Genome Editing Tools in Fungi

A special issue of Journal of Fungi (ISSN 2309-608X). This special issue belongs to the section "Fungal Genomics, Genetics and Molecular Biology".

Deadline for manuscript submissions: closed (31 March 2023) | Viewed by 8781

Special Issue Editors


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Guest Editor
Agriculture & Agri-Food Canada, Ontario, Canada
Interests: mycology; metabolomics; integrated ‘omics’; synthetic biology; bioproducts discovery; natural product chemistry

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Guest Editor
Agriculture & Agri-Food Canada, Ontario, Canada
Interests: plant–pathogen interactions; effector biology; regulation of secondary metabolism; chemical genetics
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Guest Editor
Empresa Brasileira de Pesquisa Agropecuária-EMBRAPA Genetic Resources and Biotechnology, Brasilia, Brazil
Interests: microbiology; integrated metabolomics and genomics; synthetic biology; natural polymers; biomaterials

Special Issue Information

Dear Colleagues,

Breakthrough technologies in gene editing and advances in computational biology are enabling scientists from across disciplines, such as biology, bioinformatics, and engineering, to tweak biological systems to reach desired goals. Innovations in CRISPR-Cas9 technology are rapidly advancing our knowledge of gene function at a significant speed and precision. Gene editing by the CRISPR-Cas9 system has been established in more than 40 different species of filamentous fungi (including Aspergillus, Fusarium, and Sclerotinia) and oomycetes. In addition to disrupting genes, this system has also been adapted for gene replacement and for precision editing (nucleotide changes) in fungi.

This Special Issue will focus on several key aspects and advancements in the field of gene editing in fungi. How gene editing is advancing our knowledge in inter- and intra-species interations, microbiome, microbial evolution, metabolism, and bioproduct discovery, etc., is within the scope of this Special Issue. The use of gene editing in various synthetic biology platforms will also be considered. In addition to primary research articles, we are also seeking articles that offer new perspectives in the use of this technology to further our knowledge in basic sciences and its use in synthetic biology platforms, gene and meiotic drives, etc.

Dr. David P. Overy
Dr. Rajagopal Subramaniam
Dr. Daniela Matias de Carvalho Bittencourt
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Journal of Fungi is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • gene editing
  • CRISPR
  • functional genetics
  • synthetic biology
  • integrated ‘omics’

Published Papers (5 papers)

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Research

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14 pages, 2172 KiB  
Article
Epistatic Relationship between MGV1 and TRI6 in the Regulation of Biosynthetic Gene Clusters in Fusarium graminearum
by Kristina Shostak, Dianevys González-Peña Fundora, Christopher Blackman, Tom Witte, Amanda Sproule, David Overy, Anas Eranthodi, Nehal Thakor, Nora A. Foroud and Rajagopal Subramaniam
J. Fungi 2023, 9(8), 816; https://doi.org/10.3390/jof9080816 - 02 Aug 2023
Cited by 1 | Viewed by 842
Abstract
Genetic studies have shown that the MAP kinase MGV1 and the transcriptional regulator TRI6 regulate many of the same biosynthetic gene clusters (BGCs) in Fusarium graminearum. This study sought to investigate the relationship between MGV1 and TRI6 in the regulatory hierarchy. Transgenic [...] Read more.
Genetic studies have shown that the MAP kinase MGV1 and the transcriptional regulator TRI6 regulate many of the same biosynthetic gene clusters (BGCs) in Fusarium graminearum. This study sought to investigate the relationship between MGV1 and TRI6 in the regulatory hierarchy. Transgenic F. graminearum strains constitutively expressing MGV1 and TRI6 were generated to address both independent and epistatic regulation of BGCs by MGV1 and TRI6. We performed a comparative transcriptome analysis between axenic cultures grown in nutrient-rich and secondary metabolite-inducing conditions. The results indicated that BGCs regulated independently by Mgv1 included genes of BGC52, whereas genes uniquely regulated by TRI6 included the gene cluster (BGC49) that produces gramillin. To understand the epistatic relationship between MGV1 and TRI6, CRISPR/Cas9 was used to insert a constitutive promoter to drive TRI6 expression in the Δmgv1 strain. The results indicate that BGCs that produce deoxynivalenol and fusaoctaxin are co-regulated, with TRI6 being partially regulated by MGV1. Overall, the findings from this study indicate that MGV1 provides an articulation point to differentially regulate various BGCs. Moreover, TRI6, embedded in one of the BGCs provides specificity to regulate the expression of the genes in the BGC. Full article
(This article belongs to the Special Issue Genome Editing Tools in Fungi)
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14 pages, 2235 KiB  
Article
Multiple Clonostachys rosea UDP-Glycosyltransferases Contribute to the Production of 15-Acetyl-Deoxynivalenol-3-O-Glycoside When Confronted with Fusarium graminearum
by Kelly A. Robinson, Antony D. St-Jacques, Sam W. Shields, Amanda Sproule, Zerihun A. Demissie, David P. Overy and Michele C. Loewen
J. Fungi 2023, 9(7), 723; https://doi.org/10.3390/jof9070723 - 02 Jul 2023
Viewed by 1367
Abstract
Mycotoxins, derived from toxigenic fungi such as Fusarium, Aspergillus, and Penicillium species have impacted the human food chain for thousands of years. Deoxynivalenol (DON), is a tetracyclic sesquiterpenoid type B trichothecene mycotoxin predominantly produced by F. culmorum and F. graminearum during the [...] Read more.
Mycotoxins, derived from toxigenic fungi such as Fusarium, Aspergillus, and Penicillium species have impacted the human food chain for thousands of years. Deoxynivalenol (DON), is a tetracyclic sesquiterpenoid type B trichothecene mycotoxin predominantly produced by F. culmorum and F. graminearum during the infection of corn, wheat, oats, barley, and rice. Glycosylation of DON is a protective detoxification mechanism employed by plants. More recently, DON glycosylating activity has also been detected in fungal microparasitic (biocontrol) fungal organisms. Here we follow up on the reported conversion of 15-acetyl-DON (15-ADON) into 15-ADON-3-O-glycoside (15-ADON-3G) in Clonostachys rosea. Based on the hypothesis that the reaction is likely being carried out by a uridine diphosphate glycosyl transferase (UDP-GTase), we applied a protein structural comparison strategy, leveraging the availability of the crystal structure of rice Os70 to identify a subset of potential C. rosea UDP-GTases that might have activity against 15-ADON. Using CRISPR/Cas9 technology, we knocked out several of the selected UDP-GTases in the C. rosea strain ACM941. Evaluation of the impact of knockouts on the production of 15-ADON-3G in confrontation assays with F. graminearum revealed multiple UDP-GTase enzymes, each contributing partial activities. The relationship between these positive hits and other UDP-GTases in fungal and plant species is discussed. Full article
(This article belongs to the Special Issue Genome Editing Tools in Fungi)
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20 pages, 3892 KiB  
Article
CRISPR-Cas9 Gene Editing and Secondary Metabolite Screening Confirm Fusarium graminearum C16 Biosynthetic Gene Cluster Products as Decalin-Containing Diterpenoid Pyrones
by Carmen Hicks, Thomas E. Witte, Amanda Sproule, Anne Hermans, Samuel W. Shields, Ronan Colquhoun, Chris Blackman, Christopher N. Boddy, Rajagopal Subramaniam and David P. Overy
J. Fungi 2023, 9(7), 695; https://doi.org/10.3390/jof9070695 - 23 Jun 2023
Viewed by 1544
Abstract
Fusarium graminearum is a causal organism of Fusarium head blight in cereals and maize. Although a few secondary metabolites produced by F. graminearum are considered disease virulence factors, many molecular products of biosynthetic gene clusters expressed by F. graminearum during infection and their [...] Read more.
Fusarium graminearum is a causal organism of Fusarium head blight in cereals and maize. Although a few secondary metabolites produced by F. graminearum are considered disease virulence factors, many molecular products of biosynthetic gene clusters expressed by F. graminearum during infection and their associated role in the disease are unknown. In particular, the predicted meroterpenoid products of the biosynthetic gene cluster historically designated as “C16” are likely associated with pathogenicity. Presented here are the results of CRISPR-Cas9 gene-editing experiments disrupting the polyketide synthase and terpene synthase genes associated with the C16 biosynthetic gene cluster in F. graminearum. Culture medium screening experiments using transformant strains were profiled by UHPLC-HRMS and targeted MS2 experiments to confirm the associated secondary metabolite products of the C16 biosynthetic gene cluster as the decalin-containing diterpenoid pyrones, FDDP-D and FDDP-E. Both decalin-containing diterpenoid pyrones were confirmed to be produced in wheat heads challenged with F. graminearum in growth chamber trials. The extent to which the F. graminearum C16 biosynthetic gene cluster is dispersed within the genus Fusarium is discussed along with a proposed role of the FDDPs as pathogen virulence factors. Full article
(This article belongs to the Special Issue Genome Editing Tools in Fungi)
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14 pages, 3589 KiB  
Article
Unanticipated Large-Scale Deletion in Fusarium graminearum Genome Using CRISPR/Cas9 and Its Impact on Growth and Virulence
by Adam John Foster, Emily Johnstone, Abbey Saunders, Eva Colic, Nicole Lassel and Janesse Holmes
J. Fungi 2023, 9(6), 673; https://doi.org/10.3390/jof9060673 - 14 Jun 2023
Viewed by 1638
Abstract
Fusarium graminearum, a filamentous fungus, and causal agent of Fusarium head blight (FHB) in wheat and other cereals, leads to significant economic losses globally. This study aimed to investigate the roles of specific genes in F. graminearum virulence using CRISPR/Cas9-mediated gene deletions. [...] Read more.
Fusarium graminearum, a filamentous fungus, and causal agent of Fusarium head blight (FHB) in wheat and other cereals, leads to significant economic losses globally. This study aimed to investigate the roles of specific genes in F. graminearum virulence using CRISPR/Cas9-mediated gene deletions. Illumina sequencing was used to characterize the genomic changes due to editing. Unexpectedly, a large-scale deletion of 525,223 base pairs on chromosome 2, comprising over 222 genes, occurred in two isolates. Many of the deleted genes were predicted to be involved in essential molecular functions, such as oxidoreductase activity, transmembrane transporter activity, hydrolase activity, as well as biological processes, such as carbohydrate metabolism and transmembrane transport. Despite the substantial loss of genetic material, the mutant isolate exhibited normal growth rates and virulence on wheat under most conditions. However, growth rates were significantly reduced under high temperatures and on some media. Additionally, wheat inoculation assays using clip dipping, seed inoculation, and head point inoculation methods were performed. No significant differences in virulence were observed, suggesting that these genes were not involved in infection or alternative compensatory pathways, and allow the fungi to maintain pathogenicity despite the extensive genomic deletion. Full article
(This article belongs to the Special Issue Genome Editing Tools in Fungi)
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Review

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18 pages, 1123 KiB  
Review
Advances and Challenges in CRISPR/Cas-Based Fungal Genome Engineering for Secondary Metabolite Production: A Review
by Duoduo Wang, Shunda Jin, Qianhui Lu and Yupeng Chen
J. Fungi 2023, 9(3), 362; https://doi.org/10.3390/jof9030362 - 15 Mar 2023
Cited by 8 | Viewed by 2780
Abstract
Fungi represent an important source of bioactive secondary metabolites (SMs), which have wide applications in many fields, including medicine, agriculture, human health, and many other industries. The genes involved in SM biosynthesis are usually clustered adjacent to each other into a region known [...] Read more.
Fungi represent an important source of bioactive secondary metabolites (SMs), which have wide applications in many fields, including medicine, agriculture, human health, and many other industries. The genes involved in SM biosynthesis are usually clustered adjacent to each other into a region known as a biosynthetic gene cluster (BGC). The recent advent of a diversity of genetic and genomic technologies has facilitated the identification of many cryptic or uncharacterized BGCs and their associated SMs. However, there are still many challenges that hamper the broader exploration of industrially important secondary metabolites. The recent advanced CRISPR/Cas system has revolutionized fungal genetic engineering and enabled the discovery of novel bioactive compounds. In this review, we firstly introduce fungal BGCs and their relationships with associated SMs, followed by a brief summary of the conventional strategies for fungal genetic engineering. Next, we introduce a range of state-of-the-art CRISPR/Cas-based tools that have been developed and review recent applications of these methods in fungi for research on the biosynthesis of SMs. Finally, the challenges and limitations of these CRISPR/Cas-based systems are discussed and directions for future research are proposed in order to expand their applications and improve efficiency for fungal genetic engineering. Full article
(This article belongs to the Special Issue Genome Editing Tools in Fungi)
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