Journal of Microbiology

Research Article

Simultaneous gene editing of both nuclei in a dikaryotic strain of Ganoderma lucidum using Cas9-gRNA ribonucleoprotein: Yeon-Jae Choi, Hyerang Eom, Rutuja Nandre, Minseek Kim, Youn-Lee Oh, Sinil Kim, Hyeon-Su Ro; J. Microbiol. 2025;63(1):e.2409006. Published online January 24, 2025; DOI: https://doi.org/10.71150/jm.2409006

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Abstract PDF Supplementary Material: The presence of multiple nuclei in a common cytoplasm poses a significant challenge to genetic modification in mushrooms. Here, we demonstrate successful gene editing in both nuclei of a dikaryotic strain of Ganoderma lucidum using the Cas9-gRNA ribonucleoprotein complex (RNP). The RNP targeting the pyrG gene was introduced into dikaryotic protoplasts of G. lucidum, resulting in the isolation of 31 mycelial colonies resistant to 5-fluoroorotic acid (5-FOA). Twenty-six of these isolates were confirmed as dikaryotic strains by the presence of two distinct A mating type markers, denoted as A1 and A2. All dikaryons exhibited clamp connections on their mycelial hyphae, while the remaining 5 transformants were monokaryotic. Subsequent sequence analysis of PCR amplicons targeting pyrG revealed that two dikaryons harbored disrupted pyrG in both nuclei (pyrG-/pyrG-), while 10 and 14 displayed pyrG+/pyrG- (A1/A2) and pyrG-/pyrG+ (A1/A2) configurations, respectively. The disruption was achieved through non-homologous end joining repair, involving deletion or insertion of DNA fragments at the site of the double-strand break induced by RNP. Importantly, the nuclei were stable throughout 10 serial transfers over a period of 6 months. These findings highlight the capability of RNP to target genes across multiple nuclei within the same cytoplasm.

Journal Article

Diversity of A mating type in Lentinula edodes and mating type preference in the cultivated strains: Byeongsuk Ha , Sinil Kim , Minseek Kim , Yoon Jung Moon , Yelin Song , Jae-San Ryu , Hojin Ryu , Hyeon-Su Ro; J. Microbiol. 2018;56(6):416-425. Published online June 1, 2018; DOI: https://doi.org/10.1007/s12275-018-8030-6

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Diversity of A mating type in Lentinula edodes has been assessed by analysis of A mating loci in 127 strains collected from East Asia. It was discovered that hypervariable sequence region with an approximate length of 1 kb in the A mating locus, spanning 5􍿁􀁇region of HD2-intergenic region-5􍿁􀁇region of HD1, could represent individual A mating type as evidenced by comprehensive mating analysis. The sequence analysis revealed 27 A mating type alleles from 96 cultivated strains and 48 alleles from 31 wild strains. Twelve of them commonly appeared, leaving 63 unique A mating type alleles. It was also revealed that only a few A mating type alleles such as A1, A4, A5, and A7 were prevalent in the cultivated strains, accounting for 62.5% of all A mating types. This implies preferred selection of certain A mating types in the process of strain development and suggests potential role of A mating genes in the expression of genes governing mushroom quality. Dominant expression of an A mating gene HD1 was observed from A1 mating locus, the most prevalent A allele, in A1-containing dikaryons. However, connections between HD1 expression and A1 preference in the cultivated strains remain to be verified. The A mating type was highly diverse in the wild strains. Thirty-six unique A alleles were discovered from relatively small and confined area of mountainous region in Korean peninsula. The number will further increase because no A allele has been recurrently observed in the wild strains and thus newly discovered strain will have good chances to contain new A allele. The high diversity in small area also suggests that the A mating locus has evolved rapidly and thus its diversity will further increase.

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Intraspecies Variation Offers Potential to Improve White Rot Fungi for Increasing Degradability of Lignocellulose for Ruminants
Anton S. M. Sonnenberg, Nazri Nayan, John W. Cone, Arend F. van Peer
Journal of Fungi.2024; 10(12): 858. CrossRef
Effect of a Mating Type Gene Editing in Lentinula edodes Using RNP/Nanoparticle Complex
Minseek Kim, Minji Oh, Ji-Hoon Im, Eun-Ji Lee, Hojin Ryu, Hyeon-Su Ro, Youn-Lee Oh
Journal of Fungi.2024; 10(12): 866. CrossRef
Structural Analysis of the A Mating Type Locus and Development of the Mating Type Marker of Agaricus bisporus var. bisporus
Yeon-Jae Choi, Sujin Jung, Hyerang Eom, Thimen Hoang, Hui-Gang Han, Sinil Kim, Hyeon-Su Ro
Journal of Fungi.2023; 9(3): 284. CrossRef
Determination and Analysis of Hyper-Variable A Mating Types in Wild Strains of Lentinula edodes in Korea
Mi-Jeong Park, Eunjin Kim, Yeun Sug Jeong, Mi-Young Son, Yeongseon Jang, Kang-Hyeon Ka
Mycobiology.2023; 51(1): 26. CrossRef
Haplotype-Resolved Genome Analyses Reveal Genetically Distinct Nuclei within a Commercial Cultivar of Lentinula edodes
Qi Gao, Dong Yan, Shuang Song, Yangyang Fan, Shouxian Wang, Yu Liu, Yu Huang, Chengbo Rong, Yuan Guo, Shuang Zhao, Wentao Qin, Jianping Xu
Journal of Fungi.2022; 8(2): 167. CrossRef
Mitochondrial Effects on the Physiological Characteristics of Lentinula edodes
Minseek Kim, Seong-Hyeok Yang, Hui-Gang Han, Eunbi Kim, Sinil Kim, Youn-Lee Oh, Hyeon-Su Ro
Mycobiology.2022; 50(5): 374. CrossRef
Comparative structural analysis on the mitochondrial DNAs from various strains of Lentinula edodes
Sinil Kim, Hyerang Eom, Rutuja Nandre, Yeon Jae Choi, Hwayong Lee, Hojin Ryu, Hyeon-Su Ro
Frontiers in Microbiology.2022;[Epub] CrossRef
Genetic structure and evolutionary diversity of mating-type (MAT) loci in Hypsizygus marmoreus
Gang Wang, Yuanyuan Wang, Lianfu Chen, Hongbo Wang, Lin Guo, Xuan Zhou, Meijie Dou, Baiyu Wang, Jingxian Lin, Lei Liu, Zhengchao Wang, Youjin Deng, Jisen Zhang
IMA Fungus.2021;[Epub] CrossRef
Macrosynteny analysis between Lentinula edodes and Lentinula novae-zelandiae reveals signals of domestication in Lentinula edodes
Christopher Alan Smith
Scientific Reports.2021;[Epub] CrossRef
Analysis of Genetic Diversity and Population Structure of Wild Strains and Cultivars Using Genomic SSR Markers inLentinula edodes
Hwa-Yong Lee, Suyun Moon, Hyeon-Su Ro, Jong-Wook Chung, Hojin Ryu
Mycobiology.2020; 48(2): 115. CrossRef
Structure analysis of A and B mating type loci in a representative commercial strain of Pleurotus eryngii
Yejin Ju, Sinil Kim, Minseek Kim, Jae San Ryu, Hyeon-Su Ro
Scientia Horticulturae.2020; 274: 109686. CrossRef
Molecular analysis of B mating type diversity in Lentinula edodes
Byeongsuk Ha, Yoon Jung Moon, Yelin Song, Sinil Kim, Minseek Kim, Cheol-Won Yoon, Hyeon-Su Ro
Scientia Horticulturae.2019; 243: 55. CrossRef
Variable Number Tandem Repeats in the Mitochondrial DNA of Lentinula edodes
Sinil Kim, Yelin Song, Byeongsuk Ha, Yoon Jung Moon, Minseek Kim, Hojin Ryu, Hyeon-Su Ro
Genes.2019; 10(7): 542. CrossRef
Activation of the Mating Pheromone Response Pathway ofLentinula edodesby Synthetic Pheromones
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Mycobiology.2018; 46(4): 407. CrossRef

Research Support, Non-U.S. Gov't

Novel nuclear targeting coiled-coil protein of Helicobacter pylori showing Ca2+-independent, Mg2+-dependent DNase I activity: Young Chul Kwon , Sinil Kim , Yong Seok Lee , Je Chul Lee , Myung-Je Cho , Woo-Kon Lee , Hyung-Lyun Kang , Jae-Young Song , Seung Chul Baik , Hyeon Su Ro; J. Microbiol. 2016;54(5):387-395. Published online April 20, 2016; DOI: https://doi.org/10.1007/s12275-016-5631-9

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Abstract

HP0059, an uncharacterized gene of Helicobacter pylori, encodes a 284-aa-long protein containing a nuclear localization sequence (NLS) and multiple leucine-rich heptad repeats. Effects of HP0059 proteins in human stomach cells were assessed by incubation of recombinant HP0059 proteins with the AGS human gastric carcinoma cell line. Wild-type HP0059 proteins showed cytotoxicity in AGS cells in a concentrationdependent manner, whereas NLS mutant protein showed no effect, suggesting that the cytotoxicity is attributed to host nuclear localization. AGS cells transfected with pEGFP-HP0059 plasmid showed strong GFP signal merged to the chromosomal DNA region. The chromosome was fragmented into multiple distinct dots merged with the GFP signal after 12 h of incubation. The chromosome fragmentation was further explored by incubation of AGS chromosomal DNA with recombinant HP0059 proteins, which leaded to complete degradation of the chromosomal DNA. HP0059 protein also degraded circular plasmid DNA without consensus, being an indication of DNase I activity. The DNase was activated by MgCl2, but not by CaCl2. The activity was completely blocked by EDTA. The optimal pH and temperature for DNase activity were 7.0–8.0 and 55°C, respectively. These results indicate that HP0059 possesses a novel DNase I activity along with a role in the genomic instability of human gastric cells, which may result in the transformation of gastric cells.

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