Current Technologies and Related Issues for Mushroom Transformation

Kim, Sinil,Ha, Byeong-Suk,Ro, Hyeon-Su The Korean Society of Mycology 2015 Mycobiology Vol.43 No.1

Mushroom transformation requires a series of experimental steps, including generation of host strains with a desirable selective marker, design of vector DNA, removal of host cell wall, introduction of foreign DNA across the cell membrane, and integration into host genomic DNA or maintenance of an autonomous vector DNA inside the host cell. This review introduces limitations and obstacles related to transformation technologies along with possible solutions. Current methods for cell wall removal and cell membrane permeabilization are summarized together with details of two popular technologies, Agrobacterium tumefaciens-mediated transformation and restriction enzyme-mediated integration.

Mini-review : Current Technologies and Related Issues for Mushroom Transformation

( Sinil Kim ),( Byeong Suk Ha ),( Hyeon Su Ro ) 한국균학회 2015 韓國菌學會誌 Vol.43 No.1

Mushroom transformation requires a series of experimental steps, including generation of host strains with a desirable selective marker, design of vector DNA, removal of host cell wall, introduction of foreign DNA across the cell membrane, and integration into host genomic DNA or maintenance of an autonomous vector DNA inside the host cell. This review introduces limitations and obstacles related to transformation technologies along with possible solutions. Current methods for cell wall removal and cell membrane permeabilization are summarized together with details of two popular technologies, Agrobacterium tumefaciens-mediated transformation and restriction enzyme-mediated integration.

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.

RESEARCH NOTES : Isolation of Fungal Pathogens to an Edible Mushroom, Pleurotus eryngii, and Development of Specific ITS Primers

( Sang Woo Kim ),( Sinil Kim ),( Hyun Jun Lee ),( Ju Wan Park ),( Hyeon Su Ro ) 한국균학회 2013 Mycobiology Vol.41 No.4

Fungal pathogens have caused severe damage to the commercial production of Pleurotus eryngii, the king oyster mushroom, by reducing production yield, causing deterioration of commercial value, and shortening shelf-life. Four strains of pathogenic fungi, including Trichoderma koningiopsis DC3, Phomopsis sp. MP4, Mucor circinelloides MP5, and Cladosporium bruhnei MP6, were isolated from the bottle culture of diseased P. eryngii. A species-specific primer set was designed for each fungus from the ITS1-5.8S rDNA-ITS2 sequences. PCR using the ITS primer set yielded a unique DNA band for each fungus without any cross-reaction, proving the validity of our method in detection of mushroom fungal pathogens.

안정화제를 이용한 부적합 필지 복원을 위한 현장실증시험

강신일 ( Sinil Kang ),진혜근 ( Hyegeun Jin ),김하진 ( Hajin Kim ),윤성욱 ( Sungwook Yun ),유찬 ( Chan Yu ) 한국농공학회 2011 한국농공학회 학술대회초록집 Vol.2011 No.-

광해로 인한 중금속 오염지역 중 토양이 아닌 농경지에서 생산된 농작물이 중금속(Cd)으로 오염된 부적합필지를 대상으로 오염농경지 개량복원을 위한 현장실증 시험을 수행하였다. 대상오염 농경지에 처리구를 설치하여 안정화제(석회석 3%)를 적용하였으며, 관측기간동안 처리구(무처리, 석회석 3%, 석회석 3%+복토)내 토양 및 식물체 가식부를 채취하여 이화학과 중금속 농도를 분석하여 그 특성을 비교하였다. 그 결과 중금속 성분(Cd)은 안정화제를 처리하지 않은 처리구 내 식물체 가식부에서 기준치를 초과하는 농도가 나타났으며 부적합 필지라는 사실을 확인할 수 있었다. 안정화제를 처리한 2, 3 처리구에선 기준치를 초과하지 않는 농도가 나타났으며 이는 안정화 처리시 토양의 식물이용가능성 형태의 중금속이 감소된 것으로 판단된다.

현장 pilot 대형 토조실험을 통한 단층형 매립복토시스템의 복토재로서 석탄회의 효과검토

김하진 ( Hajin Kim ),윤성욱 ( Sungwook Yun ),강신일 ( Sinil Kang ),진혜근 ( Hyegeun Jin ),유찬 ( Chan Yu ) 한국농공학회 2011 한국농공학회 학술대회초록집 Vol.2011 No.-

지역적 특성 반영 및 시공상의 어려움, 공사비가 비싸고 점토의 균열에 의한 문제점, 내구성이 취약하며, 식생조성이 어려운 기존의 복합형 최종복토시스템의 단점을 보완하기 위한 단층형 복토시스템의 복토재로서 석탄회의 활용성을 검토하기 위하여 현장 pilot 대형 토조실험을 수행하였다. 기존에 지역환경 문제로 인식되었던 산업부산물의 활용성을 검토하여, 산업부산물들을 관련 규정에 의거하여 석탄회를 일반토사와 혼합하여 현장에서 설치한 대형토조에 채워넣어 관측기간동안 (2010년 6월~2011년 7월) 단층형 복토시스템의 복토재로서 그 활용성을 일반토사만을 채운 처리구 그리고 일반토사에 건설폐기물을 혼합한 처리구와 함께 비교하였다. 그 결과 복토층 내의 식생유무에 따라 체적함수비의 차이는 석탄재, 일반토사, 건설폐기물 순으로 크게 나타났다. 또한 석탄재, 일반토사, 건설폐기물 순서로 지중침출수가 적은 것으로 나타났다. 특히 식생 유무에 따라 차이가 더 뚜렷이 나타났다. 이 같은 결과를 고려해 볼 때 건설폐기물을 복토재로서 사용하여 식생을 조성하였을 경우 발육상태가 불량한 것으로 나타났고, 석탄재를 복토재로서 사용하였을 경우 일반토사만 사용하였을 때 보다 수분저류능력이 높은 것으로 나타났다.

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.

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.

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.

Isolation and Characterization of Monokaryotic Strains of Lentinula edodes Showing Higher Fruiting Rate and Better Fruiting Body Production

Ha, Byeong-Suk,Kim, Sinil,Ro, Hyeon-Su The Korean Society of Mycology 2015 Mycobiology Vol.43 No.1

The effects of monokaryotic strains on fruiting body formation of Lentinula edodes were examined through mating and cultivation of the mated dikaryotic mycelia in sawdust medium. To accomplish this, monokaryotic strains of L. edodes were isolated from basidiospores of the commercial dikaryotic strains, Chamaram (Cham) and Sanjo701 (SJ701). A total of 703 matings (538 self-matings and 165 outcrosses) were performed, which generated 133 self-mates and 84 outcross mates. The mating rate was 25% and 50% for self-mating and outcross, respectively. The bipolarity of the outcross indicated the multi-allelic nature of the mating type genes. The mating was only dependent on the A mating type locus, while the B locus showed no effect, implying that the B locus is multi-allelic. Next, 145 selected dikaryotic mates were cultivated in sawdust medium. The self-mated dikaryotic progenies showed 51.3% and 69.5% fruiting rates for Cham and SJ701, respectively, while the fruiting rate of the outcross mates was 63.2%. The dikaryotic mates generated by mating with one of the monokaryotic strains, including A20, B2, E1, and E3, showed good fruiting performance and tended to yield high fruiting body production, while many of the monokaryotic strains failed to form fruiting bodies. Overall, these findings suggest that certain monokaryotic strains have traits enabling better mating and fruiting.