HandGun-mediated inoculation of plants with viral pathogens for mechanistic studies

Particle bombardment is an efficient method for virus inoculation of intact plants. This technique enables inoculation with full-length infectious clone cDNA, PCR products, virus from sap or virus preparation, and in vitro viral transcripts. The inoculation of some phloem-limited RNA and circular DNA viruses is also possible. The technique of bombardment without the use of vacuum permits the inoculation of soft-leaved plants that do not usually survive bombardment inoculation, the investigation of viral recombination in planta, promoter analysis, monitoring virus movement using an infectious clone bearing a reporter gene and the inoculation of large numbers of plants. The inoculation of whitefly-borne circular DNA begomoviruses is now possible due to direct genome amplification by Rolling Circle Amplification (RCA), followed by bombardment using a device that does not require a vacuum for operation. Here we describe the inoculation of intact plants with (a) RNA virus infective clones and (b) begomoviruses after direct genome amplification by RCA, using a handheld bombardment device.