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Related Experiment Videos

A versatile transreplication-based system to identify cellular proteins involved in geminivirus replication.

Gabriel Morilla1, Araceli G Castillo, Werner Preiss

  • 1Unidad de Genética, Departamento de Biología Celular, Genética, y Fisiología, Universidad de Málaga, 29071 Málaga, Spain.

Journal of Virology
|March 16, 2006
PubMed
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A novel green fluorescent protein (GFP) expression system in transgenic plants allows monitoring of Tomato yellow leaf curl Sardinia virus (TYLCSV) replication. This system visualizes viral activity, aiding in the discovery of essential host factors for geminivirus replication.

Area of Science:

  • Plant Virology
  • Molecular Biology
  • Genetics

Background:

  • Geminiviruses, like Tomato yellow leaf curl Sardinia virus (TYLCSV), are significant plant pathogens.
  • Understanding geminivirus replication mechanisms is crucial for developing effective control strategies.
  • Reporter gene systems are valuable tools for studying viral replication and host interactions.

Purpose of the Study:

  • To develop a versatile green fluorescent protein (GFP) expression cassette for monitoring TYLCSV replication in transgenic plants.
  • To investigate the role of the TYLCSV replication-associated protein (Rep) in mobilizing and replicating the cassette.
  • To establish a system for identifying host factors involved in geminivirus replication.

Main Methods:

  • Construction of a transgenic *Nicotiana benthamiana* line harboring a GFP expression cassette with TYLCSV replication origins.

Related Experiment Videos

  • Superinfection of transgenic plants with TYLCSV to mobilize the episomal replicon.
  • Virus-induced gene silencing (VIGS) to transiently inhibit host factors, such as proliferating cellular nuclear antigen (PCNA).
  • Analysis of GFP expression patterns and viral DNA levels.

    • Main Results:

    • The TYLCSV episomal replicon was mobilized and replicated in transgenic plants, leading to modified GFP expression.
    • Increased GFP fluorescence was observed in veins, stems, and roots, correlating with TYLCSV replication.
    • GFP induction and episomal replicon release were dependent on the TYLCSV Rep protein and its interaction with the intergenic region.
    • Inhibition of PCNA expression reduced GFP induction and viral DNA, confirming its role in replication.
    • The system effectively monitored TYLCSV replication status, with GFP expression induced only in tissues where Rep was present.

    Conclusions:

    • The developed GFP expression system provides a visual readout of TYLCSV replication in planta.
    • This system is dependent on the geminiviral Rep protein and host factors like PCNA.
    • Transgenic plants expressing the GFP cassette are a powerful tool for identifying novel host factors required for geminivirus replication using VIGS.