• Mycobiology
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• v.49 no.6
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• pp.599-603
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• 2021

CRISPR/Cas9 genome editing systems have been established in a broad range of eukaryotic species. Herein, we report the first method for genetic engineering in pyogo (shiitake) mushrooms (Lentinula edodes) using CRISPR/Cas9. For in vivo expression of guide RNAs (gRNAs) targeting the mating-type gene HD1 (LeA1), we identified an endogenous LeU6 promoter in the L. edodes genome. We constructed a plasmid containing the LeU6 and glyceraldehyde-3-phosphate dehydrogenase (LeGPD) promoters to express the Cas9 protein. Among the eight gRNAs we tested, three successfully disrupted the LeA1 locus. Although the CRISPR-Cas9-induced alleles did not affect mating with compatible monokaryotic strains, disruption of the transcription levels of the downstream genes of LeHD1 and LeHD2 was detected. Based on this result, we present the first report of a simple and powerful genetic manipulation tool using the CRISPR/Cas9 toolbox for the scientifically and industrially important edible mushroom, L. edodes.

• Microbiology and Biotechnology Letters
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• v.34 no.3
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• pp.211-215
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• 2006

AfsR2 is a global regulatory protein involved in the stimulation of secondary metabolite biosynthesis in various Streptomyces species including avermectin-producing S. avermitilis. Among several AfsR2-dependent genes identified from the comparative proteomics, the polyribonucleotide nucleotidyltransferase (PNP) and the glyceraldehyde-3-phosphate dehydrogenase (GPD) genes were previously proposed to regulate the actinorhodin production in S. lividans upon afsR2 over-expression positively and negatively, respectively. To show the biological significance of the PNP and GPD genes in the S. avermitilis strains, these two genes were functionally expressed in both the wild-type and the avermectin-overproducing mutant strains. The PNP gene expression stimulated secondary metabolite production in the wild-type S. avermitilis ATCC31267, but not in the avermectin-overproducing S. avermitilis ATCC31780. Interestingly, the GDP gene expression stimulated secondary metabolite production by 4-fold in the wild-type S. avermitilis ATCC31267 and by 2.5-fold in the avermectin-overproducing S. avermitilis ATCC31780, respectively. These results suggest that the biological significance of the afsR2-dependent PNP and GPD gene expressions on antibiotic biosynthetic regulation could be significantly different depending on Streptomyces species.

• The Korean Journal of Mycology
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• v.44 no.3
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• pp.201-205
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• 2016

In August 2015, leaf spot symptoms were observed on Korean gondre thistle (Cirsium setidens) in Youngwol, Korea. During the early stage, the symptoms appeared as one or more small gray-brown to brown spots on plant leaves. The spots showed extensive enlargement over time and eventually became large dark brown to black lesions on the whole leaf. Stemphylium species were consistently isolated from affected leaves. All isolates were identified as S. lycopersici, S. solani, or S. xanthosomatis based on morphological and cultural characteristics. The isolates were confirmed as S. lycopersici based on a multilocus sequence analysis using the ribosomal internal transcribed spacer (ITS) region, elongation factor 1, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and the noncoding region between the vacuolar membrane ATPase catalytic subunit A gene and a gene involved in vacuolar biogenesis. Pathogenicity was tested by spore suspension inoculation on wounded or unwounded gondre leaves. The lesions were observed on inoculated leaves within 3 days after inoculation, regardless of wound. To our knowledge, this is the first report of the leaf spot on gondre thistle caused by S. lycopersici in Korea or elsewhere.

• Mycobiology
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• v.47 no.1
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• pp.59-65
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• 2019

Agrobacterium tumefaciens-mediated transformation (ATMT), as a simple and versatile method, achieves successful transformation in the yeast-like cells (YLCs) of Tremella fuciformis with lower efficiency. Establishment of a more efficient transformation system of YLCs is important for functional genomics research and biotechnological application. In this study, an enzymolysis-assisted ATMT method was developed. The degradation degree of YLCs depends on the concentration and digestion time of Lywallzyme. Lower concentration (${\leq}0.1%$) of Lywallzyme was capable of formation of limited wounds on the surface of YLCs and has less influence on their growth. In addition, there is no significant difference of YLCs growth among groups treated with 0.1% Lywallzyme for different time. The binary vector pGEH under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter was utilized to transform the enzymolytic wounded YLCs with different concentrations and digestion time. The results of PCR, Southern blot, quantitative real-time PCR (qRT-PCR) and fluorescence microscopy revealed that the T-DNA was integrated into the YLCs genome, suggesting an efficient enzymolysis-assisted ATMT method of YLCs was established. The highest transformation frequency reached 1200 transformants per $10^6$ YLCs by 0.05% (w/v) Lywallzyme digestion for 15 min, and the transformants were genetically stable. Compared with the mechanical wounding methods, enzymolytic wounding is thought to be a tender, safer and more effective method.