A new BSMV-based vector with modified β molecule allows simultaneous and stable silencing of two genes

Abstract

Virus-induced gene silencing is an important tool for functional gene analysis and the vector based on Barley stripe mosaic virus (BSMV) is widely used for the purpose in monocots. Of the tripartite BSMV genome, currently the BSMV:γMCS molecule is used to clone a fragment of a target gene. As an alternative, the BSMV:β molecule was engineered with a unique BamHI site between the open reading frame of βc (ORF βc) and poly(A). The mixture of RNA particles α, βBamHI and γMCS was fully infectious. Barley phytoene desaturase and wheat phospholipase Dα fragments were cloned to βBamHI and γMCS. Delivery of the target gene fragment in γMCS induced stronger silencing, while delivery in βBamHI yielded more stable transcript reduction. A quantitative analysis (qRT-PCR) of the transcripts showed that the silencing induced with a fragment carried in both particles was stronger and more stable than that from a fragment placed in one particle. The modification of β enables simultaneous silencing of two genes. Quantifying the β and γ particles in virusinoculated plants revealed a 2.5-fold higher level of γ than β, while the stability of the insert was higher in β compared with γ. The possible influence of the relative quantity of β and γ particles in virus-inoculated plants on insert stability and gene silencing efficiency is discussed.

Identification and VIGS-based characterization of Bx1 ortholog in rye (Secale cereale L.)

“…It was first developed in dicots [ 25 ] and by constructing vectors derived from Barley Stripe Mosaic Virus (BSMV) the method was adapted for barley [ 26 ] and later for wheat [ 27 ], Brachypodium distachyon [ 28 ] and oat [ 29 ]. The method can be used to silence a single gene or simultaneously two different genes [ 30 ] and can be applied to genes expressed in root, leaves [ 31 ], spikes and grains [ 32 ]. VIGS approach has been used to verify gene function in biochemical pathway of morphine biosynthesis [ 33 ].…”

Recent Advancements in Gene Expression and Enabling Technologies in Crop Plants

“…When combined with wild-type RNAα and the RNAγ VIGS vector developed by Bruun-Rasmussen et al (2007; see also Table 14.1 for details) this facilitated simultaneous silencing of two unrelated genes when fragments targeting different genes were inserted into each of the cloning sites on RNAβ and RNAγ. However, delivery of a foreign gene fragment from RNAβ appeared to induce weaker silencing than when gene fragments were inserted into RNAγ (Kawalek et al 2012).…”

“…This modification also blocked translation of the BSMV γb silencing suppressor. Kawalek et al (2012) introduced a cloning site for foreign sequences at the end of the βc ORF in the plasmid representing BSMV RNAβ. When combined with wild-type RNAα and the RNAγ VIGS vector developed by Bruun-Rasmussen et al (2007; see also Table 14.1 for details) this facilitated simultaneous silencing of two unrelated genes when fragments targeting different genes were inserted into each of the cloning sites on RNAβ and RNAγ.…”

Transient Virus Expression Systems for Recombinant Protein Expression in Dicot- and Monocotyledonous Plants

“…In addition, BSMV mediated VIGS can be inherited and has been observed for up to 6 generations in wheat and barley, which not only enhances the range of phenotypes that can be explored for reasons related to the timing of developmental events, but also because progeny frequently have fewer viral symptoms [87,96]. Most recently, the  RNA has been modified to incorporate untranslated foreign gene fragments and was used with the modified  RNA vector described above to silence two endogenous genes simultaneously, or to achieve enhanced silencing of a single gene [97]. In addition to BSMV, a strain of BMV has also been engineered as a VIGS vector and has been used successfully in barley, rice and maize [98,99].…”