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Related Experiment Videos

Infectious in vitro transcripts from a plum pox potyvirus cDNA clone.

J L Riechmann1, S Laín, J A García

  • 1Centro de Biología Molecular, CSIC-UAM, Universidad Autónoma de Madrid, Spain.

Virology
|August 1, 1990
PubMed
Summary

Synthetic plum pox virus (PPV) RNA transcripts are infectious in plants when capped. Capped transcripts replicate, recover native infectivity, and lose the extra 5' G, confirming successful PPV RNA genome replication.

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Area of Science:

  • Plant Virology
  • Molecular Biology
  • RNA Synthesis

Background:

  • Plum pox virus (PPV) is a significant pathogen affecting Prunus species.
  • Understanding PPV replication is crucial for developing control strategies.
  • In vitro synthesis of infectious viral RNA is a key tool for virological studies.

Purpose of the Study:

  • To synthesize infectious full-length plum pox virus (PPV) RNA transcripts in vitro.
  • To assess the infectivity and replication characteristics of these synthetic transcripts in host plants.
  • To confirm that infections were caused by the synthetic transcripts and not by contamination.

Main Methods:

  • Cloning of the full-length PPV RNA genome into a cDNA construct.
  • In vitro transcription using T7 RNA polymerase with and without a 5' cap analog (m7GpppG).

Related Experiment Videos

  • Inoculation of Nicotiana clevelandii plants and Chenopodium foetidum with synthetic transcripts.

    • Main Results:

    • Synthesized PPV RNA transcripts were infectious in Nicotiana clevelandii plants only when a 5' cap analog was used.
    • The infectivity of synthetic transcripts was approximately 1% of native viral RNA.
    • Transcripts lost the extra 5' G during replication, recovered typical poly(A) tail distribution, and regained specific infectivity similar to native RNA.
    • Sequence heterogeneity confirmed infections originated from the transcripts.

    Conclusions:

    • In vitro synthesized PPV RNA requires 5' capping for infectivity.
    • The synthetic PPV RNA genome can replicate in plants, losing the extra 5' G and restoring native characteristics.
    • This study provides a reliable method for generating infectious PPV RNA for further research.