A novel candidate effector CcAA9-20333 from Colletotrichum coccodes: identification, expression, and functional site analysis.

Auxiliary activity family 9 (AA9) lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that oxidatively degrade a variety of polysaccharides. Despite extensive research on this class of enzymes, the essential LPMO gene involved in pathogenicity and its characterization still requires further study. Here, we obtained the candidate effector AA9 LPMOs gene CcAA9-20333 by mining transcriptome data of Colletotrichum coccodes, the causal agent of potato anthracnose. A novel CcAA9-20333 gene was cloned, prokaryotically expressed, purified, and characterized. Results indicated that the CcAA9-20333 exhibits optimal activity at 40-60 °C and has high thermostability remaining active even exposed to 60 °C for 80 min. The highest CcAA9-20333 enzymatic activity was detected at pH 6.0 and excellent pH stability was observed after incubation at pH 6.0 for 24 h at 4 °C. CcAA9-20333 crude enzyme released reducing sugars in the presence of several different metal ions and exhibited substrate specificity. Four mutants (M1-M4) were obtained with one-step point mutation methodology using pET30a(+)-CcAA9-20333 as a template. A pathogenicity assay indicated that the potential pathogenicity of wild-type to Solanaceae plants was stronger than any of the mutant strains. The gene CcAA9-20333 was cloned, heterologously expressed, and potential substrate binding sites were characterized using site-directed mutation methodology. This is the first report on the LPMO gene CcAA9-20333 from C. coccodes and its role in pathogenicity.