Purification and in vitro activities of a chitinase-modifying protein from the corn ear rot pathogen Stenocarpella maydis.

Publication date: Available online 8 December 2018Source: Physiological and Molecular Plant PathologyAuthor(s): Todd A. Naumann, Neil P.J. PriceAbstractStenocarpella maydis is the most common corn ear rot pathogen throughout the world. We previously reported that this fungus secretes a protease, Stm-cmp, which functions as a chitinase-modifying protein (CMP) to truncate corn ChitA chitinase. In the current manuscript we describe the purification of Stm-cmp from fungal cultures and characterize its activity using in vitro protease assays. Hydrophobic interaction chromatography was a key purification step as Stm-cmp bound the column under low salt conditions, allowing its separation from co-extracted proteins. The purified fraction consisted of predominantly a single protein with a mass of 100 kDa. SDS-PAGE-based protease assays in which corn ChitA chitinase was treated with the purified Stm-cmp demonstrated that it functions as an exoprotease, progressively shortening ChitA to create two truncated proteins. MALDI-TOF-based protease assays demonstrated that the protease also functions as an endoprotease to release a large peptide from the amino-terminus that is subsequently trimmed by removal of three amino acids from the amino-terminus. The complex behavior of Stm-cmp is surprising because previously characterized CMPs function as endoproteases. The biophysical characteristics of Stm-cmp observed during purification, its molecular mass, and its unique activity indicate that St...
Source: Physiological and Molecular Plant Pathology - Category: Molecular Biology Source Type: research