Infection of Citrus Plants with VirionsGenerated in Nicotiana benthamiana Plants Agroinfiltrated with a Binary Vector Based Citrus tristeza virus

Abstract

Citrus tristeza virus (CTV) of the Closteroviridae family has a plus-stranded RNA genome of approximately 20 kilobases. Purified virion RNA and in vitro- generated RNA tran- scripts from an infectious cDNA clone failed to infect citrus plants, although both RNAs initiated low levels of infection in the mesophyll protoplasts of Nicotiana benthamiana and citrus. Infection of citrus plants required successive protoplast passages of progeny virions from transcript-inocu- lated N. benthamiana protoplasts to maximize the virus titer. To improve the infection efficiency, we engineered a full-length CTV cDNA in a binary vector (pCAMBIA) that contained a 35S pro- moter with a duplicated enhancer in the 5’ end, a ribozyme and nopaline synthase polyA signal at the 3’ end, and which was transferred into Agrobacterium tumefaciens EHA105 strain for agro- inoculation studies . Initial attempts at the direct agroinfection of citrus plants were not success- ful, but the DNA of this construct produced CTV specific RNAs and virions when transfected into N. benthamiana mesophyll protoplasts. Replication and virion formation were also observed in the agroinfiltrated leaves of N. benthamiana, and citrus plants became infected when inoculated with virions partially purified from the sap expressed from agroinfiltrated N. benthamiana leaves