Plum Pox Virus Genome-Based Vector Enables the Expression of Different Heterologous Polypeptides in Nicotiana benthamiana Plants

Abstract

Plant viral vectors have become a promising tool for the rapid and cost-effective production of recombinant proteins in plants. Among the numerous genera of viruses that have been used for heterologous expression, potyviruses offer several advantages, such as polyprotein expression strategy or a broad host range. In our work, the expression vectors pAD/pAD-agro based on the plum pox virus (PPV) genome were used for the heterologous expression of different foreign polypeptides: alfalfa mosaic virus capsid protein (AMV CP), zucchini yellow mosaic virus capsid protein (ZYMV CP), the small heat-shock protein of Cronobacter sakazakii fused with hexahistidine (sHSP-his), a fragment of influenza A virus hemagglutinin (HA2-2), influenza A virus protein PB1-F2, SARS-CoV-2 nucleocapsid protein (CoN2-his), and its N- and C-terminal fragments (CoN-1-his and CoN3-his, respectively), each fused with a hexahistidine anchor. Particular proteins differed in their accumulation, tissue localization, stability, and solubility. The accumulation rate of produced polypeptides varied from low (N, hemagglutinin fragment) to relatively high (plant viral CPs, N-terminal fragment of N, PB1-F2). Some proteins preferentially accumulated in roots (sHSP, hemagglutinin fragment, PB1-F2), showing signs of proteolytic degradation in leaf tissues. Thus, each expression requires an individual approach and optimization. Here, we summarize our several-year experiments and discuss the usefulness of the pAD/pADep vector system.