Subcellular localization of alfalfa mosaic virus replicase proteins
Abstract
Alfalfa Mosaic Virus (AMV), a member of the Bromoviridae family of plant viruses, is a plus-sense RNA virus. AMV encodes only two proteins involved in replication, P1 and P2. P1 contains a helicase domain close to the C-terminus and a methyltransferase domain near the N-terminus, whereas, P2 contains an RNA polymerase domain with the conserved polymerase GDD motif. In this study, Arabdopsis thaliana protoplasts have been established as a system for studying the replication of AMV. Isolation and transfection protocols for protoplasts derived from arabidopsis cell culture have been developed to establish infection using AMV RNA or cDNAs corresponding to AMV RNA1-3 under control of the 35S promoter. Confocal laser scanning microscopy was used with living protoplasts to determine the location of GFP-fused P1 and P2 proteins. P1 localize to multivesicular bodies (MVBs) in the prevacuolar compartment (PVC) as indicated by colocalization of P1-GFP, FM4-64, and EYFP-RabF2a/Rha1. GFP-P2 did not localize to any organelle, but was throughout the cytoplasm. However, P1 recruited the cytosolic GFP-P2 protein to the PVC/MVBs compartments. P1 localized on the tonoplast in the presence of P2 and RNA3, as well as in the presence of RNA2. This suggests that P1, P2, and an RNA 3' UTR are needed, which further suggests that a fully functional replicase complex is probably required for transport of the complex to the tonoplast through the PVC compartment.
Degree
Ph.D.
Advisors
Loesch-Fries, Purdue University.
Subject Area
Cellular biology|Plant sciences|Virology
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