Comparative proteomic analysis of Fusarium oxysporum f. sp. cubense strains (Foc R1 and Foc TR4) provides better insights into mechanisms of their virulence, habitat adaptation and pathogenesis
Description
Fusarium oxysporum f. sp. cubense (Foc), a devastative soil-borne fungal pathogen causing vascular wilt (i.e. Panama disease) which leads to severe crop losses in most of the banana-growing regions of the world. The study aims to compare the proteome of the two pathogenic Foc virulent strains, Race 1 (Foc R1) and tropical race 4 (Foc TR4) that are capable of infecting the Cavendish group of bananas using 2-dimensional (2-D) gel electrophoresis, MALDI-TOF/MS and MS/MS analysis.
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Steps to reproduce
Seven-days-old mycelium of the single spore culture of Foc strains grown on potato dextrose agar (PDA) medium at 25±2 °C was further transferred to 150 mL potato dextrose liquid medium and incubated for seven days at 25±2 °C under constant shaking of 120 rpm. The mycelia were filtered through cheesecloth and washed with ice-cold 0.1 mM Phenyl methyl sulfonyl fluoride. Mycelia were then blotted dry with sterile filter paper stacks and used immediately for protein extraction. Protein extraction from air-dried Foc mycelium was performed according to Kalaiponmani et al (2017). Protein concentration was estimated according to the Bradford method [19] against Bovine Serum Albumin as standard and extracted protein. For the first dimensional isoelectric point-based separation, 250 μg of total protein was loaded on a 13 cm Immobiline dry-strip (pH 4-7, M/s. GE Healthcare Life Sciences, Amersham, UK), on a rehydration tray. For the second dimensional molecular weight-based separation, the strips were equilibrated with 10 mL of equilibration buffer I (50 mM Tris–HCl, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.002% (v/v) bromophenol blue) for 12 min containing 1% (w/v) dithiothreitol. Electrophoresed gels were visualized by colloidal Coomassie Brilliant Blue (CBB) staining (Neuhoff et al., 1988). Stained and washed gels were scanned using EPSON® Perfection 750 Pro Scanner (EPSON® Inc. USA). Careful inspection and comparison of the normalized proteome profiles from the two isolates shown qualitative and quantitative variations. Spot detection, measurement and matching were done using Hoefer™ 2-D view software (Non-linear, USA, Inc.) according to the standard protocol. Selectively excised spots from the preparative gels (stained with CBB) were sent to the proteomics facility, Molecular Biophysics Unit, Indian Institute of Science, Bengaluru, India, for MALDI-TOF/MS and MS/MS analysis. The resulted mass fingerprint and fragmentation spectra data of peptides were searched against taxonomy fungi in NCBI entries using MASCOT server (www.matrixscience.com) to classify the functions of the peptide fingerprints.