• Mycobiology
• /
• 제49권6호
• /
• pp.582-588
• /
• 2021

The interaction of mating pheromone and pheromone receptor from the B mating-type locus is the first step in the activation of the mushroom mating signal transduction pathway. The B mating-type locus of Lentinula edodes is composed of Bα and Bβ subloci, each of which contains genes for mating pheromone and pheromone receptor. Allelic variations in both subloci generate multiple B mating-types through which L. edodes maintains genetic diversity. In addition to the B mating-type locus, our genomic sequence analysis revealed the presence of a novel chromosomal locus 43.3 kb away from the B mating-type locus, containing genes for a pair of mating pheromones (PHBN1 and PHBN2) and a pheromone receptor (RCBN). The new locus (Bα-N) was homologous to the Bα sublocus, but unlike the multiallelic Bα sublocus, it was highly conserved across the wild and cultivated strains. The interactions of RcbN with various mating pheromones from the B and Bα-N mating-type loci were investigated using yeast model that replaced endogenous yeast mating pheromone receptor STE2 with RCBN. The yeast mating signal transduction pathway was only activated in the presence of PHBN1 or PHBN2 in the RcbN producing yeast, indicating that RcbN interacts with self-pheromones (PHBN1 and PHBN2), not with pheromones from the B mating-type locus. The biological function of the Bα-N locus was suggested to control the expression of A mating-type genes, as evidenced by the increased expression of two A-genes HD1 and HD2 upon the treatment of synthetic PHBN1 and PHBN2 peptides to the monokaryotic strain of L. edodes.

• 한국균학회소식:학술대회논문집
• /
• 한국균학회 2018년도 춘계학술대회 및 임시총회
• /
• pp.37-37
• /
• 2018

Mating type of Lentinula edodes is determined by two unlinked genetic loci, A and B. To better understand mating behavior of L. edodes, we investigated variations in mating type genes in129 dikaryotic strains collected from East Asia. Through sequence analysis of A locus, we discovered that hypervariable region spanning N-term of HD2-intergenic region-N-term of HD1 could represent A mating type. Mating and hypervariable region analyses revealed 70 unique A mating types: 27 from 98 cultivated strains, 53 from 31 wild strains, and 10 commonly found. It was also revealed that only a few A mating type alleles such as A1, A4, A5, and A7 were prevalent in cultivated strains. Contrarily, A mating type in wild strains was highly diverse: 23 unique A alleles were discovered in small mountainous area in Korean peninsula, suggesting rapid evolution of A mating type in nature. The B locus was assessed by allelic variations in pheromone (PHB) and pheromone receptor (RCB) pairs which constituted subloci Ba and Bb. Sequence analyses and mating assay revealed 5 alleles of RCB1 with 9 associated PHBs in Ba sublocus and 3 alleles of RCB2 with 5 associated PHBs in Bb sublocus. Each RCB was primarily associated with two PHBs. Each PHB-RCB pair was always discovered as a distinct unit. This allowed us to propose 15 B mating types via combinations of five Ba and three Bb subloci. Further investigation on 129 strains confirmed that the B locus, unlike the A locus, was indeed restricted to 15 mating types. Thus, the total number of mating types became 1,050 in L. edodes through a combination of 70 A and 15 B. This number will further increase because of rapid diversification of A mating type. Our findings provide a comprehensive and practical knowledge on mating behaviors of L. edodes.

• Mycobiology
• /
• 제46권4호
• /
• pp.407-415
• /
• 2018

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 ($A5B4{\times}A1B4$). 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.

• 한국균학회소식:학술대회논문집
• /
• 한국균학회 2014년도 추계학술대회 및 정기총회
• /
• pp.42-42
• /
• 2014

Mating of tetrapolar mushrooms is regulated by to chromosomal loci, A and B. A locus contains A gene that expresses a homeodomain protein whereas B locus contains multiple pheromones and receptor genes. In order to characterize the mating loci in Korean cultivated strains of Lentinula edodes, one hundred monokaryotic myclelia were isolated from the basidiospores of cultivated strains, including Cham-A-Ram, Sanjo701, and Sanjo707. Both mating loci were amplified using primer sets targeting conserved sequence regions for homeodomain (HD), pheromone, and receptor genes. Subsequent sequence analysis revealed that the Korean strains contained significant variations in the homeodomain of A locus, even within the same A1 or A2 mating type. Similarly, B locus was also highly diversified in the sequences of pheromones and receptors as well as gene organization. These results enabled us to design mating type-specific probes which can distinguish mating type of each strain. The specificity was confirmed by between intra- and inter-strain mating experiment.

• 한국균학회지
• /
• 제49권4호
• /
• pp.487-495
• /
• 2021

표고는 사극성의 교배계를 갖는 담자균의 일종으로, 표고의 교배형은 A와 B라 불리는 서로 독립된 두 유전자좌에 의해 결정된다. 이론적으로 하나의 이핵균주는 네 개의 서로 다른 교배형을 갖는 담자포자를 1:1:1:1의 비율로 만들 수 있다. 과거 연구 결과에 따르면, 표고 담자포자에서 교배형이 편향된 분리비로 나타남이 보고되었다. 하지만 이러한 결과들은 꺽쇠연결과 같은 형태학적 특성만을 기반으로 교배형을 결정했다는 한계가 있다. 이에 본 연구에서는 교배형의 편향된 분리비가 표고에서 일반적인 현상인지 보다 명확하게 알아보기 위해서 최근에 보고된 DNA 마커를 활용하여 세 가지 표고 품종들의 담자포자에 대한 교배형 분석을 수행하였다. 그 결과 교배형의 편향된 분리비가 과거 보고와 일치하게 균주 특이적인 특성임을 확인하였다. 분석한 세 품종 중 한 품종을 제외하고 나머지 두 품종에서 편향된 분리비가 관찰된 것이다. 다음으로는 각 담자포자 유래 단핵균사들의 생장 속도와 교배형의 상관관계를 분석하였다. 그 결과 표고 단핵균사의 생장 속도는 B 교배형과는 관계가 없고, A 교배형과 관계가 있음을 확인하였다. 따라서 A 교배형 유전자좌에 존재하는 호메오도메인 전사인자 혹은 A 교배형 유전자좌와 연관된 유전자들이 단핵균사의 생장에 영향을 줄 것으로 보인다. 버섯 신품종 육성에서 교배형의 중요성을 고려할 때, 본 연구는 효율적인 신품종 육성 전략을 세우거나 단핵균사 생장 기작을 이해하는 데 도움을 줄 것으로 기대된다.

• Journal of Microbiology and Biotechnology
• /
• 제22권9호
• /
• pp.1177-1184
• /
• 2012

In order to estimate how diverse the mating types in Pleurotus eryngii from different regions are, pairings between monokaryons derived from inter- and intra-groups were done. Sixteen and 15 alleles were identified at loci A and B from the 12 strains. In the P. eryngii KNR2312, widely used for commercial production, four mating loci, A3, A4, B3, and B4, were determined. Those loci, except A3, were found in 4 strains out of 12 strains. To improve breeding efficiency, especially in mating type determination, RAPD and BSA were performed to screen for a mating type specific marker. The SCAR marker 13-$2_{2100}$ was developed based on the RAPD-derived sequence typing B3 locus. The sequence analysis of 13-$2_{2100}$ revealed that it contained a conserved domain, the STE3 super-family, and consensus sequences like the TATA box and GC box. It seems likely that the SCAR marker region is a part of the pheromone receptor gene.

• 한국균학회소식:학술대회논문집
• /
• 한국균학회 2015년도 추계학술대회 및 정기총회
• /
• pp.35-35
• /
• 2015

Pleurotus eryngii has recently become a major cultivated mushroom; it uses tetrapolar heterothallism as a part of its reproductive process. Sexual development progresses only when the A and B mating types are compatible. Such mating incompatibility occasionally limits the efficiency of breeding programs in which crossing within loci-shared strains or backcrossing strategies are employed. Therefore, understanding the mating system in edible mushroom fungi will help provide a short cut in the development of new strains. We isolated and identified pheromone and receptor genes in the B3 locus of P. eryngii and performed a functional analysis of the genes in the mating process by transformation. A genomic DNA library was constructed to map the entire mating-type locus. The B3 locus was found to contain four pheromone precursor genes and four receptor genes. Remarkably, receptor PESTE3.3.1 has just 34 amino acid residues in its C-terminal cytoplasmic region; therefore, it seems likely to be a receptor-like gene. Real-time quantitative RT-PCR (real-time qRT-PCR) revealed that most pheromone and receptor genes showed significantly higher expression in monokaryotic cells than dikaryotic cells. The pheromone genes PEphb3.1 and PEphb3.3 and the receptor gene PESTE3.3.1 were transformed into P5 (A3B4). The transformants were mated with a tester strain (A4B4), and the progeny showed clamp connections and a normal fruiting body, which indicates the proposed role of these genes in mating and fruiting processes. This result also confirms that PESTE3.3.1 is a receptor gene. In this study, we identified pheromone and receptor genes in the B3 locus of P. eryngii and found that some of those genes appear to play a role in the mating and fruiting processes. These results might help elucidate the mechanism of fruiting differentiation and improve breeding efficiency.

• Mycobiology
• /
• 제43권1호
• /
• pp.24-30
• /
• 2015

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.

• 한국균학회지
• /
• 제48권4호
• /
• pp.389-396
• /
• 2020

표고는 한국에서 가장 많이 소비되는 식용버섯 중 하나이다. 표고는 사극성의 교배계를 따르며, 표고의 교배형은 자웅이주성의 다른 담자균류와 마찬가지로 서로 독립적인 두 유전자좌, A와 B에 의해 결정된다. 표고의 A 유전자좌에는 한 쌍의 homeodomain (HD) 전사인자가 암호화되어 있으며 이들의 N말단에서 나타나는 높은 변이가 A 교배형의 다양성에 중요한 것으로 알려져 있다. 본 연구에서는 표고 품종과 야생종에서 많이 발견되는 11종의 A 교배형을 구분할 수 있는 CAPS 마커를 개발하고자 하였다. A 유전자좌에서 변이가 큰 부분을 PCR을 통해 증폭한 뒤 두 가지 제한효소 HaeIII와 EcoRI로 절단하여 DNA 단편의 크기 및 양상을 살펴봄으로써 11종의 A 교배형을 서로 구별할 수 있었다. 또한 해당 방법이 이핵균주의 교배형을 확인하는 데 활용할 수 있는지도 살펴보았다.

• Mycobiology
• /
• 제47권2호
• /
• pp.200-206
• /
• 2019

Allelic differences in A and B mating-type loci are a prerequisite for the progression of mating in the genus Pleurotus eryngii; thus, the crossing is hampered by this biological barrier in inbreeding. Molecular markers linked to mating types of P. eryngii KNR2312 were investigated with randomly amplified polymorphic DNA to enhance crossing efficiency. An A4-linked sequence was identified and used to find the adjacent genomic region with the entire motif of the A locus from a contig sequenced by PacBio. The sequence-characterized amplified region marker $7-2_{299}$ distinguished A4 mating-type monokaryons from KNR2312 and other strains. A BLAST search of flanked sequences revealed that the A4 locus had a general feature consisting of the putative HD1 and HD2 genes. Both putative HD transcription factors contain a homeodomain sequence and a nuclear localization sequence; however, valid dimerization motifs were found only in the HD1 protein. The ACAAT motif, which was reported to have relevance to sex determination, was found in the intergenic region. The SCAR marker could be applicable in the classification of mating types in the P. eryngii breeding program, and the A4 locus could be the basis for a multi-allele detection marker.