Streamlined generation of plant virus infectious clones using the pLX mini binary vectors

Journal of Virological Methods
Fabio PasinYet-Ran Chen

Abstract

Recent metagenomic surveys have provided unprecedented amounts of data that have revolutionized our understanding of virus evolution and diversity. Infectious clones are powerful tools to aid the biological characterization of viruses. We recently described the pLX vectors, a set of mini binary T-DNA vectors (∼3 kb) that includes strong bacterial terminators and a minimal replicon from the broad-host-range plasmid pBBR1, which replicate autonomously in both Escherichia coli and Agrobacterium. In this study, a workflow that encompassed pLX binary vectors, overlap-based assembly strategies, and sequencing-by-synthesis verification steps is described and applied for the streamlined generation of infectious clones suitable for Agrobacterium-mediated delivery. The pLX-based vectors herein assembled include the first infectious clone of Wasabi mottle virus, a crucifer-infecting tobamovirus, as well as binary vectors of positive-single-stranded RNA and single- and double-stranded DNA viruses from the Potyviridae, Geminiviridae and Caulimoviridae families, respectively. Finally, the clones generated were used to agro-inoculate the model plant Arabidopsis thaliana and infections were confirmed by a multiplex RT-PCR assay. This workflow ...Continue Reading

Related Concepts

Related Feeds

Bioinformatics in Biomedicine

Bioinformatics in biomedicine incorporates computer science, biology, chemistry, medicine, mathematics and statistics. Discover the latest research on bioinformatics in biomedicine here.