A Methodological Advance of Tobacco Rattle Virus-Induced Gene Silencing for Functional Genomics in Plants

Abstract

As a promising high-throughput reverse genetic tool in plants, virus-induced gene silencing (VIGS) has already begun to fulfill some of this promise in diverse aspects. However, review of the technological advancements about widely used VIGS system, tobacco rattle virus (TRV)-mediated gene silencing, needs timely updates. Hence, this article mainly reviews viral vector construction, inoculation method advances, important influential factors, and summarizes the recent applications in diverse plant species, thus providing a better understanding and advice for functional gene analysis related to crop improvements.

Keywords: TRV-VIGS; agroinfiltration; methodology modification; secondary inoculation; vector construction.

FIGURE 1

Virus-induced gene silencing (VIGS) mechanism. Phytoene desaturase (PDS) serves as an example. Upon infection, the T-DNA carrying the viral genome is transformed into the plant by Agrobacterium and then transcribed by the host’s RNA polymerase. RNA-dependent RNA Polymerase (RdRP) (yellow) produces double-stranded RNA (dsRNA) from the single-stranded RNA (ssRNA) viral transcript. The dsRNA is then recognized by DICER-like enzyme, Dicer (green) and cleaved into short interfering RNAs (siRNAs). Antisense siRNAs are recognized by RNA-induced silencing complex, RISC (red) and melted into ssRNAs, which then serve as templates for target gene degradation. The single-stranded siRNAs are amplified and spread as mobile silencing signals throughout the plant, thus resulting in target gene silencing in plant organs distant from the site of infection, symbolized by the photo-bleaching phenotype of the entire leaf. LB: left border; RB: right border; 2 × 35S: duplicated cauliflower mosaic virus 35S promoter; CP: coat protein; MCS: multiple cloning site; PDS: PDS cDNA fragment; Rz: self-cleaving ribozyme; NOS_t: nopaline synthase terminator.

FIGURE 2

The development process of TRV vector construction. Phytoene desaturase, PDS was used as an example. (A) Genome organization of tobacco rattle virus (TRV). The TRV1 open reading frames (ORFs) correspond to 134 and 194 kDa replicases, a movement protein (MP), and a 16 kDa cysteine-rich protein (16K). The TRV2 ORFs correspond to the coat protein (CP) and 29.4 and 32.8 kDa proteins (29.4K and 32.8K). (B) TRV-based VIGS vector (Ratcliff et al., 2001). Ratcliff et al. constructed separate cDNA clones of TRV (strain PPK20) RNA1 and RNA2 under the control of cauliflower mosaic virus (CaMV) 35S promoters on the transferred (T) DNA of plant binary transformation vectors. They replaced the non-essential 29.4K and 32.8K genes with a multiple cloning site (MCS), leaving only the 5′ and 3′ untranslated regions and the viral coat protein. The cDNA clones were positioned between the left and right border (LB and RB) of the T-DNA and between CaMV 35S promoters (35S) and transcriptional terminators (T). The TRV open reading frames corresponded to the RdRp, MP, 16K, CP, and 29.4K and 32.8K proteins. (C) TRV-based VIGS vector (Liu et al., 2002a,b; Tian et al., 2014). TRV1: TRV cDNA clones were placed between the duplicated CaMV 35S promoter (2 × 35S) and the nopaline synthase terminator (NOS_t) in a T-DNA vector. Rz, self-cleaving ribozyme. TRV2: TRV cDNA clones were placed between the duplicated CaMV 35S promoter (2 × 35S) and the nopaline synthase terminator (NOS_t) in a T-DNA vector, and PDS was added to the MCS between CP and Rz. (D) Modified pTRV2 vector based on GATEWAY cloning technology containing attR1 and attR2 recombination sites (Liu et al., 2002a). The PCR products flanked by attB1 and attB2 sequences directionally recombined in vitro at the attR1 and attR2 sites contained in the plasmid when incubated with the BP CLONASE enzyme. Then, the PDS gene was cloned into the pTRV2-attR1-attR2 vector. (E) TRV-Ligation-independent cloning (LIC) vector (Dong et al., 2007). The TRV-LIC vector was created by inserting a cassette, containing adapters and two PstI sites, in two digestion and ligation reactions. Then, the CDS insely digested enzyme for digestion and ligation. (F) TRV-GFP vector (Tian et al., 2014). GFP CDS was fused with CP in the TRV2 vector to generate an easily traceable TRV vector in different parts of plants.

FIGURE 3

Schematic diagram of different VIGS inoculation methods. The phytoene desaturase, PDS gene was used as an example. Inoculation method, application range, silencing efficiency, advantages and disadvantages, and corresponding references are all included. TRV: Tobacco rattle virus; BSMV: Barley stripe mosaic virus.

FIGURE 4

Important historical milestones of VIGS system. TRV: Tobacco rattle virus; BSMV: Barley stripe mosaic virus; LIC: Ligation-independent cloning; SSA-VIGS: Seed sock agroinoculation VIGS; NbPDS: Tobacco phytoene desaturase; TaPMR5: Wheat powdery mildew resistance 5 gene; SlPDS: Tomato phytoene desaturase; GhBI-1: Cotton Bax inhibitor-1.