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 model-based 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.
Highlights
For the C. kahawae samples, different expression profiles were observed between the two genes. cat2 seemed to be expressed only in the mycelium and repressed in appressoria, both in planta and in vitro, with significant differences (Fig 4), while thr1 expression was higher in planta and in vitro appressoria and was repressed on mycellium for almost all isolates, with significant differences (Fig 3)
We evaluated the expression stability of eight candidate reference genes across several C. kahawae samples representing different growth and infection stages, with the aim of identifying the best set for data normalization of gene expression studies
New candidate reference genes were selected based on a genome wide approach (RNA-seq datasets) and among them most had expression stability similar to the typical reference genes selected in the literature
Summary
Colletotrichum kahawae J.M. Waller & P.D. Bridge, the causal agent of Coffee Berry Disease (CBD), is a highly aggressive and specialized fungal pathogen of coffee. The causal agent of Coffee Berry Disease (CBD), is a highly aggressive and specialized fungal pathogen of coffee This pathogen currently occurs in most African regions where Arabica coffee (Coffea Arabica L.) is grown, at high altitudes, ravaging plantations and causing up to 80% yield losses, if no control measures are applied [1,2,3]. C. kahawae infects several coffee organs but maximum production losses occur when infection takes place in expanding green berries, leading to their premature dropping and mummification [2,3,4]
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