Biotic Stress-Associated microRNAs: Identification, Detection, Regulation, and Functional Analysis

  • Florence Jay
  • Jean-Pierre Renou
  • Olivier Voinnet
  • Lionel Navarro
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 592)

Abstract

The methods described herein first highlight the strategies that were used to discover a biotic stress-associated miRNA. This involved (1) the selection of transcripts that were more abundant in transgenic plants expressing viral-derived suppressors of RNA silencing and transcripts that were repressed in wild-type seedlings treated with a biotic stress, (2) a 5′ RACE-derived assay to map miRNA target sites, and (3) a bioinformatic analysis to retrieve specific miRNA loci from the Arabidopsis genome. We then describe methods used to monitor (1) the levels of primary miRNA transcripts (pri-miRNAs)/mature miRNAs and (2) the transcriptional activity of miRNAs in response to a biotic stress and bacterial challenge. Furthermore, we present a strategy to identify additional biotic stress-responsive miRNA genes and get insight into their regulation. This involves (1) a microarray approach that allows detection of pri-miRNAs, coupled with (2) a promoter analysis of co-regulated miRNA genes. Finally, we describe strategies that can be used to functionally characterize individual biotic stress-associated miRNAs, or the miRNA pathway, in disease resistance.

Key words:

Biotic stress response miRNA Bioinformatics bacteria Promoter analysis 

Notes

Acknowledgments

The authors thank P. Dunoyer, S. Dharmasiri, M. Estelle and J.D.G Jones for their discussions and contributions to this work. L.N was supported by a long-term Fellowship from the Federation of European Biochemical Societies (FEBS); O.V and F.J by a grant from the trilateral Génoplante-German Plant Genome Research Program-Spanish Ministry of Research; J-P Renou by Génoplante.

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Florence Jay
    • 1
  • Jean-Pierre Renou
    • 2
  • Olivier Voinnet
    • 1
  • Lionel Navarro
    • 1
  1. 1.Institut de Biologie Moléculaire des PlantesCNRS UPR2353 – Université Louis PasteurStrasbourg CedexFrance
  2. 2.UMR Génomique Végétale INRA-CNRS-UEVEEvryFrance

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