Journal of Fungi

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20 pages, 5011 KiB

Open AccessArticle

Genomic Based Analysis of the Biocontrol Species Trichoderma harzianum: A Model Resource of Structurally Diverse Pharmaceuticals and Biopesticides

by Suhad A. A. Al-Salihi and Fabrizio Alberti

J. Fungi 2023, 9(9), 895; https://doi.org/10.3390/jof9090895 - 31 Aug 2023

Cited by 3 | Viewed by 1535

Abstract

Fungi represents a rich repository of taxonomically restricted, yet chemically diverse, secondary metabolites that are synthesised via specific metabolic pathways. An enzyme’s specificity and biosynthetic gene clustering are the bottleneck of secondary metabolite evolution. Trichoderma harzianum M10 v1.0 produces many pharmaceutically important molecules; [...] Read more.

Fungi represents a rich repository of taxonomically restricted, yet chemically diverse, secondary metabolites that are synthesised via specific metabolic pathways. An enzyme’s specificity and biosynthetic gene clustering are the bottleneck of secondary metabolite evolution. Trichoderma harzianum M10 v1.0 produces many pharmaceutically important molecules; however, their specific biosynthetic pathways remain uncharacterised. Our genomic-based analysis of this species reveals the biosynthetic diversity of its specialised secondary metabolites, where over 50 BGCs were predicted, most of which were listed as polyketide-like compounds associated clusters. Gene annotation of the biosynthetic candidate genes predicted the production of many medically/industrially important compounds including enterobactin, gramicidin, lovastatin, HC-toxin, tyrocidine, equisetin, erythronolide, strobilurin, asperfuranone, cirtinine, protoilludene, germacrene, and epi-isozizaene. Revealing the biogenetic background of these natural molecules is a step forward towards the expansion of their chemical diversification via engineering their biosynthetic genes heterologously, and the identification of their role in the interaction between this fungus and its biotic/abiotic conditions as well as its role as bio-fungicide. Full article

(This article belongs to the Special Issue Genomics Analysis of Fungi)

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13 pages, 2410 KiB

Open AccessArticle

A Whole-Genome Assembly for Hyaloperonospora parasitica, A Pathogen Causing Downy Mildew in Cabbage (Brassica oleracea var. capitata L.)

by Yuankang Wu, Bin Zhang, Shaobo Liu, Zhiwei Zhao, Wenjing Ren, Li Chen, Limei Yang, Mu Zhuang, Honghao Lv, Yong Wang, Jialei Ji, Fengqing Han and Yangyong Zhang

J. Fungi 2023, 9(8), 819; https://doi.org/10.3390/jof9080819 - 3 Aug 2023

Viewed by 1481

Abstract

Hyaloperonospora parasitica is a global pathogen that can cause leaf necrosis and seedling death, severely threatening the quality and yield of cabbage. However, the genome sequence and infection mechanisms of H. parasitica are still unclear. Here, we present the first whole-genome sequence of [...] Read more.

Hyaloperonospora parasitica is a global pathogen that can cause leaf necrosis and seedling death, severely threatening the quality and yield of cabbage. However, the genome sequence and infection mechanisms of H. parasitica are still unclear. Here, we present the first whole-genome sequence of H. parasitica isolate BJ2020, which causes downy mildew in cabbage. The genome contains 4631 contigs and 9991 protein-coding genes, with a size of 37.10 Mb. The function of 6128 genes has been annotated. We annotated the genome of H. parasitica strain BJ2020 using databases, identifying 2249 PHI-associated genes, 1538 membrane transport proteins, and 126 CAZy-related genes. Comparative analyses between H. parasitica, H.arabidopsidis, and H. brassicae revealed dramatic differences among these three Brassicaceae downy mildew pathogenic fungi. Comprehensive genome-wide clustering analysis of 20 downy mildew-causing pathogens, which infect diverse crops, elucidates the closest phylogenetic affinity between H. parasitica and H. brassicae, the causative agent of downy mildew in Brassica napus. These findings provide important insights into the pathogenic mechanisms and a robust foundation for further investigations into the pathogenesis of H. parasitica BJ2020. Full article

(This article belongs to the Special Issue Genomics Analysis of Fungi)

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14 pages, 4695 KiB

Open AccessArticle

Identification, Culture Characteristics and Whole-Genome Analysis of Pestalotiopsis neglecta Causing Black Spot Blight of Pinus sylvestris var. mongolica

by Jing Yang, Shuren Wang, Yundi Zhang, Yunze Chen, Heying Zhou and Guocai Zhang

J. Fungi 2023, 9(5), 564; https://doi.org/10.3390/jof9050564 - 12 May 2023

Cited by 3 | Viewed by 1515

Abstract

Black spot needle blight is a serious conifer disease of Pinus sylvestris var. mongolica occurring in Northeast China, which is usually caused by the plant pathogenic fungus Pestalotiopsis neglecta. From the diseased pine needles collected in Honghuaerji, the P. neglecta strain YJ-3 [...] Read more.

Black spot needle blight is a serious conifer disease of Pinus sylvestris var. mongolica occurring in Northeast China, which is usually caused by the plant pathogenic fungus Pestalotiopsis neglecta. From the diseased pine needles collected in Honghuaerji, the P. neglecta strain YJ-3 was isolated and identified as the phytopathogen, and its culture characteristics were studied. Then, we generated a highly contiguous 48.36-Mbp genome assembly (N50 = 6.62 Mbp) of the P. neglecta strain YJ-3 by combining the PacBio RS II Single Molecule Real Time (SMRT) and Illumina HiSeq X Ten sequencing platforms. The results showed that a total of 13,667 protein-coding genes were predicted and annotated using multiple bioinformatics databases. The genome assembly and annotation resource reported here will be useful for the study of fungal infection mechanisms and pathogen–host interaction. Full article

(This article belongs to the Special Issue Genomics Analysis of Fungi)

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Pestalotiopsis neglecta YJ-3 causing black spot needle blight on Pinus sylvestris var. mongolica. (A) Infected diseased pine trees; (B) Colony surface on PDA medium; (C) Conidia; (D) Disease symptoms. Scale bars = 10 μm. Full article ">Figure 2
Culture characteristics of the P. neglecta strain YJ-3. (A–C): The effects of different mediums (A), carbon sources (B) and nitrogen sources (C) on mycelial growth of the P. neglecta strain YJ-3. (D,E): The effects of different temperatures (D) and pH (E) on mycelial growth of the P. neglecta strain YJ-3. Letters (a, b, c, d) represent statistically significant differences between different conditions (p < 0.05). Full article ">Figure 3
The eggNOG functional classification diagram. Full article ">Figure 4
GO annotation of the P. neglecta strain YJ-3 genome. Full article ">Figure 5
KEGG classification of the P. neglecta strain YJ-3 genome. Full article ">Figure 6
Predicted carbohydrate active enzymes (A), pathogenically active proteins (B) and antibiotic-resistance genes (C) of Pestalotiopsis neglecta strain YJ-3. Full article ">

14 pages, 2931 KiB

Open AccessArticle

Genome Sequencing and Analysis Reveal Potential High-Valued Metabolites Synthesized by Lasiodiplodia iranensis DWH-2

by Ruiying Li, Pu Zheng, Xingyun Sun, Wenhua Dong, Ziqiang Shen, Pengcheng Chen and Dan Wu

J. Fungi 2023, 9(5), 522; https://doi.org/10.3390/jof9050522 - 28 Apr 2023

Viewed by 1734

Abstract

Lasiodiplodia sp. is a typical opportunistic plant pathogen, which can also be classified as an endophytic fungus. In this study, the genome of a jasmonic-acid-producing Lasiodiplodia iranensis DWH-2 was sequenced and analyzed to understand its application value. The results showed that the L. [...] Read more.

Lasiodiplodia sp. is a typical opportunistic plant pathogen, which can also be classified as an endophytic fungus. In this study, the genome of a jasmonic-acid-producing Lasiodiplodia iranensis DWH-2 was sequenced and analyzed to understand its application value. The results showed that the L. iranensis DWH-2 genome was 43.01 Mb in size with a GC content of 54.82%. A total of 11,224 coding genes were predicted, among which 4776 genes were annotated based on Gene Ontology. Furthermore, the core genes involved in the pathogenicity of the genus Lasiodiplodia were determined for the first time based on pathogen–host interactions. Eight Carbohydrate-Active enzymes (CAZymes) genes related to 1,3-β-glucan synthesis were annotated based on the CAZy database and three relatively complete known biosynthetic gene clusters were identified based on the Antibiotics and Secondary Metabolites Analysis Shell database, which were associated with the synthesis of 1,3,6,8-tetrahydroxynaphthalene, dimethylcoprogen, and (R)-melanin. Moreover, eight genes associated with jasmonic acid synthesis were detected in pathways related to lipid metabolism. These findings fill the gap in the genomic data of high jasmonate-producing strains. Full article

(This article belongs to the Special Issue Genomics Analysis of Fungi)

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Annotation of predicted genes in L. iranensis genome. (a) Comparison of the number of genes annotated into three major categories in the GO database, (b) GO function classification, (c) COG annotation, and (d) KEGG pathway annotation. Full article ">Figure 2
(a) Comparison of the PHI-base homologs in the genomes of L. iranensis M2017288 and L. theobromae AM2As, L. theobromae CSS_01s, and L. theobromae LA_SOL3 using OrthoVenn2. (b) Comparison of CAZymes in the genomes of L. iranensis M2017288 and L. theobromae AM2As, L. theobromae CSS_01s, and L. theobromae LA_SOL3. Full article ">Figure 3
CAZy functional classification and number of corresponding genes in L. iranensis DWH-2, L. theobromae AM2As, L. theobromae CSS_01s, and L. theobromae LA_SOL3. CBM, carbohydrate-binding module; PL, polysaccharide lyase; CE, carbohydrate esterase; GT, glycosyltransferase; AA, auxiliary activity; GH, glycoside hydrolase; double, genes containing two domains belonging to different gene families. Full article ">Figure 4
1,3-β-Glucan synthesis pathway. Genes encoding related enzymes are marked on the side of the arrow. Full article ">Figure 5
Comparison of BGCs constituents in L. iranensis with the identified BGCs involved in the biosynthesis of (a) THN, (b) dimethylcoprogen, and (c) (R)-melanin. Full article ">Figure 6
(a) Distribution map of lipid metabolism pathways in L. iranensis. (b) Genes and metabolites involved in the JA synthesis route. Annotated genes were in the orange mark, unannotated genes were in the green mark, and metabolites were in the blue mark. Full article ">Figure 7
Phylogenetic tree of the mined lipoxygenases aligned with (a) 13-LOX in plants and (b) fungal dioxygenases. Full article ">

12 pages, 1809 KiB

Open AccessArticle

Population Genetic Analysis of Phytophthora colocasiae from Taro in Japan Using SSR Markers

by Jing Zhang, Ayaka Hieno, Kayoko Otsubo, Wenzhuo Feng and Koji Kageyama

J. Fungi 2023, 9(4), 391; https://doi.org/10.3390/jof9040391 - 23 Mar 2023

Cited by 1 | Viewed by 1624

Abstract

Phytophthora colocasiae is an important pathogen that causes great economic losses in taro production in tropical and subtropical regions, especially in Japan. Understanding the genetic variations in P. colocasiae populations and their transmission patterns in Japan is essential for effective disease control. Here, [...] Read more.

Phytophthora colocasiae is an important pathogen that causes great economic losses in taro production in tropical and subtropical regions, especially in Japan. Understanding the genetic variations in P. colocasiae populations and their transmission patterns in Japan is essential for effective disease control. Here, the genetic diversity of 358 P. colocasiae isolates, including 348 from Japan, 7 from China, and 3 from Indonesia, was assessed using 11 simple sequence repeat (SSR) primer pairs with high polymorphism. The phylogenetic tree of the SSR locus showed that the isolates from Japan could be divided into 14 groups, with group A being the dominant group. Among foreign isolates, only six from mainland China were similar to those from Japan and clustered in groups B and E. Analysis of molecular variance (AMOVA), principal components analysis (PCA), and cluster analysis (K = 3) results revealed a moderate level of genetic diversity, mainly within individuals. Populations showed high heterozygosity, a lack of regional differentiation, and frequent gene flow. Analysis of mating types and ploidy levels revealed that A2 and self-fertile (SF) A2 types and tetraploids were dominant across populations. Explanations and hypotheses for the results can provide more effective strategies for disease management of taro leaf blight. Full article

(This article belongs to the Special Issue Genomics Analysis of Fungi)

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17 pages, 1389 KiB

Open AccessArticle

Kojic Acid Gene Clusters and the Transcriptional Activation Mechanism of Aspergillus flavus KojR on Expression of Clustered Genes

by Perng-Kuang Chang, Leslie L. Scharfenstein, Noreen Mahoney and Qing Kong

J. Fungi 2023, 9(2), 259; https://doi.org/10.3390/jof9020259 - 15 Feb 2023

Cited by 5 | Viewed by 2043

Abstract

Kojic acid (KA) is a fungal metabolite and has a variety of applications in the cosmetics and food industries. Aspergillus oryzae is a well-known producer of KA, and its KA biosynthesis gene cluster has been identified. In this study, we showed that nearly [...] Read more.

Kojic acid (KA) is a fungal metabolite and has a variety of applications in the cosmetics and food industries. Aspergillus oryzae is a well-known producer of KA, and its KA biosynthesis gene cluster has been identified. In this study, we showed that nearly all section Flavi aspergilli except for A. avenaceus had complete KA gene clusters, and only one Penicillium species, P. nordicum, contained a partial KA gene cluster. Phylogenetic inference based on KA gene cluster sequences consistently grouped section Flavi aspergilli into clades as prior studies. The Zn(II)₂Cys₆ zinc cluster regulator KojR transcriptionally activated clustered genes of kojA and kojT in Aspergillus flavus. This was evidenced by the time-course expression of both genes in kojR-overexpressing strains whose kojR expression was driven by a heterologous Aspergillus nidulans gpdA promoter or a homologous A. flavus gpiA promoter. Using sequences from the kojA and kojT promoter regions of section Flavi aspergilli for motif analyses, we identified a consensus KojR-binding motif to be an 11-bp palindromic sequence of 5′-CGRCTWAGYCG-3′ (R = A/G, W = A/T, Y = C/T). A CRISPR/Cas9-mediated gene-targeting technique showed that the motif sequence, 5′-CGACTTTGCCG-3′, in the kojA promoter was critical for KA biosynthesis in A. flavus. Our findings may facilitate strain improvement and benefit future kojic acid production. Full article

(This article belongs to the Special Issue Genomics Analysis of Fungi)

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Phylogenetic tree of twenty-four Aspergillus section Flavi species inferred from KA gene cluster sequences using NJ analysis. A total of 5776 conserved sites (i.e., concatenated sequences of total SNPs) from each species were used. Bootstrap values are shown at the nodes. The branch length scale is shown. Branch lengths represent genetic change; the longer the branch, the more divergence has occurred. The exact species name of ATCC12892, originally designated as A. oryzae, is not known. Full article ">Figure 2
Determination of kojR copy numbers and relative expression levels of kojR, kojA, and kojT of kojR-overexpressing strains. (A) kojR copy numbers of overexpression strains whose kojR expression was driven by the A. nidulans gpdA promoter or the A. flavus gpiA promoter. The copy numbers of kojA and kojT of the control and overexpression strains were used as single-gene-copy checks. (B) Relative expression levels of kojR to those of the control strain at 48 h and 72 h. (C) Relative expression levels of kojA and kojT to those of the ∆kojR strain, which presumably were the basal expression levels at 48 h and 72 h. Full article ">Figure 2 Cont.
Determination of kojR copy numbers and relative expression levels of kojR, kojA, and kojT of kojR-overexpressing strains. (A) kojR copy numbers of overexpression strains whose kojR expression was driven by the A. nidulans gpdA promoter or the A. flavus gpiA promoter. The copy numbers of kojA and kojT of the control and overexpression strains were used as single-gene-copy checks. (B) Relative expression levels of kojR to those of the control strain at 48 h and 72 h. (C) Relative expression levels of kojA and kojT to those of the ∆kojR strain, which presumably were the basal expression levels at 48 h and 72 h. Full article ">Figure 3
Putative KojR-binding motif identified by MEME using kojA and kojT promoter sequences of section Flavi aspergilli. The logo is the downloaded EPS (for publication) version from the MEME site, whose appearance is somewhat different from the PNG (for web) version in that all positions have the same baseline. The relative height indicates how certain it is to observe a particular nucleotide at a particular position, and high heights indicate high conservation/low uncertainty. In the MEME analysis, the maximum motif width was arbitrarily set at eleven and searched for palindromic motifs. Promoter sequences listed in <a href="#app1-jof-09-00259" class="html-app">Table S2</a> are the input sequences. Full article ">Figure 4
Identification of KojR-binding site in A. flavus kojA promoter. (A) Six indel defects in the motif of kojA, 5′- CGACTTTGCCG-3′, rendered the transformants unable to produce KA. (B) Six indel defects disrupted the identified motif of kojT, 5′-CGGCTAAGTCG-3′. However, they did not affect KA production of the transformants. The recipient strain used for the CRISPR/Cas9 work is wild-type A. flavus CA14. Wt represents wild-type sequences. Yellow-highlighted sequences are the target sites of the CRISPR/Cas9 complexes. Red trinucleotides CCG and CGG are protospacer adjacent motifs (PAM) that follow the regions targeted for cleavage by the Cas9 nuclease. Dash lines are deleted sequences. Gray-highlighted sequences are additional nucleotides inserted into respective motifs. The symbol ∆403 indicates a large deletion extending to the kojT-coding sequence (see <a href="#app1-jof-09-00259" class="html-app">Figure S3</a>). The photos above the sequences are colony morphologies of six mutants on KAM agar plates, which are shown alternatively on their front and reverse sides. Colonies were grown at 30 °C for five days in the dark. Orange-red plates are KA-producing colonies. (C) Graphic representation showing the location of the functional KojR-binding site in the kojA and kojR intergenic regions inferred from the present study. The site is 266 nucleotides from the translation start codon of kojA and 466 nucleotides from the start codon of kojR. Full article ">

14 pages, 5082 KiB

Open AccessArticle

Comparative Genomic Analysis Reveals Gene Content Diversity, Phylogenomic Contour, Putative Virulence Determinants, and Potential Diagnostic Markers within Pythium insidiosum Traits

by Weerayuth Kittichotirat, Thidarat Rujirawat, Preecha Patumcharoenpol and Theerapong Krajaejun

J. Fungi 2023, 9(2), 169; https://doi.org/10.3390/jof9020169 - 27 Jan 2023

Cited by 2 | Viewed by 1729

Abstract

Pythium insidiosum has successfully evolved into a human/animal filamentous pathogen, causing pythiosis, a life-threatening disease, worldwide. The specific rDNA-based genotype of P. insidiosum (clade I, II, or III) is associated with the different hosts and disease prevalence. Genome evolution of P. insidiosum can [...] Read more.

Pythium insidiosum has successfully evolved into a human/animal filamentous pathogen, causing pythiosis, a life-threatening disease, worldwide. The specific rDNA-based genotype of P. insidiosum (clade I, II, or III) is associated with the different hosts and disease prevalence. Genome evolution of P. insidiosum can be driven by point mutations, pass vertically to the offspring, and diverge into distinct lineages, leading to different virulence, including the ability to be unrecognized by the host. We conducted comprehensive genomic comparisons of 10 P. insidiosum strains and 5 related Pythium species using our online “Gene Table” software to investigate the pathogen’s evolutionary history and pathogenicity. In total, 245,378 genes were found in all 15 genomes and grouped into 45,801 homologous gene clusters. Gene contents among P. insidiosum strains varied by as much as 23%. Our results showed a strong agreement between the phylogenetic analysis of 166 core genes (88,017 bp) identified across all genomes and the hierarchical clustering analysis of gene presence/absence profiles, suggesting divergence of P. insidiosum into two groups, clade I/II and clade III strains, and the subsequent segregation of clade I and clade II. A stringent gene content comparison using the Pythium Gene Table provided 3263 core genes exclusively presented in all P. insidiosum strains but no other Pythium species, which could involve host-specific pathogenesis and serve as biomarkers for diagnostic purposes. More studies focusing on characterizing the biological function of the core genes (including the just-identified putative virulence genes encoding hemagglutinin/adhesin and reticulocyte-binding protein) are needed to explore the biology and pathogenicity of this pathogen. Full article

(This article belongs to the Special Issue Genomics Analysis of Fungi)

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Figure 1
A phylogenetic tree based on 166 single-copy core genes in the genomes of 10 P. insidiosum strains and 5 other Pythium species. The bootstrap values, used to indicate the reliability of the result, are shown on each branching node. Red boxes depict groups of the P. insidiosum strains assigned to rDNA-based genotype clades I, II, and III. Full article ">Figure 2
Percent sequence identities between pairs of the genomes from 10 P. insidiosum strains and 5 other Pythium species based on 166 single-copy core gene sequences. Red boxes show the P. insidiosum strains assigned to rDNA-based genotype clades I, II, and III. Color gradience indicates the degree of sequence identity (%). Full article ">Figure 3
A dendrogram showing the hierarchical clustering result based on 45,801 gene-presence profiles across 10 P. insidiosum strains and 5 Pythium species. The presence (green) or absence (black) status of 120 selected genes are shown as a heat map next to the dendrogram. The bootstrap values are shown on each dendrogram branching node. Red boxes depict groups of the P. insidiosum strains assigned to rDNA-based genotype clades I, II, and III. This result shows that P. insidiosum strains from the same clade share higher gene-content similarity. Full article ">Figure 4
All pairwise gene content comparisons of 10 P. insidiosum strains and 5 other Pythium species. The number in each parenthesis indicates the total number of non-redundant genes in the genome of each organism used in this analysis. The number in each cell shows the percentage of genes present in the genome shown on the left that is also present in the corresponding genome shown at the top of the table. Red boxes show the P. insidiosum strains assigned to rDNA-based genotype clades I, II, and III. Green boxes demonstrate the percent gene presence within the same clade. Color gradience indicates the degree of gene presence (%). Full article ">Figure 5
Core and variable genes among 10 P. insidiosum strains and 5 other Pythium species. Green boxes represent core genes (i.e., Cores 1, 2, 3, 4, and 5) present in all genomes at each level. Light blues show variable genes (i.e., Variables 1, 2, 3, 4, and 5), which present at least one but not all of the genomes at each level. Variable genes can be classified into species-specific, clade-specific, strain-specific, and unspecific groups. Full article ">Figure 6
Functional classification of Core 1 and Variable 1 genes, derived from the pangenome analysis of 10 P. insidiosum strains and 5 other Pythium species, based on clusters of orthologous groups (COG) at superfunctional (A) and functional (B) levels. Full article ">

24 pages, 7490 KiB

Open AccessArticle

Description and Genome Characterization of Three Novel Fungal Strains Isolated from Mars 2020 Mission-Associated Spacecraft Assembly Facility Surfaces—Recommendations for Two New Genera and One Species

by Atul Munish Chander, Marcus de Melo Teixeira, Nitin K. Singh, Michael P. Williams, Anna C. Simpson, Namita Damle, Ceth W. Parker, Jason E. Stajich, Christopher E. Mason, Tamas Torok and Kasthuri Venkateswaran

J. Fungi 2023, 9(1), 31; https://doi.org/10.3390/jof9010031 - 23 Dec 2022

Cited by 5 | Viewed by 3428

Abstract

National Aeronautics and Space Administration’s (NASA) spacecraft assembly facilities are monitored for the presence of any bacteria or fungi that might conceivably survive a transfer to an extraterrestrial environment. Fungi present a broad and diverse range of phenotypic and functional traits to adapt [...] Read more.

National Aeronautics and Space Administration’s (NASA) spacecraft assembly facilities are monitored for the presence of any bacteria or fungi that might conceivably survive a transfer to an extraterrestrial environment. Fungi present a broad and diverse range of phenotypic and functional traits to adapt to extreme conditions, hence the detection of fungi and subsequent eradication of them are needed to prevent forward contamination for future NASA missions. During the construction and assembly for the Mars 2020 mission, three fungal strains with unique morphological and phylogenetic properties were isolated from spacecraft assembly facilities. The reconstruction of phylogenetic trees based on several gene loci (ITS, LSU, SSU, RPB, TUB, TEF1) using multi-locus sequence typing (MLST) and whole genome sequencing (WGS) analyses supported the hypothesis that these were novel species. Here we report the genus or species-level classification of these three novel strains via a polyphasic approach using phylogenetic analysis, colony and cell morphology, and comparative analysis of WGS. The strain FJI-L9-BK-P1 isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) exhibited a putative phylogenetic relationship with the strain Aaosphaeria arxii CBS175.79 but showed distinct morphology and microscopic features. Another JPL-SAF strain, FJII-L3-CM-DR1, was phylogenetically distinct from members of the family Trichomeriaceae and exhibited morphologically different features from the genera Lithohypha and Strelitziana. The strain FKI-L1-BK-DR1 isolated from the Kennedy Space Center facility was identified as a member of Dothideomycetes incertae sedis and is closely related to the family Kirschsteiniotheliaceae according to a phylogenetic analysis. The polyphasic taxonomic approach supported the recommendation for establishing two novel genera and one novel species. The names Aaosphaeria pasadenensis (FJI-L9-BK-P1 = NRRL 64424 = DSM 114621), Pasadenomyces melaninifex (FJII-L3-CM-DR1 = NRRL 64433 = DSM 114623), and Floridaphiala radiotolerans (FKI-L1-BK-DR1 = NRRL 64434 = DSM 114624) are proposed as type species. Furthermore, resistance to ultraviolet-C and presence of specific biosynthetic gene cluster(s) coding for metabolically active compounds are unique to these strains. Full article

(This article belongs to the Special Issue Genomics Analysis of Fungi)

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20 pages, 5204 KiB

Open AccessArticle

Genomic and Metabolomic Analyses of the Medicinal Fungus Inonotus hispidus for Its Metabolite’s Biosynthesis and Medicinal Application

by Rui-qi Zhang, Xi-long Feng, Zhen-xin Wang, Tian-chen Xie, Yingce Duan, Chengwei Liu, Jin-ming Gao and Jianzhao Qi

J. Fungi 2022, 8(12), 1245; https://doi.org/10.3390/jof8121245 - 25 Nov 2022

Cited by 16 | Viewed by 2864

Abstract

Inonotus hispidus mushroom is a traditional medicinal fungus with anti-cancer, antioxidation, and immunomodulatory activities, and it is used in folk medicine as a treatment for indigestion, cancer, diabetes, and gastric illnesses. Although I. hispidus is recognized as a rare edible medicinal macrofungi, its [...] Read more.

Inonotus hispidus mushroom is a traditional medicinal fungus with anti-cancer, antioxidation, and immunomodulatory activities, and it is used in folk medicine as a treatment for indigestion, cancer, diabetes, and gastric illnesses. Although I. hispidus is recognized as a rare edible medicinal macrofungi, its genomic sequence and biosynthesis potential of secondary metabolites have not been investigated. In this study, using Illumina NovaSeq combined with the PacBio platform, we sequenced and de novo assembled the whole genome of NPCB_001, a wild I. hispidus isolate from the Aksu area of Xinjiang Province, China. Comparative genomic and phylogenomic analyses reveal interspecific differences and evolutionary traits in the genus Inonotus. Bioinformatics analysis identified candidate genes associated with mating type, polysaccharide synthesis, carbohydrate-active enzymes, and secondary metabolite biosynthesis. Additionally, molecular networks of metabolites exhibit differences in chemical composition and content between fruiting bodies and mycelium, as well as association clusters of related compounds. The deciphering of the genome of I. hispidus will deepen the understanding of the biosynthesis of bioactive components, open the path for future biosynthesis research, and promote the application of Inonotus in the fields of drug research and functional food manufacturing. Full article

(This article belongs to the Special Issue Genomics Analysis of Fungi)

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Morphologic photograph of the strain I. hispidus NPCB_001. (A) The morphologic photograph of the wild fruiting body, (B) mycelium growing on PDA for four days, (C) mature, cultivated fruiting bodies of the strain I. hispidus NPCB_001. Full article ">Figure 2
Genomic characterization, mating type loci, and comparative genomic analysis. (A) Genomic collinearity analysis between I. hispidus NPCB_001 and I. obliquus CT5. From the outside to the inside are I. Chromosome and Contigs; II&III. Gene density and GC density: the intensity of the color positively correlates with gene density; Ⅳ. Whole-genome collinearity analysis based on protein-coding genes: sequence similarity from low to high is indicated by red to purple. (B) Venn schematic of comparative genomes within Inonotus species. (C) Ks comparison within Inonotus species. (D) Structural diagram of the genes on the matA locus and matB locus of I. obliquus. Full article ">Figure 3
The evolutionary relationship and expanded and contracted gene families among Inonotus species and 45 representative Basidiomycetes. The maximum likelihood method credibility tree was inferred from 47 single-copy orthologous genes. All nodes received full bootstrap support. The divergence time is labeled as the mean crown age for each node, while the 95% highest posterior density is also given within the Inonotus clade. The black numbers at the branches indicate the corresponding divergence times in millions of years (MYA). The numbers of gene family expansion and contraction in each species are labeled with green and red symbols, respectively. The proportion of expansion and contraction in the genome of each species was displayed before its species name. The background color of each species of Agaricus indicates its corresponding order. Full article ">Figure 4
CAZymes analysis of I. hispidus and related white-rot fungi. (A) The sizes and colors (from green through purple to red) of circles in the bubble plot indicate the change in quantity, and the different colored triangles indicate different families. (B) The predicted structures of four bifunctional domain-containing CAZymes. Full article ">Figure 5
Analysis of genes involved in secondary metabolite biosynthesis. (A) Distribution of biosynthetic core genes for natural products on the chromosomes and contigs. Phylogenetic tree analysis sesquiterpene synthases (B), NRPS-likes (D), and PKSs (E) from NPCB _001 and their respective homologues. (C) The identity matrix of eight STSs. Full article ">Figure 6
Maximum likelihood method tree of 127 P450s from the strain NPCB_001. Each P450 family is shown in a separate color, and the branch reliability value of not less than 50 is marked on the corresponding branch node. Full article ">Figure 7
GNPS-based molecular network identification of metabolites from fruiting body and liquid culture of mycelium of Inonotus hispidus. Full article ">

18 pages, 32941 KiB

Open AccessArticle

Phylogenomic and Evolutionary Analyses Reveal Diversifications of SET-Domain Proteins in Fungi

by Guoqing Ding, Liqiu Shang, Wenliang Zhou, Siyi Lu, Zong Zhou, Xinyi Huang and Juan Li

J. Fungi 2022, 8(11), 1159; https://doi.org/10.3390/jof8111159 - 2 Nov 2022

Viewed by 1697

Abstract

In recent years, many publications have established histone lysine methylation as a central epigenetic modification in the regulation of chromatin and transcription. The histone lysine methyltransferases contain a conserved SET domain and are widely distributed in various organisms. However, a comprehensive study on [...] Read more.

In recent years, many publications have established histone lysine methylation as a central epigenetic modification in the regulation of chromatin and transcription. The histone lysine methyltransferases contain a conserved SET domain and are widely distributed in various organisms. However, a comprehensive study on the origin and diversification of the SET-domain-containing genes in fungi has not been conducted. In this study, a total of 3816 SET-domain-containing genes, which were identified and characterized using HmmSearch from 229 whole genomes sequenced fungal species, were used to ascertain their evolution and diversification in fungi. Using the CLANS program, all the SET-domain-containing genes were grouped into three main clusters, and each cluster contains several groups. Domain organization analysis showed that genes belonging to the same group have similar sequence structures. In contrast, different groups process domain organizations or locations differently, suggesting the SET-domain-containing genes belonging to different groups may have obtained distinctive regulatory mechanisms during their evolution. These genes that conduct the histone methylations (such as H3K4me, H3K9me, H3K27me, H4K20me, H3K36me) are mainly grouped into Cluster 1 while the other genes grouped into Clusters 2 and 3 are still functionally undetermined. Our results also showed that numerous gene duplication and loss events have happened during the evolution of those fungal SET-domain-containing proteins. Our results provide novel insights into the roles of SET-domain genes in fungal evolution and pave a fundamental path to further understanding the epigenetic basis of gene regulation in fungi. Full article

(This article belongs to the Special Issue Genomics Analysis of Fungi)

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16 pages, 3739 KiB

Open AccessArticle

Genomic Characteristics and Comparative Genomics Analysis of Parafenestella ontariensis sp. nov.

by Evgeny Ilyukhin, Svetlana Markovskaja, Abdallah M. Elgorban, Salim S. Al-Rejaie and Sajeewa S.N. Maharachchikumbura

J. Fungi 2022, 8(7), 732; https://doi.org/10.3390/jof8070732 - 14 Jul 2022

Cited by 4 | Viewed by 2786

Abstract

A new ascomycetous species of Parafenestella was isolated from Acer negundo during the survey of diseased trees in Southern Ontario, Canada. The species is morphologically similar to other taxa of Cucurbitariacea (Pleosporales). The new species is different from the extant species [...] Read more.

A new ascomycetous species of Parafenestella was isolated from Acer negundo during the survey of diseased trees in Southern Ontario, Canada. The species is morphologically similar to other taxa of Cucurbitariacea (Pleosporales). The new species is different from the extant species in the morphology of ascospores, culture characteristics and molecular data. The novel species is described as Parafenestella ontariensis sp. nov. based on morphological and multi-gene phylogenetic analyses using a combined set of ITS, LSU, tef1 and tub2 loci. Additionally, the genome of P. ontariensis was sequenced and analyzed. The phylogenomic analysis confirmed the close relationship of the species to the fenestelloid clades of Cucurbitariaceae. The comparative genomics analysis revealed that the species lifestyle appears to be multitrophic (necrotrophic or hemi-biotrophic) with a capability to turn pathogenic on a corresponding plant host. Full article

(This article belongs to the Special Issue Genomics Analysis of Fungi)

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(A) Ascomata of P. ontariensis on Acer negundo. (B) Ascus. (C–E) Ascospores. (F) Peridium. (G) Fourteen-day-old culture on MEA. Scale bars: A = 200 μm, B = 20 μm, C–E = 10 μm, F = 5 μm. Full article ">Figure 2
Phylogram of RAxML tree generated based on the analysis of combined ITS, LSU, tef1 and tub2 sequence data. Bootstrap support values for ML and MP ≥ 50% and BP ≥ 0.90 are defined as ML/MP/BP above and below the nodes. Strain of the new species is in bold. The tree is rooted to Pyrenochaeta nobilis (CBS 407.76). Full article ">Figure 3
(A) ML tree generated from the concatenated alignment of 3 145 SCOs. (B) Dot-plot graph showing syntenic blocks between scaffold sequences of F. fenestrata and P. ontariensis. (C) Graph showing the KOG gene annotations of C. berberidis (inner), F. fenestrata (middle) and P. ontariensis (outer layer). Full article ">Figure 4
(A) Overall comparison of SM backbone genes in genomes of Cucurbitariaceae species and 11 other fungi with different lifestyles. (B) Heatmap graph showing SM clusters in Cucurbitariaceae and other fungal species with different lifestyles. Full article ">Figure 5
(A) Overall comparison of CAZY complements in genomes of Cucurbitariaceae species and fungi with different lifestyles. (B) Heatmap graph showing CAZY expansion in Cucurbitariaceae and 11 other fungal species related to different lifestyles. Full article ">Figure 6
Distribution of selected plant cell wall degrading enzymes among Cucurbitariaceae species and other fungi with different lifestyles. Full article ">

20 pages, 3807 KiB

Open AccessArticle

Comparative Genomics of Three Aspergillus Strains Reveals Insights into Endophytic Lifestyle and Endophyte-Induced Plant Growth Promotion

by Minyu Jing, Xihui Xu, Jing Peng, Can Li, Hanchao Zhang, Chunlan Lian, Yahua Chen, Zhenguo Shen and Chen Chen

J. Fungi 2022, 8(7), 690; https://doi.org/10.3390/jof8070690 - 29 Jun 2022

Cited by 9 | Viewed by 2489

Abstract

Aspergillus includes both plant pathogenic and beneficial fungi. Although endophytes beneficial to plants have high potential for plant growth promotion and improving stress tolerance, studies on endophytic lifestyles and endophyte-plant interactions are still limited. Here, three endophytes belonging to Aspergillus, AS31, AS33, [...] Read more.

Aspergillus includes both plant pathogenic and beneficial fungi. Although endophytes beneficial to plants have high potential for plant growth promotion and improving stress tolerance, studies on endophytic lifestyles and endophyte-plant interactions are still limited. Here, three endophytes belonging to Aspergillus, AS31, AS33, and AS42, were isolated. They could successfully colonize rice roots and significantly improved rice growth. The genomes of strains AS31, AS33, and AS42 were sequenced and compared with other Aspergillus species covering both pathogens and endophytes. The genomes of AS31, AS33, and AS42 were 36.8, 34.8, and 35.3 Mb, respectively. The endophytic genomes had more genes encoding carbohydrate-active enzymes (CAZymes) and small secreted proteins (SSPs) and secondary metabolism gene clusters involved in indole metabolism than the pathogens. In addition, these endophytes were able to improve Pi (phosphorus) accumulation and transport in rice by inducing the expression of Pi transport genes in rice. Specifically, inoculation with endophytes significantly increased Pi contents in roots at the early stage, while the Pi contents in inoculated shoots were significantly increased at the late stage. Our results not only provide important insights into endophyte-plant interactions but also provide strain and genome resources, paving the way for the agricultural application of Aspergillus endophytes. Full article

(This article belongs to the Special Issue Genomics Analysis of Fungi)

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Identification of strains AS31, AS33, and AS42. (A) Colony morphology of the three strains isolated from the roots of healthy Phytolacca americana L. (B) Phylogenetic relationships between the three strains and related species showing the position of strains AS31, AS33, and AS42 within the genus Aspergillus. The maximum likelihood (ML) phylogenetic tree is shown. The ML bootstrap values based on 1000 replications are indicated above the branches. Asterisks indicate the strains isolated in this study. Bar, 0.05 substitutions per nucleotide position. Full article ">Figure 2
Colonization of strains AS31, AS33, and AS42 in rice roots and shoots after 7 and 14 days of inoculation. (A) Magenta staining was used to monitor the inoculation sites on rice roots and shoots. Bars = 100 µm. The three fungi successfully colonized rice roots and produced a large number of hyphae, but these fungi did not colonize rice leaves. (B) Isolation of strains AS31, AS33, and AS42 from the inoculated rice roots and shoots. (C,D) Fungal colonization index at 7 (C) days and 14 (D) days after inoculation. Data represent the means ± SD of three biological replicates per treatment. Different letters indicate significant differences according to Duncan’s multiple range test. Values of p < 0.05 are considered significant. Full article ">Figure 3
Differential rice responses to inoculation with strains AS31, AS33, or AS42 at the phenotypic level. (A,B) Promotion of rice growth by the three strains at 7 (A) and 14 (B) days after inoculation. (C–G) The shoot height (C), fresh weight (D), dry weight (E), root fresh weight (F), and the content of chlorophyll (G) of rice inoculated with strains AS31, AS33, or AS42 for 7 days. (H–L) The shoot height (H), fresh weight (I), dry weight (J), root fresh weight (K), and the content of chlorophyll (L) of rice inoculated with strains AS31, AS33, or AS42 for 14 days. Data represent the means ± SD of three biological replicates per treatment. Different letters indicate significant differences according to Duncan’s multiple range test. Values of p < 0.05 are considered significant. Full article ">Figure 4
Promotions of rice growth by inoculation of strains AS31, AS33, or AS42 in soils. Differential rice responses to inoculation with strains AS31, AS33, or AS42 in the pot experiments are shown. (A) Promotion of rice growth by the three strains at 60 days after inoculation. (B–G) The shoot height (B), fresh weight (D), dry weight (F), and root length (C), root fresh weight (E), root dry weight (G) of rice inoculated with strains AS31, AS33, or AS42 for 60 days in the pot experiments. Data represent the means ± SD of six biological replicates per treatment. Different letters indicate significant differences according to Duncan’s multiple range test. Values of p < 0.05 are considered significant. Full article ">Figure 5
Comparative analysis of CAZyme and secretome encoded in the genomes of AS31, AS33, AS42, and related species. (A) Maximum likelihood (ML) phylogenetic tree showing the evolutionary relationships of six beneficial (green) species, three pathogenic (orange) species, and one other (blue) species. The names in brackets refer to the abbreviation of each species. (B) Numbers of CAZymes and SSPs encoded in the 10 genomes according to A. CBM, carbohydrate-binding module; CE, carbohydrate esterase; GH, glycoside hydrolase; GT, glycosyl transferase; PL, polysaccharide lyases; AA, auxiliary activity; TOTAL, total number of CAZymes; SSPs, small secreted proteins. (C) Principal coordinates analysis (PCoA) of CAZyme and secretome based on gene numbers. The abbreviation of each species is the same as A. Full article ">Figure 6
Numbers of genes related to CAZyme from the genomes of AS31, AS33, and AS42. (A) numbers of genes dominated in categories of glycoside hydrolase (GH); (B) numbers of genes dominated in categories of polysaccharide lyases (PL). (C) numbers of genes dominated in categories of glycosyl transferase (GT); (D) numbers of genes dominated in categories of carbohydrate esterase (CE); (E) numbers of genes dominated in categories of carbohydrate-binding module (CBM); (F) numbers of genes dominated in categories of auxiliary activity (AA). Full article ">Figure 7
Pi accumulation and expression of Pi transport-related genes in rice in response to the inoculation of strains AS31, AS33, or AS42 after 7 and 14 days. (A,B) Pi concentrations in shoots (A) and roots (B) of rice inoculated with each strain after 7 days; (C,D) Pi concentrations of shoots (C) and roots (D) of rice inoculated with each strain after 14 days. Different letters indicate significant differences according to Duncan’s multiple range test. Values of p < 0.05 are considered significant. (E) heat map showing the expression (log2-fold change of inoculated vs. uninoculated samples) of Pi transport-related genes in both the roots and shoots of rice at 7 and 14 days after inoculation with strains AS31, AS33, or AS42. The gene expression was obtained by quantitative real-time PCR (qRT-PCR), and the primers used in the qRT-PCR are listed in <a href="#app1-jof-08-00690" class="html-app">Table S1</a>. Red, increase in gene expressions; blue, decrease in gene expressions. Asterisks indicate significant differences (p < 0.05). Full article ">

16 pages, 3336 KiB

Open AccessArticle

The Lysine Demethylases KdmA and KdmB Differently Regulate Asexual Development, Stress Response, and Virulence in Aspergillus fumigatus

by Yong-Ho Choi, Min-Woo Lee and Kwang-Soo Shin

J. Fungi 2022, 8(6), 590; https://doi.org/10.3390/jof8060590 - 31 May 2022

Cited by 3 | Viewed by 2203

Abstract

Histone demethylases govern diverse cellular processes, including growth, development, and secondary metabolism. In the present study, we investigated the functions of two lysine demethylases, KdmA and KdmB, in the opportunistic human pathogenic fungus Aspergillus fumigatus. Experiments with mutants harboring deletions of genes [...] Read more.

Histone demethylases govern diverse cellular processes, including growth, development, and secondary metabolism. In the present study, we investigated the functions of two lysine demethylases, KdmA and KdmB, in the opportunistic human pathogenic fungus Aspergillus fumigatus. Experiments with mutants harboring deletions of genes encoding KdmA (ΔkdmA) and KdmB (ΔkdmB) showed that KdmA is necessary for normal growth and proper conidiation, whereas KdmB negatively regulates vegetative growth and conidiation. In both mutant strains, tolerance to H₂O₂ was significantly decreased, and the activities of both conidia-specific catalase (CatA) and mycelia-specific catalase (Cat1) were decreased. Both mutants had significantly increased sensitivity to the guanine nucleotide synthesis inhibitor 6-azauracil (6AU). The ΔkdmA mutant produced more gliotoxin (GT), but the virulence was not changed significantly in immunocompromised mice. In contrast, the production of GT and virulence were markedly reduced by the loss of kdmB. Comparative transcriptomic analyses revealed that the expression levels of developmental process-related genes and antioxidant activity-related genes were downregulated in both mutants. Taken together, we concluded that KdmA and KdmB have opposite roles in vegetative growth, asexual sporulation, and GT production. However, the two proteins were equally important for the development of resistance to 6AU. Full article

(This article belongs to the Special Issue Genomics Analysis of Fungi)

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Molecular structures of KdmA and KdmB. (A) Schematic presentation of the domain structures of the KdmA and KdmB proteins using SMART (<a href="http://smart.embl-heidelberg.de" target="_blank">http://smart.embl-heidelberg.de</a>, accessed on 4 April 2021). (B) Multiple sequence alignment of the JmjC domains of demethylases from A. nidulans (AN), A. fumigatus (Afu), A. niger (An), A. flavus (AFL), and A. oryzae (AO). Conserved residues responsible for α-ketoglutarate and Fe (II) binding sites are marked in green and blue, respectively. (C) A phylogenetic tree of demethylase-like proteins in various Aspergillus species constructed based on the matrix of neighbor-joining distances between the JmjC domain sequences. (D) Western blot with antibody specific to histone H3, H3K4me3, and H3K36me3 antibodies. Relative intensities are shown below. Statistical significance of differences was assessed by Student’s t-test: ** p < 0.01. Full article ">Figure 2
KdmA and KdmB regulate normal growth and asexual development in different ways. (A) Colony photographs and growth of WT, ΔkdmA, and ΔkdmB strains point-inoculated on various solid media and grown in solid glucose minimal medium with 0.1% yeast extract (MMY), 10 g/L yeast extract + 30 g/L glucose (YG), or potato dextrose agar (PDA) for 3 days and determined colony diameter. (B) Conidia numbers produced by each strain per plate. (C) Transcript levels of the key asexual developmental regulators in the mutants relative to the corresponding level in the WT strain at 3 days determined by quantitative RT-PCR (RT-qPCR). Dot line indicates the level of WT transcript. Fungal cultures were grown in MMY, and mRNA levels were normalized to the expression level of the ef1α gene. Statistical significance of differences was assessed by Student’s t-test: * p < 0.05, ** p < 0.01. Full article ">Figure 3
Distinct responses of ΔkdmA and ΔkdmB mutants to various kinds of oxidative stress. (A) Heatmap of altered expression levels of genes encoding oxidative stress-related proteins in ΔkdmA and ΔkdmB mutants. (B) Colony appearance and radial growth inhibition after inoculation of 1 × 105 conidia on solid YG medium containing oxidative stressors. (C) Catalase activity of the WT and mutant strains. (D) SOD activity of the WT and mutant strains shown in non-denaturing polyacrylamide gels. Relative intensities of each enzyme’s activity are shown below. Statistical significance of differences between WT and mutant strains was evaluated using Student’s t-test: ** p < 0.01. Full article ">Figure 4
Increased sensitivity of the ΔkdmA and ΔkdmB mutants to 6AU. (A) Radial growth of the WT and mutant strains in the presence of chromatin-targeting inhibitors. (B) RT-qPCR analysis of expression levels of the 6AU resistance-related genes imd2 and sdt2 in the WT and mutant strains in the presence of 100 μg/mL of AU. Dot line indicates the level of WT transcript. Statistical significance of differences between WT and mutant strains was evaluated using Student’s t-test: ** p < 0.01. Full article ">Figure 5
Roles of KdmA and KdmB in the production of gliotoxin (GT) and other putative secondary metabolites. (A) Heatmap of altered expression levels of genes encoding secondary metabolite biosynthesis in mutant strains. (B) Determination of GT production and several secondary metabolites in the WT and mutant strains. Standard GT concentration was 10 μg (left side) and 5 μg (right side). Left: a representative thin-layer chromatogram of the culture supernatant of each strain extracted with chloroform. Right: a graph of relative intensities of individual chromatogram bands in culture supernatants from different strains. (C) RT-qPCR analysis of changes the laeA and gliZ gene expression levels in mutant strains compared to that in the WT strain. Dot line indicates the level of WT transcript. Statistical significance of differences between WT and mutant strains was assessed by Student’s t-test: * p < 0.05, ** p < 0.01. Full article ">Figure 6
Effects of KdmA and KdmB on A. fumigatus virulence. (A) Survival curves of mice intranasally administered with PBS or with conidia of the WT or one of the mutant strains (n = 10/group). (B) Representative lung sections of mice from different experimental groups stained with hematoxylin and eosin (H&E) or periodic acid–Schiff reagent (PAS). Arrows indicate fungal mycelium. Scale bar = 50 µm. (C) Fungal burden in the lungs of mice infected with the WT or one of the mutant strains. Statistical significance of differences between WT and mutant strains was evaluated by Student’s t-test: ** p < 0.01. Full article ">Figure 7
Genome-wide expression analyses of the ΔkdmA and ΔkdmB. (A) Volcano plot showing the fold change (x-axis) and p-value (y-axis) of genes sequenced in ΔkdmA (upper) and ΔkdmB (lower). Red and green dots denote up- and downregulated genes, respectively. (B) Functional annotation histograms of DEGs in ΔkdmA (upper) and ΔkdmB (lower). The red bars represent genes whose mRNA levels increased in the mutant, whereas the green bars represent those genes whose mRNA levels decreased in the mutant strain. Full article ">

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