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 한국균학회 2022 Mycobiology Vol.50 No.5

In the mating of filamentous basidiomycetes, dikaryotic mycelia are generated through the reciprocal movement of nuclei to a monokaryotic cytoplasm where a nucleus of compatible mating type resides, resulting in the establishment of two different dikaryotic strains having the same nuclei but different mitochondria. To better understand the role of mitochondria in mushrooms, we created four sets of dikaryotic strains of Lentinula edodes, including B2 E13 (B2 side) and B2 E13 (E13 side), B5 E13 (B5 side) and B5 E13 (E13 side), E8 H3 (E8 side) and E8 H3 (H3 side), and K3 H3 (K3 side) and K3 H3 (H3 side). The karyotypes and mitochondrial types of the dikaryotic strains were successfully identified by the A mating type markers and the mitochondrial variable length tandem repeat markers, respectively. Comparative analyses of the dikaryotic strains on the mycelial growth, substrate browning, fruiting characteristics, and mitochondrial gene expression revealed that certain mitochondria are more effective in the mycelial growth and the production of fruiting body, possibly through the activated energy metabolism. Our findings indicate that mitochondria affect the physiology of dikaryotic strains having the same nuclear information and therefore a selection strategy aimed at mitochondrial function is needed in the development of new mushroom strain.

Enhancement of gene correction efficiency in Shiitake mushrooms with nanoparticles

Minseek Kim,Hojin Ryu,Min ji Oh,Ji-Hoon Im,Yunsang Lee,Youn-Lee Oh 한국버섯학회 2022 버섯 Vol.26 No.1

Growth Characteristics of Polyporales Mushrooms for the Mycelial Mat Formation

( Bin Bae ),( Minseek Kim ),( Sinil Kim ),( Hyeon-su Ro ) 한국균학회 2021 Mycobiology Vol.49 No.3

Mushroom strains of Polyporales from the genera Coriolus, Trametes, Pycnoporus, Ganoderma, and Formitella were explored in terms of mycelial growth characteristics for the application of mushroom mycelia as alternative sources of materials replacing fossil fuel-based materials. Among the 64 strains of Polyporales, G. lucidum LBS5496GL was selected as the best candidate because it showed fast mycelial growth with high mycelial strength in both the sawdust- based solid medium and the potato dextrose liquid plate medium. Some of the Polyporales in this study have shown good mycelial growth, however, they mostly formed mycelial mat of weak physical strength. The higher physical strength of mycelial mat by G. lucidum LBS5496GL was attributed to its thick hyphae with the diameter of 13 mm as revealed by scanning electron microscopic analysis whereas the hyphae of others exhibited less than 2 mm. Glycerol and skim milk supported the best mycelial growth of LBS5496GL as a carbon and a nitrogen source, respectively.

Activation of the Mating Pheromone Response Pathway of Lentinula edodes by Synthetic Pheromones

( Byeongsuk Ha ),( Sinil Kim ),( Minseek Kim ),( Hyeon-su Ro ) 한국균학회 2018 Mycobiology Vol.46 No.4

Pheromone (PHB)-receptor (RCB) interaction in the mating pheromone response pathway of Lentinula edodes was investigated using synthetic PHBs. Functionality of the C-terminally carboxymethylated synthetic PHBs was demonstrated by concentration-dependent induction of a mating-related gene (znf2) expression and by pseudoclamp formation in a monokaryotic strain S1-11 of L. edodes. Treatment with synthetic PHBs activated the expression of homeodomain genes (HDs) residing in the A mating type locus, and of A-regulated genes, including znf2, clp1, and priA, as well as genes in the B mating type locus, including pheromone (phb) and receptor (rcb) genes. The synthetic PHBs failed to discriminate self from non-self RCBs. PHBs of the B4 mating type (B4 PHBs) were able to activate the mating pheromone response pathway in both monokaryotic S1-11 and S1-13 strains, whose B mating types were B4 (self) and B12 (non-self), respectively. The same was true for B12 PHBs in the B4 (non-self) and B12 (self) mating types. The synthetic PHBs also promoted the mating of two monokaryotic strains carrying B4-common incompatible mating types (A5B4_A1B4). However, the dikaryon generated by this process exhibited abnormally high content of hyphal branching and frequent clamp connections and, more importantly, was found to be genetically unstable due to overexpression of mating-related genes such as clp1. Although synthetic PHBs were unable to discriminate self from non-self RCBs, they showed a higher affinity for non-self RCBs, through which the mating pheromone response pathway in non-self cells may be preferentially activated.

양송이 원형질체 분리와 PEG 형질전환법의 최적화

김민식,장갑열,이윤상,오민지,임지훈,오연이,Kim, Minseek,Jang, Kab-yeul,Lee, Yun-Sang,Oh, Min Ji,Im, Ji-Hoon,Oh, Youn-Lee 한국버섯학회 2021 한국버섯학회지 Vol.19 No.3

현재 양송이 품종의 개발은 1980년대에 개발된 방법에 의존하여 진행되고 있다. 유전자가위를 이용한 유전자교정 기술이 다양한 분야에서 각광받고 있고, 이 기술을 버섯 육종에도 적용하기 위하여 진행된 이 연구에서는, CRISPR/Cas9 활용에 필수적인 원형질체 분리 효율을 1.0 × 10⁸/mL까지 안정적으로 끌어올렸고, spermidine을 이용하여 PEG 형질전환의 효율 또한 기존 방법에 비해 100배가량 끌어올렸음을 보고한다. Currently, button mushroom, Agaricus bisporus is one of the most consumed mushroom in the world. However, despite of its importance in food market, molecular genetic modification method for breeding of A. bisporus is not well established. In this study, we optimized yield of A. bisporus protoplast with Lysing enzyme, Chimax-N and cellulase. With this composition, 1.0 × 10⁸/mL of protoplasts were obtained reliably. PEG-mediated transformation with spermidine showed almost 100-fold higher yield than non-spermidine method.

Nucleus-Selective Expression of Laccase Genes in the Dikaryotic Strain of Lentinula edodes

( Byeongsuk Ha ),( Sieun Lee ),( Sinil Kim ),( Minseek Kim ),( Yoon Jung Moon ),( Yelin Song ),( Hyeon-su Ro ) 한국균학회 2017 Mycobiology Vol.45 No.4

In mating of Lentinula edodes, dikaryotic strains generated from certain monokaryotic strains such as the B2 used in this study tend to show better quality of fruiting bodies regardless of the mated monokaryotic strains. Unlike B2, dikaryotic strains generated from B16 generally show low yields, with deformed or underdeveloped fruiting bodies. This indicates that the two nuclei in the cytoplasm do not contribute equally to the physiology of dikaryotic L. edodes, suggesting an expression bias in the allelic genes of the two nuclei. To understand the role of each nucleus in dikaryotic strains, we investigated single nucleotide polymorphisms (SNPs) in laccase genes of monokaryotic strains to reveal nuclear origin of the expressed mRNAs in dikaryotic strain. We performed reverse transcription PCR (RT-PCR) analysis using total RNAs extracted from dikaryotic strains (A5B2, A18B2, and A2B16) as well as from compatible monokaryotic strains (A5, A18, and B2 for A5B2 and A18B2; A2 and B16 for A2B16). RT-PCR results revealed that Lcc1, Lcc2, Lcc4, Lcc7, and Lcc10 were the mainly expressed laccase genes in the L. edodes genome. To determine the nuclear origin of these laccase genes, the genomic DNA sequences in monokaryotic strains were analyzed, thereby revealing five SNPs in Lcc4 and two in Lcc7. Subsequent sequence analysis of laccase mRNAs expressed in dikaryotic strains revealed that these were almost exclusively expressed from B2-originated nuclei in A5B2 and A18B2 whereas B16 nucleus did not contribute to laccase expression in A2B16 strain. This suggests that B2 nucleus dominates the expression of allelic genes, thereby governing the physiology of dikaryons.

Molecular analysis of <i>B</i> mating type diversity in <i>Lentinula edodes</i>

Ha, Byeongsuk,Moon, Yoon Jung,Song, Yelin,Kim, Sinil,Kim, Minseek,Yoon, Cheol-Won,Ro, Hyeon-Su Elsevier 2019 Scientia horticulturae Vol.243 No.-

<P><B>Abstract</B></P> <P>Pheromone and pheromone receptor genes in <I>B</I> mating type locus constitute <I>B</I> mating type of fungi belonging to Basidiomycota. In <I>Lentinula edodes</I>, multiple <I>B</I> mating types have been suggested based on genome sequence analysis and mating assay. Here we report finding of new alleles of pheromones (<I>phb</I>s) and pheromone receptors (<I>rcb</I>s) to constitute five alleles of <I>rcb1</I> (<I>rcb1-1</I>∼<I>rcb1-5</I>) with nine associated <I>phbs</I> in <I>Bα</I> sublocus and three alleles of <I>rcb2</I> (<I>rcb2-1</I>∼<I>rcb2-3</I>) with five associated <I>phbs</I> in <I>Bβ</I> sublocus. Each <I>rcb</I> was primarily associated with two <I>phbs</I>. Each <I>phb</I>-<I>rcb</I> pair was recurrently discovered as a distinct unit in various monokarytotic and dikaryotic strains, regardless of subloci. This allowed us to propose 15 <I>B</I> mating types through combinations of five <I>Bα</I> and three <I>Bβ</I> subloci. PCR analyses with primer sets probing alleles of <I>rcb1</I> and <I>rcb2</I> demonstrated that all monokaryotic strains contained one of the five <I>rcb1</I>s and one of the three <I>rcb2</I>s representing <I>Bα</I> and <I>Bβ</I> subloci, respectively, thereby enabling designation of their <I>B</I> mating types via combinations of <I>Bα</I> and <I>Bβ</I>. PCR analyses also revealed the presence of one or two alleles of <I>rcb1</I> and <I>rcb2</I> representing <I>B</I> mating type of each nuclei in dikaryotic cytoplasm. Further investigation of 111 dikaryotic strains, including 83 cultivated and 28 wild strains collected from East Asia, revealed that <I>B8</I> and <I>B11</I> mating types constructed by <I>rcb1-3</I> + <I>rcb2-2</I> and <I>rcb1-4</I> + <I>rcb2-2</I>, respectively, were more prevalent in cultivated strains. In <I>B</I> mating type pairs representing each nucleus in dikaryons, <I>B2B12</I>, <I>B4B8</I>, and <I>B8B11</I> were frequently occurring pairs in cultivated strains while none of them was found in wild strains. Such prevalence indicates that certain nuclei have been preferentially selected in the generation of cultivated strains. Our findings shed new light on the construction of <I>B</I> mating type in <I>L. edodes</I> and provide practical tools in the breeding of new cultivars.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The <I>B</I> mating type locus of <I>L. edodes</I> consists of multiallelic pheromone and receptor genes. </LI> <LI> 5 <I>rcb1</I>s and 3 <I>rcb2</I>s constitute <I>Bα</I> and <I>Bβ</I> subloci, respectively, with 14 <I>phb</I>s. </LI> <LI> Each <I>rcb</I> pairs with distinct <I>phb</I>(s) and thus found as a <I>phb-rcb</I> unit in sublocus. </LI> <LI> Combinatorial assortment of <I>Bα</I> and <I>Bβ</I> subloci generates fifteen <I>B</I> mating types. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

납축전지 열화 원인 분석 및 수명 연장을 위한 전기적 방법 특성 비교 분석

이제림(Jelim Lee),박진형(Jinhyeong Park),성용준(Yongjun Sung),최민식(Minseek Choi),이수준(Soojun Lee),김종훈(Jonghoon Kim) 전력전자학회 2022 전력전자학술대회 논문집 Vol.2013 No.11

흰색 팽이 신품종 '설한' 육성

임지훈,오민지,오연이,김민식,이윤상,Im, Ji-Hoon,Oh, Minji,Oh, Youn-Lee,Kim, Minseek,Lee, Yun-Sang 한국버섯학회 2021 한국버섯학회지 Vol.19 No.4

팽이(Flammulina velutipes)는 주름버섯목(Agaricales), 뽕나무버섯과(Physalacriaceae)에 속하는 담자균류로서, 국내 생산량은 연간 31,818톤('19)으로 전체 생산량의 약 18%이며 버섯 전체 수출액의 약 36%를 차지할 정도로 대표적인 식용버섯이다. 그러나 국내 재배되고 있는 팽이 국산품종의 비중이 31.6%(20')로 매우 낮으므로 국제 경쟁력이 우수한 국내육성 품종확보가 필요하며 수출 버섯으로서 보다 품질이 우수한 품종 육성이 필요하다. 이에 본 연구는 농진청에서 육성한 '백승'과 '우리1호'의 단핵균주를 확보하여 mon-mon교잡을 통해 '설한' 품종을 육성하였다. 균사배양 특성은 25℃에서 균사생장이 가장 좋았으며 20, 25℃에서 대조품종보다 균사 생장속도가 빨랐다. 병재배 시 배양 최적온도는 15~17℃로 배지온도가 20℃이상 되지 않게 관리해야하며 생육온도는 7~8℃로 대조품종에 비해 다소 높다. 또한 자실체 수량은 병당(850 mL) 226.5±8.0 g으로 대조품종에 비해 18.4% 증수하였으며 초발이일수가 2일 빨라 전체적인 생육기간이 단축되었다. 또한 설한의 외형은 대조품종보다 갓이 작고 대는 굵었으며 갓 색이 더 밝았다. 대치배양과 유전적 다형성 검정 결과, 설한이 다른 흰색 품종들과 유전적으로 구별되는 것을 확인하였다. Flammulina velutipes is the representative edible mushroom in Korea. It makes up around 18% of the total mushroom production and accounts for about 36% of the total mushroom export. Despite being one of the most industrially important mushroom in Korea, the distribution rate of domestic cultivar was low (~31.6%) in 2020. Therefore, we need a new domestic cultivar with high quality as the main export mushroom. We developed a new white domestic cultivar, 'Seolhan', bred by mating two monokaryotic strains isolated from Baekseung and Woori-1ho. The optimum mycelial growth temperature was 25℃ and Seolhan had higher mycelial growth than the control at 20 and 25℃. In bottle cultivation, the optimum temperature of incubation was from 15℃ to 17℃ and the growth temperature of Seolhan was 7-8℃, which was slightly higher than that of the control. The yield of Seolan was 226.5±8.0 g, an increase of 18.4% compared to the control. The period of primodia formation also was a couple of days ahead. In morphological traits, the pileus of Seolhan was smaller than that of the control, the stipe was thicker, and the color brighter. As the results of replacement culture and genetic diversity test, it was confirmed that Seolhan was genetically different from other white varieties.