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Mutation of key amino acids in the polygalacturonase-inhibiting proteins CkPGIP1 and GhPGIP1 improves resistance to Verticillium wilt in cotton.

Verticillium wilt, one of the most devastating diseases of cotton (Gossypium hirsutum), causes severe yield and quality losses. Given the effectiveness of plant polygalacturonase-inhibiting proteins (PGIPs) in reducing fungal polygalacturonase (PG) activity, it is necessary to uncover the key functional amino acids to enhance cotton resistance to Verticillium dahliae. To identify novel antifungal proteins, the selectivity of key amino acids was investigated by screening against a panel of relevant PG-binding residues. Based on the obtained results, homologous models of the mutants were established. The docking models showed that hydrogen bonds and structural changes in the convex face in the conserved portion of leucine-rich repeats (LRRs) may be essential for enhanced recognition of PG. Additionally, we successfully constructed Cynanchum komarovii PGIP1 (CkPGIP1) mutants Asp176Val, Pro249Gln, and Asp176Val/Pro249Gln and G. hirsutum PGIP1 (GhPGIP1) mutants Glu169Val, Phe242Gln, and Glu169Val/Phe242Gln with site-directed mutagenesis. The proteins of interest can effectively inhibit VdPG1 activity and V. dahliae mycelial growth in a dose-dependent manner. Importantly, mutants that overproduced PGIP in Arabidopsis and cotton showed enhanced resistance to V. dahliae, with reduced Verticillium-associated chlorosis and wilting. Furthermore, the lignin content was measured in mutant-overexpressing plants, and the results showed enhanced lignification of the xylem, which blocked the spread of V. dahliae. Thus, using site-directed mutagenesis assays, we showed that mutations in CkPGIP1 and GhPGIP1 give rise to PGIP versatility, which allows evolving recognition specificities for PG and is required to promote Verticillium resistance in cotton by restricting the growth of invasive fungal pathogens.