Geminivirus vectors for high‐level expression of foreign proteins in plant cells

Abstract

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins.

DNA Replicons for Plant Genome Engineering

“…LIR and SIR sequences were derived from the mild BeYDV strain (Halley-Stott et al, 2007) (GenBank accession number DQ458791). The boundaries of the LIR and SIR sequences were defined following previously described methods (Mor et al, 2003). The cauliflower mosaic virus promoter (P35S) was positioned within the replicon and upstream of the LIR.…”

“…Lack of cell-to-cell movement can be compensated using Agrobacterium or particle bombardment to deliver viral vectors to cells. This approach has been successfully used to generate replicons based on the bean yellow dwarf virus (BeYDV) for expression of reporter proteins, vaccine proteins and monoclonal antibodies (Mor et al, 2003;Zhang and Mason, 2006;Huang et al, 2009Huang et al, , 2010Regnard et al, 2010). Currently, there is no obvious upper size limit for replicons that do not move from cell to cell (Laufs et al, 1990;Matzeit et al, 1991;Shen and Hohn, 1994;Huang et al, 2009).…”

Agroinfection of tobacco by croton yellow vein mosaic virus and designing of a replicon vector for expression of foreign gene in plant

“…Deletion of coat protein in some bipartite begomoviruses has been employed to insert foreign sequence up to 800 nt [22]. Geminivirus replicon vector was also developed by deleting AV2/V2 and CP genes of other geminiviruses [11,21,24,25]. In this study, we deleted the V2 and V1 gene of CYVMV and retained the elements required for rolling-circle replication.…”

Transient Virus Expression Systems for Recombinant Protein Expression in Dicot- and Monocotyledonous Plants

“…Maize streak virus is an example of a single component virus which has been converted into an expression vector [67]. Bean yellow dwarf virus (BeYDV) has been developed into a single and dual-component replicon system that permits simultaneous, efficient replication of two DNA replicons and thus high-level accumulation of one or two recombinant proteins in the same plant cell [68,69]. This system has been used to produce express immunoglobulin proteins and human papilloma virus HPV-16 and the p24 protein of HIV-1 [70].…”