Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi

Hwang, In-Sul; Ahn, Il-Pyung

doi:10.5423/ppj.oa.12.2015.0263

Cited by 10 publications

(12 citation statements)

References 33 publications

(40 reference statements)

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“…Pathogenicity was assessed by plant height in the present study. It has been reported that fumonisin producers do not induce the typical symptoms induced by gibberellin in rice, such as stem elongation, but instead cause root rot and stunting (24,34). Gene disruption of Fusarium cyclin C1 (FCC1) or FUM1, which play an important role in fumonisin biosynthesis in the fumonisin-producing strain B14, resulted in a significant reduction in stunting-type symptoms (24,35).…”

Section: Discussionmentioning

confidence: 99%

Genetic Differentiation Associated with Fumonisin and Gibberellin Production in Japanese Fusarium fujikuroi

Suga

Arai

Fukasawa

et al. 2019

Appl Environ Microbiol

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Fusarium fujikuroi is a pathogenic fungus that infects rice. It produces several important mycotoxins, such as fumonisins. Fumonisin production has been detected in strains of maize, strawberry, and wheat, whereas it has not been detected in strains from rice seedlings infested with bakanae disease in Japan. We investigated the genetic relationships, pathogenicity, and resistance to a fungicide, thiophanate-methyl (TM), in 51 fumonisin-producing strains and 44 nonproducing strains. Phylogenetic analyses based on amplified fragment length polymorphism (AFLP) markers and two specific genes (a combined sequence of translation elongation factor 1α [TEF1α] and RNA polymerase II second-largest subunit [RPB2]) indicated differential clustering between the fumonisin-producing and -nonproducing strains. One of the AFLP markers, EATMCAY107, was specifically present in the fumonisin-producing strains. A specific single nucleotide polymorphism (SNP) between the fumonisin-producing and nonproducing strains was also detected in RPB2, in addition to an SNP previously found in TEF1α. Gibberellin production was higher in the nonproducing than in the producing strains according to an in vitro assay, and the nonproducing strains had the strongest pathogenicity with regard to rice seedlings. TM resistance was closely correlated with the cluster of fumonisin-nonproducing strains. The results indicate that intraspecific evolution in Japanese F. fujikuroi is associated with fumonisin production and pathogenicity. Two subgroups of Japanese F. fujikuroi, designated G group and F group, were distinguished based on phylogenetic differences and the high production of gibberellin and fumonisin, respectively. IMPORTANCE Fusarium fujikuroi is a pathogenic fungus that causes rice bakanae disease. Historically, this pathogen has been known as Fusarium moniliforme, along with many other species based on a broad species concept. Gibberellin, which is currently known as a plant hormone, is a virulence factor of F. fujikuroi. Fumonisin is a carcinogenic mycotoxin posing a serious threat to food and feed safety. Although it has been confirmed that F. fujikuroi produces gibberellin and fumonisin, production varies among strains, and individual production has been obscured by the traditional appellation of F. moniliforme, difficulties in species identification, and variation in the assays used to determine the production of these secondary metabolites. In this study, we discovered two phylogenetic subgroups associated with fumonisin and gibberellin production in Japanese F. fujikuroi.

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Section: Discussionmentioning

confidence: 99%

Genetic Differentiation Associated with Fumonisin and Gibberellin Production in Japanese Fusarium fujikuroi

Suga

Arai

Fukasawa

et al. 2019

Appl Environ Microbiol

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“…30 The Fusarium cyclin C1 gene (fcc1) was first chosen as the target gene, because previous research has demonstrated that fcc1-disrupted F. f ujikuroi strains exhibited significantly more red pigment accumulation on PDA compared with the control. 25 In order to verify whether this phenomenon is also present in our strain, we constructed an fcc1-disruption cassette and disrupted the fcc1 gene via homologous recombination (Figure 3). Interestingly, when grown on PDA, the fcc1-disrupted F. fujikuroi accumulated more purple pigment rather than red (Figure 3).…”

Section: Modification Of the Protoplast-based Polyethylenementioning

confidence: 95%

CRISPR/Cas9-Based Genome Editing in the Filamentous Fungus Fusarium fujikuroi and Its Application in Strain Engineering for Gibberellic Acid Production

et al. 2019

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The filamentous fungus Fusarium fujikuroi is well-known for its production of natural plant growth hormones: a series of gibberellic acids (GAs). Some GAs, including GA1, GA3, GA4, and GA7, are biologically active and have been widely applied in agriculture. However, the low efficiency of traditional genetic tools limits the further research toward making this fungus more efficient and able to produce tailor-made GAs. Here, we established an efficient CRISPR/Cas9-based genome editing tool for F. fujikuroi. First, we compared three different nuclear localization signals (NLS) and selected an efficient NLS from histone H2B (HTB NLS ) to enable the import of the Cas9 protein into the fungal nucleus. Then, different sgRNA expression strategies, both in vitro and different promoter-based in vivo strategies, were explored. The promoters of the U6 small nuclear RNA and 5S rRNA, which were identified in F. f ujikuroi, had the highest editing efficiency. The 5S rRNA-promoter-driven genome editing efficiency reached up to 79.2%. What's more, multigene editing was also explored and showed good results. Finally, we used the developed genome editing tool to engineer the metabolic pathways responsible for the accumulation of a series GAs in the filamentous fungus F. f ujikuroi, and successfully changed its GA product profile, from GA3 to tailor-made GA4 and GA7 mixtures. Since these mixtures are more efficient for agricultural use, especially for fruit growth, the developed strains will greatly improve industrial GA production.

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“…This vector was used to disrupt the gene encoding P450-3 , thus yielding the final disruption mutant Δ P450-3 . Subsequently, sgRNA and f FuCas9 vector were introduced into F. fujikuroi through the modified protoplast-based polyethylene glycol and Ca 2+ transformation method ( Hwang and Ahn, 2016 ). Additionally, overexpression of two key genes encoding copalyl diphosphate synthase/kaurene synthase ( Cps/Ks ) and a truncated HMG-CoA reductase ( tHmgR ) were also performed separately and simultaneously to explore the possibility of increasing the amount of GA4/GA7 mixture produced based on previous research ( Albermann et al, 2013 ).…”

Section: Specific Application Of Crispr/cas9 In Secondary Metabolitesmentioning

confidence: 99%

Applications of CRISPR/Cas9 in the Synthesis of Secondary Metabolites in Filamentous Fungi

Jiang

et al. 2021

Front. Microbiol.

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Filamentous fungi possess the capacity to produce a wide array of secondary metabolites with diverse biological activities and structures, such as lovastatin and swainsonine. With the advent of the post-genomic era, increasing amounts of cryptic or uncharacterized secondary metabolite biosynthetic gene clusters are continually being discovered. However, owing to the longstanding lack of versatile, comparatively simple, and highly efficient genetic manipulation techniques, the broader exploration of industrially important secondary metabolites has been hampered thus far. With the emergence of CRISPR/Cas9-based genome editing technology, this dilemma may be alleviated, as this advanced technique has revolutionized genetic research and enabled the exploitation and discovery of new bioactive compounds from filamentous fungi. In this review, we introduce the CRISPR/Cas9 system in detail and summarize the latest applications of CRISPR/Cas9-mediated genome editing in filamentous fungi. We also briefly introduce the specific applications of the CRISPR/Cas9 system and CRISPRa in the improvement of secondary metabolite contents and discovery of novel biologically active compounds in filamentous fungi, with specific examples noted. Additionally, we highlight and discuss some of the challenges and deficiencies of using the CRISPR/Cas9-based genome editing technology in research on the biosynthesis of secondary metabolites as well as future application of CRISPR/Cas9 strategy in filamentous fungi are highlighted and discussed.

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Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi

Cited by 10 publications

References 33 publications

Genetic Differentiation Associated with Fumonisin and Gibberellin Production in Japanese Fusarium fujikuroi

Genetic Differentiation Associated with Fumonisin and Gibberellin Production in Japanese Fusarium fujikuroi

CRISPR/Cas9-Based Genome Editing in the Filamentous Fungus Fusarium fujikuroi and Its Application in Strain Engineering for Gibberellic Acid Production

Applications of CRISPR/Cas9 in the Synthesis of Secondary Metabolites in Filamentous Fungi

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