Characterization of the Neurospora crassa DHN melanin biosynthetic pathway in developing ascospores and peridium cells

Ao, Jie; Bandyopadhyay, Sumit; Free, Stephen J.

doi:10.1016/j.funbio.2018.10.005

Cited by 21 publications

(27 citation statements)

References 28 publications

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“…Nonetheless, these two possibilities remain hypothetical and require further experimental proof. While involvement of non-reducing PKSs in cell wall formation have been reported [6][7][8][9][10]31 . Our study is the first to report a role for a reducing PKS in cell wall formation.…”

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…We performed PEG-mediated protoplast transformation as previously described 8 . For complementation of Δpks15, pks15 mutant was transformed with pCas9sgBar and/or the donor DNA pCR-PKS15-1 k. For each transformation, a conidial suspension, as harvested from a 7-day old PDA culture, was used as inoculum of 50 ml PDB and shaken at 150 rpm, 25 °C for 16 h. Young mycelia were harvested by centrifugation at 3,000 × g. To generate protoplasts, the mycelia were incubated with the wall-lysing enzyme VinoTaste® Pro (Novozymes, Denmark) at a final concentration of 35 mg ml −1 in 1.2 M MgSO 4 , 10 mM sodium phosphate buffer, pH 5.8.…”

Section: Peg-mediated Protoplast Transformation Of ∆Pks15 Mutantmentioning

confidence: 99%

“…Unfortunately, little is known regarding the relationship between polyketides and the fungal wall. In a few reports, melanin, the metabolite of a non-reducing PKS and other enzymes in melanin biosynthetic pathway, has been found in the cell walls of Aspergillus fumigatus 6 , Colletotrichum lagenarium 7 , Neurospora crassa 8 and Pestalotiopsis microspora 9 . The P. microsopora PKS1 is also important for wall integrity and conidial germination in this endophytic fungus 9 .…”

Section: Introductionmentioning

confidence: 99%

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The polyketide synthase PKS15 has a crucial role in cell wall formation in Beauveria bassiana

Udompaisarn

Toopaang

Sae-Ueng

et al. 2020

Sci Rep

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Entomopathogenic fungi utilize specific secondary metabolites to defend against insect immunity, thereby enabling colonization of their specific hosts. We are particularly interested in the polyketide synthesis gene pks15, which is involved in metabolite production, and its role in fungal virulence. targeted disruption of pks15 followed by genetic complementation with a functional copy of the gene would allow for functional characterization of this secondary metabolite biosynthesis gene. Using a Beauveria bassiana ∆pks15 mutant previously disrupted by a bialophos-resistance (bar) cassette, we report here an in-cis complementation at bar cassette using CRISPR/Cas9 gene editing. A barspecific short guide RNA was used to target and cause a double-strand break in bar, and a donor DnA carrying a wild-type copy of pks15 was co-transformed with the guide RNA. Isolate G6 of ∆pks15 complemented with pks15 was obtained and verified by PCR, Southern analyses and DNA sequencing. Compared to ∆pks15 which showed a marked reduction in sporulation and insect virulence, the complementation in G6 restored with insect virulence, sporulation and conidial germination to wildtype levels. Atomic force and scanning electron microscopy revealed that G6 and wild-type conidial wall surfaces possessed the characteristic rodlet bundles and rough surface while ∆pks15 walls lacked the bundles and were relatively smoother. Conidia of ∆pks15 were larger and more elongated than that of G6 and the wild type, indicating changes in their cell wall organization. Our data indicate that PKS15 and its metabolite are likely not only important for fungal virulence and asexual reproduction, but also cell wall formation. Beauveria bassiana, an entomopathogenic fungus, has a broad host spectrum and is considered to have high potential for insect biocontrol in agriculture. While B. bassiana can cause mycosis in several insect species 1,2 , insect killing is fairly slow due to several limiting factors, particularly in the field. The fungus is also vulnerable to environmental stress factors such as UV radiation, high temperature and drought 3. A better understanding of the biological and physiological characteristics of this entomopathogen should allow us to improve its virulence and stress tolerance. Secondary metabolites are abundant in entomopathogenic fungi and include polyketides, nonribosomal peptides, terpenes and alkaloids that play important roles in various aspects of the fungal life cycle. B. bassiana BCC2660, a widely used biocontrol fungus in Thailand, has 12 polyketide synthase (PKS) genes in its genome 4. Two PKS genes, pks15 and pks14, have crucial roles in virulence against insects, as previously demonstrated by targeted gene deletion 4,5. The pks15 mutant exhibits loss in phagocytic survival ability, a phenotype likely associated with changes in the cell wall, the outermost layer of fungal conidia. Unfortunately, little is known regarding the relationship between polyketides and the fungal wall. In a few reports, melanin, the metabolite of a n...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Peg-mediated Protoplast Transformation Of ∆Pks15 Mutantmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

The polyketide synthase PKS15 has a crucial role in cell wall formation in Beauveria bassiana

Udompaisarn

Toopaang

Sae-Ueng

et al. 2020

Sci Rep

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“…All of the enzymes involved in the synthesis of DHN are found encoded in the N. crassa genome. Mutants affected in the ability to produce DHN are unable to melanize their ascospores and perithecia, demonstrating that the pathway is responsible for melanizing these structures (Howe and Benson, 1974; Howe, 1976; Johnson, 1977; McCluskey et al, 2011; Ao et al, 2019). The genome has two paralogs for the heptaketide hydrolase and the THN reductase steps in the pathway, and a single gene for the other steps.…”

Section: Melaninmentioning

confidence: 99%

The Genetics and Biochemistry of Cell Wall Structure and Synthesis in Neurospora crassa, a Model Filamentous Fungus

Patel

Free

2019

Front. Microbiol.

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This review discusses the wealth of information available for the N. crassa cell wall. The basic organization and structure of the cell wall is presented and how the wall changes during the N. crassa life cycle is discussed. Over forty cell wall glycoproteins have been identified by proteomic analyses. Genetic and biochemical studies have identified many of the key enzymes needed for cell wall biogenesis, and the roles these enzymes play in cell wall biogenesis are discussed. The review includes a discussion of how the major cell wall components (chitin, β-1,3-glucan, mixed β-1,3-/ β-1,4- glucans, glycoproteins, and melanin) are synthesized and incorporated into the cell wall. We present a four-step model for how cell wall glycoproteins are covalently incorporated into the cell wall. In N. crassa, the covalent incorporation of cell wall glycoproteins into the wall occurs through a glycosidic linkage between lichenin (a mixed β-1,3-/β-1,4- glucan) and a “processed” galactomannan that has been attached to the glycoprotein N-linked oligosaccharides. The first step is the addition of the galactomannan to the N-linked oligosaccharide. Mutants affected in galactomannan formation are unable to incorporate glycoproteins into their cell walls. The second step is carried out by the enzymes from the GH76 family of α-1,6-mannanases, which cleave the galactomannan to generate a processed galactomannan. The model suggests that the third and fourth steps are carried out by members of the GH72 family of glucanosyltransferases. In the third step the glucanosyltransferases cleave lichenin and generate enzyme/substrate intermediates in which the lichenin is covalently attached to the active site of the glucanosyltransferases. In the final step, the glucanosyltransferases attach the lichenin onto the processed galactomannans, which creates new glycosidic bonds and effectively incorporates the glycoproteins into the cross-linked cell wall glucan/chitin matrix.

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Biological control of melanin biosynthesis pathway on prolific and pleochromatic induction of Lasiodiplodia theobromae

Liu

Chen

et al. 2023

Arch Microbiol

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Characterization of the Neurospora crassa DHN melanin biosynthetic pathway in developing ascospores and peridium cells

Cited by 21 publications

References 28 publications

The polyketide synthase PKS15 has a crucial role in cell wall formation in Beauveria bassiana

The polyketide synthase PKS15 has a crucial role in cell wall formation in Beauveria bassiana

The Genetics and Biochemistry of Cell Wall Structure and Synthesis in Neurospora crassa, a Model Filamentous Fungus

Biological control of melanin biosynthesis pathway on prolific and pleochromatic induction of Lasiodiplodia theobromae

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