Comparative validation of conventional and RNA-seq data-derived reference genes for qPCR expression studies of Colletotrichum kahawae

Ana Vieira, Ana Lúcia Tinoco Cabral, Joana Fino, Helena G. Azinheira, Andreia Loureiro, Pedro Talhinhas, Ana Sofia Pires, Vitor Varzea, Pilar Moncada, Helena Oliveira, Maria Do Céu Silva, Octávio S. Paulo, Dora Batista

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Colletotrichum kahawae is an emergent fungal pathogen causing severe epidemics of Coffee Berry Disease on Arabica coffee crops in Africa. Currently, the molecular mechanisms underlying the Coffea arabica-C. kahawae interaction are still poorly understood, as well as the differences in pathogen aggressiveness, which makes the development of functional studies for this pathosystem a crucial step. Quantitative real time PCR (qPCR) has been one of the most promising approaches to perform gene expression analyses. However, proper data normalization with suitable reference genes is an absolute requirement. In this study, a set of 8 candidate reference genes were selected based on two different approaches (literature and Illumina RNA-seq datasets) to assess the best normalization factor for qPCR expression analysis of C. kahawae samples. The gene expression stability of candidate reference genes was evaluated for four isolates of C. kahawae bearing different aggressiveness patterns (Ang29, Ang67, Zim12 and Que2), at different stages of fungal development and key time points of the plant-fungus interaction process. Gene expression stability was assessed using the pairwise method incorporated in geNorm and the modelbased method used by NormFinder software. For C. arabica-C. kahawae interaction samples, the best normalization factor included the combination of PP1, Act and ck34620 genes, while for C. kahawae samples the combination of PP1, Act and ck20430 revealed to be the most appropriate choice. These results suggest that RNA-seq analyses can provide alternative sources of reference genes in addition to classical reference genes. The analysis of expression profiles of bifunctional catalase-peroxidase (cat2) and trihydroxynaphthalene reductase (thr1) genes further enabled the validation of the selected reference genes. This study provides, for the first time, the tools required to conduct accurate qPCR studies in C. kahawae considering its aggressiveness pattern, developmental stage and host interaction.

Original languageEnglish
Article numbere0150651
JournalPlosOne
Volume11
Issue number3
DOIs
Publication statusPublished - 1 Mar 2016

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Colletotrichum
Genes
RNA
Gene Expression
Coffea arabica
genes
Coffea
Gene expression
aggression
Coffee
Pathogens
gene expression
Bearings (structural)
Colletotrichum kahawae
pathogens
Fungi
sampling
Catalase
Peroxidase
Crops

Cite this

Vieira, Ana ; Tinoco Cabral, Ana Lúcia ; Fino, Joana ; Azinheira, Helena G. ; Loureiro, Andreia ; Talhinhas, Pedro ; Pires, Ana Sofia ; Varzea, Vitor ; Moncada, Pilar ; Oliveira, Helena ; Do Céu Silva, Maria ; Paulo, Octávio S. ; Batista, Dora. / Comparative validation of conventional and RNA-seq data-derived reference genes for qPCR expression studies of Colletotrichum kahawae. In: PlosOne. 2016 ; Vol. 11, No. 3.
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abstract = "Colletotrichum kahawae is an emergent fungal pathogen causing severe epidemics of Coffee Berry Disease on Arabica coffee crops in Africa. Currently, the molecular mechanisms underlying the Coffea arabica-C. kahawae interaction are still poorly understood, as well as the differences in pathogen aggressiveness, which makes the development of functional studies for this pathosystem a crucial step. Quantitative real time PCR (qPCR) has been one of the most promising approaches to perform gene expression analyses. However, proper data normalization with suitable reference genes is an absolute requirement. In this study, a set of 8 candidate reference genes were selected based on two different approaches (literature and Illumina RNA-seq datasets) to assess the best normalization factor for qPCR expression analysis of C. kahawae samples. The gene expression stability of candidate reference genes was evaluated for four isolates of C. kahawae bearing different aggressiveness patterns (Ang29, Ang67, Zim12 and Que2), at different stages of fungal development and key time points of the plant-fungus interaction process. Gene expression stability was assessed using the pairwise method incorporated in geNorm and the modelbased method used by NormFinder software. For C. arabica-C. kahawae interaction samples, the best normalization factor included the combination of PP1, Act and ck34620 genes, while for C. kahawae samples the combination of PP1, Act and ck20430 revealed to be the most appropriate choice. These results suggest that RNA-seq analyses can provide alternative sources of reference genes in addition to classical reference genes. The analysis of expression profiles of bifunctional catalase-peroxidase (cat2) and trihydroxynaphthalene reductase (thr1) genes further enabled the validation of the selected reference genes. This study provides, for the first time, the tools required to conduct accurate qPCR studies in C. kahawae considering its aggressiveness pattern, developmental stage and host interaction.",
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Vieira, A, Tinoco Cabral, AL, Fino, J, Azinheira, HG, Loureiro, A, Talhinhas, P, Pires, AS, Varzea, V, Moncada, P, Oliveira, H, Do Céu Silva, M, Paulo, OS & Batista, D 2016, 'Comparative validation of conventional and RNA-seq data-derived reference genes for qPCR expression studies of Colletotrichum kahawae' PlosOne, vol. 11, no. 3, e0150651. https://doi.org/10.1371/journal.pone.0150651

Comparative validation of conventional and RNA-seq data-derived reference genes for qPCR expression studies of Colletotrichum kahawae. / Vieira, Ana; Tinoco Cabral, Ana Lúcia; Fino, Joana; Azinheira, Helena G.; Loureiro, Andreia; Talhinhas, Pedro; Pires, Ana Sofia; Varzea, Vitor; Moncada, Pilar; Oliveira, Helena; Do Céu Silva, Maria; Paulo, Octávio S.; Batista, Dora.

In: PlosOne, Vol. 11, No. 3, e0150651, 01.03.2016.

Research output: Contribution to journalArticle

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T1 - Comparative validation of conventional and RNA-seq data-derived reference genes for qPCR expression studies of Colletotrichum kahawae

AU - Vieira, Ana

AU - Tinoco Cabral, Ana Lúcia

AU - Fino, Joana

AU - Azinheira, Helena G.

AU - Loureiro, Andreia

AU - Talhinhas, Pedro

AU - Pires, Ana Sofia

AU - Varzea, Vitor

AU - Moncada, Pilar

AU - Oliveira, Helena

AU - Do Céu Silva, Maria

AU - Paulo, Octávio S.

AU - Batista, Dora

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N2 - Colletotrichum kahawae is an emergent fungal pathogen causing severe epidemics of Coffee Berry Disease on Arabica coffee crops in Africa. Currently, the molecular mechanisms underlying the Coffea arabica-C. kahawae interaction are still poorly understood, as well as the differences in pathogen aggressiveness, which makes the development of functional studies for this pathosystem a crucial step. Quantitative real time PCR (qPCR) has been one of the most promising approaches to perform gene expression analyses. However, proper data normalization with suitable reference genes is an absolute requirement. In this study, a set of 8 candidate reference genes were selected based on two different approaches (literature and Illumina RNA-seq datasets) to assess the best normalization factor for qPCR expression analysis of C. kahawae samples. The gene expression stability of candidate reference genes was evaluated for four isolates of C. kahawae bearing different aggressiveness patterns (Ang29, Ang67, Zim12 and Que2), at different stages of fungal development and key time points of the plant-fungus interaction process. Gene expression stability was assessed using the pairwise method incorporated in geNorm and the modelbased method used by NormFinder software. For C. arabica-C. kahawae interaction samples, the best normalization factor included the combination of PP1, Act and ck34620 genes, while for C. kahawae samples the combination of PP1, Act and ck20430 revealed to be the most appropriate choice. These results suggest that RNA-seq analyses can provide alternative sources of reference genes in addition to classical reference genes. The analysis of expression profiles of bifunctional catalase-peroxidase (cat2) and trihydroxynaphthalene reductase (thr1) genes further enabled the validation of the selected reference genes. This study provides, for the first time, the tools required to conduct accurate qPCR studies in C. kahawae considering its aggressiveness pattern, developmental stage and host interaction.

AB - Colletotrichum kahawae is an emergent fungal pathogen causing severe epidemics of Coffee Berry Disease on Arabica coffee crops in Africa. Currently, the molecular mechanisms underlying the Coffea arabica-C. kahawae interaction are still poorly understood, as well as the differences in pathogen aggressiveness, which makes the development of functional studies for this pathosystem a crucial step. Quantitative real time PCR (qPCR) has been one of the most promising approaches to perform gene expression analyses. However, proper data normalization with suitable reference genes is an absolute requirement. In this study, a set of 8 candidate reference genes were selected based on two different approaches (literature and Illumina RNA-seq datasets) to assess the best normalization factor for qPCR expression analysis of C. kahawae samples. The gene expression stability of candidate reference genes was evaluated for four isolates of C. kahawae bearing different aggressiveness patterns (Ang29, Ang67, Zim12 and Que2), at different stages of fungal development and key time points of the plant-fungus interaction process. Gene expression stability was assessed using the pairwise method incorporated in geNorm and the modelbased method used by NormFinder software. For C. arabica-C. kahawae interaction samples, the best normalization factor included the combination of PP1, Act and ck34620 genes, while for C. kahawae samples the combination of PP1, Act and ck20430 revealed to be the most appropriate choice. These results suggest that RNA-seq analyses can provide alternative sources of reference genes in addition to classical reference genes. The analysis of expression profiles of bifunctional catalase-peroxidase (cat2) and trihydroxynaphthalene reductase (thr1) genes further enabled the validation of the selected reference genes. This study provides, for the first time, the tools required to conduct accurate qPCR studies in C. kahawae considering its aggressiveness pattern, developmental stage and host interaction.

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