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Reference Gene Selection and Validation for the Early Responses to Downy Mildew Infection in Susceptible and Resistant Vitis vinifera Cultivars

The pivotal role of cultivated grapevine (Vitis vinifera L.) in many countries economy is compromised by its high susceptibility to Plasmopara viticola, the causal agent of downy mildew disease. Recent research has identified a set of genes related to resistance which may be used to track downy mild...

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Autores principales: Monteiro, Filipa, Sebastiana, Mónica, Pais, Maria Salomé, Figueiredo, Andreia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3762845/
https://www.ncbi.nlm.nih.gov/pubmed/24023800
http://dx.doi.org/10.1371/journal.pone.0072998
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author Monteiro, Filipa
Sebastiana, Mónica
Pais, Maria Salomé
Figueiredo, Andreia
author_facet Monteiro, Filipa
Sebastiana, Mónica
Pais, Maria Salomé
Figueiredo, Andreia
author_sort Monteiro, Filipa
collection PubMed
description The pivotal role of cultivated grapevine (Vitis vinifera L.) in many countries economy is compromised by its high susceptibility to Plasmopara viticola, the causal agent of downy mildew disease. Recent research has identified a set of genes related to resistance which may be used to track downy mildew infection. Quantification of the expression of these resistance genes requires normalizing qPCR data using reference genes with stable expression in the system studied. In this study, a set of eleven genes (VATP16, 60 S, UQCC, SMD3, EF1α, UBQ, SAND, GAPDH, ACT, PsaB, PTB2) was evaluated to identify reference genes during the first hours of interaction (6, 12, 18 and 24 hpi) between two V. vinifera genotypes and P. viticola. Two analyses were used for the selection of reference genes: direct comparison of susceptible, Trincadeira, and resistant, Regent, V. vinifera cultivars at 0 h, 6, 12, 18 and 24 hours post inoculation with P. viticola (genotype effect); and comparison of each genotype with mock inoculated samples during inoculation time-course (biotic stress effect). Three statistical methods were used, GeNorm, NormFinder, and BestKeeper, allowing to identify UBQ, EF1α and GAPDH as the most stable genes for the genotype effect. For the biotic stress effect, EF1α, SAND and SMD3 were the most constant for the susceptible cultivar Trincadeira and EF1α, GAPDH, UBQ for the resistant cultivar Regent. In addition, the expression of three defense-related transcripts, encoding for subtilisin-like protein, CYP and PR10, was analysed, for both datasets, during inoculation time-course. Taken together, our results provide guidelines for reference gene(s) selection towards a more accurate and widespread use of qPCR to study the first hours of interaction between different grapevine cultivars and P. viticola.
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spelling pubmed-37628452013-09-10 Reference Gene Selection and Validation for the Early Responses to Downy Mildew Infection in Susceptible and Resistant Vitis vinifera Cultivars Monteiro, Filipa Sebastiana, Mónica Pais, Maria Salomé Figueiredo, Andreia PLoS One Research Article The pivotal role of cultivated grapevine (Vitis vinifera L.) in many countries economy is compromised by its high susceptibility to Plasmopara viticola, the causal agent of downy mildew disease. Recent research has identified a set of genes related to resistance which may be used to track downy mildew infection. Quantification of the expression of these resistance genes requires normalizing qPCR data using reference genes with stable expression in the system studied. In this study, a set of eleven genes (VATP16, 60 S, UQCC, SMD3, EF1α, UBQ, SAND, GAPDH, ACT, PsaB, PTB2) was evaluated to identify reference genes during the first hours of interaction (6, 12, 18 and 24 hpi) between two V. vinifera genotypes and P. viticola. Two analyses were used for the selection of reference genes: direct comparison of susceptible, Trincadeira, and resistant, Regent, V. vinifera cultivars at 0 h, 6, 12, 18 and 24 hours post inoculation with P. viticola (genotype effect); and comparison of each genotype with mock inoculated samples during inoculation time-course (biotic stress effect). Three statistical methods were used, GeNorm, NormFinder, and BestKeeper, allowing to identify UBQ, EF1α and GAPDH as the most stable genes for the genotype effect. For the biotic stress effect, EF1α, SAND and SMD3 were the most constant for the susceptible cultivar Trincadeira and EF1α, GAPDH, UBQ for the resistant cultivar Regent. In addition, the expression of three defense-related transcripts, encoding for subtilisin-like protein, CYP and PR10, was analysed, for both datasets, during inoculation time-course. Taken together, our results provide guidelines for reference gene(s) selection towards a more accurate and widespread use of qPCR to study the first hours of interaction between different grapevine cultivars and P. viticola. Public Library of Science 2013-09-04 /pmc/articles/PMC3762845/ /pubmed/24023800 http://dx.doi.org/10.1371/journal.pone.0072998 Text en © 2013 Monteiro et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Monteiro, Filipa
Sebastiana, Mónica
Pais, Maria Salomé
Figueiredo, Andreia
Reference Gene Selection and Validation for the Early Responses to Downy Mildew Infection in Susceptible and Resistant Vitis vinifera Cultivars
title Reference Gene Selection and Validation for the Early Responses to Downy Mildew Infection in Susceptible and Resistant Vitis vinifera Cultivars
title_full Reference Gene Selection and Validation for the Early Responses to Downy Mildew Infection in Susceptible and Resistant Vitis vinifera Cultivars
title_fullStr Reference Gene Selection and Validation for the Early Responses to Downy Mildew Infection in Susceptible and Resistant Vitis vinifera Cultivars
title_full_unstemmed Reference Gene Selection and Validation for the Early Responses to Downy Mildew Infection in Susceptible and Resistant Vitis vinifera Cultivars
title_short Reference Gene Selection and Validation for the Early Responses to Downy Mildew Infection in Susceptible and Resistant Vitis vinifera Cultivars
title_sort reference gene selection and validation for the early responses to downy mildew infection in susceptible and resistant vitis vinifera cultivars
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3762845/
https://www.ncbi.nlm.nih.gov/pubmed/24023800
http://dx.doi.org/10.1371/journal.pone.0072998
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