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

Authors

  • Tsafrir S. Mor,

    1. Boyce Thompson Institute for Plant Research Inc., Cornell University, Tower Road, Ithaca, New York 14853; telephone: 607-254-1365; fax: 607-254-2958
    Current affiliation:
    1. Department of Plant Biology, Arizona State University, POB 1601, Tempe, Arizona 85287
    Search for more papers by this author
  • Yong-Sun Moon,

    1. Boyce Thompson Institute for Plant Research Inc., Cornell University, Tower Road, Ithaca, New York 14853; telephone: 607-254-1365; fax: 607-254-2958
    Search for more papers by this author
  • Kenneth E. Palmer,

    1. Boyce Thompson Institute for Plant Research Inc., Cornell University, Tower Road, Ithaca, New York 14853; telephone: 607-254-1365; fax: 607-254-2958
    Current affiliation:
    1. Large Scale Biology Corporation, 3333 Vaca Valley Parkway, Vacaville, California 95688
    Search for more papers by this author
  • Hugh S. Mason

    Corresponding author
    1. Boyce Thompson Institute for Plant Research Inc., Cornell University, Tower Road, Ithaca, New York 14853; telephone: 607-254-1365; fax: 607-254-2958
    Current affiliation:
    1. Department of Plant Biology, Arizona State University, POB 1601, Tempe, Arizona 85287
    • Boyce Thompson Institute for Plant Research Inc., Cornell University, Tower Road, Ithaca, New York 14853; telephone: 607-254-1365; fax: 607-254-2958
    Search for more papers by this author

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. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 430–437, 2003.

Ancillary