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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N et al (2006) A Plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science 312:436–439
Taganov KD, Boldin MP, Chang KJ, Baltimore D (2006) NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc Natl Acad Sci U S A 103:12481–12486
Jagadeeswaran G, Saini A, Sunkar R (2009) Biotic and abiotic stress down-regulate miR398 expression in Arabidopsis. Plant 229:1009–1014
Chapman EJ, Prokhnevsky AI, Gopinath K, Dolja VV, Carrington JC (2004) Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes Dev 18:1179–1186
Dunoyer P, Lecellier CH, Parizotto EA, Himber C, Voinnet O (2004) Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing. Plant Cell 16:1235–1250
Kanz C, Aldebert P, Althorpe N, Baker W, Baldwin A et al (2005) The EMBL nucleotide sequence database. Nucleic Acids Res 33:D29–D33
Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5: 199–206
Lurin C, Andres C, Aubourg S, Bellaoui M, Bitton F et al (2004) Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell 16:2089–2103
Yuan J, He SY (1996) The Pseudomonas syringae Hrp regulation and secretion system controls the production and secretion of multiple extracellular proteins. J Bacteriol 178:6399–6402
He P, Shan L, Lin NC, Martin GB, Kemmerling B et al (2006) Specific bacterial suppressors of MAMP signaling upstream of MAPKKK in Arabidopsis innate immunity. Cell 125:563–575
Li X, Lin H, Zhang W, Zou Y, Zhang J et al (2005) Flagellin induces innate immunity in nonhost interactions that is suppressed by Pseudomonas syringae effectors. Proc Natl Acad Sci U S A 6(102):12990–12995
Navarro L, Zipfel C, Rowland O, Keller I, Robatzek S et al (2004) The transcriptional innate immune response to flg22. Interplay and overlap with Avr gene-dependent defense responses and bacterial pathogenesis. Plant Physiol 135:1113–1128
Dharmasiri N, Dharmasiri S, Weijers D, Lechner E, Yamada M et al (2005) Plant development is regulated by a family of auxin receptor F box proteins. Dev Cell 9:109–119
Parizotto EA, Dunoyer P, Rahm N, Himber C, Voinnet O (2004) In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. Genes Dev 18:2237–2242
Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M, Dunoyer P, Yamamoto YY et al (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320(5880):1185–1190
Xie Z, Allen E, Fahlgren N, Calamar A, Givan SA, Carrington JC (2005) Expression of Arabidopsis MIRNA genes. Plant Physiol 138:2145–2154
Franco-Zorrilla JM, Valli A, Todesco M, Mateos I, Puga MI et al (2007) Target mimicry provides a new mechanism for regulation of microRNA activity. Nat Genet 39:1033–1037
Katiyar-Agarwal S, Gao S, Vivian-Smith A, Jin H (2007) A novel class of bacteria-induced small RNAs in Arabidopsis. Genes Dev 21:3123–3134
Katiyar-Agarwal S, Morgan R, Dahlbeck D, Borsani O, Villegas A Jr et al (2006) A pathogen-inducible endogenous siRNA in plant immunity. Proc Natl Acad Sci U S A 103: 18002–18007
Mi S, Cai T, Hu Y, Chen Y, Hodges E et al (2008) Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5’ terminal nucleotide. Cell 133: 116–127.
Montgomery TA, Howell MD, Cuperus JT, Li D, Hansen JE et al (2008) Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation.
Jones-Rhoades MW, Bartel DP (2004) Computational identification of plant microRNAs and their targets, including a stress-induced miRNA.
Pall GS, Hamilton AJ (2008) Improved northern blot method for enhanced detection of small RNA. Nature protocols 3: 1077–1084
Gagnot S, Tamby JP, Martin-Magniette ML, Bitton F, Taconnat L et al (2008) CAT db: a public access to Arabidopsis transcriptome data from the URGV-CATMA platform. Nucleic Acids Research 36:D986–D990
Navarro L, Jay F, Nomura K, He SY, Voinnet O (2008) Suppression of the miRNA pathway by bacterial effector proteins. Science 321:964–967
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Jay, F., Renou, JP., Voinnet, O., Navarro, L. (2010). Biotic Stress-Associated microRNAs: Identification, Detection, Regulation, and Functional Analysis. In: Meyers, B., Green, P. (eds) Plant MicroRNAs. Methods in Molecular Biology, vol 592. Humana Press. https://doi.org/10.1007/978-1-60327-005-2_13
Download citation
DOI: https://doi.org/10.1007/978-1-60327-005-2_13
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-004-5
Online ISBN: 978-1-60327-005-2
eBook Packages: Springer Protocols