Development of expression vectors based on pepino mosaic virus

Agroinfectious clones were produced from two different PepMV genotypes (European and Chilean), and these were able to initiate typical PepMV infections. We explored several strategies for vector development including coat protein (CP) replacement, duplication of the CP subgenomic promoter (SGP) and the creation of a fusion protein using the foot-and-mouth disease virus (FMDV) 2A catalytic peptide. We found that CP replacement vectors were unable to move systemically and that vectors with duplicated SGPs (even heterologous SGPs) suffered from significant transgene instability. The fusion protein incorporating the FMDV 2A catalytic peptide gave by far the best results, maintaining stability through serial passages and allowing the accumulation of GFP to 0.2-0.4 g per kg of leaf tissue. The possible use of PepMV as a virus-induced gene silencing vector to study gene function was also demonstrated. Protocols for the use of this vector are described.A stable PepMV vector was generated by expressing the transgene as a CP fusion using the sequence encoding the foot-and-mouth disease virus (FMDV) 2A catalytic peptide to separate them. We have generated a novel tool for the expression of recombinant proteins in plants and for the functional analysis of virus and plant genes. Our experiments have also highlighted virus requirements for replication in single cells as well as intercellular and long-distance movement.Pepino mosaic virus (PepMV) is a widespread plant virus that causes a major disease of tomato crops worldwide [1-11]. PepMV belongs to the genus Potexvirus (family Flexiviridae) and, like other members of this genus, has virions that are non-enveloped flexuous rods 508 nm in length [12]. The PepMV genome is a single-stranded RNA molecule approximately 6.4 kb in length, comprising five open reading frames (ORFs) flanked by 5' and 3' untranslated regions (UTRs) with a 5'-cap and a 3' poly-A tail. The ORFs encode a 164-kDa RNA-dependent RNA polymerase (RdRp); three tri