An unprecedented number of viruses have been discovered by leveraging advances in high-throughput sequencing . Infectious clone technology is a universal approach that facilitates the study of biology and role in disease of viruses . In recent years homology-based cloning methods such as Gibson assembly have been used to generate virus infectious clones . We detail herein the preparation of home-made cloning materials for Gibson assembly . The home-made materials were used in one-step generation of the infectious cDNA clone of a plant RNA virus into a T-DNA binary vector . The clone was verified by a single Illumina reaction and a de novo read assembly approach that required no primer walking, custom primers or reference sequences . Clone infectivity was finally confirmed by Agrobacterium -mediated delivery to host plants . We anticipate that the convenient home-made materials, one-step cloning and Illumina verification strategies described herein will accelerate characterization of viruses and their role in disease development.
Index: Agro-infection, Gibson assembly, Home-made cloning reagents, Illumina sequencing, Potyvirus, Tobacco vein mottling virus, Virus infectious clone