Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression

Abstract

We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein expression, based on Alternanthera mosaic virus (AltMV), for infection of a wide range of host plants including Nicotiana benthamiana and Arabidopsis thaliana by either mechanical inoculation of in vitro transcripts or via agroinfiltration. In vivo transcripts produced by co-agroinfiltration of bacteriophage T7 RNA polymerase resulted in T7-driven AltMV infection from a binary vector in the absence of the Cauliflower mosaic virus 35S promoter. An artificial bipartite viral vector delivery system was created by separating the AltMV RNA-dependent RNA polymerase and Triple Gene Block (TGB)123-Coat protein (CP) coding regions into two constructs each bearing the AltMV 5' and 3' non-coding regions, which recombined in planta to generate a full-length AltMV genome. Substitution of TGB1 L(88)P, and equivalent changes in other potexvirus TGB1 proteins, affected RNA silencing suppression efficacy and suitability of the vectors from protein expression to VIGS.

Mutation of a chloroplast-targeting signal in Alternanthera mosaic virus TGB3 impairs cell-to-cell movement and eliminates long-distance virus movement

“…AltMV TGB3 was amplified and inserted in place of eGFP in AltMVeGFPDTGB3 to form AltMVDTGB3(TGB3+); AltMVDTGB3(TGB3-GFP+), expressing TGB3-GFP as an added gene, was created by replacing eGFP with TGB3-GFP, amplified from pHVLG-TGB3 (see below), into BamHI/NheI-digested AltMV-eGFP. AltMV TGB3 was amplified and inserted as a BamHI/MluI fragment into infectious clone PVX-MCS, forming PVX(TGB3 AltMV +); PVX-MCS is a derivative of PVX infectious clone pPC2S, described elsewhere (Lim et al, 2010b Full-length cDNA clones were linearized by using XbaI (AltMV clones) or SpeI (PVX clones) before in vitro transcription of infectious RNA for N. benthamiana inoculation, using T7 RNA polymerase as described by Petty et al (1989). Q-RT-PCR to quantify TGB3 RNA was performed as described by Bae et al (2006) using TGB3-specific primers.…”

Selective Interaction Between Chloroplast β-ATPase and TGB1L88 Retards Severe Symptoms Caused by Alternanthera mosaic virus Infection

“…Alternanthera mosaic virus (AltMV) is emerging as another model potexvirus with some significant differences from PVX (Jang et al, 2013; Lim et al, 2010a), in which a single amino acid difference in TGB1 affects not only the relative efficiency of TGB1 as a suppressor of RNA silencing (Lim et al, 2010b, c), but also both the cell wall and nuclear localization of TGB1 (Lim et al, 2010b, c; Nam et al, 2013). A naturally occurring variant with proline (P) at TGB1 residue 88 has significantly reduced silencing suppression efficiency, whereas AltMV differing only by having the typical leucine (L) at this position accumulates to approximately 40-fold higher levels (Lim et al, 2010b, c, and unpublished).…”

Possible role of the Nt-4/1 protein in macromolecular transportin vascular tissue

“…6 Detection of GuS was performed according to Jefferson et al In this work we report evidence for a possible role of 4/1 protein in the long-distance vascular movement of a molecular pathogen, potato spindle tuber viroid (PSTVd). 6 PSTVd was inoculated onto N. benthamiana plants where the level of endogenous 4/1 mRNA was downregulated by virus-induced gene silencing (VIGS) employing a previously described Alternanthera mosaic potexvirus (AltMV) silencing construct 7 carrying a fragment of Nt-4/1 cDNA. Despite the fact that accumulation of PSTVd in inoculated leaves of 4/1-silenced plants was significantly lower than in control AltMV-infected plants, the level of viroid RNA accumulation in the 4th, 6th and 7th systemic leaves above the PSTVd-inoculated leaf was significantly higher in 4/1-silenced plants compared with control plants (Fig.…”