Three-Dimensional Structural Heteromorphs of Mating-Type Proteins in Hirsutella sinensis and the Natural Cordyceps sinensis Insect–Fungal Complex

The MAT1-1-1 and MAT1-2-1 proteins are essential for the sexual reproduction of <i>Ophiocordyceps sinensis</i>. Although <i>Hirsutella sinensis</i> has been postulated to be the sole anamorph of <i>O. sinensis</i> and to undergo self-fertilization under homothallism or pseudohomothallism, little is known about the three-dimensional (3D) structures of the mating proteins in the natural <i>Cordyceps sinensis</i> insect-fungal complex, which is a valuable therapeutic agent in traditional Chinese medicine. However, the alternative splicing and differential occurrence and translation of the <i>MAT1-1-1</i> and <i>MAT1-2-1</i> genes have been revealed in <i>H. sinensis</i>, negating the self-fertilization hypothesis but rather suggesting the occurrence of self-sterility under heterothallic or hybrid outcrossing. In this study, the MAT1-1-1 and MAT1-2-1 proteins in 173 <i>H. sinensis</i> strains and wild-type <i>C. sinensis</i> isolates were clustered into six and five clades in the Bayesian clustering trees and belonged to 24 and 21 diverse AlphaFold-predicted 3D structural morphs, respectively. Over three-quarters of the strains/isolates contained either MAT1-1-1 or MAT1-2-1 proteins but not both. The diversity of the heteromorphic 3D structures of the mating proteins suggested functional alterations of the proteins and provided additional evidence supporting the self-sterility hypothesis under heterothallism and hybridization for <i>H. sinensis</i>, Genotype #1 of the 17 genome-independent <i>O. sinensis</i> genotypes. The heteromorphic stereostructures and mutations of the MAT1-1-1 and MAT1-2-1 proteins in the wild-type <i>C. sinensis</i> isolates and natural <i>C. sinensis</i> insect-fungi complex suggest that there are various sources of the mating proteins produced by two or more cooccurring heterospecific fungal species in natural <i>C. sinensis</i> that have been discovered in mycobiotic, molecular, metagenomic, and metatranscriptomic studies, which may inspire future studies on the biochemistry of mating and pheromone receptor proteins and the reproductive physiology of <i>O. sinensis</i>.