Protein QID74 protects the cell wall of Trichoderma from degradation caused by its own chitinase, which lacks a carbohydrate-binding module

ABSTRACT The fungal cell wall, formed by a chitin backbone, is critical for cellular integrity and environmental fitness. Mycoparasitic fungi of the genus Trichoderma deploy an arsenal of potent chitinases to dismantle the cell walls of prey fungi. However, the mechanisms that safeguard the mycoparasite from self-inflicted enzymatic damage remain poorly understood. While most chitinases possess carbohydrate-binding modules (CBMs) to facilitate substrate recognition, the critical group A chitinase of Trichoderma harzianum is conspicuously CBM-free. In this study, we investigated the evolutionary and functional rationale for this structural omission by comparing native Chit46 with engineered CBM-fused variants. Results demonstrate that while native Chit46 promotes the hyphal growth of T. harzianum , the fusion of a CBM shifts the enzymatic activity from highly specific to self-destructive. CBM-fused variants significantly enhanced the degradation of host cell walls but simultaneously triggered the hydrolysis of Trichoderma ’s own hyphae, a phenotype absent in the native enzyme. These findings suggest an evolutionary selection against CBMs in specific chitinases to prevent deleterious autocatalytic activity. Furthermore, QID74 was identified as a critical cell wall-associated “shield” protein that specifically binds Chit46 to prevent self-hydrolysis. Structural characterization revealed that QID74 is composed of 13 tandem 59-residue repeats containing conserved tridecapeptide motifs (NGKQCVCPKGQVW). Biochemical validation via His pull-down and isothermal titration calorimetry confirmed that these repetitive units are essential and sufficient for chitinase binding. Our results illuminate a sophisticated “arrow-and-shield” mechanism that balances offensive mycoparasitic capabilities with the maintenance of self-integrity, providing new insights into fungal self-recognition and the optimization of microbial biocontrol agents. IMPORTANCE This study uncovers a unique survival strategy in Trichoderma . By lacking a carbohydrate-binding module, Trichoderma ’s chitinase can effectively degrade host cell walls while minimizing self-damage. The protective protein QID74 further safeguards the cell wall by binding to the chitinase. This research offers novel insights into fungal biology and ecology, with potential applications in developing sustainable biocontrol agents.