In situ/subcellular localization of arabinogalactan protein expression by fluorescent in situ hybridization (FISH)
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2020
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Humana Press Inc. Springer
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da Costa ML, Solís MT, Testillano PS, Coimbra S. In Situ/Subcellular Localization of Arabinogalactan Protein Expression by Fluorescent In Situ Hybridization (FISH). Methods Mol Biol. 2020;2149:403-427. doi: 10.1007/978-1-0716-0621-6_23. PMID: 32617948.
Abstract
The arabinogalactan proteins are highly glycosylated and ubiquitous in plants. They are involved in several aspects of plant development and reproduction; however, the mechanics behind their function remains for the most part unclear, as the carbohydrate moiety, covering the most part of the protein core, is poorly characterized at the individual protein level. Traditional immunolocalization using antibodies that recognize the glycosidic moiety of the protein cannot be used to elucidate individual proteins’ distribution, function, or interactors. Indirect approaches are typically used to study these proteins, relying on reverse genetic analysis of null mutants or using a reporter fusion system. In the method presented here, we propose the use of RNA probes to assist in the localization of individual AGPs expression/mRNAs in tissues of Arabidopsis by fluorescent in situ hybridization, FISH. An extensive description of all aspects of this technique is provided, from RNA probe synthesis to the hybridization, trying to overcome the lack of specific antibodies for the protein core of AGPs.
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This work was financed by FEDER through the COMPETE program, and by Portuguese National funds through FCT, Fundaçao para a Ciência e Tecnologia (Project PTDC/AGR-GPL/115358/ 2009 and FCT - 02-SAICT-2017 – POCI-01-0145-FEDER027839) and PhD grant SFRH/BD/111781/2015), and received support from Spanish–Portuguese Joint Project Nº E 30/12. EU project 690946 ‘SexSeed’ (Sexual Plant Reproduction – Seed Formation) funded by H2020-MSCA-RISE-2015.