Secondary Metabolic Gene Clusters Research Articles

Genome sequencing of Elaeocarpus spp. stem blight pathogen Pseudocryphonectria elaeocarpicola reveals potential adaptations to colonize woody bark

BackgroundElaeocarpus spp. stem blight, caused by Pseudocryphonectria elaeocarpicola, is a destructive disease, which will significantly reduce the productivity and longevity of Elaeocarpus spp. plants, especially in the Guangdong Province of China. However, few information is available for P. elaeocarpicola. To unravel the potential adaptation mechanism of stem adaptation, the whole genome of P. elaeocarpicola was sequenced by using the DNBSEQ and PacBio platforms.ResultsP. elaeocarpicola harbors 44.49 Mb genome with 10,894 predicted coding genes. Genome analysis revealed that the P. elaeocarpicola genome encodes a plethora of pathogenicity-related genes. Analysis of carbohydrate-active enzymes (CAZymes) revealed a rich variety of enzymes participated in plant cell wall degradation, which could effectively degrade cellulose, hemicellulose and xyloglucans in the plant cell wall and promote the invasion of the host plant. There are 213 CAZyme families found in P. elaeocarpicola, among which glycoside hydrolase (GH) family has the largest number, far exceeding other tested fungi by 53%. Besides, P. elaeocarpicola has twice as many genes encoding chitin and cellulose degradation as Cryphonectria parasitica, which belong to the same family. The predicted typical secreted proteins of P. elaeocarpicola are numerous and functional, including many known virulence effector factors, indicating that P. elaeocarpicola has great potential to secrete virulence effectors to promote pathogenicity on host plants. AntiSMASH revealed that the genome encoded 61 secondary metabolic gene clusters including 86 secondary metabolic core genes which was much higher than C. parasitica (49). Among them, two gene cluster of P. elaeocarpicola, cluster12 and cluster52 showed 100% similarity with the mycotoxins synthesis clusters from Aspergillus steynii and Alternaria alternata, respectively. In addition, we annotated cytochrome P450 related enzymes, transporters, and transcription factors in P. elaeocarpicola, which are important virulence determinants of pathogenic fungi.ConclusionsTaken together, our study represents the first genome assembly for P. elaeocarpicola and reveals the key virulence factors in the pathogenic process of P. elaeocarpicola, which will promote our understanding of its pathogenic mechanism. The acquired knowledge lays a foundation for further exploration of molecular interactions with the host and provide target for management strategies in future research.

Unveiling the Arsenal of Apple Bitter Rot Fungi: Comparative Genomics Identifies Candidate Effectors, CAZymes, and Biosynthetic Gene Clusters in Colletotrichum Species.

The bitter rot of apple is caused by Colletotrichum spp. and is a serious pre-harvest disease that can manifest in postharvest losses on harvested fruit. In this study, we obtained genome sequences from four different species, C. chrysophilum, C. noveboracense, C. nupharicola, and C. fioriniae, that infect apple and cause diseases on other fruits, vegetables, and flowers. Our genomic data were obtained from isolates/species that have not yet been sequenced and represent geographic-specific regions. Genome sequencing allowed for the construction of phylogenetic trees, which corroborated the overall concordance observed in prior MLST studies. Bioinformatic pipelines were used to discover CAZyme, effector, and secondary metabolic (SM) gene clusters in all nine Colletotrichum isolates. We found redundancy and a high level of similarity across species regarding CAZyme classes and predicted cytoplastic and apoplastic effectors. SM gene clusters displayed the most diversity in type and the most common cluster was one that encodes genes involved in the production of alternapyrone. Our study provides a solid platform to identify targets for functional studies that underpin pathogenicity, virulence, and/or quiescence that can be targeted for the development of new control strategies. With these new genomics resources, exploration via omics-based technologies using these isolates will help ascertain the biological underpinnings of their widespread success and observed geographic dominance in specific areas throughout the country.

Histone H2B lysine 122 and lysine 130, as the putative targets of Penicillium oxalicum LaeA, play important roles in asexual development, expression of secondary metabolite gene clusters, and extracellular glycoside hydrolase synthesis.

Core histones in the nucleosome are subject to a wide variety of posttranslational modifications (PTMs), such as methylation, phosphorylation, ubiquitylation, and acetylation, all of which are crucial in shaping the structure of the chromatin and the expression of the target genes. A putative histone methyltransferase LaeA/Lae1, which is conserved in numerous filamentous fungi, functions as a global regulator of fungal growth, virulence, secondary metabolite formation, and the production of extracellular glycoside hydrolases (GHs). LaeA's direct histone targets, however, were not yet recognized. Previous research has shown that LaeA interacts with core histone H2B. Using S-adenosyl-L-methionine (SAM) as a methyl group donor and recombinant human histone H2B as the substrate, it was found that Penicillium oxalicum LaeA can transfer the methyl groups to the C-terminal lysine (K) 108 and K116 residues in vitro. The H2BK108 and H2BK116 sites on recombinant histone correspond to P. oxalicum H2BK122 and H2BK130, respectively. H2BK122A and H2BK130A, two mutants with histone H2B K122 or K130 mutation to alanine (A), were constructed in P. oxalicum. The mutants H2BK122A and H2BK130A demonstrated altered asexual development and decreased extracellular GH production, consistent with the findings of the laeA gene deletion strain (ΔlaeA). The transcriptome data showed that when compared to wild-type (WT) of P. oxalicum, 38 of the 47 differentially expressed (fold change ≥ 2, FDR ≤ 0.05) genes that encode extracellular GHs showed the same expression pattern in the three mutants ΔlaeA, H2BK122A, and H2BK130A. The four secondary metabolic gene clusters that considerably decreased expression in ΔlaeAalso significantly decreased in H2BK122A or H2BK130A. The chromatin of promotor regions of the key cellulolytic genes cel7A/cbh1 and cel7B/eg1 compacted in the ΔlaeA, H2BK122A, and H2BK130A mutants, according to the results of chromatin accessibility real-time PCR (CHART-PCR). The chromatin accessibility index dropped. The histone binding pocket of the LaeA-methyltransf_23 domain is compatible with particular histone H2B peptides, providing appropriate electrostatic and steric compatibility to stabilize these peptides, according to molecular docking. The findings of the study demonstrate that H2BK122 and H2BK130, which are histone targets of P. oxalicum LaeA in vitro, are crucial for fungal conidiation, the expression of gene clusters encoding secondary metabolites, and the production of extracellular GHs.

Analysis of the complete genome sequence of Paenibacillus sp. lzh-N1 reveals its antagonistic ability

BackgroundPlant diseases caused by pathogenic fungi are devastating. However, commonly used fungicides are harmful to the environment, and some are becoming ineffective due to fungal resistance. Therefore, eco-friendly biological methods to control pathogenic fungi are urgently needed.ResultsIn this study, a strain, Paenibacillus sp. lzh-N1, that could inhibit the growth of the pathogenic fungus Mycosphaerella sentina (Fr) Schrorter was isolated from the rhizosphere soil of pear trees, and the complete genome sequence of the strain was obtained, annotated, and analyzed to reveal the genetic foundation of its antagonistic ability. The entire genome of this strain contained a circular chromosome of 5,641,488 bp with a GC content of 45.50%. The results of species identification show that the strain belongs to the same species as P. polymyxa Sb3-1 and P. polymyxa CJX518. Sixteen secondary metabolic biosynthetic gene clusters were predicted by antiSMASH, including those of the antifungal peptides fusaricidin B and paenilarvins. In addition, biofilm formation-related genes containing two potential gene clusters for cyclic lactone autoinducer, a gene encoding S-ribosylhomocysteine lyase (LuxS), and three genes encoding exopolysaccharide biosynthesis protein were identified.ConclusionsAntifungal peptides and glucanase biosynthesized by Paenibacillus sp. lzh-N1 may be responsible for its antagonistic effect. Moreover, quorum sensing systems may influence the biocontrol activity of this strain directly or indirectly.

The jet-like chromatin structure defines active secondary metabolism in fungi.

Eukaryotic genomes are spatially organized within the nucleus in a nonrandom manner. However, fungal genome arrangement and its function in development and adaptation remain largely unexplored. Here, we show that the high-order chromosome structure of Fusarium graminearum is sculpted by both H3K27me3 modification and ancient genome rearrangements. Active secondary metabolic gene clusters form a structure resembling chromatin jets. We demonstrate that these jet-like domains, which can propagate symmetrically for 54 kb, are prevalent in the genome and correlate with active gene transcription and histone acetylation. Deletion of GCN5, which encodes a core and functionally conserved histone acetyltransferase, blocks the formation of the domains. Insertion of an exogenous gene within the jet-like domain significantly augments its transcription. These findings uncover an interesting link between alterations in chromatin structure and the activation of fungal secondary metabolism, which could be a general mechanism for fungi to rapidly respond to environmental cues, and highlight the utility of leveraging three-dimensional genome organization in improving gene transcription in eukaryotes.

Genomic insights into Penicillium chrysogenum adaptation to subseafloor sedimentary environments

BackgroundPenicillium chrysogenum is a filamentous fungal species with diverse habitats, yet little is known about its genetics in adapting to extreme subseafloor sedimental environments.ResultsHere, we report the discovery of P. chrysogenum strain 28R-6-F01, isolated from deep coal-bearing sediments 2306 m beneath the seafloor. This strain possesses exceptional characteristics, including the ability to thrive in extreme conditions such as high temperature (45 °C), high pressure (35 Mpa), and anaerobic environments, and exhibits broad-spectrum antimicrobial activity, producing the antibiotic penicillin at a concentration of 358 μg/mL. Genome sequencing and assembly revealed a genome size of 33.19 Mb with a GC content of 48.84%, containing 6959 coding genes. Comparative analysis with eight terrestrial strains identified 88 unique genes primarily associated with penicillin and aflatoxins biosynthesis, carbohydrate degradation, viral resistance, and three secondary metabolism gene clusters. Furthermore, significant expansions in gene families related to DNA repair were observed, likely linked to the strain’s adaptation to its environmental niche.ConclusionsOur findings provide insights into the genomic and biological characteristics of P. chrysogenum adaptation to extreme anaerobic subseafloor sedimentary environments, such as high temperature and pressure.

Global Marine Cold Seep Metagenomes Reveal Diversity of Taxonomy, Metabolic Function, and Natural Products

Abstract Cold seeps in the deep sea are closely linked to energy exploration as well as global climate change. The alkane-dominated chemical energy-driven model makes cold seeps an oasis of deep-sea life, showcasing an unparalleled reservoir of microbial genetic diversity. By analyzing 113 metagenomes collected from 14 global sites across 5 cold seep types, we present a comprehensive Cold Seep Microbiomic Database (CSMD) to archive the genomic and functional diversity of cold seep microbiome. The CSMD included over 49 million non-redundant genes and 3175 metagenome-assembled genomes (MAGs), which represented 1895 species spanning 105 phyla. In addition, beta diversity analysis indicated that both the sampling site and cold seep type had a substantial impact on the prokaryotic microbiome community composition. Heterotrophic and anaerobic metabolisms were prevalent in microbial communities, accompanied by considerable mixotrophs and facultative anaerobes, highlighting the versatile metabolic potential in cold seeps. Furthermore, secondary metabolic gene cluster analysis indicated that at least 98.81% of the sequences potentially encoded novel natural products, with ribosomal processing peptides being the predominant type widely distributed in archaea and bacteria. Overall, the CSMD represents a valuable resource that would enhance the understanding and utilization of global cold seep microbiomes.

A fungal sesquiterpene biosynthesis gene cluster critical for mutualist-pathogen transition in Colletotrichum tofieldiae

Plant-associated fungi show diverse lifestyles from pathogenic to mutualistic to the host; however, the principles and mechanisms through which they shift the lifestyles require elucidation. The root fungus Colletotrichum tofieldiae (Ct) promotes Arabidopsis thaliana growth under phosphate limiting conditions. Here we describe a Ct strain, designated Ct3, that severely inhibits plant growth. Ct3 pathogenesis occurs through activation of host abscisic acid pathways via a fungal secondary metabolism gene cluster related to the biosynthesis of sesquiterpene metabolites, including botrydial. Cluster activation during root infection suppresses host nutrient uptake-related genes and changes mineral contents, suggesting a role in manipulating host nutrition state. Conversely, disruption or environmental suppression of the cluster renders Ct3 beneficial for plant growth, in a manner dependent on host phosphate starvation response regulators. Our findings indicate that a fungal metabolism cluster provides a means by which infectious fungi modulate lifestyles along the parasitic–mutualistic continuum in fluctuating environments.

Comparative genomic and phenotypic analyses of pathogenic fungi Neoscytalidium dimidiatum and Bipolaris papendorfii isolated from human skin scraping.

Neoscytalidium dimidiatum and Bipolaris species are fungal plant pathogens that have been reported to cause human diseases. Recently, we have isolated numerous N. dimidiatum and Bipolaris species from the skin scrapings and nails of different patients. In this work, we have sequenced the genome of one strain of N. dimidiatum. The sequenced genome was compared to that of a previously reported Bipolaris papendorfii genome for a better understanding of their complex lifestyle and broad host-range pathogenicity. Both N. dimidiatum UM 880 (~ 43Mb) and B. papendorfii UM 226 (~ 33Mb) genomes include 11,015-12,320 putative coding DNA sequences, of which 0.51-2.49% are predicted transposable elements. Analysis of secondary metabolism gene clusters revealed several genes involved in melanin biosynthesis and iron uptake. The arsenal of CAZymes related to plants pathogenicity is comparable between the species, including genes involved in hemicellulose and pectin decomposition. Several important gene encoding keratinolytic peptidases were identified in N. dimidiatum and B. papendorfii, reflecting their potential pathogenic role in causing skin and nail infections. In this study, additional information on the metabolic features of these two species, such as nutritional profiling, pH tolerance, and osmotolerant, are revealed. The genomic characterization of N. dimidiatum and B. papendorfii provides the basis for the future functional studies to gain further insights as to what makes these fungi persist in plants and why they are pathogenic to humans.

Characteristics of staphylococcal cassette chromosome mec and lugdunin operon genes in the complete genome of Staphylococcus lugdunensis.

Characteristics of staphylococcal cassette chromosome mec and lugdunin operon genes in the complete genome of Staphylococcus lugdunensis.

High-Quality Genome Resource of Fusarium pseudograminearum Isolate Fp22-2 by Oxford Nanopore Long-Read Sequencing.

Fusarium crown rot (FCR), caused by Fusarium pseudograminearum, results in severe yield and quality losses of cereal crops in many arid and semiarid areas of the world. Limited information about the genome of F. pseudograminearum restricts the pathogenesis research and breeding of disease-resistant wheat varieties. In this study, a high-quality genome assembly of F. pseudograminearum isolate Fp22-2 was generated using Oxford Nanopore long-read sequencing technology. The assembled nuclear genome of Fp22-2 is 37.33 Mb with a repeat content of 3.69% and is divided into four contigs with a k-mer completeness score of 97.2% and a base quality accuracy of >99.99%. A total of 14,475 protein-coding genes (BUSCO completeness score, 99.9%) were predicted and functionally annotated. Moreover, genes encoding pathogenic proteins, including effector proteins and carbohydrate-active enzymes, and secondary metabolic gene clusters were identified. Overall, the high-quality genome assembly and gene annotation provided here will allow further investigation of the biology of F. pseudograminearum and lead to the development of new control options for FCR.

Polyphase taxonomy and genome analysis reveal the adaptability of Luteolibacter rhizosphaerae sp. nov. to the rhizosphere soil of Ulmus pumila L.

A Gram-stain-negative, rod-shaped, non-flagellated, pale-yellow bacterium, designated GHJ8T, was isolated from the rhizosphere soil of Ulmus pumila L., Shanxi Province, China. Growth occurred at 20-37°C (optimum, 28°C), pH 6.0-11.0 (optimum, pH 8.0), and 0-1% NaCl (optimum, 0%). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain GHJ8T was related to members of the genus Luteolibacter, and close to Luteolibacter flavescens GKXT (98.5%), Luteolibacter luteus G-1-1-1T (97.3%), Luteolibacter arcticus MC 3726T (97.2%), and Luteolibacter marinus NBU1238T (96.0%). The genome size of strain GHJ8T was 6.2 Mbp, with a G + C content of 62.5%. Genomic mining revealed that the strain contained antibiotic resistance genes and secondary metabolic gene clusters, indicating that it had adaptation mechanisms to environmental stress. Comparative genomic analyses clearly separated strain GHJ8T from the recognized species of the genus Luteolibacter based on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values below the thresholds for species delineation. The major cellular fatty acids were iso-C14:0 (30.8%), C16:1 ω9c (23.0%), C16:0 (17.3%), and C14:0 (13.4%). The quinone system was composed of the major menaquinones MK-8, MK-9, and MK-10, and the principal polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, an unidentified aminophospholipid, an unidentified glycolipid, two unidentified phospholipids, and three unidentified lipids. Based on its phenotypic and genotypic properties and phylogenetic inference, strain GHJ8T is a novel species of the genus Luteolibacter, for which the name Luteolibacter rhizosphaerae sp. nov. is proposed. The type strain is GHJ8T (= GDMCC 1.2160T = KCTC 82452T = JCM 34400T).

Genome sequencing, assembly, and characterization of Pichia fermentans Z9Y-3 as a non-Saccharomyces yeast with aroma enhancing potential

Pichia fermentans is an important non-Saccharomyces yeast applied in mixed fermentation to enhance fruity and floral traits of wine aroma. In this study, genome sequencing of P. fermentans Z9Y-3 was performed using the PacBio single-molecule real-time sequencing platform, and was further assembled using MECAT. The sequenced genes are annotated by non-redundant protein (Nr), Gene Ontology (GO), Cluster of Orthologous Groups of proteins (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG) and carbohydrate-active enzymes (CAZymes) database. Results indicated that the genome size was about 13.5 Mb with 42.09% GC content and 4730 protein coding genes. The 95.90% genes were annotated to the non-redundant protein (Nr) database, and then annotated to GO database. The 62.64% genes annotated to the COG database revealed 25 functional groups. The 58.22% genes annotated to the KEGG database revealed 5 groups. The 586 genes were annotated to CAZymes, in which two genes bglH and bglC encoding beta-glucosidase were found and further analyzed its motifs. A terpene and a nonribosomal peptides like secondary metabolic gene clusters were predicted using antiSMASH 7.0.0. This study benefits us to understand physiological and biochemical behavior of P. fermentans Z9Y-3 at a molecular level to better use P. fermentans Z9Y-3 in aroma enhancing winemaking.

Challenges and advancements in bioprocess intensification of fungal secondary metabolite: kojic acid

Kojic acid is a fungal secondary metabolite commonly known as a tyrosinase inhibitor, that acts as a skin-whitening agent. Its applications are widely distributed in the area of cosmetics, medicine, food, and chemical synthesis. Renewable resources are the alternative feedstocks that can fulfill the demand for free sugars which are fermented for the production of kojic acid. This review highlights the current progress and importance of bioprocessing of kojic acid from various types of competitive and non-competitive renewable feedstocks. The bioprocessing advancements, secondary metabolic pathway networks, gene clusters and regulations, strain improvement, and process design have also been discussed. The importance of nitrogen sources, amino acids, ions, agitation, and pH has been summarized. Two fungal species Aspergillus flavus and Aspergillus oryzae are found to be extensively studied for kojic acid production due to their versatile substrate utilization and high titer ability. The potential of A. flavus to be a competitive industrial strain for large-scale production of kojic acid has been studied.

Comprehensive analysis of 84 Faecalibacterium prausnitzii strains uncovers their genetic diversity, functional characteristics, and potential risks.

Faecalibacterium prausnitzii is a beneficial human gut microbe and a candidate for next-generation probiotics. With probiotics now being used in clinical treatments, concerns about their safety and side effects need to be considered. Therefore, it is essential to obtain a comprehensive understanding of the genetic diversity, functional characteristics, and potential risks of different F. prausnitzii strains. In this study, we collected the genetic information of 84 F . prausnitzii strains to conduct a pan-genome analysis with multiple perspectives. Based on single-copy genes and the sequences of 16S rRNA and the compositions of the pan-genome, different phylogenetic analyses of F. prausnitzii strains were performed, which showed the genetic diversity among them. Among the proteins of the pan-genome, we found that the accessory clusters made a greater contribution to the primary genetic functions of F. prausnitzii strains than the core and specific clusters. The functional annotations of F. prausnitzii showed that only a very small number of proteins were related to human diseases and there were no secondary metabolic gene clusters encoding harmful products. At the same time, complete fatty acid metabolism was detected in F. prausnitzii. In addition, we detected harmful elements, including antibiotic resistance genes, virulence factors, and pathogenic genes, and proposed the probiotic potential risk index (PPRI) and probiotic potential risk score (PPRS) to classify these 84 strains into low-, medium-, and high-risk groups. Finally, 15 strains were identified as low-risk strains and prioritized for clinical application. Undoubtedly, our results provide a comprehensive understanding and insight into F. prausnitzii, and PPRI and PPRS can be applied to evaluate the potential risks of probiotics in general and to guide the application of probiotics in clinical application.

Characteristics and adaptability of Flavobacterium panici BSSL-CR3 in tidal flat revealed by comparative genomic and enzymatic analysis

Tidal flat microbes play an important ecological role by removing organic pollutants and providing an energy source. However, bacteria isolated from tidal flatsand their genomes have been scarcely reported, making it difficult to elucidate which genes and pathways are potentially involved in the above roles. In this study, strain BSSL-CR3, the third reported species among the tidal flat Flavobacterium was analyzed using whole-genome sequencing to investigate its adaptability and functionality in tidal flats. BSSL-CR3 is comprised of a circular chromosome of 5,972,859bp with a GC content of 33.84%. Genome annotation and API ZYM results showed that BSSL-CR3 has a variety of secondary metabolic gene clusters and enzyme activities including α-galactosidase. BSSL-CR3 had more proteins with a low isoelectric point (pI) than terrestrial Flavobacterium strains, and several genes related to osmotic regulation were found in thegenomic island(GI). Comparative genomic analysis with other tidal flat bacteria also revealed that BSSL-CR3 had the largest number of genes encoding Carbohydrate Active EnZymes (CAZymes) which are related to algae degradation. This study will provide insight into the adaptability of BSSL-CR3 to the tidal flats and contribute to facilitating future comparative analysis of bacteria in tidal flats.

Comparative genomics of five Valsa species gives insights on their pathogenicity evolution.

Valsa is a genus of ascomycetes within the Valsaceae family. This family includes many wood destructive pathogens such as the well known Valsa mali and Valsa pyri which cause canker diseases in fruit trees and threaten the global fruit production. Lack of genomic information of this family is impeding our understandings about their evolution and genetic basis of their pathogenicity divergence. Here, we report genome assemblies of Valsa malicola, Valsa persoonii, and Valsa sordida which represent close relatives of Valsa mali and Valsa pyri with different host preferences. Comparative genomics analysis revealed that segmental rearrangements, inversions, and translocations frequently occurred among Valsa spp. genomes. Gene families that exhibited gene copy expansions tended to be associated with secondary metabolism, transmembrane transport, and pyrophosphatase activities. Orthologous genes in regions lost synteny exhibited significantly higher rate of synonymous substitution (KS) than those in regions retained synteny. Moreover, among these genes, membrane transporter families associated with antidrug (MFS, DHA) activities and nutrient transportation (SP and APCs) activities were significantly over-represented. Lineage specific synonymous substitution (KS) and nonsynonymous substitution (KA) analysis based on the phylogeny constructed from 11 fungal species identified a set of genes with selection signatures in Valsa clade and these genes were significantly enriched in functions associated with fatty acid beta-oxidation, DNA helicase activity, and ATPase activity. Furthermore, unique genes that possessed or retained by each of the five Valsa species are more likely part of the secondary metabolic (SM) gene clusters. SM gene clusters conserved across five Valsa species showed various degrees of diversification in both identity and completeness. All 11 syntenically conserved SM clusters showed differential expression during the infection of apple branch with Valsa mali suggesting involvements of secondary metabolism in the pathogenicity of Valsa species.

Gap-Free Nuclear and Mitochondrial Genomes of Ustilaginoidea virens JS60-2, a Fungal Pathogen Causing Rice False Smut

Rice false smut (RFS), caused by Ustilaginoidea virens, has become a major disease in recent years, and mycotoxins produced by U. virens often threaten food safety. To study fungal pathogenesis and identify potential targets for developing new fungicides, gap-free nuclear and complete mitochondrial genomes of U. virens JS60-2 were sequenced and assembled. Using the second and third generation sequencing data, we assembled a 38.02-Mb genome that consists of seven contigs with the contig N50 being 6.32-Mb. In total, 8,486 protein-coding genes were annotated in the genome, including 21 secondary metabolism gene clusters. We also assembled the complete mitochondrial genome, which is 102,498 bp, with 28% GC content. The JS60-2 genomes assembled in this study will facilitate research on U. virens and contribute to RFS control. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Identification and Mechanism of Action of the Global Secondary Metabolism Regulator SaraC in Stereum hirsutum.

DNA methylation is an important factor in the regulation of gene expression. In analyzing genomic data of Stereum hirsutum FP-91666, we found a hypothetical bifunctional transcription regulator/O6Meguanine-DNA methyltransferase (named SaraC), which is widely present in both bacteria and fungi, and confirmed that its function in bacteria is mainly for DNA reparation. In this paper, we confirmed that SaraC has the function of DNA binding and demethylation through surface plasma resonance and reaction experiments in vitro. Then, we achieved the overexpression of SaraC (OES) in S. hirsutum, sequenced the methylation and transcription levels of the whole-genome, and further conducted untargeted metabolomics analyses of the OES transformants and the wild type (WT). The results confirmed that the overall-methylation levels of the transformants were significantly downregulated, and various genes related to secondary metabolism were upregulated. Through comparative untargeted metabolomic analyses, it showed that OES SA6 transformant produced a greater number of hybrid polyketides, and we identified 2 novel hybrid polyketides from the fermentation products of SA6. Our results show that overexpression SaraC can effectively stimulate the expression of secondary-metabolism-related genes, which could be a broad-spectrum tool for discovery of metabolites due to its cross-species conservation. IMPORTANCE Fungi are one of the important sources of active compounds. However, in fungi, most of the secondary metabolic biosynthetic gene clusters are weakly expressed or silenced under conventional culture conditions. How to efficiently excavate potential new compounds contained in fungi is becoming a research hot spot in the world. In this study, we found a DNA demethylation protein (SaraC) and confirmed that it is a global secondary metabolism regulator in Stereum hirsutum FP-91666. In the past, SaraC-like proteins were mainly regarded as DNA repair proteins, but our findings proved that it will be a powerful tool for mining secondary metabolites for overexpression of SaraC, which can effectively stimulate the expression of genes related to secondary metabolism.

Transcription Factor VdCf2 Regulates Growth, Pathogenicity, and the Expression of a Putative Secondary Metabolism Gene Cluster in Verticillium dahliae.

Transcription factors (TFs) bind to the promoters of target genes to regulate gene expression in response to different stimuli. The functions and regulatory mechanisms of transcription factors (TFs) in Verticillium dahliae are, however, still largely unclear. This study showed that a C2H2-type zinc finger TF, VdCf2 (V. dahliae chorion transcription factor 2), plays key roles in V. dahliae growth, melanin production, and virulence. Transcriptome sequencing analysis showed that VdCf2 was involved in the regulation of expression of genes encoding secreted proteins, pathogen-host interaction (PHI) homologs, TFs, and G protein-coupled receptors (GPCRs). Furthermore, VdCf2 positively regulated the expression of VdPevD1 (VDAG_02735), a previously reported virulence factor. VdCf2 thus regulates the expression of several pathogenicity-related genes that also contribute to virulence in V. dahliae. VdCf2 also inhibited the transcription of the Vd276-280 gene cluster and interacted with two members encoding proteins (VDAG_07276 and VDAG_07278) in the gene cluster. IMPORTANCE Verticillium dahliae is an important soilborne phytopathogen which can ruinously attack numerous host plants and cause significant economic losses. Transcription factors (TFs) were reported to be involved in various biological processes, such as hyphal growth and virulence of pathogenic fungi. However, the functions and regulatory mechanisms of TFs in V. dahliae remain largely unclear. In this study, we identified a new transcription factor, VdCf2 (V. dahliae chorion transcription factor 2), based on previous transcriptome data, which participates in growth, melanin production, and virulence of V. dahliae. We provide evidence that VdCf2 regulates the expression of the pathogenicity-related gene VdPevD1 (VDAG_02735) and Vd276-280 gene cluster. VdCf2 also interacts with VDAG_07276 and VDAG_07278 in this gene cluster based on a yeast two-hybrid and bimolecular fluorescence complementation assay. These results revealed the regulatory mechanisms of a pivotal pathogenicity-related transcription factor, VdCf2 in V. dahliae.