Potato virus X-based expression vectors are stabilized for long-term production of proteins and larger inserts.

Plus-strand RNA viruses such as Potato virus X (PVX) are often used as high-yielding expression vectors in plants, because they tolerate extra transgene insertion and expression without disrupting normal virus functions. However, sequence redundancy due to promoter duplication often leads to genetic instability. Although heterologous subgenomic promoter-like sequences (SGPs) have been successfully used in Tobacco mosaic virus vectors, only homologous SGP duplications have been used in PVX vectors. We stabilized PVX-based vectors by combining heterologous SGPs from related potexviruses with an N-terminal coat protein (CP) deletion. We selected two SGPs with core sequences homologous to PVX, from Bamboo mosaic virus (BaMV) and Cassava common mosaic virus, as well as a SGP with a heterologous core sequence from Foxtail mosaic virus (FoMV). We found that only the BaMV and CsCMV SGPs were utilized by the PVX replicase. However, the transgene remained unstable, due to the presence of an additional region with strong sequence similarity at the 5' end of the cp gene. The BaMV SGP combined with an N-terminal CP deletion achieved high PVX vector stability. This new expression vector is particularly useful for long-term production of proteins and for larger inserts. The improved PVX-based vectors are suitable for the systemic expression of any gene of interest in PVX host plants. The PVX-based vector can be advantageous for the overexpression of proteins, to analyze protein functions in planta or as a system for virus-induced gene silencing.