In situ/Subcellular localization of arabinogalactan protein expression by fluorescent in situ hybridization, FISH

31 p. Methods Mol Biol 2149:403-427The 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.This work was financed by FEDER through the COMPETE program, and by Portuguese National funds through FCT, Fundação para a Ciência eTecnologia (Project PTDC/AGR-GPL/115358/2009 and FCT - 02-SAICT-2017 – POCI-01-0145-FEDER-027839) 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.Peer reviewe

Genome-wide association study reveals a set of genes associated with resistance to the Mediterranean corn borer (Sesamia nonagrioides L.) in a maize diversity panel

[Background] Corn borers are the primary maize pest; their feeding on the pith results in stem damage and yield losses. In this study, we performed a genome-wide association study (GWAS) to identify SNPs associated with resistance to Mediterranean corn borer in a maize diversity panel using a set of more than 240,000 SNPs.[Results] Twenty five SNPs were significantly associated with three resistance traits: 10 were significantly associated with tunnel length, 4 with stem damage, and 11 with kernel resistance. Allelic variation at each significant SNP was associated with from 6 to 9% of the phenotypic variance. A set of genes containing or physically close to these SNPs are proposed as candidate genes for borer resistance, supported by their involvement in plant defense-related mechanisms in previously published evidence. The linkage disequilibrium decayed (r2 < 0.10) rapidly within short distance, suggesting high resolution of GWAS associations.[Conclusions] Most of the candidate genes found in this study are part of signaling pathways, others act as regulator of expression under biotic stress condition, and a few genes are encoding enzymes with antibiotic effect against insects such as the cystatin1 gene and the defensin proteins. These findings contribute to the understanding the complex relationship between plant-insect interactions.This work was supported by the National Plan for Research and Development of Spain (projects AGL2012-33415). L.F. Samayoa acknowledges a contract JAE-Predoc from the Spanish Council for Scientific Research (CSIC).Peer reviewe