ABSTRACT
BACKGROUND: Since the 2000's, plants have been used as bioreactors for the transient production of molecules of interest such as vaccines. To improve protein yield, "amplicon" vectors based on plant viruses are used. These viral constructs, engineered to carry the gene of interest replicate strongly once introduced into the plant cell, allowing significant accumulation of the protein. Here, we evaluated the suitability of the monocot-infecting RNA virus Rice yellow mottle virus (RYMV) as an amplicon vector. The promastigote surface antigen (PSA) of the protozoan Leishmania was considered as a protein of interest due to its vaccine properties against canine leishmaniasis. RESULTS: Since P1 (ORF1) and CP (ORF3) proteins are not strictly necessary for viral replication, ORF1 was deleted and the PSA gene was substituted to ORF3 in the RYMV-based vector. We evaluated its expression in the best described plant bioreactor system, Nicotiana benthamiana which, unlike rice, allows transient transformation by Agrobacterium. Despite not being its natural host, we demonstrated a low level of RYMV-based vector replication in N. benthamiana leaves. Under optimized ratio, we showed that the P19 silencing suppressor in combination with the missing viral CP ORF significantly enhanced RYMV amplicon replication in N. benthamiana. Under these optimized CP/P19 conditions, we showed that the RYMV amplicon replicated autonomously in the infiltrated N. benthamiana cells, but was unable to move out of the infiltrated zones. Finally, we showed that when the RYMV amplicon was expressed under the optimized conditions we set up, it allowed enhanced PSA protein accumulation in N. benthamiana compared to the PSA coding sequence driven by the 35S promoter without amplicon background. CONCLUSION: This work demonstrates that a non-dicot-infecting virus can be used as an amplicon vector for the efficient production of proteins of interest such as PSA in N. benthamiana leaves.
