Structural Diversity and Differential Natural Pairings of MAT1-1-1 and MAT1-2-1 Proteins Essential for Sexual Reproduction in Ophiocordyceps sinensis Strains

The MAT1-1-1 and MAT1-2-1 proteins perform essential DNA-binding activities and regulation of the transcription of genes governing sexual reproduction in Ophiocordyceps sinensis. Previous studies have documented differential occurrence, alternative splicing, and transcriptional divergence of MAT1-1-1, MAT1-2-1, and pheromone receptor genes in Hirsutella sinensis (Genotype #1 among 17 genome-independent genotypes of O. sinensis fungi). In the present study, we analyzed the natural pairing patterns of structurally variant MAT1-1-1 and MAT1-2-1 proteins simultaneously produced by each of 20 purportedly homogenous O. sinensis strains, based on AlphaFold-predicted 3D structural models and pairwise structural superposition analyses. The differentially naturally paired mating proteins exhibited distinct heteromorphic stereostructures across strains. Specifically, the MATα_HMGbox domain of MAT1-1-1 and the HMG-box_ROX1-like domain of MAT1-2-1 displayed variable N- and/or C-terminal truncations, 1–4 amino acid substitutions at distinct sites, and concomitant alterations in hydrophobic properties and secondary/tertiary structural configurations. Thus, the differentially naturally paired but structurally divergent mating proteins support heterogeneous fungal origins within the analyzed O. sinensis strains and are inconsistent with a strictly self-fertilization reproductive model. Our findings suggest that O. sinensis adopts a self-sterile reproductive strategy, potentially involving heterothallic mating or hybrid reproduction during the lifecycle of the LEVEL-II protected Cordyceps sinensis insect–fungal complex endemic to the Qinghai-Tibet Plateau.