Preassembled Cas9 Ribonucleoprotein-Mediated Gene Deletion Identifies the Carbon Catabolite Repressor and Its Target Genes in Coprinopsis cinerea.

Cre1 is an important transcription factor that regulates carbon catabolite repression (CCR) and is widely conserved across fungi. The cre1 gene has been extensively studied in several Ascomycota species, whereas its role in gene expression regulation in the Basidiomycota species remains poorly understood. Here, we identified and investigated the role of cre1 in Coprinopsis cinerea, a basidiomycete model mushroom that can efficiently degrade lignocellulosic plant wastes. We used a rapid and efficient gene deletion approach based on PCR-amplified split-marker DNA cassettes together with in vitro assembled Cas9-guide RNA ribonucleoproteins (Cas9 RNPs) to generate C. cinerea cre1 gene deletion strains. Gene expression profiling of two independent C. cinerea cre1 mutants showed significant deregulation of carbohydrate metabolism, plant cell wall degrading enzymes (PCWDEs), plasma membrane transporter-related and several transcription factor-encoding genes, among others. Our results support the notion that, like reports in the ascomycetes, Cre1 of C. cinerea orchestrates CCR through a combined regulation of diverse genes, including PCWDEs, transcription factors that positively regulate PCWDEs, and membrane transporters which could import simple sugars that can induce the expression of PWCDEs. Somewhat paradoxically, though in accordance with other Agaricomycetes, genes related to lignin degradation were mostly downregulated in cre1 mutants, indicating they fall under different regulation than other PCWDEs. The gene deletion approach and the data presented here will expand our knowledge of CCR in the Basidiomycota and provide functional hypotheses on genes related to plant biomass degradation. IMPORTANCE Mushroom-forming fungi include some of the most efficient lignocellulosic plant biomass degraders. They degrade dead plant materials by a battery of lignin-, cellulose-, hemicellulose-, and pectin-degrading enzymes, the encoding genes of which are under tight transcriptional control. One of the highest-level regulations of these metabolic enzymes is known as carbon catabolite repression, which is orchestrated by the transcription factor Cre1, and ensures that costly lignocellulose-degrading enzyme genes are expressed only when simple carbon sources (e.g., glucose) are not available. Here, we identified the Cre1 ortholog in a litter decomposer Agaricomycete, Coprinopsis cinerea, knocked it out, and characterized transcriptional changes in the mutants. We identified several dozen lignocellulolytic enzyme genes as well as membrane transporters and other transcription factors as putative target genes of C. cinerea cre1. These results extend knowledge on carbon catabolite repression to litter decomposer Basidiomycota.

Transcriptomic atlas of mushroom development reveals conserved genes behind complex multicellularity in fungi.

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.

Comparative genomics reveals unique wood-decay strategies and fruiting body development in the Schizophyllaceae.

Agaricomycetes are fruiting body-forming fungi that produce some of the most efficient enzyme systems to degrade wood. Despite decades-long interest in their biology, the evolution and functional diversity of both wood-decay and fruiting body formation are incompletely known. We performed comparative genomic and transcriptomic analyses of wood-decay and fruiting body development in Auriculariopsis ampla and Schizophyllum commune (Schizophyllaceae), species with secondarily simplified morphologies, an enigmatic wood-decay strategy and weak pathogenicity to woody plants. The plant cell wall-degrading enzyme repertoires of Schizophyllaceae are transitional between those of white rot species and less efficient wood-degraders such as brown rot or mycorrhizal fungi. Rich repertoires of suberinase and tannase genes were found in both species, with tannases restricted to Agaricomycetes that preferentially colonize bark-covered wood, suggesting potential complementation of their weaker wood-decaying abilities and adaptations to wood colonization through the bark. Fruiting body transcriptomes revealed a high rate of divergence in developmental gene expression, but also several genes with conserved expression patterns, including novel transcription factors and small-secreted proteins, some of the latter which might represent fruiting body effectors. Taken together, our analyses highlighted novel aspects of wood-decay and fruiting body development in an important family of mushroom-forming fungi.

Genomes of fungi and relatives reveal delayed loss of ancestral gene families and evolution of key fungal traits.

Fungi are ecologically important heterotrophs that have radiated into most niches on Earth and fulfil key ecological services. Despite intense interest in their origins, major genomic trends of their evolutionary route from a unicellular opisthokont ancestor to derived multicellular fungi remain poorly known. Here we provide a highly resolved genome-wide catalogue of gene family changes across fungal evolution inferred from the genomes of 123 fungi and relatives. We show that a dominant trend in early fungal evolution has been the gradual shedding of protist genes and the punctuated emergence of innovation by two main gene duplication events. We find that the gene content of non-Dikarya fungi resembles that of unicellular opisthokonts in many respects, owing to the conservation of protist genes in their genomes. The most rapidly duplicating gene groups included extracellular proteins and transcription factors, as well as ones linked to the coordination of nutrient uptake with growth, highlighting the transition to a sessile osmotrophic feeding strategy and subsequent lifestyle evolution as important elements of early fungal history. These results suggest that the genomes of pre-fungal ancestors evolved into the typical filamentous fungal genome by a combination of gradual gene loss, turnover and several large duplication events rather than by abrupt changes. Consequently, the taxonomically defined Fungi represents a genomically non-uniform assemblage of species.

Assessment of the dual role of Lyonia ovalifolia (Wall.) Drude in inhibiting AGEs and enhancing GLUT4 translocation through LC-ESI-QTOF-MS/MS determination and in silico studies.

Introduction: Diabetes mellitus (DM) is a metabolic disorder that results in glucose accumulation in the blood, accompanied by the production of advanced glycation end products (AGEs) through glycation of cellular proteins. These AGEs interfere with insulin signaling and prevent GLUT4 membrane translocation, thereby promoting the accumulation of more glucose in the blood and causing post-diabetic complications. Methods: In this study, we examine the anti-diabetic potential of Lyonia ovalifolia (Wall.) Drude, a well-known ethnomedicinal plant of the Indian Himalayas. Considering its various medicinal properties, we analyzed its ethanolic extract and various solvent fractions for in vitro antiglycation activity and antidiabetic potential, i.e., stimulation of GLUT4 translocation. Result and Discussions: The results showed that the extract and fractions exhibited increased antiglycation activity and an increased level of GLUT4 translocation. Analysis of a further 12 bioactive compounds of ethanolic extract, identified through LC-ESI-QTOF-MS/MS, revealed the presence of three new compounds: leucothol B, rhodoterpenoids A, and leucothol A. Moreover, we performed molecular docking of identified compounds against key proteins of diabetes mellitus: the sirtuin family of NAD (+)-dependent protein deacetylases 6 (SIRT6), aldose reductase (AR), and tyrosine kinase (TK). The results showed that flavonoid luteolin showed the best binding affinity ((-12.3 kcal/mol), followed by eriodictyol, astilbin, and syringaresinol. An ADMET study showed that luteolin, eriodictyol, astilbin, and syringaresinol may be promising drug candidates belonging to the flavonoid class of compounds, with no harmful effects and complying with all the drug-likeness guidelines. Furthermore, molecular dynamics (MD) simulations on a 50 ns timescale revealed that AR protein was most stable with luteolin throughout the simulation period. Therefore, this study reveals for the first time that L. ovalifolia plays an important role in insulin homeostasis, as shown in in vitro and in silico studies.

Dual RNA-Seq Profiling Unveils Mycoparasitic Activities of Trichoderma atroviride against Haploid Armillaria ostoyae in Antagonistic Interaction Assays.

Armillaria ostoyae, a species among the destructive forest pathogens from the genus Armillaria, causes root rot disease on woody plants worldwide. Efficient control measures to limit the growth and impact of this severe underground pathogen are under investigation. In a previous study, a new soilborne fungal isolate, Trichoderma atroviride SZMC 24276 (TA), exhibited high antagonistic efficacy, which suggested that it could be utilized as a biocontrol agent. The dual culture assay results indicated that the haploid A. ostoyae-derivative SZMC 23085 (AO) (C18/9) is highly susceptible to the mycelial invasion of TA. In the present study, we analyzed the transcriptome of AO and that of TA in in vitro dual culture assays to test the molecular arsenal of Trichoderma antagonism and the defense mechanisms of Armillaria. We conducted time-course analysis and functional annotation and analyzed enriched pathways and differentially expressed genes including biocontrol-related candidate genes from TA and defense-related candidate genes from AO. The results indicated that TA deployed several biocontrol mechanisms when confronted with AO. In response, AO initiated multiple defense mechanisms to protect against the fungal attack. To our knowledge, the present study offers the first transcriptome analysis of a biocontrol fungus attacking AO. Overall, this study provides insights that aid the further exploration of plant pathogen-biocontrol agent interaction mechanisms. IMPORTANCE Armillaria species can survive for decades in the soil on dead woody debris, develop rapidly under favorable conditions, and harmfully infect newly planted forests. Our previous study found Trichoderma atroviride to be highly effective in controlling Armillaria growth; therefore, our current work explored the molecular mechanisms that might play a key role in Trichoderma-Armillaria interactions. Direct confrontation assays combined with time course-based dual transcriptome analysis provided a reliable system for uncovering the interactive molecular dynamics between the fungal plant pathogen and its mycoparasitic partner. Furthermore, using a haploid Armillaria isolate allowed us to survey the deadly prey-invading activities of the mycoparasite and the ultimate defensive strategies of its prey. Our current study provides detailed insights into the essential genes and mechanisms involved in Armillaria defense against Trichoderma and the genes potentially involved in the efficiency of Trichoderma to control Armillaria. In addition, using a sensitive haploid Armillaria strain (C18/9), with its complete genome data already available, also offers the opportunity to test possible variable molecular responses of Armillaria ostoyae toward diverse Trichoderma isolates with various biocontrol abilities. Initial molecular tests of the dual interactions may soon help to develop a targeted biocontrol intervention with mycoparasites against plant pathogens.

Vertical and horizontal gene transfer shaped plant colonization and biomass degradation in the fungal genus Armillaria.

The fungal genus Armillaria contains necrotrophic pathogens and some of the largest terrestrial organisms that cause tremendous losses in diverse ecosystems, yet how they evolved pathogenicity in a clade of dominantly non-pathogenic wood degraders remains elusive. Here we show that Armillaria species, in addition to gene duplications and de novo gene origins, acquired at least 1,025 genes via 124 horizontal gene transfer events, primarily from Ascomycota. Horizontal gene transfer might have affected plant biomass degrading and virulence abilities of Armillaria, and provides an explanation for their unusual, soft rot-like wood decay strategy. Combined multi-species expression data revealed extensive regulation of horizontally acquired and wood-decay related genes, putative virulence factors and two novel conserved pathogenicity-induced small secreted proteins, which induced necrosis in planta. Overall, this study details how evolution knitted together horizontally and vertically inherited genes in complex adaptive traits of plant biomass degradation and pathogenicity in important fungal pathogens.

Evolutionary Morphogenesis of Sexual Fruiting Bodies in Basidiomycota: Toward a New Evo-Devo Synthesis.

The development of sexual fruiting bodies is one of the most complex morphogenetic processes in fungi. Mycologists have long been fascinated by the morphological and developmental diversity of fruiting bodies; however, evolutionary developmental biology of fungi still lags significantly behind that of animals or plants. Here, we summarize the current state of knowledge on fruiting bodies of mushroom-forming Basidiomycota, focusing on phylogenetic and developmental biology. Phylogenetic approaches have revealed a complex history of morphological transformations and convergence in fruiting body morphologies. Frequent transformations and convergence is characteristic of fruiting bodies in contrast to animals or plants, where main body plans are highly conserved. At the same time, insights into the genetic bases of fruiting body development have been achieved using forward and reverse genetic approaches in selected model systems. Phylogenetic and developmental studies of fruiting bodies have each yielded major advances, but they have produced largely disjunct bodies of knowledge. An integrative approach, combining phylogenetic, developmental, and functional biology, is needed to achieve a true fungal evolutionary developmental biology (evo-devo) synthesis for fungal fruiting bodies.

Hallmarks of Basidiomycete Soft- and White-Rot in Wood-Decay -Omics Data of Two Armillaria Species.

Wood-decaying Basidiomycetes are among the most efficient degraders of plant cell walls, making them key players in forest ecosystems, global carbon cycle, and in bio-based industries. Recent insights from -omics data revealed a high functional diversity of wood-decay strategies, especially among the traditional white-rot and brown-rot dichotomy. We examined the mechanistic bases of wood-decay in the conifer-specialists Armillaria ostoyae and Armillaria cepistipes using transcriptomic and proteomic approaches. Armillaria spp. (Fungi, Basidiomycota) include devastating pathogens of temperate forests and saprotrophs that decay wood. They have been discussed as white-rot species, though their response to wood deviates from typical white-rotters. While we observed an upregulation of a diverse suite of plant cell wall degrading enzymes, unlike white-rotters, they possess and express an atypical wood-decay repertoire in which pectinases and expansins are enriched, whereas lignin-decaying enzymes (LDEs) are generally downregulated. This combination of wood decay genes resembles the soft-rot of Ascomycota and appears widespread among Basidiomycota that produce a superficial white rot-like decay. These observations are consistent with ancestral soft-rot decay machinery conserved across asco- and Basidiomycota, a gain of efficient lignin-degrading ability in white-rot fungi and repeated, complete, or partial losses of LDE encoding gene repertoires in brown- and secondarily soft-rot fungi.

Extraction, fractionation and re-fractionation of Artemisia nilagirica for anticancer activity and HPLC-ESI-QTOF-MS/MS determination.

ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal plants used in traditional medicines are affordable, easily accessible, safer, less toxic and considered as a rich or efficient source of bioactive molecules for modern therapeutics. Artemisia nilagirica (AR) has a long history of use in Indian traditional medicine to combat a wide variety of diseases including cancer. AIM OF THE STUDY: Considering the vast potential of traditional healing plants to deliver safer, less toxic and efficient chemotherapeutics, we have examined anticancer activity of ethanolic extract, bioactive fractions and sub-fractions of AR against different human cancer cell lines along with their phytochemical analysis to understand the insights of novel anticancer activities for further preclinical studies. MATERIALS AND METHODS: Fresh plant material of AR was procured from the wild, dried and ground. The grinded materials was extracted in ethanol (AR-01) and fractionated into butanol (AR-02), ethyl acetate (AR-03), hexane (AR-04) and water (AR-05). The cytotoxicity was evaluated against three different human cancer cell lines, i.e. colon (DLD-1), lung (A-549), and breast (MCF-7) using Sulforhodamine B (SRB) assay along with non-cancerous VERO cells as control and doxorubicin (DOX) as positive control. As we observed strong cytotoxicity of AR-03 and AR-04 fractions against tested cells and marked cytotoxic effects particularly in colon cancer cell lines, we further re-fractionated, AR-03 into (AR-03A, AR-03B, AR-03C, AR-03D, AR-03E) and AR-04 into (AR-04A, AR-04B, AR-04C) sub-fractions by column chromatography and investigated against the same panel of cell lines in addition to one more colon cancer cell line (HT-29). Phytochemical analysis was performed through HPLC-ESI-QTOF-MS/MS fragmentation. RESULTS: Ethyl acetate (AR-03) and hexane (AR-04) fractions were found to be the most cytotoxic against all the tested cell lines. Further, AR-03E and AR-04A sub-fractions were found more specific cytotoxic selectively against DLD-1 cancer cell lines at 100µg/ml concentration. HPLC-ESI-QTOF-MS/MS determination revealed the presence of 17 compounds in AR-01. Among them, 4 compounds were reported for the first time in this species. However, 3 identified compounds (artemorin, ß-santonin and caryophyllene oxide) in AR-03E sub-fraction were commonly present in each bioactive fraction and may be considered as potential and safest cytotoxic agents for anticancer activity. CONCLUSIONS: Experimental evidences reported in this paper for anticancer activity validate the traditional wisdom of Artemisia nilagirica as an anticancer herbal drug. To our knowledge, this is our first novel observation of cytotoxicity and selectivity of ethyl acetate and hexane sub-fraction of AR-01 i.e. AR-03E and AR-04A respectively against DLD-1 human cancer cell lines. HPLC-ESI-QTOF-MS/MS determination attributes the identification of cytotoxic compounds which may be used for further preclinical studies.

Draft Genome Sequence of Pseudomonas sp. Strain BMS12, a Plant Growth-Promoting and Protease-Producing Bacterium, Isolated from the Rhizosphere Sediment of Phragmites karka of Chilika Lake, India.

We report the 4.51 Mb draft genome of Pseudomonas sp. strain BMS12, a Gram-negative bacterium in the class of Gammaproteobacteria, isolated from the rhizospheric sediment of Phragmites karka, an invasive weed in Chilika Lake, Odisha, India. The Pseudomonas sp. strain BMS12 is capable of producing proteases and is also an efficient plant growth promoter that can be useful for various phytoremedial and industrial applications.

Draft Genome Sequence of Halobacillus sp. Strain KGW1, a Moderately Halophilic and Alkaline Protease-Producing Bacterium Isolated from the Rhizospheric Region of Phragmites karka from Chilika Lake, Odisha, India.

Halobacillus sp. strain KGW1 is a moderately halophilic, rod shaped, Gram-positive, yellow pigmented, alkaline protease-producing bacterium isolated from a water sample from Chilika Lake, Odisha, India. Sequencing of bacterial DNA assembled a 3.68-Mb draft genome. The genome annotation analysis showed various gene clusters for tolerance to stress, such as elevated pH, salt concentration, and toxic metals.

Draft Genome Sequence of Acinetobacter sp. Strain BMW17, a Cellulolytic and Plant Growth-Promoting Bacterium Isolated from the Rhizospheric Region of Phragmites karka of Chilika Lake, India.

We report the 3.16 Mb draft genome of Acinetobacter sp. strain BMW17, a Gram-negative bacterium in the class of Gammaproteobacteria, isolated from the rhizospheric region of Phragmites karka, an invasive weed in Chilika Lake, Odisha, India. The strain BMW17(T) is capable of degrading cellulose and is also an efficient plant growth promoter that can be useful for various phytoremedial and commercial applications.