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

Authors: Naumann, Todd; Price, Neil

Submitted to: Physiological and Molecular Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/5/2018
Publication Date: 12/14/2018
Citation: Naumann, T.A., Price, N.P.J. 2018. Purification and in vitro activities of a chitinase-modifying protein from the corn ear rot pathogen Stenocarpella maydis. Physiological and Molecular Plant Pathology 106:74-80. https://doi.org/10.1016/j.pmpp.2018.12.004.
DOI: https://doi.org/10.1016/j.pmpp.2018.12.004

Interpretive Summary: Diplodia ear rot is a common fungal corn disease that decreases yields, contaminates grains with mycotoxins, and causes diplodiosis, a fatal disease of sheep and cattle. In previous research scientists at the National Center for Agricultural Utilization Research, Peoria, IL, discovered that the fungus that causes diplodia ear rot inactivates a plant defense protein called ChitA. They further found that a single nucleotide change in the corn genome gives ChitA resistance to this inactivation. How the fungus inactivates this plant defense protein was a mystery. In order to guide efforts to enhance corn resistance to disease, we isolated the fungal protein that inactivates ChitA and determined how it works. These results will facilitate the development of novel methods to improve diplodia ear-rot resistance, which will increase corn yields and reduce mycotoxins in harvested grains.

Technical Abstract: Stenocarpella 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 Stm-cmp is unrelated to the three previously described types of CMPs. The diversity of CMP proteases suggests that ChitA and related plant proteins play an important role in plant defense.