Establishment of a Genetic Transformation System in Guanophilic Fungus Amphichorda guana

Liang, Min; Li, Wei; Qi, Landa; Chen, Guocan; Cai, Lei; Yin, Wen‐Bing

doi:10.3390/jof7020138

Cited by 10 publications

(8 citation statements)

References 40 publications

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“…Stable osmotic pressure is the critical factor for protoplasts to regenerate cell walls [ 31 ]. In this study, the osmotic pressure stabilizers 1 M sucrose and 1.2 M sorbitol were tested for protoplast regeneration in ANM regeneration medium with the optimized protoplast formation system.…”

Section: Resultsmentioning

confidence: 99%

“…Taking the economy and efficiency into consideration, we found that 1 M sucrose is more suitable as the osmotic pressure stabilizer for the protoplast regeneration of both strains. Stable osmotic pressure is the critical factor for protoplasts to regenerate cell walls [31]. In this study, the osmotic pressure stabilizers 1 M sucrose and 1.2 M sorbitol were tested for protoplast regeneration in ANM regeneration medium with the optimized protoplast formation system.…”

Section: Establishment Of Protoplast Preparation and Transformationmentioning

confidence: 99%

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Development of the CRISPR-Cas9 System for the Marine-Derived Fungi Spiromastix sp. SCSIO F190 and Aspergillus sp. SCSIO SX7S7

Chen

Cai

Yang

et al. 2022

JoF

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Marine-derived fungi are emerging as attractive producers of structurally novel secondary metabolites with diverse bioactivities. However, the lack of efficient genetic tools limits the discovery of novel compounds and the elucidation of biosynthesis mechanisms. Here, we firstly established an effective PEG-mediated chemical transformation system for protoplasts in two marine-derived fungi, Spiromastix sp. SCSIO F190 and Aspergillus sp. SCSIO SX7S7. Next, we developed a simple and versatile CRISPR-Cas9-based gene disruption strategy by transforming a target fungus with a single plasmid. We found that the transformation with a circular plasmid encoding cas9, a single-guide RNA (sgRNA), and a selectable marker resulted in a high frequency of targeted and insertional gene mutations in both marine-derived fungal strains. In addition, the histone deacetylase gene rpd3 was mutated using the established CRISPR-Cas9 system, thereby activating novel secondary metabolites that were not produced in the wild-type strain. Taken together, a versatile CRISPR-Cas9-based gene disruption method was established, which will promote the discovery of novel natural products and further biological studies.

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Section: Resultsmentioning

confidence: 99%

Section: Establishment Of Protoplast Preparation and Transformationmentioning

confidence: 99%

Development of the CRISPR-Cas9 System for the Marine-Derived Fungi Spiromastix sp. SCSIO F190 and Aspergillus sp. SCSIO SX7S7

Chen

Cai

Yang

et al. 2022

JoF

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“…This transformation efficiency was prominent in the entire mushroom transformation system (Table 1). High-quality protoplasts lay the foundation for successful transformation [37]. During the enzymatic hydrolysis of hyphae, lysozyme reduces protoplast viability, especially when using excessive amounts of lysozyme or extended reaction time.…”

Section: Discussionmentioning

confidence: 99%

Establishment of an Efficient Polyethylene Glycol (PEG)-Mediated Transformation System in Pleurotus eryngii var. ferulae Using Comprehensive Optimization and Multiple Endogenous Promoters

Zhang

Zhao

Shen

et al. 2022

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Pleurotus eryngii var. ferulae, a fungus of the genus Pleurotus, efficiently degrades lignin, especially during co-cultivation with other fungi. However, low transformation efficiency and heterologous gene expression restrict systematic studies of the molecular mechanisms and metabolic control of natural products in this mushroom. In this study, the homologous resistance marker carboxin (cbx) was used to establish a polyethylene glycol-mediated transformation (PMT) system in P. eryngii var. ferulae. Optimization of the transformation process greatly improved the number of positive transformants. In particular, we optimized: (i) protoplast preparation and regeneration; (ii) screening methods; and (iii) transformation-promoting factors. The optimized transformation efficiency reached 72.7 CFU/μg, which is higher than the average level of Pleurotus sp. (10–40 CFU/μg). Moreover, three endogenous promoters (Ppfgpd1, Ppfgpd2, and Ppfsar1) were screened and evaluated for different transcription initiation characteristics. A controllable overexpression system was established using these three promoters that satisfied various heterologous gene expression requirements, such as strong or weak, varied, or stable expression levels. This study lays the foundation for recombinant protein expression in P. eryngii var. ferulae and provides a method to investigate the underlying molecular mechanisms and secondary metabolic pathway modifications.

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“…Protoplast preparation and PEG−mediated transformation were performed as protocols as previously described [32] with some modifications. The purified protoplasts were resuspended in 3 mL 1× STC sorbitol and estimated using a hemocytometer (XB.…”

Section: Generation Of Gene Deletion Mutantsmentioning

confidence: 99%

Mitogen-Activated Protein Kinases SvPmk1 and SvMps1 Are Critical for Abiotic Stress Resistance, Development and Pathogenesis of Sclerotiophoma versabilis

Felix

Kuang

Biregeya

et al. 2023

JoF

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Mitogen-activated protein kinase (MAPK) signaling pathways are evolutionarily conserved in eukaryotes and modulate responses to both internal and external stimuli. Pmk1 and Mps MAPK pathways regulate stress tolerance, vegetative growth and cell wall integrity in Saccharomyces cerevisiae and Pyricularia oryzae. Here, we deployed genetic and cell biology strategies to investigate the roles of the orthologs of Pmk1 and Mps1 in Sclerotiophoma versabilis (herein referred to as SvPmk1 and SvMps1, respectively). Our results showed that SvPmk1 and SvMps1 are involved in hyphal development, asexual reproduction and pathogenesis in S. versabilis. We found that ∆Svpmk1 and ∆Svmps1 mutants have significantly reduced vegetative growths on PDA supplemented with osmotic stress-inducing agents, compared to the wild type, with ∆Svpmps1 being hypersensitive to hydrogen peroxide. The two mutants failed to produce pycnidia and have reduced pathogenicity on Pseudostellaria heterophylla. Unlike SvPmk1, SvMps1 was found to be indispensable for the fungal cell wall integrity. Confocal microscopic analyses revealed that SvPmk1 and SvMps1 are ubiquitously expressed in the cytosol and nucleus. Taken together, we demonstrate here that SvPmk1 and SvMps1 play critical roles in the stress resistance, development and pathogenesis of S. versabilis.

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Establishment of a Genetic Transformation System in Guanophilic Fungus Amphichorda guana

Cited by 10 publications

References 40 publications

Development of the CRISPR-Cas9 System for the Marine-Derived Fungi Spiromastix sp. SCSIO F190 and Aspergillus sp. SCSIO SX7S7

Development of the CRISPR-Cas9 System for the Marine-Derived Fungi Spiromastix sp. SCSIO F190 and Aspergillus sp. SCSIO SX7S7

Establishment of an Efficient Polyethylene Glycol (PEG)-Mediated Transformation System in Pleurotus eryngii var. ferulae Using Comprehensive Optimization and Multiple Endogenous Promoters

Mitogen-Activated Protein Kinases SvPmk1 and SvMps1 Are Critical for Abiotic Stress Resistance, Development and Pathogenesis of Sclerotiophoma versabilis

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