Mutations and Differential Transcription of Mating-Type and Pheromone Receptor Genes in Hirsutella sinensis and the Natural Cordyceps sinensis Insect-Fungi Complex

The natural Cordyceps sinensis insect-fungal complex is one of the most highly valued therapeutic agents in traditional Chinese medicine. The sexual reproduction of Ophiocordyceps sinensis is controlled by the expression of mating-type genes within the MAT1-1 and MAT1-2 idiomorphs and a-/alpha-pheromone receptor genes. This study revealed the differential expression of these genes in Hirsutella sinensis (Genotype #1 among the 17 genotypes of O. sinensis) and alternative splicing of the MAT1-2-1 gene, characterized by an unspliced intron I that contains stop codons. Mutations of the mating and pheromone receptor proteins were also identified, potentially leading to changes in the secondary structures and functionalities of the proteins. These results indicate the requirement of mating partners under either heterothallism or hybridization during the sexual life of natural C. sinensis but do not support the "self-fertilization" hypothesis for O. sinensis under homothallism or pseudohomothallism. Sexual reproduction in ascomycetes is controlled by the mating-type (MAT) locus. (Pseudo)homothallic reproduction has been hypothesized on the basis of genetic data from Hirsutella sinensis (Genotype #1 of Ophiocordyceps sinensis). However, the differential occurrence and differential transcription of mating-type genes in the MAT1-1 and MAT1-2 idiomorphs were found in the genome and transcriptome assemblies of H. sinensis, and the introns of the MAT1-2-1 transcript were alternatively spliced with an unspliced intron I that contains stop codons. These findings reveal that O. sinensis reproduction is controlled at the genetic, transcriptional, and coupled transcriptional-translational levels. This study revealed that mutant mating proteins could potentially have various secondary structures. Differential occurrence and transcription of the a-/alpha-pheromone receptor genes were also found in H. sinensis. The data were inconsistent with self-fertilization under (pseudo)homothallism but suggest the self-sterility of H. sinensis and the requirement of mating partners to achieve O. sinensis sexual outcrossing under heterothallism or hybridization. Although consistent occurrence and transcription of the mating-type genes of both the MAT1-1 and MAT1-2 idiomorphs have been reported in natural and cultivated Cordyceps sinensis insect-fungi complexes, the mutant MAT1-1-1 and alpha-pheromone receptor transcripts in natural C. sinensis result in N-terminal or middle-truncated proteins with significantly altered overall hydrophobicity and secondary structures of the proteins, suggesting heterogeneous fungal source(s) of the proteins and hybridization reproduction because of the co-occurrence of multiple genomically independent genotypes of O. sinensis and >90 fungal species in natural C. sinensis.