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Helvella bachu, a prized edible and medicinal fungus, is primarily found in the forests of Populus euphratica, an ancient and endangered species crucial to desert riparian ecosystems. Despite extensive efforts, the isolation of pure cultures and cultivation of fruiting bodies of H. bachu have remained elusive. While some species within the Helvella genus have been confirmed as ectomycorrhizal fungi, others have been considered either saprotrophic or mycorrhizal. By integrating field observations of H. bachu habitat, macro- and micro-anatomical examination of plant root tips, and molecular data from fruiting bodies, mycorrhizae, and host plants, it has been confirmed that H. bachu forms ectomycorrhizal associations with Populus trees. The mycorrhiza of H. bachu displays a light earth color with a curved smooth cylindrical shape. It features a thick mantle and the presence of a Hartig net, accompanied by a small amount of epitaxy mycelia. Morphological observation of the root tips requires meticulous handling, and the paraffin section technique has yielded noteworthy results. Host plants encompass four Populus species, including P. euphratica, P. pruinosa, P. nigra, and P. alba var. pyramidalis (synonym Populus bolleana). A conservation area was established within the young P. euphratica forest at Tarim University, resulting in a 14.75% increase in the quantity of fruiting bodies during the second year. Establishing a conservation area and in situ propagation of H. bachu holds economic and ecological implications. This study will contribute to the conservation of resources related to H. bachu and P. euphratica. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cordyceps militaris is a popular edible fungus with important economic value worldwide. In this study, an efficient CRISPR/Cas9 genome‐editing system based on an autonomously replicating plasmid with an AMA1 sequence was constructed. Further, a precisely targeted gene deletion via homology‐directed repair was effectively introduced in C. militaris. Gene editing was successful, with efficiencies of 55.1% and 89% for Cmwc‐1 and Cmvvd, respectively. Precisely targeted gene deletion was achieved at an efficiency of 73.9% by a single guide RNA supplementation with donor DNAs. Double genes, Cmwc‐1 and Cmvvd, were edited simultaneously with an efficiency of 10%. Plasmid loss was observed under non‐selective culture conditions, which could permit recycling of the selectable marker and avoid the adverse effects of the CRISPR/Cas9 system on the fungus, which is beneficial for the generation of new cultivars. RNA Pol III promoters, endogenous tRNAPro of C. militaris, and chimeric AfU6‐tRNAGly can be used to improve the efficiency. Polyethylene glycol‐mediated protoplast transformation was markedly more efficient than Agrobacterium tumefaciens‐mediated transformation of C. militaris. To our knowledge, this is the first description of genome editing and precisely targeted gene deletion in mushrooms based on AMA1 plasmids. Our findings will enable the modification of multiple genes in both functional genomics research and strain breeding. [ABSTRACT FROM AUTHOR]

Light is necessary for primordium differentiation and fruiting body development for most edible fungi; however, light perception and signal transduction have only been well studied in model fungi. In this study, a hierarchical network of transcriptional response to light in Cordyceps militaris, one of the edible fungi, has been described on a genome-wide scale using dynamic transcriptome analysis. It was shown that light regulated the transcript of 1722 genes, making up 18% of the whole genome of C. militaris. Analysis of light-responsive genes in C. militaris identified 4 categories: immediate-early, early, late, and continuous light-responsive genes, and the gene number increased distinctly with prolonged light exposure. Light-responsive genes with distinct functional categories showed specific time-dependent regulation. The target genes of CmWC-1, the most important photoreceptor, were revealed by ChIP-seq. A total of 270 significant peaks corresponding to 427 genes were identified to be directly regulated by CmWC-1, among which 143 genes respond to light. Based on 270 ChIP-seq peaks, the binding site for CmWC-1 was identified as AAATCAGACCAC/GTGGTCTGATTT, differing from the binding site by the homolog in Neurospora crassa. Elucidating the mechanisms of light perception and signal transduction will be helpful for further research on the fruiting body development in edible fungi. [ABSTRACT FROM AUTHOR]

Summary: Fruiting body development and carotenoid production are light‐induced in Cordyceps militaris. Our previous studies have shown that two blue‐light receptors, CmWC‐1 and CmCRY‐DASH, regulate fruiting body development and secondary metabolism. However, the photosensory system of C. militaris remains unclear. Here, gene deletion of Cmvvd, coding for another blue‐light receptor, resulted in reduced conidiation level and significant promotion of carotenoid content. Cmvvd transcription levels at fruiting body stages were higher than at other stages, and fruiting bodies could not develop normally in ΔCmvvd strains, indicating that Cmvvd might play an important role in fruiting body development. Rhythm loops were not affected in ΔCmvvd strains but were regulated by Cmwc‐1, and the expression of the rhythm regulator gene Cmfrq was dependent on CmWC‐1. Chromatin immunoprecipitation assay confirmed that Cmvvd is the direct target of CmWC‐1 in this fungus. Our results also revealed interdependent transcriptional relationships between Cmwc‐1 and Cmvvd, and between Cmwc‐1 and Cmcry‐DASH. Cmcry‐DASH expression was affected by Cmvvd, and the function‐loss of Cmcry‐DASH might be compensated by the high transcription of Cmvvd. This is the first report of the transcriptional linkage among the three blue‐light receptors in edible fungi and will be helpful for studies of multicellular development in this fungus. [ABSTRACT FROM AUTHOR]

Sparassis latifolia is a valuable edible fungus cultivated in East Asia that is rich in β-glucans. Understanding the mating system and sexual life cycle is important not only for breeding programs to improve strains but also for studies on speciation and population structures. In the present study, mating experiments using monokaryons derived from two different parental strains were performed. Chi-squared test indicated satisfied Mendel segregation, which supported a tetrapolar mating system. A search in the genome for homologs to the well-defined homeodomain and pheromone/receptors, as well as frequently found flanking genes, resulted in the identification of known mating-type loci previously identified in tetrapolar basidiomycetes, each represented by two idiomorphic alleles on separate contigs. Deficiency of the β-flanking protein in S. latifolia and S. crispa around the MAT-A locus may be explained by the locus being rich in transposable elements adjacent to HD genes. Monokaryotic mycelia are characterized by a slower growth rate and a relative lack of aerial mycelia compared with the parental strain. Chlamydospores can be produced in both monokaryotic and dikaryotic mycelial stages. We provide genetic and molecular evidence for the mating system of S. latifolia , a finding that will be helpful for the cross-breeding of this mushroom. • Both genetic and molecular evidence supports the tetrapolar system of Sparassis latifolia. • β-Flanking protein is absent around the MAT-A locus in S. latifolia and Sparassis crispa. • Three transposable elements are adjacent to HD genes in S. latifolia and S. crispa. [ABSTRACT FROM AUTHOR]