Rapid generation of inoculum of a plant RNA virus using overlap PCR.

We have developed an efficient method to rapidly generate infectious inoculum of a plant RNA virus and confirmed its infectivity by mechanical inoculation. The method takes advantage of overlap PCR to bypass the cloning steps, which makes it relatively simple, rapid, and inexpensive compared to the traditional methods. Using this approach, inoculum of a tobamovirus, Turnip vein clearing virus (TVCV), was generated. PCR products specific for the 35S promoter and TVCV genome were used as templates for overlap PCR to form a single product containing the full-length TVCV cDNA under the control of the double 35S promoter, and the entire process took only 8 h. This inoculum was infectious in Nicotiana benthamiana, and its infectivity was ca. 67% compared to 0% and 100% with negative and positive controls, respectively. Thus, this rapid method generates efficient infectious inoculum for a plant RNA virus.