Biochemical and Molecular Study of Trichoderma harzianum Enriched Secretome Protein Profiles Using Lectin Affinity Chromatography

• Published in: Applied biochemistry and biotechnology 2019-01, Vol.187 (1), p.1-13
• Main Authors: Nauom, Stephanie, da Silva Neto, Benedito Rodrigues, Ribeiro, Marcela Suriani, Pedersoli, Wellington Ramos, Ulhoa, Cirano José, Silva, Roberto N., Monteiro, Valdirene Neves
• Format: Article
• Language: English
• Subjects: Affinity; Affinity chromatography; Ascomycota - metabolism; Biochemistry; Biological control; Biotechnology; Carbohydrates; Cell Wall - metabolism; Cell walls; Chemistry; Chemistry and Materials Science; Chromatography, Affinity - methods; Concanavalin A; Concanavalin A - chemistry; Databases, Protein; Electrophoresis, Polyacrylamide Gel; Enrichment; Fungal Proteins - genetics; Fungal Proteins - metabolism; Fungi; Fusarium - metabolism; Fusarium oxysporum; Galactose; Galactose oxidase; Gel electrophoresis; Gene expression; Gene Expression Regulation, Fungal; Gene regulation; Genes, Fungal; Glycoproteins; Glycoproteins - genetics; Glycoproteins - metabolism; Glycosylation; Glycosyltransferase; Mass Spectrometry; Molecular chains; Post-translation; Proteins; Real-Time Polymerase Chain Reaction; Secretome; Sodium lauryl sulfate; Spectrometry; Trichoderma - enzymology; Trichoderma - genetics; Trichoderma - metabolism; Trichoderma harzianum
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-018-2795-2

Protein glycosylation is one of the most studied post-translational modifications and has received considerable attention for its critical role in the cell biology of eukaryotic cells. The genus Trichoderma has been extensively studied in the biocontrol of soil-borne fungal phytopathogens. The aim of this study was to identify the proteins secreted from Trichoderma harzianum after interacting with the cell walls of two phytopathogens, Sclerotinia sclerotiorum and Fusarium oxysporum . This study used N-glycoprotein enrichment with a concanavalin A (Con A) affinity column, staining detection differential SDS-PAGE, sequencing by mass spectrometric, and protein identification by comparison with the NCBI database to detect the protein expression of the two resulting secretome samples. The majority of the proteins found in both enriched secretomes belonged to a specific class of carbohydrate-active enzymes (CAZymes), within which glycosyl hydrolases (GHs), glycosyltransferases (GTs), and auxiliary activities (AAs) were identified. In this study was described two proteins that have not been previously reported in the secretomes of Trichoderma, a glycosyltransferase (six-harpin) and a galactose oxidase, belonging to the class of auxiliary activities (AA), classified as an AA subfamily AA5-2.The expression pattern of gene encoding to 17 identified proteins, evaluated by real-time quantitative PCR (RT-qPCR), showed significant difference of expression of some GHs and proteases, suggesting a specific gene expression regulation by T. harzianum in presence of different cell walls of two phytopathogens.