Transcriptomic Analysis of the White-rot Basidiomycete Lentinus Squarrosulus to Provide Insights Into Its Lignocellulose Biodegradation Ability

BackgroundBasidiomycetes are of special interest in biotechnological research for their versatile potential in the degradation of lignocellulosic biomass, chiefly attributed to ligninolytic enzymes along with exo, endo β-glucanases, xylanases, esterases, pectinases, mannanases, cellobiohydrolases, polysaccharide monooxygenases. Relatively little is known about the metabolic process and the subsequent polysaccharide degradation. Transcriptomic analysis of lignicolous fungi grown on different substrates, although attempted by researchers, has focused on a fairly small group of species reporting the expression of fungal genes in response to lignocellulosic biomass as a substrate. This study accordingly reports analysis of transcriptome of a white-rot Basidiomycete L.squarrosulus grown in simple potato dextrose broth supplemented with aromatic compound, reactive black dye to gain an insight into the degradation ability of the fungus. RNA was sequenced using Illumina NextSeq 500 to obtain 6,679,162 high-quality paired-end reads that were assembled de novo using CLC assembly cells to generate 25,244 contigs. Putative functions were assigned for the 10,494 transcripts based on sequence similarities through BLAST2GO 5.2 and Function annotator.ResultsFunctional assignments revealed enhanced oxidoreductase activity through the expression of diverse biomass-degrading enzymes and their corresponding coregulators. CAZyme analysis through dbCAN and CUPP revealed the presence of 6 families of polysaccharide lyases, 51 families of glycoside hydrolases, 23 families of glycoside transferases, 7 families of carbohydrate esterases and 10 families of auxiliary activities. Genes encoding ligninolytic enzymes and auxiliary activities among the transcript sequences were identified through gene prediction by AUGUSTUS and FGENESH. Biochemical analysis of several biomass-degrading enzymes substantiated the functional predictions.ConclusionIn essence, L. squarrosulus grown in a simple medium devoid of lignocellulosic substrate demonstrated the presence of a repertoire of lignocellulose-degrading enzymes, simplying that a source of lignocellulose is not required for the expression of these biomass-degrading enzymes. This study on the transcriptome analysis of L. squarrosulus revealed significant facts on this front and will definitely enhance the knowledge about the biodegradative ability of this fungus, potentially paving the way for efficient biotechnological applications utilizing its potency in biomass degradation and its future functional exploitation in biomass conversion applications.

Conducting genetic interaction analysis with CRISPR-Cas9-based strategies in Candida albicans

Candida albicans is an opportunistic fungal pathogen found in the oral mucosa, the gut, the vaginal mucosa, and humans' skin. While C. albicans can cause superficial infections, severe invasive infections can occur in immunocompromised individuals. Understanding the survival mechanisms and pathogenesis of C. albicans is critical for novel antifungal drug discovery. Determining the relationships between different genes can create a genetic interaction map, which can identify complementary gene sets, central to C. albicans survival, as potential drug targets in combination therapy. A genetic approach using the CRISPR-Cas9-based genome editing platform will focus on genetic interaction analysis of C. albicans stress response genes. The ultimate goal is to create a stress response gene deletion library to study its pathogen survival role. This library of single and double stress response gene mutants will be screened under diverse growth conditions to assess their relative fitness. Genetic interaction analysis will help map out epistatic interactions between fungal genes involved in growth, survival, and pathogenesis and uncover putative targets for combination antifungal therapy based on negative or synthetic lethal genetic interactions.

Carbon assimilating fungi from surface ocean to subseafloor revealed by coupled phylogenetic and stable isotope analysis

Fungi are ubiquitous in the ocean and hypothesized to be important members of marine ecosystems, but their roles in the marine carbon cycle are poorly understood. Here, we use 13C DNA stable isotope probing coupled with phylogenetic analyses to investigate carbon assimilation within diverse communities of planktonic and benthic fungi in the Benguela Upwelling System (Namibia). Across the redox stratified water column and in the underlying sediments, assimilation of 13C-labeled carbon from diatom extracellular polymeric substances (13C-dEPS) by fungi correlated with the expression of fungal genes encoding carbohydrate-active enzymes. Phylogenetic analysis of genes from 13C-labeled metagenomes revealed saprotrophic lineages related to the facultative yeast Malassezia were the main fungal foragers of pelagic dEPS. In contrast, fungi living in the underlying sulfidic sediments assimilated more 13C-labeled carbon from chemosynthetic bacteria compared to dEPS. This coincided with a unique seafloor fungal community and dissolved organic matter composition compared to the water column, and a 100-fold increased fungal abundance within the subseafloor sulfide-nitrate transition zone. The subseafloor fungi feeding on 13C-labeled chemolithoautotrophs under anoxic conditions were affiliated with Chytridiomycota and Mucoromycota that encode cellulolytic and proteolytic enzymes, revealing polysaccharide and protein-degrading fungi that can anaerobically decompose chemosynthetic necromass. These subseafloor fungi, therefore, appear to be specialized in organic matter that is produced in the sediments. Our findings reveal that the phylogenetic diversity of fungi across redox stratified marine ecosystems translates into functionally relevant mechanisms helping to structure carbon flow from primary producers in marine microbiomes from the surface ocean to the subseafloor.

Cross-Kingdom Gene Coexpression Analysis Using a Stemphylium botryosum–Lens ervoides System Revealed Plasticity of Intercommunication Between the Pathogen Secretome and the Host Immune Systems

Necrotrophic pathogens are responsible for significant declines in crop yield and quality worldwide. During the infection process, a pathogen releases a series of secretory proteins to counteract the plant immune system, and this interaction of pathogen and host molecules determines whether the pathogen will successfully invade the host plant tissues. In this study, we adopted co-transcriptomic approaches to analyze the Lens ervoides–Stemphylium botryosum system, with a focus on 1,216 fungal genes coding for secretory proteins and 8,810 disease-responsive genes of the host 48, 96, and 144 h postinoculation, captured in two F9 recombinant inbred lines (RILs) displaying contrasting disease responses. By constructing in planta gene coexpression networks (GCNs) for S. botryosum, we found that the pathogen tended to co-upregulate genes regulating cell wall degradation enzymes, effectors, oxidoreductases, and peptidases to a much higher degree in the susceptible host LR-66-577 than in the resistant RIL LR-66-637, indicating that the promotion of these digestive enzymes and toxins increased S. botryosum virulence. Construction of cross-kingdom GCNs between pathogen and plant for the two RILs revealed that the co-upregulation of these fungal digestive enzymes and toxins simultaneously promoted a series of defense responses such as redox change, expression of membrane-related genes and serine/threonine kinase, and stress and disease responses in the susceptible RIL which was not observed in the resistant RIL, indicating that these activities exacerbated susceptibility to S. botryosum. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Gcorn fungi: A Web Tool for Detecting Biases between Gene Evolution and Speciation in Fungi

(1) Background: Fungi contain several millions of species, and the diversification of fungal genes has been achieved by speciation, gene duplication, and horizontal gene transfer. Although several databases provide information on orthologous and paralogous events, these databases show no information on biases between gene mutation and speciation. Here, we designed the Gcorn fungi database to better understand such biases. (2) Methods: Amino acid sequences of fungal genes in 249 species, which contain 2,345,743 sequences, were used for this database. Homologous genes were grouped at various thresholds of the homology index, which was based on the percentages of gene mutations. By grouping genes that showed highly similar homology indices to each other, we showed functional and evolutionary traits in the phylogenetic tree depicted for the gene of interest. (3) Results: Gcorn fungi provides well-summarized information on the evolution of a gene lineage and on the biases between gene evolution and speciation, which are quantitatively identified by the Robinson–Foulds metric. The database helps users visualize these traits using various depictions. (4) Conclusions: Gcorn fungi is an open access database that provides a variety of information with which to understand gene function and evolution.

Novel Genetic Dysregulations and Oxidative Damage in Fusarium graminearum Induced by Plant Defense Eliciting Psychrophilic Bacillus atrophaeus TS1

This study elaborates inter-kingdom signaling mechanisms, presenting a sustainable and eco-friendly approach to combat biotic as well as abiotic stress in wheat. Fusarium graminearum is a devastating pathogen causing head and seedling blight in wheat, leading to huge yield and economic losses. Psychrophilic Bacillus atrophaeus strain TS1 was found as a potential biocontrol agent for suppression of F. graminearum under low temperature by carrying out extensive biochemical and molecular studies in comparison with a temperate biocontrol model strain Bacillus amyloliquefaciens FZB42 at 15 and 25 °C. TS1 was able to produce hydrolytic extracellular enzymes as well as antimicrobial lipopeptides, i.e., surfactin, bacillomycin, and fengycin, efficiently at low temperatures. The Bacillus strain-induced oxidative cellular damage, ultrastructural deformities, and novel genetic dysregulations in the fungal pathogen as the bacterial treatment at low temperature were able to downregulate the expression of newly predicted novel fungal genes potentially belonging to necrosis inducing protein families (fgHCE and fgNPP1). The wheat pot experiments conducted at 15 and 25 °C revealed the potential of TS1 to elicit sudden induction of plant defense, namely, H2O2 and callose enhanced activity of plant defense-related enzymes and induced over-expression of defense-related genes which accumulatively lead to the suppression of F. graminearum and decreased diseased leaf area.

A plant host, Nicotiana benthamiana, enables the production and study of fungal lignin-degrading enzymes

Lignin has significant potential as an abundant and renewable source for commodity chemicals yet remains vastly underutilized. Efforts towards engineering a biochemical route to the valorization of lignin are currently limited by the lack of a suitable heterologous host for the production of lignin-degrading enzymes. Here, we show that expression of fungal genes in Nicotiana benthamiana enables production of members from seven major classes of enzymes associated with lignin degradation (23 of 35 tested) in soluble form for direct use in lignin activity assays. We combinatorially characterized a subset of these enzymes in the context of model lignin dimer oxidation, revealing that fine-tuned coupling of peroxide-generators to peroxidases results in more extensive C-C bond cleavage compared to direct addition of peroxide. Comparison of peroxidase isoform activity revealed that the extent of C-C bond cleavage depends on peroxidase identity, suggesting that peroxidases are individually specialized in the context of lignin oxidation. We anticipate the use of N. benthamiana as a platform to rapidly produce a diverse array of fungal lignin-degrading enzymes will facilitate a better understanding of their concerted role in nature and unlock their potential for lignin valorization, including within the plant host itself.

Detection and Identification of Novel Genes from Fungi Isolated from Wastewater

The environment in Mosul city is very rich, containing a wide variety of microorganisms which have not been recognised for a long time. Five new fungal genes were identified and registered for the first time in the gene bank. These included Fusarium falciforme 2020-06-MIK-F1 genes for 5.8S rRNA with Accession no. LC555741, Nectriaceae sp. 2020-06-MIK-F2 genes for ITS1 with Accession no. LC555742, Trichoderma asperellum MIK3 genes for 5.8S rRNA with Accession no. LC575020, Penecillum sp. MIK4 genes for 5.8S rRNA with Accession no. LC575021, and Neurospora crassa MIK5 genes for 5.8S rRNA with Accession no. LC575022. These fungal genes were isolated from wastewater of Khosr river in Mosul city/ Iraq, which has many contamination sources.

Deliberations on Natural Products and Future Directions in the Pharmaceutical Industry

Natural Products (NPs) are molecular' special equipment ' that impart survival benefits on their producers in nature. Due to their evolved functions to modulate biology these privileged metabolites are substantially represented in the drug market and are continuing to contribute to the discovery of innovative medicines such as the recently approved semi-synthetic derivative of the bacterial alkaloid staurosporin in oncology indications. The innovation of low molecular weight compounds in modern drug discovery is built on rapid progress in chemical, molecular biological, pharmacological and data sciences, which together provide a rich understanding of disease-driving molecular interactions and how to modulate them. NPs investigated in these pharmaceutical research areas create new perspectives on their chemical and biological features and thereby new chances to advance medical research. New methods in analytical chemistry linked with searchable NP-databases solved the issue of reisolation and enabled targeted and efficient access to novel molecules from nature. Cheminformatics delivers high resolution descriptions of NPs and explores the substructures that systematically map NP-chemical space by sp3-enriched fragments. Whole genome sequencing has revealed the existence of collocated gene clusters that form larger functional entities together with proximate resistance factors thus avoiding self-inhibition of the encoded metabolites. The analysis of bacterial and fungal genes provides tantalizing glimpses of new compound-target pairs of therapeutic value. Furthermore, a dedicated investigation of structurally unique, selectively active NPs in chemical biology demonstrates their extraordinary power as shuttles between new biological target spaces of pharmaceutical relevance.

Developing Functional Genomics Platforms for Fungi

Fungi are responsible for diseases that result in the deaths of over a million individuals each year and devastating crop infestations that threaten global food supplies. However, outside of a select few model organisms, the majority of fungal genes are uncharacterized.