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Viruses 2017, 9(11), 332; doi:10.3390/v9110332

Rapid Construction of Complex Plant RNA Virus Infectious cDNA Clones for Agroinfection Using a Yeast-E. coli-Agrobacterium Shuttle Vector

1
State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China
2
Yunnan Academy of Tobacco Agricultural Sciences, Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Kunming 650021, China
3
Sichuan Plant Protection Station, Chengdu 610041, China
*
Author to whom correspondence should be addressed.
Academic Editor: K. Andrew White
Received: 27 September 2017 / Revised: 2 November 2017 / Accepted: 3 November 2017 / Published: 7 November 2017
(This article belongs to the Section Viruses of Plants, Fungi and Protozoa)
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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. View Full-Text
Keywords: infectious cDNA clone; agroinfection; yeast-E.coli-Agrobacterium shuttle vector; yeast homologous recombination; potyvirus; potato virus Y; rhabdovirus; sonchus yellow net virus infectious cDNA clone; agroinfection; yeast-E.coli-Agrobacterium shuttle vector; yeast homologous recombination; potyvirus; potato virus Y; rhabdovirus; sonchus yellow net virus
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Sun, K.; Zhao, D.; Liu, Y.; Huang, C.; Zhang, W.; Li, Z. Rapid Construction of Complex Plant RNA Virus Infectious cDNA Clones for Agroinfection Using a Yeast-E. coli-Agrobacterium Shuttle Vector. Viruses 2017, 9, 332.

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