title: Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning.
abstract: Papaya leaf distortion mosaic virus (PLDMV) is becoming a threat to papaya and transgenic papaya resistant to the related pathogen, papaya ringspot virus (PRSV). The generation of infectious viral clones is an essential step for reverse-genetics studies of viral gene function and cross-protection. In this study, a sequence- and ligation-independent cloning system, the In-Fusion(®) Cloning Kit (Clontech, Mountain View, CA, USA), was used to construct intron-less or intron-containing full-length cDNA clones of the isolate PLDMV-DF, with the simultaneous scarless assembly of multiple viral and intron fragments into a plasmid vector in a single reaction. The intron-containing full-length cDNA clone of PLDMV-DF was stably propagated in Escherichia coli. In vitro intron-containing transcripts were processed and spliced into biologically active intron-less transcripts following mechanical inoculation and then initiated systemic infections in Carica papaya L. seedlings, which developed similar symptoms to those caused by the wild-type virus. However, no infectivity was detected when the plants were inoculated with RNA transcripts from the intron-less construct because the instability of the viral cDNA clone in bacterial cells caused a non-sense or deletion mutation of the genomic sequence of PLDMV-DF. To our knowledge, this is the first report of the construction of an infectious full-length cDNA clone of PLDMV and the splicing of intron-containing transcripts following mechanical inoculation. In-Fusion cloning shortens the construction time from months to days. Therefore, it is a faster, more flexible, and more efficient method than the traditional multistep restriction enzyme-mediated subcloning procedure.
authors.affiliation: Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China. tuodecai@itbb.org.cn.
authors.forename: Decai
authors.initials: D
authors.lastname: Tuo
authors.affiliation: Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China. dnaswt@hotmail.com.
authors.forename: Wentao
authors.initials: W
authors.lastname: Shen
authors.affiliation: Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China. yanpu@itbb.org.cn.
authors.forename: Pu
authors.initials: P
authors.lastname: Yan
authors.affiliation: Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China. lixiaoying@itbb.org.cn.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China. zhoupeng@itbb.org.cn.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhou
mesh_terms: D029441:Carica / Q000821:virology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D016679:Genome, Viral; D042822:Genomic Instability; D010935:Plant Diseases / Q000821:virology*; D010957:Plasmids; D017800:Potyvirus / Q000235:genetics* / Q000502:physiology*; D059386:Reverse Genetics / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D004279:DNA, Viral
keywords: In-Fusion; PLDMV; infectious cDNA clone; intron; papaya