Next Article in Journal
Potential for Improving Potency and Specificity of Reovirus Oncolysis with Next-Generation Reovirus Variants
Previous Article in Journal
Identification of a New Ribonucleoside Inhibitor of Ebola Virus Replication
Open AccessCommunication

Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning

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
*
Authors to whom correspondence should be addressed.
Academic Editor: Thomas Hohn
Viruses 2015, 7(12), 6241-6250; https://doi.org/10.3390/v7122935
Received: 7 July 2015 / Revised: 18 November 2015 / Accepted: 19 November 2015 / Published: 1 December 2015
(This article belongs to the Section Viruses of Plants, Fungi and Protozoa)
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. View Full-Text
Keywords: PLDMV; infectious cDNA clone; In-Fusion; intron; papaya PLDMV; infectious cDNA clone; In-Fusion; intron; papaya
Show Figures

Graphical abstract

MDPI and ACS Style

Tuo, D.; Shen, W.; Yan, P.; Li, X.; Zhou, P. Rapid Construction of Stable Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning. Viruses 2015, 7, 6241-6250.

Show more citation formats Show less citations formats

Article Access Map by Country/Region

1
Search more from Scilit
 
Search
Back to TopTop