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Discusses a study which examined the medicinal properties of Coprinoid mushrooms. Nutritional contents of Coprinoid mushrooms; Effectiveness of the mushrooms as antibiotics; Physiological characteristics of the mushrooms.

Abstract Background Verticillium longisporum is one of the most important pathogens of Brassicaceae that remains strictly in the xylem during most stages of its development. It has been suggested that disease symptoms are associated with clogging of xylem vessels. The aim of our study was to investigate extracellular defence reactions induced by V. longisporum in the xylem sap and leaf apoplast of Brassica napus var. napus in relation to the development of disease symptoms, photosynthesis and nutrient status. Results V. longisporum (strain VL43) did not overcome the hypocotyl barrier until 3 weeks after infection although the plants showed massive stunting of the stem and mild leaf chlorosis. During this initial infection phase photosynthetic carbon assimilation, transpiration rate and nutrient elements in leaves were not affected in VL43-infected compared to non-infected plants. Proteome analysis of the leaf apoplast revealed 170 spots after 2-D-protein separation, of which 12 were significantly enhanced in response to VL43-infection. LS-MS/MS analysis and data base searches revealed matches of VL43-responsive proteins to an endochitinase, a peroxidase, a PR-4 protein and a β-1,3-glucanase. In xylem sap three up-regulated proteins were found of which two were identified as PR-4 and β-1,3-glucanase. Xylem sap of infected plants inhibited the growth of V. longisporum. Conclusion V. longisporum infection did not result in drought stress or nutrient limitations. Stunting and mild chlorosis were, therefore, not consequences of insufficient water and nutrient supply due to VL43-caused xylem obstruction. A distinct array of extracellular PR-proteins was activated that might have limited Verticillium spreading above the hypocotyl. In silico analysis suggested that ethylene was involved in up-regulating VL43-responsive proteins.

Apoptotic-like programmed cell death (PCD) is one of the main strategies for fungi to resist environmental stresses and maintain homeostasis. The apoptosis-inducing factor (AIF) has been shown in different fungi to trigger PCD through upregulating reactive oxygen species (ROS). This study identified a mitochondrial localized AIF homolog, CcAIF1, from Coprinopsis cinerea monokaryon Okayama 7. Heterologous overexpression of CcAIF1 in Saccharomyces cerevisiae caused apoptotic-like PCD of the yeast cells. Ccaif1 was increased in transcription when C. cinerea interacted with Gongronella sp. w5, accompanied by typical apoptotic-like PCD in C. cinerea, including phosphatidylserine externalization and DNA fragmentation. Decreased mycelial ROS levels were observed in Ccaif1 silenced C. cinerea transformants during cocultivation, as well as reduction of the apoptotic levels, mycelial growth, and asexual sporulation. By comparison, Ccaif1 overexpression led to the opposite phenotypes. Moreover, the transcription and expression levels of laccase Lcc9 decreased by Ccaif1 silencing but increased firmly in Ccaif1 overexpression C. cinerea transformants in coculture. Thus, in conjunction with our previous report that intracellular ROS act as signal molecules to stimulate defense responses, we conclude that CcAIF1 is a regulator of ROS to promote apoptotic-like PCD and laccase expression in fungal-fungal interactions. In an axenic culture of C. cinerea, CcAIF1 overexpression and H 2 O 2 stimulation together increased laccase secretion with multiplied production yield. The expression of two other normally silent isozymes, Lcc8 and Lcc13, was unexpectedly triggered along with Lcc9. KEY POINTS: • Mitochondrial CcAIF1 induces PCD during fungal-fungal interactions • CcAIF1 is a regulator of ROS to trigger the expression of Lcc9 for defense • CcAIF1 overexpression and H 2 O 2 stimulation dramatically increase laccase production., (© 2024. The Author(s).)

Laccase, a copper-containing polyphenol oxidase, is an important green biocatalyst. In this study, Laccase Lcc5 was homologous recombinantly expressed in Coprinopsis cinerea and a novel strategy of silencing chitinase gene expression was used to enhance recombinant Lcc5 extracellular yield. Two critical chitinase genes, ChiEn1 and ChiE2, were selected by analyzing the transcriptome data of C. cinerea FA2222, and their silent expression was performed by RNA interference (RNAi). It was found that silencing either ChiEn1 or ChiE2 reduced sporulation and growth rate, and increased cell wall sensitivity, but had no significant effect on mycelial branching. Among them, the extracellular laccase activity of the ChiE2-silenced engineered strain Cclcc5-antiChiE2-5 and the control Cclcc5-13 reached the highest values (38.2 and 25.5 U/mL, respectively) at 250 and 150 rpm agitation speeds, corresponding to productivity of 0.35 and 0.19 U/mL·h, respectively, in a 3-L fermenter culture. Moreover, since Cclcc5-antiChiE2-5 could withstand greater shear forces, its extracellular laccase activity was 2.6-fold higher than that of Cclcc5-13 when the agitation speed was all at 250 rpm. To our knowledge, this is the first report of enhanced recombinant laccase production in C. cinerea by silencing the chitinase gene. This study will pave the way for laccase industrial production and accelerate the development of a C. cinerea high-expression system. KEY POINTS: • ChiEn1 and ChiE2 are critical chitinase genes in C. cinerea FA2222 genome. • Chitinase gene silencing enhanced the tolerance of C. cinerea to shear forces. • High homologous production of Lcc5 is achieved by fermentation in a 3-L fermenter., (© 2024. The Author(s).)

Forests provide invaluable economic, ecological, and social services. At the same time, they are exposed to several threats, such as fragmentation, changing climatic conditions, or increasingly destructive pests and pathogens. Trees, the inherent species of forests, cannot be viewed as isolated organisms. Manifold (micro)organisms are associated with trees playing a pivotal role in forest ecosystems. Of these organisms, fungi may have the greatest impact on the life of trees. A multitude of molecular and genetic methods are now available to investigate tree species and their associated organisms. Due to their smaller genome sizes compared to tree species, whole genomes of different fungi are routinely compared. Such studies have only recently started in forest tree species. Here, we summarize the application of molecular and genetic methods in forest conservation genetics, tree breeding, and association genetics as well as for the investigation of fungal communities and their interrelated ecological functions. These techniques provide valuable insights into the molecular basis of adaptive traits, the impacts of forest management, and changing environmental conditions on tree species and fungal communities and can enhance tree-breeding cycles due to reduced time for field testing. It becomes clear that there are multifaceted interactions among microbial species as well as between these organisms and trees. We demonstrate the versatility of the different approaches based on case studies on trees and fungi. KEY POINTS: • Current knowledge of genetic methods applied to forest trees and associated fungi. • Genomic methods are essential in conservation, breeding, management, and research. • Important role of phytobiomes for trees and their ecosystems., (© 2023. The Author(s).)

Frequently, laccases are triggered during fungal cocultivation for overexpression. The function of these activated laccases during coculture has not been clarified. Previously, we reported that Gongronella sp. w5 (w5) (Mucoromycota, Mucoromycetes) specifically triggered the laccase Lcc9 overexpression in Coprinopsis cinerea (Basidiomycota, Agaricomycetes). To systematically analyze the function of the overexpressed laccase during fungal interaction, C. cinerea mycelia before and after the initial Lcc9 overexpression were chosen for transcriptome analysis. Results showed that accompanied by specific utilization of fructose as carbohydrate substrate, oxidative stress derived from antagonistic compounds secreted by w5 appears to be a signal critical for laccase production in C. cinerea . A decrease in reactive oxygen species (ROS) in the C. cinerea wild-type strain followed the increase in laccase production, and then lcc9 transcription and laccase activity stopped. By comparison, increased H 2 O 2 content and mycelial ROS levels were observed during the entire cocultivation in lcc9 silenced C. cinerea strains. Moreover, lcc9 silencing slowed down the C. cinerea mycelial growth, affected hyphal morphology, and decreased the asexual sporulation in coculture. Our results showed that intracellular ROS acted as signal molecules to stimulate defense responses by C. cinerea with the expression of oxidative stress response regulator Skn7 and various detoxification proteins. Lcc9 takes part in a defense strategy to eliminate oxidative stress during the interspecific interaction with w5. IMPORTANCE The overproduction of laccase during interspecific fungal interactions is well known. However, the exact role of the upregulated laccases remains underexplored. Based on comparative transcriptomic analysis of C. cinerea and gene silencing of laccase Lcc9, here we show that oxidative stress derived from antagonistic compounds secreted by Gongronella sp. w5 was a signal critical for laccase Lcc9 production in Coprinopsis cinerea. Intracellular ROS acted as signal molecules to stimulate defense responses by C. cinerea with the expression of oxidative stress response regulator Skn7 and various detoxification proteins. Ultimately, Lcc9 takes part in a defense strategy to eliminate oxidative stress and help cell growth and development during the interspecific interaction with Gongronella sp. w5. These findings deepened our understanding of fungal interactions in their natural population and communities.

In heterothallic basidiomycete fungi, sexual compatibility is restricted by mating types, typically controlled by two loci: PR , encoding pheromone precursors and pheromone receptors, and HD , encoding two types of homeodomain transcription factors. We analysed the single mating-type locus of the commercial button mushroom variety, Agaricus bisporus var. bisporus , and of the related variety burnettii . We identified the location of the mating-type locus using genetic map and genome information, corresponding to the HD locus, the PR locus having lost its mating-type role. We found the mip1 and β-fg genes flanking the HD genes as in several Agaricomycetes, two copies of the β-fg gene, an additional HD2 copy in the reference genome of A. bisporus var. bisporus and an additional HD1 copy in the reference genome of A. bisporus var. burnettii. We detected a 140 kb-long inversion between mating types in an A. bisporus var. burnettii heterokaryon, trapping the HD genes, the mip1 gene and fragments of additional genes. The two varieties had islands of transposable elements at the mating-type locus, spanning 35 kb in the A. bisporus var. burnettii reference genome. Linkage analyses showed a region with low recombination in the mating-type locus region in the A. bisporus var. burnettii variety. We found high differentiation between β-fg alleles in both varieties, indicating an ancient event of recombination suppression, followed more recently by a suppression of recombination at the mip1 gene through the inversion in A. bisporus var. burnettii and a suppression of recombination across whole chromosomes in A. bisporus var. bisporus , constituting stepwise recombination suppression as in many other mating-type chromosomes and sex chromosomes.

Plants secrete purple acid phosphatases (PAPs) under phosphorus (P) shortage but the contribution of plant PAPs to P acquisition is not well understood. The goals of this study were to investigate comprehensively the transcription patterns of PAPs under P shortage in poplar (Populus × canescens), to identify secreted PAPs and to characterize their contribution to mobilize organic P. Phylogenetic analyses of the PAP family revealed 33 putative members. In this study, distinct, tissue-specific P responsive expression patterns could be shown for 23 PAPs in roots and leaves. Root-associated PAP activities were localized on the root surface by in-vivo staining. The activities of root-surface PAPs increased significantly under low P availability, but were suppressed by a PAP inhibitor and corresponded to elevated P uptake from ATP as an organic P source. By proteomic analyses of the root apoplast, we identified three newly secreted proteins under P shortage: PtPAP1 (Potri.005G233400) and two proteins with unknown functions (Potri.013G100800 and Potri.001G209300). Our results, based on the combination of transcriptome and proteome analyses with phosphatase activity assays, support that PtPAP1 plays a central role in enhanced P acquisition from organic sources, when the phosphate concentrations in soil are limited., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Two reference strains have been sequenced from the mushroom Coprinopsis cinerea , monokaryon Okayama 7/#130 (OK130) and the self-compatible homokaryon AmutBmut. An adenine-auxotrophy in OK130 ( ade8-1 ) and a para -aminobenzoic acid (PABA)-auxotrophy in AmutBmut ( pab1-1 ) offer selection markers for transformations. Of these two strains, homokaryon AmutBmut had been transformed before to PABA-prototrophy and with the bacterial hygromycin resistance marker hph , respectively., Results: Gene ade8 encodes a bifunctional enzyme with an N-terminal glycinamide ribonucleotide synthase (GARS) and a C-terminal aminoimidazole ribonucleotide synthase (AIRS) domain required for steps 2 and 5 in the de novo biosynthesis of purines, respectively. In OK130, a missense mutation in ade8-1 rendered residue N231 for ribose recognition by the A loop of the GARS domain into D231. The new ade8 + vector p Cc Ade8 complements the auxotrophy of OK130 in transformations. Transformation rates with p Cc Ade8 in single-vector and co-transformations with ade8 + -selection were similarly high, unlike for trp1 + plasmids which exhibit suicidal feedback-effects in single-vector transformations with complementation of tryptophan synthase defects. As various other plasmids, unselected p Cc Ade8 helped in co-transformations of trp1 strains with a trp1 + -selection vector to overcome suicidal effects by transferred trp1 + . Co-transformation rates of p Cc Ade8 in OK130 under adenine selection with nuclear integration of unselected DNA were as high as 80% of clones. Co-transformation rates of expressed genes reached 26-42% for various laccase genes and up to 67% with lcc9 silencing vectors. The bacterial gene hph can also be used as another, albeit less efficient, selection marker for OK130 transformants, but with similarly high co-transformation rates. We further show that the pab1-1 defect in AmutBmut is due to a missense mutation which changed the conserved PIKGT motif for chorismate binding in the C-terminal PabB domain to PIEGT in the mutated 4-amino-4-deoxychorismate synthase., Conclusions: ade8-1 and pab1-1 auxotrophic defects in C. cinerea reference strains OK130 and AmutBmut for complementation in transformation are described. p Cc Ade8 is a new transformation vector useful for selection in single and co-transformations of the sequenced monokaryon OK130 which was transformed for the first time. The bacterial gene hph can also be used as an additional selection marker in OK130, making in combination with ade8 + successive rounds of transformation possible., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2020.)

Mycopathogens are serious threats to the crops in commercial mushroom cultivations. In contrast, little is yet known on their occurrence and behaviour in nature. Cobweb infections by a conidiogenous Cladobotryum-type fungus identified by morphology and ITS sequences as Hypomyces odoratus were observed in the year 2015 on primordia and young and mature fruiting bodies of Agaricus xanthodermus in the wild. Progress in development and morphologies of fruiting bodies were affected by the infections. Infested structures aged and decayed prematurely. The mycoparasites tended by mycelial growth from the surroundings to infect healthy fungal structures. They entered from the base of the stipes to grow upwards and eventually also onto lamellae and caps. Isolated H. odoratus strains from a diseased standing mushroom, from a decaying overturned mushroom stipe and from rotting plant material infected mushrooms of different species of the genus Agaricus while Pleurotus ostreatus fruiting bodies were largely resistant. Growing and grown A. xanthodermus and P. ostreatus mycelium showed degrees of resistance against the mycopathogen, in contrast to mycelium of Coprinopsis cinerea. Mycelial morphological characteristics (colonies, conidiophores and conidia, chlamydospores, microsclerotia, pulvinate stroma) and variations of five different H. odoratus isolates are presented. In pH-dependent manner, H. odoratus strains stained growth media by pigment production yellow (acidic pH range) or pinkish-red (neutral to slightly alkaline pH range).

The secretome complexity and lignocellulose degrading capacity of Pleurotus ostreatus monokaryons mkPC9 and mkPC15 and mated dikaryon dkN001 were studied in submerged liquid cultures containing wood, glucose, and wood plus glucose as carbon sources. The study revealed that this white-rot basidiomycete attacks all the components of the plant cell wall. P. ostreatus secretes a variety of glycoside hydrolases, carbohydrate esterases, and polysaccharide lyases, especially when wood is the only carbon source. The presence of wood increased the secretome complexity, whereas glucose diminished the secretion of enzymes involved in cellulose, hemicellulose and pectin degradation. In contrast, the presence of glucose did not influence the secretion of redox enzymes or proteases, which shows the specificity of glucose on the secretion of cellulolytic enzymes. The comparison of the secretomes of monokaryons and dikaryons reveals that secretome complexity is unrelated to the nuclear composition of the strain.

White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here, we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood-decaying activity within the Basidiomycota genus Pycnoporus. We observed a strong conservation in the genome structures and the repertoires of protein-coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analysed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H2O2-producing enzymes with H2O2-consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 lytic polysaccharide monooxygenase gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood-decaying process., (© The Author(s) 2020. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

Engineering of fungal laccases with optimum catalytic activity at alkaline pH has been a long-lasting challenge. In this study, a mutant library containing 3000 clones was obtained by error-prone PCR to adapt the optimum pH of a fungal laccase Lcc9 from the basidiomycete Coprinopsis cinerea. After three rounds of functional screening, a mutant with three amino acid changes (E116K, N229D, I393T) named PIE5 was selected. PIE5 showed an optimum pH of 8.5 and 8.0 against guaiacol and 2,6-DMP when expressed in Pichia pastoris, representing the first fungal laccase that possesses an optimum pH at an alkaline condition. Site directed mutagenesis disclosed that N229D contributed the most to the optimum pH increment. A single N229D mutation caused an increase in optimum pH by 1.5 units. When used in indigo dye decolorization, PIE5 efficiently decolorized 87.1 ± 1.1% and 90.9 ± 0.3% indigo dye at the optimum conditions of pH 7.0-7.5 and 60 °C, and with either methyl 3,5-dimethoxy-4-hydroxybenzoate or 2,2'-azino-bis(3-ethylbenzothazoline-6-sulfonate) as the mediator. In comparison, the commercially available fungal laccase TvLac from Trametes villosa decolorized 84.3 ± 1.8% of indigo dye under its optimum conditions (opt. pH 5.0 and 60 °C). The properties of an alkaline-dependent activity and the high indigo dye decolorization ability (1.3-fold better than the parental Lcc9) make the new fungal laccase PIE5 an alternative for specific industrial applications.

The evolution of complex multicellularity has been one of the major transitions in the history of life. In contrast to simple multicellular aggregates of cells, it has evolved only in a handful of lineages, including animals, embryophytes, red and brown algae, and fungi. Despite being a key step toward the evolution of complex organisms, the evolutionary origins and the genetic underpinnings of complex multicellularity are incompletely known. The development of fungal fruiting bodies from a hyphal thallus represents a transition from simple to complex multicellularity that is inducible under laboratory conditions. We constructed a reference atlas of mushroom formation based on developmental transcriptome data of six species and comparisons of >200 whole genomes, to elucidate the core genetic program of complex multicellularity and fruiting body development in mushroom-forming fungi (Agaricomycetes). Nearly 300 conserved gene families and >70 functional groups contained developmentally regulated genes from five to six species, covering functions related to fungal cell wall remodeling, targeted protein degradation, signal transduction, adhesion, and small secreted proteins (including effector-like orphan genes). Several of these families, including F-box proteins, expansin-like proteins, protein kinases, and transcription factors, showed expansions in Agaricomycetes, many of which convergently expanded in multicellular plants and/or animals too, reflecting convergent solutions to genetic hurdles imposed by complex multicellularity among independently evolved lineages. This study provides an entry point to studying mushroom development and complex multicellularity in one of the largest clades of complex eukaryotic organisms., Competing Interests: The authors declare no conflict of interest.

When sucrose was used as the carbon source, the Basidiomycete Coprinopsis cinerea showed poor growth and low laccase activity in pure culture, but greatly enhanced the level of laccase activity (>1800 U/L) during coculture with the Mucoromycete Gongronella sp. w5. As a result, the mechanism of laccase overproduction in coculture was investigated by starting from clarifying the function of sucrose. Results demonstrated that Gongronella sp. w5 in the coculture system hydrolyzed sucrose to glucose and fructose by an intracellular invertase. Fructose rather than glucose was supplied by Gongronella sp. w5  as the readily available carbon source for C. cinerea, and contributed to an alteration of its growth behavior and a basal laccase secretion of 110.6 ± 3.3 U/L. On the other hand, separating Gongronella sp. w5 of C. cinerea by transfer into dialysis tubes yielded the same level of laccase activity as without separation, indicating that enhanced laccase production probably resulted from the metabolites in the fermentation broth. Further investigation showed that the ethyl acetate-extracted metabolites generated by Gongronella sp. w5 induced C. cinerea laccase production. One of the laccase-inducing compounds namely p-hydroxybenzoic acid (HBA) was purified and identified from the extract. When using HBA as the inducer and fructose as the carbon source in monoculture, C. cinerea observed similar high laccase activity to that in coculture, and zymograms revealed the same expression of laccase Lcc9 as the main and Lcc1 and Lcc5 as the minor enzymes. Overall, our experiments verified that Gongronella sp. w5 elevates Coprinopsis cinerea laccase production by carbon source syntrophism and secondary metabolite induction.

Laccase production and pellet formation of transformants of Coprinopsis cinerea strain FA2222 of C. cinerea laccase gene lcc1 subcloned behind the gpdII-promoter from Agaricus bisporus were compared with a control transformant carrying no extra laccase gene. At the optimum growth temperature of 37 °C, maximal laccase yields of 2.9 U/ml were obtained by the best lcc1 transformant pYSK7-26 in liquid shake flask cultures. Reduction in temperature to 25 °C increased laccase yields up to 9.2 U/ml. The control transformant had no laccase activities at 37 °C but native activity at 25 °C (3.5 U/ml). Changing the temperature had severe effects on the morphology of the mycelial pellets formed during cultivation, but links of distinct pellet morphologies to native or recombinant laccase production could not be established. Automated image analysis was used to characterise pellet formation and morphological parameters (pellet area, diameter, convexity and mycelial structure). Cross sections of selected pellets showed that they differentiated in an outer rind and an inner medulla of loosened hyphae. Pellets at 25 °C had a small and dense outer zone and adopted with time a smooth surface. Pellets at 37 °C had a broader outer zone and a fringy surface due to generation of more and larger protuberances in the rind that when released can serve for production of further pellets.