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Journal of Virology, April 2006, p. 3624-3633, Vol. 80, No. 7
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.7.3624-3633.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A Versatile Transreplication-Based System To Identify Cellular Proteins Involved in Geminivirus Replication

Gabriel Morilla,1,{dagger} Araceli G. Castillo,1,{ddagger} Werner Preiss,2 Holger Jeske,2 and Eduardo R. Bejarano1*

Unidad de Genética, Departamento de Biología Celular, Genética, y Fisiología, Universidad de Málaga, 29071 Málaga, Spain,1 Biologisches Institut, Abteilung für Molekularbiologie und Virologie der Pflanzen, Universität Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart, Germany2

Received 6 October 2005/ Accepted 17 December 2005

A versatile green fluorescent protein (GFP) expression cassette containing the replication origins of the monopartite begomovirus Tomato yellow leaf curl Sardinia virus (TYLCSV) is described. Transgenic Nicotiana benthamiana plants containing one copy of the cassette stably integrated into their genome were superinfected with TYLCSV, which mobilized and replicated the cassette as an episomal replicon. The expression of the reporter gene (the GFP gene) was thereby modified. Whereas GFP fluorescence was dimmed in the intercostal areas, an increase of green fluorescence in veins of all leaves placed above the inoculation site, as well as in transport tissues of roots and stems, was observed. The release of episomal trans replicons from the transgene and the increase in GFP expression were dependent on the cognate geminiviral replication-associated protein (Rep) and required interaction between Rep and the intergenic region of TYLCSV. This expression system is able to monitor the replication status of TYLCSV in plants, as induction of GFP expression is only produced in those tissues where Rep is present. To further confirm this notion, the expression of a host factor required for geminivirus replication, the proliferating cellular nuclear antigen (PCNA) was transiently silenced. Inhibition of PCNA prevented GFP induction in veins and reduced viral DNA. We propose that these plants could be widely used to easily identify host factors required for geminivirus replication by virus-induced gene silencing.

* Corresponding author. Mailing address: Unidad de Genética, Departamento de Biología Celular, Genética, y Fisiología, Universidad de Málaga, 29071 Málaga, Spain. Phone: 34-952131677. Fax: 34-9522000. E-mail: Edu_rodri{at}uma.es.

{dagger} Present address: Institut de Biologie Moleculaire des Plantes du CNRS, 12, rue du General Zimmer, 67084 Strasbourg Cedex, France.

{ddagger} Present address: Wellcome Trust Centre for Cell Biology ICMB, Mayfield Road, 6.33 M. Swann Building, EH9 3JR Edinburgh, United Kingdom.

Journal of Virology, April 2006, p. 3624-3633, Vol. 80, No. 7
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.7.3624-3633.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Jin, M., Li, C., Shi, Y., Ryabov, E., Huang, J., Wu, Z., Fan, Z., Hong, Y. (2008). A single amino acid change in a geminiviral Rep protein differentiates between triggering a plant defence response and initiating viral DNA replication. J. Gen. Virol. 89: 2636-2641 [Abstract] [Full Text]  
  • Peretz, Y., Mozes-Koch, R., Akad, F., Tanne, E., Czosnek, H., Sela, I. (2007). A Universal Expression/Silencing Vector in Plants. Plant Physiol. 145: 1251-1263 [Abstract] [Full Text]  


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