A novel betapartitivirus isolated from Cordyceps militaris, an edible-medicinal mushroom.

In this study, we identified a novel partitivirus, named "Cordyceps militaris partitivirus 1" (CmPV1), in Cordyceps militaris strain RCEF7506. The complete genome of CmPV1 comprises two segments, dsRNA1 and dsRNA2, each encoding a single protein. dsRNA1 (2,206 bp) encodes an RNA-dependent RNA polymerase (RdRp), and dsRNA2 (2,256 bp) encodes a coat protein (CP). Sequence analysis revealed that dsRNA1 has the highest similarity to that of Bipolaris maydis partitivirus 2 (BmPV2), whereas dsRNA2 shows the highest similarity to human blood-associated partitivirus (HuBPV). Phylogenetic analysis based on RdRp sequences suggests that CmPV1 is a new member of the genus Betapartitivirus of the family Partitiviridae. This is the first documentation of a betapartitivirus infecting the entomopathogenic fungus C. militaris.

Effects of mating-type ratio imbalance on the degeneration of Cordyceps militaris subculture and preventative measures.

The rapid degeneration of Cordyceps militaris strains during subculture represents a bottleneck problem that affects production stability. This study explored the mechanism underlying this degeneration in three production and three wild-type strains of Cordyceps militaris, isolating single-conidium strains from each. The effects of subculturing on fructification in both original and single mating-type strains were compared. Changes in the ratio of the two mating types were analyzed in both original and degenerated strains. Based on these findings, the two mating strains were paired in different ratios to determine their effects on fruiting. The resulting five strains were heterokaryotic strains with both MAT1-1 and MAT1-2 mating-type genes. Strain jb-2 was a single mating type (MAT1-1) mutant strain that produced stable fruiting bodies but failed to produce ascospores. It was found that the loss of or imbalance in mating types was the main reason for the rapid degeneration of fruiting traits during subculture and that this occurred randomly in the MAT1-1 and MAT1-2 types. The strains differed significantly in their stability during subculture. Fruiting was stable in the single mating-type Jb-2 strain, and the eleventh-generation fruited normally. There were differences in yield between the production and wild strains after inoculation with spawn containing different proportions of mating types. The production strain was more stable when inoculated with strains with mating-type ratios of 1:9 to 9:1 without affecting the yield. However, the yield of the wild-type strain xf-1 was positively correlated with the proportion of the MAT1-2 type, while the other two strains showed no correlations. Subculturing single mating-type mycelia separately and mixing them before production effectively mitigated degeneration during subculture. For Cordyceps militaris breeding, selecting strains containing both mating types, which are insensitive to the proportion of mating-type genes, enhanced stability in subculture and reduced the risk of mating-type loss. Direct breeding of specific single-mating type strains to induce fruiting is thus an effective breeding strategy.

Identification of a novel member of the genus Laulavirus (family Phenuiviridae) from the entomopathogenic ascomycete fungus Cordyceps javanica.

The virus family Phenuiviridae (order Hareavirales, comprising segmented negative-sense single stranded RNA viruses) has highly diverse members that are known to infect animals, plants, protozoans, and fungi. In this study, we identified a novel phenuivirus infecting a strain of the entomopathogenic fungus Cordyceps javanica isolated from a small brown plant hopper (Laodelphax striatellus), and this virus was tentatively named "Cordyceps javanica negative-strand RNA virus 1" (CjNRSV1). The CjNRSV1 genome consists of three negative-sense single stranded RNA segments (RNA1-3) with lengths of 7252, 2401, and 1117 nt, respectively. The 3'- and 5'-terminal regions of the RNA1, 2, and 3 segments have identical sequences, and the termini of the RNA segments are complementary to each other, reflecting a common characteristic of viruses in the order Hareavirales. RNA1 encodes a large protein (∼274 kDa) containing a conserved domain for the bunyavirus RNA-dependent RNA polymerase (RdRP) superfamily, with 57-80% identity to the RdRP encoded by phenuiviruses in the genus Laulavirus. RNA2 encodes a protein (∼79 kDa) showing sequence similarity (47-63% identity) to the movement protein (MP, a plant viral cell-to-cell movement protein)-like protein (MP-L) encoded by RNA2 of laulaviruses. RNA3 encodes a protein (∼28 kDa) with a conserved domain of the phenuivirid nucleocapsid protein superfamily. Phylogenetic analysis using the RdRPs of various phenuiviruses and other unclassified phenuiviruses showed CjNRSV1 to be grouped with established members of the genus Laulavirus. Our results suggest that CjNRSV1 is a novel fungus-infecting member of the genus Laulavirus in the family Phenuiviridae.

A rho-type GTPase activating protein affects the growth and development of Cordyceps cicadae.

Cordyceps cicadae is recognized for its medicinal properties, attributed to bioactive constituents like polysaccharides and adenosine, which have been shown to improve kidney and liver functions and possess anti-tumor properties. Rho GTPase activating proteins (Rho GAPs) serve as inhibitory regulators of Rho GTPases in eukaryotic cells by accelerating the GTP hydrolysis of Rho GTPases, leading to their inactivation. In this study, we explored the function of the CcRga8 gene in C. cicadae, which encodes a Rho-type GTPase activating protein. Our study found that the knockout of CcRga8 resulted in a decrease in polysaccharide levels and an increase in adenosine concentration. Furthermore, the mutants exhibited altered spore yield and morphology, fruiting body development, decreased infectivity, reduced resistance to hyperosmotic stress, oxidative conditions, and cell wall inhibitors. These findings suggest that CcRga8 plays a crucial role in the development, stress response, and bioactive compound production of C. cicadae.

Cordyceps militaris: A novel mushroom platform for metabolic engineering.

Cordyceps militaris, widely recognized as a medicinal and edible mushroom in East Asia, contains a variety of bioactive compounds, including cordycepin (COR), pentostatin (PTN) and other high-value compounds. This review explores the potential of developing C. militaris as a cell factory for the production of high-value chemicals and nutrients. This review comprehensively summarizes the fermentation advantages, metabolic networks, expression elements, and genome editing tools specific to C. militaris and discusses the challenges and barriers to further research on C. militaris across various fields, including computational biology, existing DNA elements, and genome editing approaches. This review aims to describe specific and promising opportunities for the in-depth study and development of C. militaris as a new chassis cell. Additionally, to increase the practicability of this review, examples of the construction of cell factories are provided, and promising strategies for synthetic biology development are illustrated.

Genomic and Transcriptome Analysis Reveals the Biosynthesis Network of Cordycepin in Cordyceps militaris.

Cordycepin is the primary active compound of Cordyceps militaris. However, the definitive genetic mechanism governing cordycepin synthesis in fruiting body growth and development remains elusive, necessitating further investigation. This study consists of 64 C. militaris strains collected from northeast China. The high-yielding cordycepin strain CMS19 was selected for the analysis of cordycepin production and the genetic basis of cordycepin anabolism. First, the whole-genome sequencing of CMS19 yielded a final size of 30.96 Mb with 8 contigs and 9781 protein-coding genes. The genome component revealed the presence of four additional secondary metabolite gene clusters compared with other published genomes, suggesting the potential for the production of new natural products. The analyses of evolutionary and genetic differentiation revealed a close relationship between C. militaris and Beauveria bassiana. The population of strains distributed in northeast China exhibited the significant genetic variation. Finally, functional genes associated with cordycepin synthesis were identified using a combination of genomic and transcriptomic analyses. A large number of functional genes associated with energy and purine metabolism were significantly enriched, facilitating the reconstruction of a hypothetical cordycepin metabolic pathway. Therefore, our speculation of the cordycepin metabolism pathway involved 24 genes initiating from the glycolysis and pentose phosphate pathways, progressing through purine metabolism, and culminating in the core region of cordycepin synthesis. These findings could offer fundamental support for scientific utilizations of C. militaris germplasm resources and standardized cultivation for cordycepin production.

Enhanced polysaccharide production through quorum sensing system in Cordyceps militaris.

This study aimed to enhance extracellular polysaccharide (EPS) production in Cordyceps militaris by constructing a quorum sensing (QS) system to regulate the expression of biosynthetic enzyme genes, including phosphoglucomutase, hexokinase, phosphomannomutase, polysaccharide synthase, and UDP-glucose 4-epimerase genes. The study found higher EPS concentrations in seven recombinant strains compared to the wild-type C. militaris, indicating that the overexpression of key enzyme genes increased EPS production. Among them, the CM-pgm-2 strain exhibited the highest EPS production, reaching a concentration of 3.82 ± 0.26 g/L, which was 1.52 times higher than the amount produced by the wild C. militaris strain. Additionally, the regulatory effects of aromatic amino acids on the QS system of the CM-pgm-2 strain were investigated. Under the influence of 45 mg/L tryptophan, the EPS production in CM-pgm-2 reached 4.75 ± 0.20 g/L, representing a 1.90-fold increase compared to wild C. militaris strains. This study provided an effective method for the large-scale production of EPSs in C. militaris, and opened up new avenues for research into fungal QS mechanisms.

Holistic transcriptional responses of Cordyceps militaris to different culture temperatures.

Cordyceps militaris is a medicinal entomopathogenic fungus containing valuable biometabolites for pharmaceutical applications. Its genetic inheritance and environmental factors play a crucial role in the production of biomass enriched with cordycepin. While temperature is a crucial controlled parameter for fungal cultivation, its impacts on growth and metabolite biosynthesis remains poorly characterized. This study aimed to investigate the metabolic responses and cordycepin production of C. militaris strain TBRC6039 under various temperature conditions through transcriptome analysis. Among 9599 expressed genes, 576 genes were significantly differentially expressed at culture temperatures of 15 and 25 °C. The changes in the transcriptional responses induced by these temperatures were found in several metabolisms involved in nutrient assimilation and energy source, including amino acids metabolism (e.g., glycine, serine and threonine metabolism) and lipid metabolism (e.g., biosynthesis of unsaturated fatty acids and steroid biosynthesis). At the lower temperature (15 °C), the biosynthetic pathways of lipids, specifically ergosterol and squalene, were the target for maintaining membrane function by transcriptional upregulation. Our study revealed the responsive mechanisms of C. militaris in acclimatization to temperature conditions that provide an insight on physiological manipulation for the production of metabolites by C. militaris.

Construction of a New Agrobacterium tumefaciens-Mediated Transformation System based on a Dual Auxotrophic Approach in Cordyceps militaris.

Cordyceps militaris is a significant edible fungus that produces a variety of bioactive compounds. We have previously established a uridine/uracil auxotrophic mutant and a corresponding Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic characterization in C. militaris using pyrG as a screening marker. In this study, we constructed an ATMT system based on a dual pyrG and hisB auxotrophic mutant of C. militaris. Using the uridine/uracil auxotrophic mutant as the background and pyrG as a selection marker, the hisB gene encoding imidazole glycerophosphate dehydratase, required for histidine biosynthesis, was knocked out by homologous recombination to construct a histidine auxotrophic C. militaris mutant. Then, pyrG in the histidine auxotrophic mutant was deleted to construct a ΔpyrG ΔhisB dual auxotrophic mutant. Further, we established an ATMT transformation system based on the dual auxotrophic C. militaris by using GFP and DsRed as reporter genes. Finally, to demonstrate the application of this dual transformation system for studies of gene function, knock out and complementation of the photoreceptor gene CmWC-1 in the dual auxotrophic C. militaris were performed. The newly constructed ATMT system with histidine and uridine/uracil auxotrophic markers provides a promising tool for genetic modifications in the medicinal fungus C. militaris.

Biological characteristics of Cordyceps militaris single mating-type strains.

Cordyceps militaris has been extensively cultivated as a model cordyceps species for commercial purposes. Nevertheless, the problems related to strain degeneration and breeding technologies remain unresolved. This study assessed the physiology and fertility traits of six C. militaris strains with distinct origins and characteristics, focusing on single mating-type strains. The results demonstrated that the three identified strains (CMDB01, CMSY01, and CMJB02) were single mating-type possessing only one mating-type gene (MAT1-1). In contrast, the other three strains (CMXF07, CMXF09, and CMMS05) were the dual mating type. The MAT1-1 strains sourced from CMDB01, CMSY01, and CMJB02 consistently produced sporocarps but failed to generate ascospores. However, when paired with MAT1-2 strains, the MAT1-1 strains with slender fruiting bodies and normal morphology were fertile. The hyphal growth rate of single mating-type strains (CMDB01, CMSY01, and CMJB02) typically surpassed that of dual mating-type strains (CMXF07, CMXF09, and CMMS05). The growth rates of MAT1-2 and MAT1-1 strains were proportional to their ratios, such that a single mating-type strain with a higher ratio exhibited an increased growth rate. As C. militaris matured, the adenosine content decreased. In summary, the C. militaris strains that consistently produce sporocarps and have a single mating type are highly promising for production and breeding.

Selection and validation of reference genes for RT-qPCR in ophiocordyceps sinensis under different experimental conditions.

The Chinese caterpillar mushroom, Ophiocordyceps sinensis (O. sinensis), is a rarely medicinal fungus in traditional chinese herbal medicine due to its unique medicinal values, and the expression stability of reference genes is essential to normalize its gene expression analysis. In this study, BestKeeper, NormFinder and geNorm, three authoritative statistical arithmetics, were applied to evaluate the expression stability of sixteen candidate reference genes (CRGs) in O. sinensis under different stress [low temperature (4°C), light treatment (300 lx), NaCl (3.8%)] and different development stages (mycelia, primordia and fruit bodies) and formation of morphologic mycelium (aeriasubstrate, hyphae knot mycelium). The paired variation values indicated that two genes could be enough to accurate standardization exposed to different conditions of O.sinensis. Among these sixteen CRGs, 18S ribosomal RNA (18S rRNA) and beta-Tubulin (ß-TUB) showed the topmost expression stability in O.sinensis exposed to all conditions, while glutathione hydrolase proenzym (GGT) and Phosphoglucose isomerase (PGI) showed the least expression stability. The optimal reference gene in different conditions was various. ß-TUB and Ubiquitin (UBQ) were identified as the two most stable genes in different primordia developmental stage, while phosphoglucomutase (PGM) with elongation factor 1-alpha (EF1-α) and 18S rRNA with UBQ were the most stably expressed for differentially morphologic mycelium stages and different stresses, respectively. These results will contribute to more accurate evaluation of the gene relative expression levels in O.sinensis under different conditions using the optimal reference gene in real-time quantitative PCR (RT-qPCR) analysis.

First report of emerging fungal pathogens of Cordyceps militaris in Vietnam.

Cultivation of Cordyceps militaris, a valuable medicinal and edible fungus, has dramatically increased in Vietnam since 2010. During industrial production, parasitic white molds were found to infect the mycelia and fruiting bodies of C. militaris causing significant quality and yield losses. Two different fungal strains were obtained from the mycelia and fruiting bodies of C. militaris in Danang mushroom farms and were characterized by morphological and multiple DNA markers analysis. The sequence alignment of ITS, LSU and rpb2 markers revealed that the pathogens are related to the type species Lecanicillium coprophilum and Calcarisporium cordycipiticola with more than 99% sequence identities. The growth characteristics and pathogenic activities of the two isolated species on their host C. militaris were also investigated. The phylogenetic analysis based on the ITS sequences showed that L. coprophilum WF2611 is closer to its host C. militaris than C. cordycipiticola NT1504. To our knowledge, this is the first worldwide report of C. militaris infected by L. coprophilum which would be an useful information on prevention and control of the disease and be helpful for the industrial cultivation of C. militaris.

Safe-Harbor-Targeted CRISPR/Cas9 System and Cmhyd1 Overexpression Enhances Disease Resistance in Cordyceps militaris.

Fungal disease of mushroomCordyceps militaris (CM) caused byCalcarisporium cordycipiticola (CC) is destructive to fruiting body cultivation, resulting in significant economic loss and potential food safety risks. CRISPR/Cas9 genome editing has proven to be a powerful tool for crop improvement but seldom succeeded in mushrooms. Here, the first genomic safe-harbor site, CmSH1 locus, was identified in the CM genome. A safe-harbor-targeted CRISPR/Cas9 system based on an autonomously replicating plasmid was designed to facilitate alien gene integration at the CmSH1 locus. Cmhyd1, one of the hydrophobin genes, was confirmed as a defensive factor against CC infection, and Cmhyd1 overexpression by this system showed enhancement of disease resistance with negligible effect on the agronomic traits of CM. No off-target events and residues of plasmid sequence were tested by PCR and genome resequencing. This study provided the first safe harbor site for genetic manipulations, a safe harbor-targeted CRISPR/Cas9 system, and the first disease-resistant gene-editing breeding system in mushrooms.

Efficient disruption of CmHk1 using CRISPR/Cas9 ribonucleoprotein delivery in Cordyceps militaris.

Cordyceps militaris, an entomopathogenic ascomycete, produces edible medicinal mushrooms known to have medicinal and therapeutic functions. To develop the genetic transformation system in C. militaris, green fluorescent protein (GFP) mutants of C. militaris were generated by PEG-mediated protoplast transformation. The CRISPR/Cas9 ribonucleoprotein (RNP) targeting the class III histidine kinase of C. militaris (CmHk1) was then delivered into protoplasts of C. militaris through the transformation system. Mutations induced by the RNP in selected mutants were detected: 1 nt deletion (6 mutants), 3 nt deletion with substitution of 1 nt (1 mutant), insertion of 85 nts (1 mutant), 41 nts (2 mutants), and 35 nts (5 mutants). An in vitro sensitivity assay of the mutants indicated that knockout of CmHk1 reduced sensitivity to two fungicides, iprodione and fludioxonil, but increased sensitivity to osmotic stresses compared to the wild type. Summing up, the CRISPR/Cas9 RNP delivery system was successfully developed, and our results revealed that CmHk1 was involved in the fungicide resistance and osmotic stress in C. militaris.

[Differential transcription of mating-type genes during sexual reproduction of natural Cordyceps sinensis].

Natural Cordyceps sinensis as an insect-fungal complex, which is developed after Ophiocordyceps sinensis infects a larva of Hepialidae family. Seventeen genotypes of O. sinensis have been identified in natural C. sinensis. This paper summarized the literature reports and GenBank database regarding occurrence and transcription of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs in natural C. sinensis, in Hirsutella sinensis(GC-biased Genotype #1 of O. sinensis), to infer the mating pattern of O. sinensis in the lifecycle of natural C. sinensis. The mating-type genes and transcripts of MAT1-1 and MAT1-2 idiomorphs were identified in the metagenomes and metatranscriptomes of natural C. sinensis. However, their fungal sources are unclear because of co-colonization of several genotypes of O. sinensis and multiple fungal species in natural C. sinensis. The mating-type genes of MAT1-1 and MAT1-2 idiomorphs were differentially present in 237 H. sinensis strains, constituting the genetic control of the O. sinensis reproduction. Transcriptional control of the O. sinensis reproduction includes: differential transcription or silencing of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs, and the MAT1-2-1 transcript with unspliced intron I that contains 3 stop codons. Research on the H. sinensis transcriptome demonstrated differential and complementary transcriptions of the mating-type genes of MAT1-1 and MAT1-2 idiomorphs in Strains L0106 and 1229, which may become mating partners to accomplish physiological heterothallism. The differential occurrence and transcription of the mating-type genes in H. sinensis are inconsistent with the self-fertilization hypothesis under homothallism or pseudohomothallism, but instead indicate the need of mating partners of the same H. sinensis species, either monoecious or dioecious, for physiological heterothallism, or heterospecific species for hybridization. Multiple GC-and AT-biased genotypes of O. sinensis were identified in the stroma, stromal fertile portion(densely covered with numerous ascocarps) and ascospores of natural C. sinensis. It needs to be further explored if the genome-independent O. sinensis genotypes could become mating partners to accomplish sexual reproduction. S. hepiali Strain FENG experienced differential transcription of the mating-type genes with a pattern complementary to that of H. sinensis Strain L0106. Additional evidence is needed to explore a hybridization possibility between S. hepiali and H. sinensis, whether they are able to break the interspecific reproductive isolation. Genotypes #13～14 of O. sinensis feature large DNA segment reciprocal substitutions and genetic material recombination between 2 heterospecific parental fungi, H. sinensis and an AB067719-type fungus, indicating a possibility of hybridization or parasexuality. Our analysis provides important information at the genetic and transcriptional levels regarding the mating-type gene expression and reproduction physiology of O. sinensis in the sexual life of natural C. sinensis and offers crucial reproductive physiology evidence, to assist in the design of the artificial cultivation of C. sinensis to supplement the increasing scarcity of natural resource.

Altered GC- and AT-biased genotypes of Ophiocordyceps sinensis in the stromal fertile portions and ascospores of natural Cordyceps sinensis.

OBJECTIVE: To examine multiple genotypes of Ophiocordyceps sinensis in a semi-quantitative manner in the stromal fertile portion (SFP) densely covered with numerous ascocarps and ascospores of natural Cordyceps sinensis and to outline the dynamic alterations of the coexisting O. sinensis genotypes in different developmental phases. METHODS: Mature Cordyceps sinensis specimens were harvested and continuously cultivated in our laboratory (altitude 2,254 m). The SFPs (with ascocarps) and fully and semi-ejected ascospores were collected for histological and molecular examinations. Biochip-based single nucleotide polymorphism (SNP) MALDI-TOF mass spectrometry (MS) was used to genotype multiple O. sinensis mutants in the SFPs and ascospores. RESULTS: Microscopic analysis revealed distinct morphologies of the SFPs (with ascocarps) before and after ascospore ejection and SFP of developmental failure, which, along with the fully and semi-ejected ascospores, were subjected to SNP MS genotyping analysis. Mass spectra showed the coexistence of GC- and AT-biased genotypes of O. sinensis that were genetically and phylogenetically distinct in the SFPs before and after ejection and of developmental failure and in fully and semi-ejected ascospores. The intensity ratios of MS peaks were dynamically altered in the SFPs and the fully and semi-ejected ascospores. Mass spectra also showed transversion mutation alleles of unknown upstream and downstream sequences with altered intensities in the SFPs and ascospores. Genotype #5 of AT-biased Cluster-A maintained a high intensity in all SFPs and ascospores. An MS peak with a high intensity containing AT-biased Genotypes #6 and #15 in pre-ejection SFPs was significantly attenuated after ascospore ejection. The abundance of Genotypes #5‒6 and #16 of AT-biased Cluster-A was differentially altered in the fully and semi-ejected ascospores that were collected from the same Cordyceps sinensis specimens. CONCLUSION: Multiple O. sinensis genotypes coexisted in different combinations with altered abundances in the SFPs prior to and after ejection, the SFP of developmental failure, and the two types of ascospores of Cordyceps sinensis, demonstrating their genomic independence. Metagenomic fungal members present in different combinations and with dynamic alterations play symbiotic roles in different compartments of natural Cordyceps sinensis.

Diversity of anamorphic Cordyceps (formerly Isaria) isolated from Brazilian agricultural sites.

A total of 53 anamorphic strains of Brazilian Cordyceps species currently maintained in a government-owned culture collection, were reassessed for diversity and species identity using multi-loci-based phylogenetic methods. The strains used in this study were originally obtained from soil samples or were isolated from insects of the orders Hemiptera, Lepidoptera, Coleoptera and Diptera, mostly from agricultural sites. A Bayesian phylogenetic tree was constructed based on a concatenation of five loci (ITS, LSU, RPB1, RPB2 and TEF). In a few cases of ambiguity, morphological traits were also considered for species delimitations. Considerable variability within the set of strains was detected and six Cordyceps species were identified: C. amoenerosea, C. fumosorosea, C. javanica, C. tenuipes and, for the first time, C. brevistroma and C. spegazzinii are reported in Brazil. Four other taxonomically equivocal groups, closely related to other known taxa (C. amoenerosea, C. cateniannulata, C. polyarthra and C. spegazzinii), were also recognized, although further studies will be required to confirm their identifications or their descriptions as new species. Cordyceps javanica was the most common species in our dataset, originally isolated from soil and several different insect orders, and includes 17 strains from the whitefly, Bemisia tabaci. Interestingly, strains previously identified as C. fumosorosea based on morphology and growth characteristics, were shown to be C. javanica, including the active ingredients of some commercial mycoinsecticides. Cordyceps farinosa, usually mentioned in the literature as occurring in Brazil, was not found in our study. Since most strains were from insect crop pests, further studies with hosts from non-agricultural settings or from environmental samples would be advisable for a deeper understanding of the occurrence of anamorphic Cordyceps in Brazil.

Heterologous expression of the lectin CmRlec from Cordyceps militaris (Cordycipitaceae, Ascomycota) in Escherichia coli.

Ascomycete lectins may play an important role in their life cycle. In this report, we mined a ricin B-type lectin, named CmRlec, from the Cordyceps militaris genome by homology search. Furthermore, we succeeded in the soluble expression of CmRlec using ß-glucuronidase as a solubilization tag and demonstrated that this lectin is a novel chitin-recognizing lectin.

Stable reference gene selection for Ophiocordyceps sinensis gene expression studies under different developmental stages and light-induced conditions.

The molecular mechanism of Chinese cordyceps formation has received a substantial amount of attention because of its usage as traditional Chinese medicine. The formation process of Chinese cordyceps includes two parts: asexual proliferation (Ophiocordyceps sinensis proliferates in the hemolymph of Thitarodes armoricanus larvae) and sexual development (formation and development of fruiting bodies). Therefore, validation of reference genes under different development stages and experimental conditions is crucial for RT-qPCR analysis. However, there is no report on stable reference genes at the development stage of O. sinensis fruiting body. In this study, 10 candidate reference genes, Actin, Cox5, Tef1, Ubi, 18s, Gpd, Rpb1, Try, Tub1 and Tub2, were selected and calculated their expression stability using four methods: geNorm, NormFinder, BestKeeper, and Comparative △Ct. After comprehensive analysis of the results of these four methods with RefFinder, we determined that the most stable reference genes during asexual reproduction of O. sinensis were Tef1 and Tub1, while the most stable reference genes during fruiting body development were Tyr and Cox5, and the most stable reference genes under light-induced conditions were Tyr and Tef1. Our study provides a guidance for reference genes selections at different proliferation processes with light stress of O. sinensis, and represents a foundation for studying the molecular mechanism of Chinese cordyceps formation.

Uncovering Gene Expression Profiles of Three Morphologic Mycelium Forms of the Chinese Caterpillar Mushroom Ophiocordyceps sinensis (Ascomycota) Using High-Throughput Sequencing.

The asexual form of Ophiocordyceps sinensis has been controversial, but various morphologic mycelium appeared when O. sinensis was cultured under experimental conditions. To explore the generation mechanism of morphologic mycelium, developmental transcriptomes were analyzed from three kinds of mycelium (aerial mycelium, hyphae knot, and substrate mycelium). The results showed that diameter and morphology of these three kinds of mycelium were obviously different. KEGG functional enrichment analysis showed that the differential expressed genes (DEGs) of substrate mycelium were enriched in ribosomes and peroxisomes, indicating that prophase culture was rich in nutrients and the metabolism of substrate mycelium cells was vigorous in the stage of nutrient absorption. The up-DEGs of hyphae knot were mainly enriched in the oxidative phosphorylation pathway, indicating that oxidative phosphorylation was the main energy source for mycelium formation in the stage of nutrient accumulation and reproductive transformation. The up-DEGs of aerial mycelium were mainly enriched in the synthesis and degradation pathways of valine, leucine, and isoleucine, suggesting that the occurrence of aerial mycelium was related to amino acid metabolism at the later stage of culture, and nutritional stress accelerated the reproduction of asexual spores. In addition, the important roles of mycelium formation related genes were verified by combined analysis of qRT-PCR and transcriptome sequencing. Collectively, this study will provide theoretical guidance for inhibiting the occurrence of aerogenous mycelium and promoting the development of mycelium into pinhead primordia in the culture of O. sinensis in the future.