-induced gene silencing (VIGS), has been demonstrated only in dicot plant species, where it has become an important tool for functional genomics. Barley stripe mosaic virus (BSMV) is a tripartite, positive-sense RNA virus that infects many agriculturally important monocot species including barley, oats, wheat and maize. To demonstrate VIGS in a monocot host, we modi®ed BSMV to express untranslatable foreign inserts downstream of the gb gene, in either sense or antisense orientations. Phytoene desaturase (PDS) is required for synthesizing carotenoids, compounds that protect chlorophyll from photo-bleaching. A partial PDS cDNA ampli®ed from barley was 90, 88 and 74% identical to PDS cDNAs from rice, maize and Nicotiana benthamiana, respectively. Barley infected with BSMV expressing barley, rice or maize PDS fragments became photo-bleached and accumulated phytoene (the substrate for PDS) in a manner similar to plants treated with the chemical inhibitor of PDS, nor¯urazon. In contrast, barley infected with wild-type BSMV, or BSMV expressing either N. benthamiana PDS or antisense green¯uorescent protein (GFP), did not photo-bleach or accumulate phytoene. Thus BSMV silencing of the endogenous PDS was homology-dependent. Deletion of the coat protein enhanced the ability of BSMV to silence PDS. This is the ®rst demonstration of VIGS in a monocot, and suggests that BSMV can be used for functional genomics and studies of RNA-silencing mechanisms in monocot plant species.

The phenotypic expression of QTLs for partial resistance to barley leaf rust during plant development

“…Moreover, multiple disease evaluations can be conducted for Rphq4 in individual plants by inoculation of the fourth leaf to the Xag leaf, respectively, enabling a more reliable evaluation of Rphq4 in a single plant. Barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) is an important tool for the analysis of gene function in barley (Holzberg et al 2002;Lacomme et al 2003) and has been successfully used to deWne the function of important genes (Hein et al 2005;Shen et al 2007). In BSMV-VIGS system, photobleaching symptoms were usually observed approximately 3 weeks after the viral inoculation.…”

The wheat NB‐LRR gene TaRCR1 is required for host defence response to the necrotrophic fungal pathogen Rhizoctonia cerealis

“…At the three‐leaf stage, at least 20 plants of CI12633 were inoculated with BSMV:TaRCR1 or BSMV:00 (as a control) following Holzberg et al . (2002). At 10 day after virus infection, the fourth leaves were collected to monitor BSMV infection and to evaluate the transcript changes of TaRCR1 .…”

RNA interference for wheat functional gene analysis

“…The upper limit for RNAi trigger size is less defined, with successful examples in wheat of up to 683 bp (Li et al 2005) and in barley VIGS of up to 1,215 bp (Holzberg et al 2002). The determination of the upper limit for RNAi trigger size may be also related to the number of targets that can be effectively included in a single RNAi construct to knockdown multiple target transcripts.…”

“…To date, VIGS has had broad application in dicots (Burch-Smith et al 2004) and has also proven to be feasible in barley when using engineered Barley Stripe Mosaic Virus (BSMV) vectors (Holzberg et al 2002). Using PDS and the visible photobleaching phenotype, Holzberg et al (2002) showed that the BSMV-VIGS system elicits target-directed gene silencing in barley in a sequence dependent manner.…”

“…Using PDS and the visible photobleaching phenotype, Holzberg et al (2002) showed that the BSMV-VIGS system elicits target-directed gene silencing in barley in a sequence dependent manner. Similar results were extended recently to common wheat (Scofield et al 2005).…”