Dataset Information

A Versatile Plant Rhabdovirus-Based Vector for Gene Silencing, miRNA Expression and Depletion, and Antibody Production.

ABSTRACT: Plant virus vectors are ideal tools for delivery of genetic cargo into host cells for functional genomics studies and protein overexpression. Although a vast number of plant virus vectors have been developed for different purposes, the utility of a particular virus vector is generally limited. Here, we report a multipurpose plant rhabdovirus-based vector system suitable for a wide range of applications in Nicotiana benthamiana. We engineered sonchus yellow net rhabdovirus (SYNV)-based gene silencing vectors through expressing a sense, antisense, or double-stranded RNAs of target genes. Robust target gene silencing was also achieved with an SYNV vector expressing a designed artificial microRNA. In addition, ectopic expression of a short tandem target mimic RNA using the SYNV vector led to a significant depletion of the target miR165/166 and caused abnormal leaf development. More importantly, SYNV was able to harbor two expression cassettes that permitted simultaneous RNA silencing and overexpression of large reporter gene. This dual capacity vector also enabled systemic expression of a whole-molecule monoclonal antibody consisting of light and heavy chains. These results highlight the utility of the SYNV vector system in gene function studies and agricultural biotechnology and provide a technical template for developing similar vectors of other economically important plant rhabdoviruses.

SUBMITTER: Peng X

PROVIDER: S-EPMC7835261 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A Versatile Plant Rhabdovirus-Based Vector for Gene Silencing, miRNA Expression and Depletion, and Antibody Production.

Peng Xingxing X Ma Xiaonan X Lu Shuting S Li Zhenghe Z

Frontiers in plant science 20210112

Plant virus vectors are ideal tools for delivery of genetic cargo into host cells for functional genomics studies and protein overexpression. Although a vast number of plant virus vectors have been developed for different purposes, the utility of a particular virus vector is generally limited. Here, we report a multipurpose plant rhabdovirus-based vector system suitable for a wide range of applications in <i>Nicotiana benthamiana</i>. We engineered sonchus yellow net rhabdovirus (SYNV)-based gen ...[more]

PMID: 33510764

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Project description:Positive-stranded RNA virus movement proteins (MPs) generally lack sequence-specific nucleic acid-binding activities and display cross-family movement complementarity with related and unrelated viruses. Negative-stranded RNA plant rhabdoviruses encode MPs with limited structural and functional relatedness with other plant virus counterparts, but the precise mechanisms of intercellular transport are obscure. In this study, we first analyzed the abilities of MPs encoded by five distinct rhabdoviruses to support cell-to-cell movement of two positive-stranded RNA viruses by using trans-complementation assays. Each of the five rhabdovirus MPs complemented the movement of MP-defective mutants of tomato mosaic virus and potato X virus. In contrast, movement of recombinant MP deletion mutants of sonchus yellow net nucleorhabdovirus (SYNV) and tomato yellow mottle-associated cytorhabdovirus (TYMaV) was rescued only by their corresponding MPs, i.e., SYNV sc4 and TYMaV P3. Subcellular fractionation analyses revealed that SYNV sc4 and TYMaV P3 were peripherally associated with cell membranes. A split-ubiquitin membrane yeast two-hybrid assay demonstrated specific interactions of the membrane-associated rhabdovirus MPs only with their cognate nucleoproteins (N) and phosphoproteins (P). More importantly, SYNV sc4-N and sc4-P interactions directed a proportion of the N-P complexes from nuclear sites of replication to punctate loci at the cell periphery that partially colocalized with the plasmodesmata. Our data show that cell-to-cell movement of plant rhabdoviruses is highly specific and suggest that cognate MP-nucleocapsid core protein interactions are required for intra- and intercellular trafficking.IMPORTANCE Local transport of plant rhabdoviruses likely involves the passage of viral nucleocapsids through MP-gated plasmodesmata, but the molecular mechanisms are not fully understood. We have conducted complementation assays with MPs encoded by five distinct rhabdoviruses to assess their movement specificity. Each of the rhabdovirus MPs complemented the movement of MP-defective mutants of two positive-stranded RNA viruses that have different movement strategies. In marked contrast, cell-to-cell movement of two recombinant plant rhabdoviruses was highly specific in requiring their cognate MPs. We have shown that these rhabdovirus MPs are localized to the cell periphery and associate with cellular membranes, and that they interact only with their cognate nucleocapsid core proteins. These interactions are able to redirect viral nucleocapsid core proteins from their sites of replication to the cell periphery. Our study provides a model for the specific inter- and intracellular trafficking of plant rhabdoviruses that may be applicable to other negative-stranded RNA viruses.

Project description:The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA) or microRNA (miRNA) and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen) whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage. Finally, we determined that both isoforms of the AT-Rich Interacting Domain 4B (ARID4B) protein could induce G1 arrest when overexpressed. As new technologies emerge, the vectors in this collection can be easily modified and adapted without the need for extensive recloning.

Dataset Information

A Versatile Plant Rhabdovirus-Based Vector for Gene Silencing, miRNA Expression and Depletion, and Antibody Production.

Publications

A Versatile Plant Rhabdovirus-Based Vector for Gene Silencing, miRNA Expression and Depletion, and Antibody Production.

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