DEVELOPMENTALLY REGULATED PLASMA MEMBRANE PROTEIN of Nicotiana benthamiana contributes to potyvirus movement and transports to plasmodesmata via the early secretory pathway and the actomyosin system.

The intercellular movement of plant viruses requires both viral and host proteins. Previous studies have demonstrated that the frame-shift protein P3N-PIPO (for the protein encoded by the open reading frame [ORF] containing 5'-terminus of P3 and a +2 frame-shift ORF called Pretty Interesting Potyviridae ORF and embedded in the P3) and CYLINDRICAL INCLUSION (CI) proteins were required for potyvirus cell-to-cell movement. Here, we provide genetic evidence showing that a Tobacco vein banding mosaic virus (TVBMV; genus Potyvirus) mutant carrying a truncated PIPO domain of 58 amino acid residues could move between cells and induce systemic infection in Nicotiana benthamiana plants; mutants carrying a PIPO domain of seven, 20, or 43 amino acid residues failed to move between cells and cause systemic infection in this host plant. Interestingly, the movement-defective mutants produced progeny that eliminated the previously introduced stop codons and thus restored their systemic movement ability. We also present evidence showing that a developmentally regulated plasma membrane protein of N. benthamiana (referred to as NbDREPP) interacted with both P3N-PIPO and CI of the movement-competent TVBMV. The knockdown of NbDREPP gene expression in N. benthamiana impeded the cell-to-cell movement of TVBMV. NbDREPP was shown to colocalize with TVBMV P3N-PIPO and CI at plasmodesmata (PD) and traffic to PD via the early secretory pathway and the actomyosin motility system. We also show that myosin XI-2 is specially required for transporting NbDREPP to PD. In conclusion, NbDREPP is a key host protein within the early secretory pathway and the actomyosin motility system that interacts with two movement proteins and influences virus movement.

Development of new potato virus X-based vectors for gene over-expression and gene silencing assay.

Multiple plant viruses, including potato virus X (PVX), have been modified as vectors for expressing heterologous genes or silencing endogenous genes in plants. PVX-based vectors facilitate the functional analysis of genes in plant. However, they can only express one protein in a time. In this paper we report the construction of new vectors based on a 35S promoter-driven PVX infectious clone, pCaPVX100. Vector pCaPVX440 contains two additional subgenomic promoters and can be utilized to express two foreign genes at the same time. Plasmid pCaPVX760 is a CP minus vector and can be used to express foreign proteins through the gene substitution strategy. In addition, plasmid pCaPVX100 was engineered into a gene silencing vector (pCaPVX440-LIC) by introducing a ligation independent cloning (LIC) site into the vector. These results indicate that the newly developed PVX vectors are competent for multiple research purposes.