Biotic stress-associated microRNAs: identification, detection, regulation, and functional analysis.
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.
Jay
Florence
F
Institut de Biologie Moléculaire des Plantes, CNRS UPR2353-Université Louis Pasteur, Strasbourg Cedex, France.
Renou
Jean-Pierre
JP
Voinnet
Olivier
O
Navarro
Lionel
L
D017360:Arabidopsis / Q000235:genetics* / Q000382:microbiology*; D001483:Base Sequence; D019295:Computational Biology / Q000379:methods; D018506:Gene Expression Regulation, Plant / Q000235:genetics / Q000502:physiology; D035683:MicroRNAs / Q000235:genetics*; D008969:Molecular Sequence Data; D044224:Pseudomonas syringae / Q000472:pathogenicity / Q000502:physiology; D017423:Sequence Analysis, RNA; D012689:Sequence Homology, Nucleic Acid
D035683:MicroRNAs