Three-Dimensional Structural Heteromorphs of Mating-Type Proteins in Hirsutella sinensis and the Natural Cordyceps...

The MAT1-1-1 and MAT1-2-1 proteins are essential for the sexual reproduction of Ophiocordyceps sinensis. Although Hirsutella sinensis has been postulated to be the sole anamorph of O. sinensis 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 Cordyceps sinensis insect-fungal complex, which is a valuable therapeutic agent in traditional Chinese medicine. However, the alternative splicing and differential occurrence and translation of the MAT1-1-1 and MAT1-2-1 genes have been revealed in H. sinensis, 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 H. sinensis strains and wild-type C. sinensis 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 H. sinensis, Genotype #1 of the 17 genome-independent O. sinensis genotypes. The heteromorphic stereostructures and mutations of the MAT1-1-1 and MAT1-2-1 proteins in the wild-type C. sinensis isolates and natural C. sinensis insect-fungi complex suggest that there are various sources of the mating proteins produced by two or more cooccurring heterospecific fungal species in natural C. sinensis 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 O. sinensis.