Rapid Construction of Complex Plant RNA Virus Infectious cDNA Clones for Agroinfection Using a Yeast-E. coli-Agrobacterium Shuttle Vector
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...
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| Format: | Article |
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MDPI AG
2017-11-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/9/11/332 |
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| author | Kai Sun Danyang Zhao Yong Liu Changjun Huang Wei Zhang Zhenghe Li |
| author_facet | Kai Sun Danyang Zhao Yong Liu Changjun Huang Wei Zhang Zhenghe Li |
| author_sort | Kai Sun |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-11T08:50:47Z |
| format | Article |
| id | doaj.art-af72acc7cc0d4d2b8477243890de1cd1 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-12-11T08:50:47Z |
| publishDate | 2017-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-af72acc7cc0d4d2b8477243890de1cd12022-12-22T01:14:02ZengMDPI AGViruses1999-49152017-11-0191133210.3390/v9110332v9110332Rapid Construction of Complex Plant RNA Virus Infectious cDNA Clones for Agroinfection Using a Yeast-E. coli-Agrobacterium Shuttle VectorKai Sun0Danyang Zhao1Yong Liu2Changjun Huang3Wei Zhang4Zhenghe Li5State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, ChinaState Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, ChinaYunnan Academy of Tobacco Agricultural Sciences, Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Kunming 650021, ChinaYunnan Academy of Tobacco Agricultural Sciences, Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Kunming 650021, ChinaSichuan Plant Protection Station, Chengdu 610041, ChinaState Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, ChinaThe 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.https://www.mdpi.com/1999-4915/9/11/332infectious cDNA cloneagroinfectionyeast-E.coli-Agrobacterium shuttle vectoryeast homologous recombinationpotyviruspotato virus Yrhabdovirussonchus yellow net virus |
| spellingShingle | Kai Sun Danyang Zhao Yong Liu Changjun Huang Wei Zhang Zhenghe Li Rapid Construction of Complex Plant RNA Virus Infectious cDNA Clones for Agroinfection Using a Yeast-E. coli-Agrobacterium Shuttle Vector Viruses infectious cDNA clone agroinfection yeast-E.coli-Agrobacterium shuttle vector yeast homologous recombination potyvirus potato virus Y rhabdovirus sonchus yellow net virus |
| title | Rapid Construction of Complex Plant RNA Virus Infectious cDNA Clones for Agroinfection Using a Yeast-E. coli-Agrobacterium Shuttle Vector |
| title_full | Rapid Construction of Complex Plant RNA Virus Infectious cDNA Clones for Agroinfection Using a Yeast-E. coli-Agrobacterium Shuttle Vector |
| title_fullStr | Rapid Construction of Complex Plant RNA Virus Infectious cDNA Clones for Agroinfection Using a Yeast-E. coli-Agrobacterium Shuttle Vector |
| title_full_unstemmed | Rapid Construction of Complex Plant RNA Virus Infectious cDNA Clones for Agroinfection Using a Yeast-E. coli-Agrobacterium Shuttle Vector |
| title_short | Rapid Construction of Complex Plant RNA Virus Infectious cDNA Clones for Agroinfection Using a Yeast-E. coli-Agrobacterium Shuttle Vector |
| title_sort | rapid construction of complex plant rna virus infectious cdna clones for agroinfection using a yeast e coli agrobacterium shuttle vector |
| topic | infectious cDNA clone agroinfection yeast-E.coli-Agrobacterium shuttle vector yeast homologous recombination potyvirus potato virus Y rhabdovirus sonchus yellow net virus |
| url | https://www.mdpi.com/1999-4915/9/11/332 |
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