Simultaneous equimolar expression of multiple proteins in plants from a disarmed potyvirus vector

The use of viral vectors for expression of heterologous proteins in plants is hampered by some limitations including the amount of exogenous genetic information that can be incorporated, difficulties with coexpression of stoichiometric amounts of multiple polypeptides and the risk that infectious clones could escape to environment. Here, a new plant viral vector is described which overcomes these limitations. The technology is based on the replacement of the viral RNA polymerase (NIb) cistron of a potyvirus by a cassette for the coexpression of multiple heterologous proteins. The heterologous proteins are flanked by specific cleavage motifs of a viral protease that mediate their efficient release from the viral polyprotein. The vector only replicates and moves systemically in plants where the viral NIb activity is supplied in trans by a transgene or another viral vector. The vector allowed for simultaneous expression of three fluorescent reporter proteins in the same subcellular location or two interacting transcription factors inducing anthocyanin accumulation. The vector had sufficient stability throughout the infectious cycle and the NIb deletion prevented infection of wild-type plants which improves biosecurity.