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Title: Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression

item Lim, Hyoun-Sub
item VAIRA, ANNA MARIA - National Research Council - Italy
item Domier, Leslie
item LEE, SUNG CURL - University Of California
item KIM, HONG GI - Chungnam National University
item Hammond, John

Submitted to: Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/10/2010
Publication Date: 6/20/2010
Citation: Lim, H.S., Vaira, A.M., Domier, L.L., Lee, S.C., Kim, H.G., Hammond, J. 2010. Efficiency of VIGS and gene expression in a novel bipartite potevirus vector delivery system as a function of strength of TGB1 silencing suppression. Virology. 402:149-163.

Interpretive Summary: Most previously-developed plant viral-based vectors have had limited applicability; a particular vector system is typically more suitable either for protein expression, or for Virus-Induced Gene Silencing (VIGS). Particular vectors have also been developed either for initiation of infection from DNA (inoculation fy plasmids, or by Agrobacterium-mediated infection) or separate constructs for inoculation of RNA transcripts produced in a test tube. A newly developed plant viral vector based on Alternanthera mosaic virus (AltMV) offers greater flexibility from a single system; infection can by established from either DNA or RNA from the same construct, and variants of the same vector can be used for efficient protein expression or for VIGS. A novel adaptation of the vector system allows efficient high throughput insertion of genes for either protein expression or VIGS, further increasing the utility of this vector system. This was achieved by separating the replication functions and the movement functions of the viral vector for separate delivery to the plant, in which the two parts of the virus was shown to recombine to regenerate the wild-type virus. Variants suitable for protein expression or for VIGS differ only in the efficiency of gene silencing suppression, with high efficiency suited for protein expression, and lower efficiency suited for VIGS. A particular amino acid mutation was found to strongly influence the efficacy of silencing suppression. The interations affected by the mutation were examined, and an equivalent mutation demonstrated to have similar effect on the efficacy silencing suppression in a Potato virus X viral vector system. These results will increase the utility of palnt viral vector for expression of desirable proteins, and for VIGS applications for understanding gene function.

Technical Abstract: We have developed a versatile plant viral-based vector system based on Alternanthera mosaic virus (AltMV), suitable for infection by either agroinfiltration or invivo T7 transcripts from the same infectious clone; infection via agroinfiltration is signaficantly enchanced by coinfiltration of a T7 RNA polymerase construct. Variants of this vector system are adapted for efficient protein expression or for virus-induced gene silencing (VIGS), based on a specific amino acid substitution (L88P) in the triple gene block 1 (TGB1) protein. We have further demonstrated separation of the replicase (RdRp) functions from the movement and encapsidation (TGB and coat protein, CP) functions of AltMV delivered separately by agroinfiltration, and developed a bipartite delivery system for AltMV, resulting in precise recombination of RdRp and TGB-CP constructs in planta. The bipartite delivery system has potential for high throughout cloning for both protein expression and VIGS with the appropriate TGB1 variant, for hosts including Nicotiana benthamiana and Arabidopsis thaliana. Equivalant Leu>Pro substitutions in the TGB1 protein of two other potexviruses reduced the efficacy of RNA silencing suppression as for AltMV; an infectious clone of Potato virus X (PVX) into which PVX TGB1 p86 was substituted replicated to a lower level and produced milder symptoms than with wild-type PVX TGB1 L86. The differences in silencing suppression efficiency of TGB1 mutants was further examined through subcellular localization of GFP:TGB1 fusions; AltMV TGB1 L88 and mutants retaining efficient silencing suppresion induced discrete intranuclear aggregates, whereas TGB1 P88 and other forms with reduced silencing suppression activity accumulated at the nuclear periphery.