Hydrophobins are a family of small proteins exclusively secreted by fungi, and play a variety of roles in the life cycle. Cmhyd1, one of the hydrophobin class II members in Cordyceps militaris, has been shown to have a high transcript level during fruiting body development. Here, deletion of Cmhyd1 results in reduction in aerial mycelia, conidiation, hydrophobicity and infection ability, and complete inhibition of pigmentation and primordium differentiation. Cmhyd1 plays roles in conidiation and cuticle-bypassing infection by regulating the transcripts of frequency clock protein, Cmfrq, and velvet protein, Cmvosa, as well as primordium formation via the mitogen-activated protein kinase signaling pathway. Cmhyd1 also participates in stress response, including tolerance of mycelia to osmotic and oxidative stresses, and conidia to high or low temperatures. CmAreA, a transcription factor of nitrogen regulatory, is recruited to the promoter of Cmhyd1 and activates the transcription of Cmhyd1 with coactivator CmOTam using electrophoretic mobility shift assays and transient luciferase expression in tobacco. Furthermore, CmHYD1 is proved to regulate the transcription of Cmarea at different developmental stages via a positive feedback loop. These results reveal the diverse roles and regulation of Cmhyd1 in C. militaris, and provide insights into the developmental regulatory mechanism of mushrooms.

Hydrophobins are a family of small secreted proteins found exclusively in fungi, and they play various roles in the life cycle. In the present study, genome wide analysis and transcript profiling of the hydrophobin family in Cordyceps militaris, a well-known edible and medicinal mushroom, were studied. The distribution of hydrophobins in ascomycetes with different lifestyles showed that pathogenic fungi had significantly more hydrophobins than saprotrophic fungi, and class II members accounted for the majority. Phylogenetic analysis of hydrophobin proteins from the species of Cordyceps s.l. indicated that there was more variability among the class II members than class I. Only a few hydrophobin-encoding genes evolved by duplication in Cordyceps s.l., which was inconsistent with the important role of gene duplication in basidiomycetes. Different transcript patterns of four hydrophobin-encoding genes during the life cycle indicated the possible different functions for each. The transcripts of Cmhyd2, 3 and 4 can respond to light and were related with the photoreceptors. CmQHYD, with four hydrophobin II domains, was first found in C. militaris, and multi-domain hydrophobins were only distributed in the species of Cordycipitaceae and Clavicipitaceae. These results could be helpful for further function research of hydrophobins and could provide valuable information for the evolution of hydrophobins.

A deep understanding of the mechanism of fruiting body development is important for mushroom breeding and cultivation. Hydrophobins, small proteins exclusively secreted by fungi, have been proven to regulate the fruiting body development in many macro fungi. In this study, the hydrophobin gene Cmhyd4 was revealed to negatively regulate the fruiting body development in Cordyceps militaris, a famous edible and medicinal mushroom. Neither the overexpression nor the deletion of Cmhyd4 affected the mycelial growth rate, the hydrophobicity of the mycelia and conidia, or the conidial virulence on silkworm pupae. There was also no difference between the micromorphology of the hyphae and conidia in WT and ΔCmhyd4 strains observed by SEM. However, the ΔCmhyd4 strain showed thicker aerial mycelia in darkness and quicker growth rates under abiotic stress than the WT strain. The deletion of Cmhyd4 could promote conidia production and increase the contents of carotenoid and adenosine. The biological efficiency of the fruiting body was remarkably increased in the ΔCmhyd4 strain compared with the WT strain by improving the fruiting body density, not the height. It was indicated that Cmhyd4 played a negative role in fruiting body development. These results revealed that the diverse negative roles and regulatory effects of Cmhyd4 were totally different from those of Cmhyd1 in C. militaris and provided insights into the developmental regulatory mechanism of C. militaris and candidate genes for C. militaris strain breeding.

Calcarisporium cordycipiticola is the pathogen in the white mildew disease of Cordyceps militaris, one of the popular mushrooms. This disease frequently occurs and there is no effective method for disease prevention and control. In the present study, C. militaris is found to be the only host of C. cordycipiticola, indicating strict host specificity. The infection process was monitored by fluorescent labeling and scanning and transmission electron microscopes. C. cordycipiticola can invade into the gaps among hyphae of the fruiting bodies of the host and fill them gradually. It can degrade the hyphae of the host by both direct contact and noncontact. The parasitism is initially biotrophic, and then necrotrophic as mycoparasitic interaction progresses. The approximate chromosome-level genome assembly of C. cordycipiticola yielded an N50 length of 5.45 Mbp and a total size of 34.51 Mbp, encoding 10,443 proteins. Phylogenomic analysis revealed that C. cordycipiticola is phylogenetically close to its specific host, C. militaris. A comparative genomic analysis showed that the number of CAZymes of C. cordycipiticola was much less than in other mycoparasites, which might be attributed to its host specificity. Secondary metabolite cluster analysis disclosed the great biosynthetic capabilities and potential mycotoxin production capability. This study provides insights into the potential pathogenesis and interaction between mycoparasite and its host.

Grifola frondosa is a valuable edible fungus with high nutritional and medicinal values. The mating systems of fungi not only offer practical strategies for breeding, but also have far-reaching effects on genetic variability. Grifola frondosa has been considered as a sexual species with a tetrapolar mating system based on little experimental data. In the present study, one group of test crosses and six groups of three-round mating experiments from two parental strains were conducted to determine the mating system in G. frondosa. A chi-squared test of the results of the test-cross mating experiments indicated that they satisfied Mendelian segregation, while a series of three-round mating experiments showed that Mendelian segregation was not satisfied, implying a segregation distortion phenomenon in G. frondosa. A genomic map of the G. frondosa strain, y59, grown from an LMCZ basidiospore, with 40.54 Mb and 12 chromosomes, was generated using genome, transcriptome and Hi-C sequencing technology. Based on the genomic annotation of G. frondosa, the mating-type loci A and B were located on chromosomes 1 and 11, respectively. The mating-type locus A coded for the β-fg protein, HD1, HD2 and MIP, in that order. The mating-type locus B consisted of six pheromone receptors (PRs) and five pheromone precursors (PPs) in a crossed order. Moreover, both HD and PR loci may have only one sublocus that determines the mating type in G. frondosa. The nonsynonymous SNP and indel mutations between the A₁B₁ and A₂B₂ mating-type strains and the reference genome of y59 only occurred on genes HD2 and PR1/2, preliminarily confirming that the mating type of the y59 strain was A₁B₂ and not A₁B₁. Based on the genetic evidence and the more reliable molecular evidence, the results reveal that the mating system of G. frondosa is tetrapolar. This study has important implications for the genetics and hybrid breeding of G. frondosa.

Grifola frondosa is a valuable medicinal and edible mushroom whose industrial cultivation and developmental mechanisms remain poorly understood. In this study, we systematically investigated the optimal cultivation parameters and molecular basis of fruiting body development using the white strain Gr0001+3 through integrated physiological and transcriptomic approaches. The results showed that the optimal liquid medium composition was glucose (28.5 g/L), yeast extract (11.5 g/L), and MgSO₄ (2 g/L), with a C/N ratio of 10:1. This composition achieved a mycelial biomass of 2.333 g/L via an orthogonal design. Ideal culture conditions were 100 mL/250 mL liquid volume, 10% inoculum size, and pH 4.0 in single-factor experiments. The fruiting body developmental transcriptomes were analyzed in four stages: early primordia (EP), middle primordia (MP), late primordia (LP), and mature fruiting body (FB). Principal component analysis revealed distinct transcriptional profiles, with greater similarities among later developmental stages. Differential gene expression peaked during the LP vs. FB transition. Functional enrichment (GO/KEGG) showed conserved biological processes in the MP-LP-FB transitions. Heat shock proteins (hsp_78/hsp_82) and the cAMP signaling pathway component (PKAC) were involved in fruiting body development, based on RT-qPCR. This work establishes practical cultivation parameters and offers fundamental insights into the molecular regulation of G. frondosa development, providing a comprehensive foundation for advancing the industrial production of this mushroom.