Abstract
The availability of infectious full-length clone is indispensable for reverse genetics studies of virus biology, pathology and construction of viral vectors. However, for RNA viruses with large genome sizes or those exhibiting inherent cloning difficulties, procedure to generate biologically active complementary DNA (cDNA) clones can be time-consuming or technically challenging. Here we have constructed a yeast-Escherichia coli-Agrobacterium shuttle vector that enables highly efficient homologous recombination in yeast for assembly of Agrobacterium compatible plant virus clones. Using this vector, we show that infectious cDNA clones of a plant negative-stranded RNA virus, sonchus yellow net rhabdovirus, can be rapidly assembled. In addition, one-step assembly of infectious clones of potato virus Y in yeast, either with or without intron, was readily achieved from as many as eight overlapping DNA fragments. More importantly, the recovered yeast plasmids can be transformed directly into Agrobacterium for inoculation, thereby obviating the E. coli cloning steps and associated toxicity issues. This method is rapid, highly efficient and cost-effective and should be readily applicable to a broad range of plant viruses.
Highlights
The availability of RNA virus manipulation tools has resulted in enormous advances in our understanding of virus life cycle and pathogenesis
We show that functional full-length sonchus yellow net virus (SYNV) antigenome complementary DNA (cDNA) clone can be efficiently assembled by yeast homologous recombination (HR)
To construct a shuttle plasmid able to replicate in yeast, Agrobacterium and E. coli, a small binary plasmid pCB301-hepatitis delta virus (HDV), containing the cauliflower mosaic virus (CaMV) 35S promoter followed by an HDV antigenomic ribozyme and the Nos terminator within the T-DNA region, was chosen as the vector backbone (Figure 1a)
Summary
The availability of RNA virus manipulation tools has resulted in enormous advances in our understanding of virus life cycle and pathogenesis. To avoid in vitro transcription steps, Mori et al [5] demonstrated that recombinant RNA virus can be recovered after mechanical delivery of cloned plasmid DNAs into plant tissues, if viral cDNA was positioned downstream of a cauliflower mosaic virus (CaMV) 35S promoter. In contrast to positive-stranded RNA viruses, whose naked RNAs are infectious after introduction into susceptible cells, obtaining recombinant plant NSVs from cloned cDNA presents additional challenging problems [4] These difficulties include delivery of nearly exact copies of viral RNA genomes into cells and a requirement for expression of viral core proteins in the same cells for in vivo assembly of biologically active RNPs, which are the minimal infectious units of NSVs [4]. The procedure is highly efficient, can be completed within two weeks and requires no expensive reagents
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