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Journal of Virology, December 2003, p. 13315-13322, Vol. 77, No. 24
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.24.13315-13322.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Novel System for the Simultaneous Analysis of Geminivirus DNA Replication and Plant Interactions in Nicotiana benthamiana

Yiguo Hong,^1* John Stanley,² and Rene van Wezel¹

Horticulture Research International, East Malling, West Malling, Kent ME19 6BJ,¹ Department of Disease and Stress Biology, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom²

Received 28 May 2003/ Accepted 12 September 2003

The origin of replication of African cassava mosaic virus (ACMV) and a gene expression vector based on Potato virus X were exploited to devise an in planta system for functional analysis of the geminivirus replication-associated protein (Rep) in transgenic Nicotiana benthamiana line pOri-2. This line contains an integrated copy of a tandem repeat of the ACMV origin of replication flanking nonviral sequences that can be mobilized and replicated by Rep as an episomal replicon. A Rep-GFP fusion protein can also mobilize and amplify the replicon, facilitating Rep detection in planta. The activity of Rep and its mutants, Rep-mediated host response, and the correlation between Rep intracellular localization and biological functions could be effectively assessed by using this in planta system. Our results indicate that modification of amino acid residues R₂, R₅, R₇ and K₁₁ or H₅₆, L₅₇ and H₅₈ prevent Rep function in replication. This defect correlates with possible loss of Rep nuclear localization and inability to trigger the host defense mechanism resembling a hypersensitive response.

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* Corresponding author. Mailing address: Horticulture Research International, East Malling, West Malling, Kent ME19 6BJ, United Kingdom. Phone: 44 173 284 3833. Fax: 44 173 284 9067. E-mail: yiguo.hong{at}hri.ac.uk.

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Journal of Virology, December 2003, p. 13315-13322, Vol. 77, No. 24
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.24.13315-13322.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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