Ma Ting, Yang Chengde, Jin Mengjun, Cai Fengfeng, Zhang Cuiwen, Osei Richard
College of Plant Protection, Biocontrol Engineering Laboratory of Crop Diseases and Pests of Gansu Province, Gansu Agricultural University, Lanzhou, 730070, China.
College of Plant Protection, Biocontrol Engineering Laboratory of Crop Diseases and Pests of Gansu Province, Gansu Agricultural University, Lanzhou, 730070, China.
Microb Pathog. 2025 Sep;206:107834. doi: 10.1016/j.micpath.2025.107834. Epub 2025 Jun 22.
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.
辅助活性家族9（AA9）裂解多糖单加氧酶（LPMO）是一类氧化降解多种多糖的单铜酶。尽管对这类酶进行了广泛研究，但参与致病性的关键LPMO基因及其特性仍需进一步研究。在此，我们通过挖掘马铃薯炭疽病病原菌球炭疽菌的转录组数据，获得了候选效应子AA9 LPMO基因CcAA9 - 20333。克隆了一个新的CcAA9 - 20333基因，进行了原核表达、纯化及特性分析。结果表明，CcAA9 - 20333在40 - 60℃表现出最佳活性，具有较高的热稳定性，即使在60℃下暴露80分钟仍保持活性。在pH 6.0时检测到CcAA9 - 20333的最高酶活性，在4℃下于pH 6.0孵育24小时后观察到其具有优异的pH稳定性。CcAA9 - 20333粗酶在几种不同金属离子存在下能释放还原糖，并表现出底物特异性。以pET30a(+) - CcAA9 - 20333为模板，采用一步点突变方法获得了四个突变体（M1 - M4）。致病性测定表明，野生型对茄科植物的潜在致病性强于任何一个突变菌株。克隆了CcAA9 - 20333基因，进行了异源表达，并采用定点突变方法对潜在的底物结合位点进行了特性分析。这是关于来自球炭疽菌的LPMO基因CcAA9 - 20333及其在致病性中作用的首次报道。
