Computational protocol: A first insight into the involvement of phytohormones pathways in coffee resistance and susceptibility to Colletotrichum kahawae

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Protocol publication

[…] Within the targeted phytohormone pathways, sixteen genes related with biosynthesis, reception and responsiveness [,,] were selected for expression analysis in Coffea spp. after C. kahawae challenge (). For SA, the following genes were included: Isochlorismate synthase 2 (ICS2); Phenylalanine ammonia-lyase (PAL); Non-expressor of pathogenesis-related 1 (NPR1); Pathogenesis-related (PR1). For JA the following genes were included: 12-oxoplytodienotae reductase 1-like (OPR3); Coronatine insensitive 1 (COI1); Pathogenesis-related 10 (PR10). For ET the following genes were included: 1-aminocyclepropane-1-carboxylic acid synthase 5 (ACS5); 1-aminocyclepropane-1-carboxylic acid oxidase 2 (ACO2); Ethylene resistant 1 (ETR1); Ethylene insensitive 2 (EIN2); Constitutive triple response 1 (CTR1); Ethylene-responsive factor 1 (ERF1). With the exception of PAL and PR10 genes, that were previously described in coffee [], the remaining fourteen genes were retrieved from a coffee RNA-seq database [] as being orthologous of previously described genes in Arabidopsis thaliana (TAIR database:www.arabidopsis.org). Tubulin beta-9 (β-Tub9)/ ribosomal protein S24 (S24) and Insuline Degrading Enzyme (IDE)/S24 were used as reference genes for susceptible and resistant varieties samples, respectively []. Coffee specific primers () were designed with PrimerSelect version 5.0 (DNAStar, Inc., USA) using the following parameters: amplicon length 70 and 200 bp; size between 17 and 22 bp; annealing temperature (Ta) between 58 and 62°C and GC content± 50%. [...] The qPCR experiments were carried out using SYBR Green Supermix (BioRad) in an iQ5 real-time thermal cycler (Bio-Rad, USA). Each 25μl reaction comprised 4μl cDNA template (2.5μg/μl), 12.5μl SYBR Green Supermix (Bio-Rad, USA), 0.4μl of each primer (10μM) and 0.7μl of sterile distilled water. Thermal cycling started with a denaturation step at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 15s and annealing at the respective temperature for each gene () for 30s. Each set of reactions included a negative control with no template. The amplification efficiency for each gene of interest was determined using the LinRegPCR version 2013.0. Dissociation curves () and agarose gel electrophoresis were used to analyze non-specific PCR products. Three biological replicates and two technical replicates were used for each sample. Relative gene expression (fold change) was calculated according to Hellemans et al., (2007) []. The gene expression data were further visualized using the software MeV viewer (http://www.tm4.org). [...] For statistical analysis of cytological data, Student’s t was applied using IBM®SPSS® Statistics version 20.0 (SPSS Inc., USA) software and arcsine-transformed percentages were used where appropriate. Statistical significance (p≤0.05) of gene expression between the two coffee varieties was determined by the non-parametric Mann–Whitney U test using IBM®SPSS® Statistics version 20.0 (SPSS Inc., USA) software. […]

Pipeline specifications

Software tools PrimerSelect, LinRegPCR, TM4, SPSS
Databases TAIR
Applications Miscellaneous, RNA-seq analysis, qPCR
Organisms Fungi
Chemicals Salicylic Acid