Rice yellow mottle virus is a suitable amplicon vector for an efficient production of an anti-leishmianiasis vaccine in Nicotiana benthamiana leaves.

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.

Development of an attenuated potato virus Y mutant carrying multiple mutations in helper-component protease for cross-protection.

Tobacco (Nicotiana tabacum) is one of the major cash crops in China. Potato virus Y (PVY), a representative member of the genus Potyvirus, greatly reduces the quality and yield of tobacco leaves by inducing veinal necrosis. Mild strain-mediated cross-protection is an attractive method of controlling diseases caused by PVY. Currently, there is a lack of effective and stable attenuated PVY mutants. Potyviral helper component-protease (HC-Pro) is a likely target for the development of mild strains. Our previous studies showed that the residues lysine at positions 124 and 182 (K124 and K182) in HC-Pro were involved in PVY virulence, and the conserved KITC motif in HC-Pro was involved in aphid transmission. In this study, to improve the stability of PVY mild strains, K at position 50 (K50) in KITC motif, K124, and K182 were separately substituted with glutamic acid (E), leucine (L), and arginine (R), resulting in a triple-mutant PVY-HCELR. The mutant PVY-HCELR had attenuated virulence and did not induce leaf veinal necrosis symptoms in tobacco plants and could not be transmitted by Myzus persicae. Furthermore, PVY-HCELR mutant was genetically stable after six serial passages, and only caused mild mosaic symptoms in tobacco plants even at 90 days post inoculation. The tobacco plants cross-protected by PVY-HCELR mutant showed high resistance to the wild-type PVY. This study showed that PVY-HCELR mutant was a promising mild mutant for cross-protection to control PVY.

Expression of the crucifer-infecting TMV-Cg movement protein in tobacco plants complements in trans a TMV-U1 trafficking-deficient mutant

Tobamovirus movement proteins play a determinant role in the establishment of infections in plants, allowing the local movement of viral RNA genome through plasmodesmatas. We expressed the movement protein (MP) of the crucifer- and garlic-infecting Tobacco Mosaic Virus strain Cg (TMV-Cg) in both resistant Xanthi NN and sensitive Xanthi nn Nicotiana tabacum plants. MP-Cg function was assayed by inoculating transgenic plants with a trafficking-deficient mutant of TMV strain U1. Following infection, local necrotic lesions were developed in resistant transgenic plants, and a systemic infection was produced in sensitive tobaccos. Thus, movement function of the mutant virus was complemented in trans by MP-Cg expressed in transgenic plants, causing the same symptoms as wild-type strain. We demonstrated that the function of MP-U1 could be replaced efficiently by MP-Cg, even though these proteins share only 36% of identity. Similar hydrophobic patterns of MP-Cg and MP-U1 suggests structure and function conservations of both proteins. This work is an example of how two tobamoviruses differing in their host range help to understand viral movement mechanism during the infection.