Virus-induced gene silencing in rice using a vector derived from a DNA virus

Abstract

Virus-induced gene silencing (VIGS) is a method of rapid and transient gene silencing in plants using viral vectors. A VIGS vector for gene silencing in rice has been developed from Rice tungro bacilliform virus (RTBV), a rice-infecting virus containing DNA as the genetic material. A full-length RTBV DNA cloned as a partial dimer in a binary plasmid accumulated in rice plants when inoculated through Agrobacterium (agroinoculation) within 2 weeks and produced detectable levels of RTBV coat protein. Deletion of two of the four viral ORFs did not compromise the ability of the cloned RTBV DNA to accumulate in rice plants. To modify the cloned RTBV DNA as a VIGS vector (pRTBV-MVIGS), the tissue-specific RTBV promoter was replaced by the constitutively expressed maize ubiquitin promoter, sequences comprising the tRNA-binding site were incorporated to ensure reverse transcription-mediated replication, sequences to ensure optimal context for translation initiation of the viral genes were added and a multi-cloning site for the ease of cloning DNA fragments was included. The silencing ability of pRTBV-MVIGS was tested using the rice phytoene desaturase (pds) gene on rice. More than half of the agroinoculated rice plants showed white streaks in leaves within 21 days post-inoculation (dpi), which continued to appear in all emerging leaves till approximately 60-70 dpi. Compared to control samples, real-time PCR showed only 10-40% accumulation of pds transcripts in the leaves showing the streaks. This is the first report of the construction of a VIGS vector for rice which can be introduced by agroinoculation.

Foxtail Mosaic Virus-Induced Gene Silencing in Monocot Plants

“…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.…”

Recent Advances on Virus Induced Gene Silencing (VIGS): Plant Functional Genomics

“…Rice tungro bacilliform virus (RTBV) has been developed as a rice-infecting virus containing DNA as thegenetic material. Agrobacterium (agroinoculation) mediated transfection accumulated a full-length RTBV DNA cloned as apartial dimer in a binary plasmid and produced detectable RTBV coat proteins within 2 weeks of infection [8]. Cucumber mosaic virus vector (CMV-A1) was used to analyseVIGS containing partial sequence of AINTEGUMENTA gene of Antirrhinum majus (Am-ANT) that inhibited the expression level of Am-ANT mRNA when infected by the vector.…”

Assessment of Banana Streak MY virus-based infectious clone vectors in Musa ssp.

“…To improve its infectivity, additional modifications replaced the native RTBV tissue-specific viral promoter with the constitutive maize ubiquitin promoter to allow expression in all plant tissues. Other sequences such as the tRNA-binding site to allow reverse transcription during replication and those that were necessary for optimal translation initiation of the viral genes were also added (Purkayastha et al, 2010).…”

“…RTBV has been modified as an expression vector by deleting ORF 1 and 2 which have been demonstrated to be dispensable for infection (Purkayastha et al, 2010).…”

“…Other sequences such as the tRNA-binding site to allow reverse transcription during replication and those that were necessary for optimal translation initiation of the viral genes were also added (Purkayastha et al, 2010 (Dawson et al, 1986;Dawson et al, 1988;Purkayastha et al, 2010;Scholthof et al, 1996). As such, research into the effects of gene deletion on the infectivity and symptom development on modified viral vectors is needed.…”