Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes

Plant Pathology Laboratory, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan.
Virology (Impact Factor: 3.28). 03/2009; 386(2):407-16. DOI: 10.1016/j.virol.2009.01.039
Source: PubMed

ABSTRACT Apple latent spherical virus (ALSV) vectors were evaluated for virus-induced gene silencing (VIGS) of endogenous genes among a broad range of plant species. ALSV vectors carrying partial sequences of a subunit of magnesium chelatase (SU) and phytoene desaturase (PDS) genes induced highly uniform knockout phenotypes typical of SU and PDS inhibition on model plants such as tobacco and Arabidopsis thaliana, and economically important crops such as tomato, legume, and cucurbit species. The silencing phenotypes persisted throughout plant growth in these plants. In addition, ALSV vectors could be successfully used to silence a meristem gene, proliferating cell nuclear antigen and disease resistant N gene in tobacco and RCY1 gene in A. thaliana. As ALSV infects most host plants symptomlessly and effectively induces stable VIGS for long periods, the ALSV vector is a valuable tool to determine the functions of interested genes among a broad range of plant species.

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    ABSTRACT: Apple latent spherical virus (ALSV) is an efficient virus-induced gene silencing vector in functional genomics analyses of a broad range of plant species. Here, an Agrobacterium-mediated inoculation (agroinoculation) system was developed for the ALSV vector, and virus-induced transcriptional gene silencing (VITGS) is described in plants infected with the ALSV vector. The cDNAs of ALSV RNA1 and RNA2 were inserted between the cauliflower mosaic virus 35S promoter and the NOS-T sequences in a binary vector pCAMBIA1300 to produce pCALSR1 and pCALSR2-XSB or pCALSR2-XSB/MN. When these vector constructs were agroinoculated into Nicotiana benthamiana plants with a construct expressing a viral silencing suppressor, the infection efficiency of the vectors was 100%. A recombinant ALSV vector carrying part of the 35S promoter sequence induced transcriptional gene silencing of the green fluorescent protein gene in a line of N. benthamiana plants, resulting in the disappearance of green fluorescence of infected plants. Bisulfite sequencing showed that cytosine residues at CG and CHG sites of the 35S promoter sequence were highly methylated in the silenced generation zero plants infected with the ALSV carrying the promoter sequence as well as in progeny. The ALSV-mediated VITGS state was inherited by progeny for multiple generations. In addition, induction of VITGS of an endogenous gene (chalcone synthase-A) was demonstrated in petunia plants infected with an ALSV vector carrying the native promoter sequence. These results suggest that ALSV-based vectors can be applied to study DNA methylation in plant genomes, and provide a useful tool for plant breeding via epigenetic modification.
    Frontiers in Microbiology 11/2014; 5:595. DOI:10.3389/fmicb.2014.00595 · 3.94 Impact Factor
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    ABSTRACT: The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process. Citation: Kaido M, Abe K, Mine A, Hyodo K, Taniguchi T, et al. (2014) GAPDH-A Recruits a Plant Virus Movement Protein to Cortical Virus Replication Complexes to Facilitate Viral Cell-to-Cell Movement. PLoS Pathog 10(11): e1004505. doi:10.1371/journal.ppat.1004505 Copyright: ß 2014 Kaido et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported in part by a Grant-in-Aid (22580047) for Scientific Research C and a Grant-in-Aid (25450058) for Scientific Research C from the Japan Society for the Promotion of Science ( The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
    PLoS Pathogens 11/2014; DOI:10.1371/journal.ppat.1004505 · 8.06 Impact Factor
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    ABSTRACT: Apple latent spherical virus (ALSV) vectors have been shown to effectively induce stable virus-induced gene silencing (VIGS) in a wide range of plant species, including rosaceous fruit tree species such as apple (Malus x domestica Borkh.), pear (Pyrus communis L.), and Japanese pear (P. pyrifolia Nakai). In this study, we attempted to develop a VIGS-based gene evaluation system for two Prunus fruit tree species, apricot and Japanese apricot, using ALSV vectors. A partial sequence of the P armeniaca PHYTOENE DESATURASE (ParPDS) gene was cloned and ligated into the T-DNA region of a binary vector, pBICAL2, designed based on RNA2 of ALSV. The resultant pBICAL2-ParPDS was introduced into a disarmed Agrobacterium strain, EHA105. pBICAL1, a binary plasmid for the expression of ALSV RNA1 in plants, was also introduced into EHA105. Leaves of Nicotiana benthamiana were infected with pBICAL1/EHA105 and pBICAL2-ParPDS/EHA105 simultaneously to produce and amplify recombinant ALSV particles. The amplified ParPDS-ALSV in N. benthamiana was isolated and infected into the cotyledons of apricot and Japanese apricot seedlings by particle bombardment. Although our attempts to infect wild and recombinant ALSVs into Japanese apricot seedlings were unsuccessful, uniform discoloration of the upper leaves, a typical phenotype of PBS knock down, was observed several weeks after inoculation in apricot seedlings. We discuss the possible use of this VIGS-based gene evaluation system in Prunus.
    Journal- Japanese Society for Horticultural Science 01/2014; 83(1):23-31. DOI:10.2503/jjshs1.CH-091 · 0.82 Impact Factor

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