Polyglycine hydrolases secreted by Pleosporineae fungi that target the linker region of plant class IV chitinases*

Authors: Naumann, Todd; Wicklow, Donald; Price, Neil

Submitted to: Biochemical Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/17/2014
Publication Date: 3/17/2014
Citation: Naumann, T.A., Wicklow, D.T., Price, N.P. 2014. Polyglycine hydrolases secreted by Pleosporineae fungi that target the linker region of plant class IV chitinases. Biochemical Journal. 460(2):187-198.

Interpretive Summary: Plant chitinases are proteins involved in defense against fungal pathogens. Previously, we discovered fungalysin protease proteins which truncate specific chitinases during corn ear rot. In this research we describe a different type of fungal protease, polyglycine hydrolases, which also truncate these chitinases. Polyglycine hydrolases target a different part of the chitinase than fungalysins and appear to be used by a specific group of plant pathogenic fungi, the Pleosporineae. The part of the corn chitinase that polyglycine hydrolases target only exists in chitinases from cereal crops and related plants. We therefore predict that this activity is important for diseases of cereal crops—like corn, sorghum, rice, sugarcane, and wheat—that are caused by Pleosporineae fungi. This information is important because it increases our understanding of how fungi cause corn ear rot and other plant diseases. Moreover, the reported polyglycine hydrolase activity has not been observed previously in any branch of biology, and its description is valuable to all scientists who study proteases.

Technical Abstract: Chitinase modifying proteins (cmps) are fungal proteases that truncate plant class IV chitinases by cleaving near their amino termini. We previously described Fv-cmp, a fungalysin protease that cleaves a conserved glycine-cysteine bond within the hevein domain. Here we describe a new type of cmp—polyglycine hydrolases—as proteases that selectively cleave glycine-glycine peptide bonds within the polyglycine linker of plant class IV chitinases. Polyglycine hydrolases were purified from Cochliobolus carbonum (syn. Bipolaris zeicola, Bz-cmp) and Epicoccum sorghi (syn. Phoma sorghina, Es-cmp) and shown to cleave three different maize class IV chitinase substrates. The proteolytic cleavage sites were assessed by sodium-dodecyl sulate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and indicated cleavage of multiple peptide bonds within the polyglycine linker regions. Site-directed mutagenesis was used to produce mutants of maize ChitB chitinase in which two serine residues in its linker were systematically modified to glycines. Serine to glycine changes in the ChitB linker resulted in higher susceptibility to truncation by Bz-cmp and Es-cmp and altered substrate specificity, such that different glycine-glycine peptide bonds were cleaved. Our findings demonstrate that plant class IV chitinases with polyglycine linkers are targeted for truncation by selective polyglycine hydrolases that are secreted by plant pathogenic fungi. This novel proteolysis of polyglycine motifs is previously unreported, but the specificity is similar to that of bacterial lysostaphin proteases, which cleave pentaglycine crosslinks from peptidoglycan.