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.

Generation of stable infectious clones of plant viruses by using Rhizobium radiobacter for both cloning and inoculation

“…The reasons may be the different sequences of viral genomes between the two isolates, or the use of different vectors. Several other potyviruses also have proved to be instable in E. coli (Bedoya and Daros, 2010;Gao et al, 2012;Johansen, 1996;Lopez-Moya and Garcia, 2000;Olsen and Johansen, 2001;Tuo et al, 2015;Yang et al, 1998). When the viral genome-encoding plasmids assembled in vitro were transformed into R. radiobacter, the colonies with correct viral genome were obtained with high efficiency.…”

“…However, full-length clones of many viruses have been proved to be difficult or even impossible to perform molecular manipulation in E. coli due to their toxicity to E. coli. The instability in E. coli has been reported for an array of viruses belonging to potyviruses (Bedoya and Daros, 2010;Gao et al, 2012;Johansen, 1996;Lopez-Moya and Garcia, 2000;Olsen and Johansen, 2001;Tuo et al, 2015;Yang et al, 1998), tobraviruses (Constantin et al, 2004;Ratcliff et al, 2001), flaviviruses (Aubry et al, 2015), coronaviruses (Almazan et al, 2000), picornaviruses (Zibert et al, 1990) and pestiviruses (Rasmussen et al, 2010). Several methods have been developed to circumvent this problem, including the in vitro ligation method involving two plasmid, mutating cryptic prokaryotic promoter sites in the viral genome, and using intron insertions, low copy number plasmids, specific E. coli strains and low growth temperature (Aubry et al, 2015;Edmonds et al, 2013;Johansen and Lund, 2008;Pu et al, 2011;Siridechadilok et al, 2013;Yamshchikov et al, 2001).…”

“…Several methods have been developed to circumvent this problem, including the in vitro ligation method involving two plasmid, mutating cryptic prokaryotic promoter sites in the viral genome, and using intron insertions, low copy number plasmids, specific E. coli strains and low growth temperature (Aubry et al, 2015;Edmonds et al, 2013;Johansen and Lund, 2008;Pu et al, 2011;Siridechadilok et al, 2013;Yamshchikov et al, 2001). For generation of infectious clones of plant virus that are instable in E. coli, insertion of introns in viral genome to terminate expression of undesired toxic proteins in E. coli is the most extensively used approach so far (Desbiez et al, 2012;Gao et al, 2012;Johansen, 1996;Johansen and Lund, 2008;Lopez-Moya and Garcia, 2000;Tuo et al, 2015;Yang et al, 1998). However, determination of the numbers and locations of intron insertions required to stabilize infectious clones is time-consuming and laborious.…”

“…However, determination of the numbers and locations of intron insertions required to stabilize infectious clones is time-consuming and laborious. Some sequence-independent cloning methods were also developed to assemble infectious clones of plant viruses in vivo or in vitro, such as yeast recombination system (Desbiez et al, 2012;Youssef et al, 2011), Gibson assembly and In-Fusion cloning method (Blawid and Nagata, 2015;Bordat et al, 2015;Tuo et al, 2015), but the assembly products require E. coli transformation for propagation, which means those methods just make easier viral genome assembly, not solving the problem of viral genome instability in E. coli. Thus for plant viruses that cannot maintain stability in E. coli, generation of stable infectious clones by these assembly methods still depends on the using of intron insertions (Desbiez et al, 2012;Tuo et al, 2015), which remains a major hurdle in the research of these viruses.…”

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

“…Molecules assembled by In-Fusion are not covalently joined and are sealed in vivo. A potyvirus infectious clone for in vitro transcription (Tuo et al, 2015) and a binary vector for agroinoculation of a trichovirus have been generated by this method (Zhang and Jelkmann, 2017). In-Fusion was also used to engineer the first binary vector for plant delivery of a negative-stranded RNA virus (Wang et al, 2015).…”