Biology subjects

Lee, Y.-Y.

Publications and source records attributed to Lee, Y.-Y..

8 recordsLinked to original sources

bioRxiv2025

Establishment of a Congenic Strain for the Oyster mushroom reveals the Structure and Evolution of Mating-Type Loci

Pleurotus ostreatus, a widely cultivated edible oyster mushroom, is an ecologically versatile species with applications in biotechnology, agriculture, and food production. It functions as a decomposer and in nutrient-limited conditions it enhances its survival by using a potent toxin to prey on nematodes. Its adaptability is further regulated by sexual reproduction, which follows a tetrapolar mating system governed by two unlinked, multiallelic loci, matA and matB. The two mating-compatible monokaryotic strains PC9 and PC15, derived from the parental dikaryon strain N001, exhibit significant physiological differences. PC9 grows robustly in laboratory conditions, whereas PC15 grows more slowly, making PC9 the preferred strain for research. To advance P. ostreatus as a genetic model, we characterized the mating-type (MAT) loci of both monokaryon strains and developed a congenic strain. We analyzed the MAT loci in multiple P. ostreatus strains, and identified 11 A and 12 B alleles among twelve haplotypes, confirming their multiallelic nature. Using genetic backcrosses, we introgressed the matA and matB loci from PC15 into the PC9 genetic background to generate the congenic strain PC9.15. After sequencing and assembling a high-quality and contiguous genome for PC9.15, we confirmed that the genomes of PC9.15 and PC9 are 99% similar, with the only major difference placed at the matA and matB loci. Author SummaryMushrooms are not just decomposers-they can also be predators. The oyster mushroom Pleurotus ostreatus is an ecologically adaptable species that thrives by breaking down plant material and preying on nematodes using a potent toxin that triggers rapid paralysis of nematode prey. In addition to its ability to switch from saprophyte to predator, another feature contributing to the ecological success of Pleurotus is its reproductive strategy. P. ostreatus reproduces sexually through a tetrapolar mating-type system, governed by two unlinked mating loci, matA and matB. These loci determine mating compatibility and contribute to genetic diversity, yet their precise structure and allelic diversity remain unclear. In this study, we characterized the matA and matB loci in P. ostreatus strains PC9 and PC15 and analyzed MAT loci across multiple strains, confirming their multiallelic nature. To enhance P. ostreatus as a genetic model, we developed the congenic strain PC9.15 by introgressing MAT alleles from PC15 into the PC9 genetic background through genetic backcrosses. Genome sequencing confirmed that PC9.15 closely matches the genome of PC9, except at the MAT loci. This study expands our understanding of mating-type diversity in P. ostreatus, introduces PC9.15 as a valuable tool for genetic studies and controlled crosses, and facilitates the development of P. ostreatus as a model system for fungal biology.

genetics↗

bioRxiv2025

Directed evolution of compact RNA-guided nucleases for homology-directed repair in mammalian cells

RNA-guided nucleases enable DNA editing and offer promise for treating genetic diseases, particularly when used for precise sequence replacement. However, many of the most effective enzymes, such as S. pyogenes Cas9, are too large for delivery using vectors like adeno-associated virus (AAV). This has prompted interest in smaller alternatives from the Cas12f and TnpB families. Yet, these nucleases often show low activity in mammalian cells, limiting their utility. To address this, we used directed evolution in human cells to select variants with greatly improved activity. The resulting variants, Cas12f1Super and TnpBSuper, exhibited up to 11-fold increase in editing efficiency without increased off-target effects. When tested as a base editor, Cas12f1Super showed up to 10-fold improvement relative to the previously engineered CasMINI, suggesting utility beyond nuclease-related activities. These compact and efficient genome editors expand the current toolkit and hold promise for both research and therapeutic use in mammalian systems.

bioengineering↗

bioRxiv2021

Genetic compensation of triacylglycerol biosynthesis in the green microalga Chlamydomonas reinhardtii

Genetic compensation through transcriptional adaptation has been proposed to explain phenotypic differences between gene knockouts and knockdowns. With the rapid development of reverse genetic tools such as CRISPR/Cas9 and RNAi in microalgae, it is increasingly important to assess whether genetic compensation affects the phenotype of engineered algal mutants. While exploring triacylglycerol (TAG) biosynthesis pathways in Chlamydomonas reinhardtii, it was discovered that knockout of certain genes catalyzing rate-limiting steps of TAG biosynthesis, type-2 diacylglycerol acyltransferase genes (DGTTs), triggered genetic compensation under abiotic stress conditions. Genetic compensation of a DGTT1 null mutation by a related PDAT gene was observed regardless of the strain background or mutagenesis approach, e.g., CRISPR/Cas 9 or insertional mutagenesis. However, no compensation was found in the PDAT knockout mutant. The effect of PDAT knockout was evaluated in a {Delta}vtc1 mutant, in which PDAT was up-regulated under stress. Knockout of PDAT in the {Delta}vtc1 background induced a 12.8-fold upregulation of DGTT1 and a 272.3% increase in TAG content in {Delta}vtc1/pdat1 cells, while remaining viable. These data suggest that genetic compensation contributes to the genetic robustness of microalgal TAG biosynthetic pathways, maintaining lipid and redox homeostasis in the knockout mutants under abiotic stress with a mechanism distinct from that found in plants. This work demonstrates examples of genetic compensation in microalgae, implies the physiological relevance of genetic compensation in TAG biosynthesis, and provides guidance for future genetic engineering and mutant characterization efforts.

plant biology↗

bioRxiv2020

Forward genetic screens identified mutants with defects in trap morphogenesis in the nematode-trapping fungus Arthrobotrys oligospora

Nematode-trapping fungi (NTF) are carnivorous fungi that prey on nematodes under nutrient-poor conditions via specialized hyphae that function as traps. The molecular mechanisms involved in the interactions between nematode-trapping fungi and their nematode prey are largely unknown. In this study, we conducted forward genetic screens to identify potential genes and pathways that are involved in trap morphogenesis and predation in the NTF Arthrobotrys oligospora. Using Ethyl methanesulfonate and UV as the mutagens, we generated 5552 randomly-mutagenized A. oligospora strains and identified 15 mutants with strong defects in trap morphogenesis. Whole genome sequencing and bioinformatic analyses revealed mutations in genes with roles in signaling, transcription or membrane transport that may contribute to the defects of trap morphogenesis in these mutants. We further conducted functional analyses on a candidate gene, YBP-1, and demonstrate that mutation of that gene was causative of the phenotypes observed in one of the mutants. The methods established in this study might provide helpful insights for establishing forward genetic screening methods for other non-model fungal species.

molecular biology↗

bioRxiv2020

Genome sequence of the oyster mushroom Pleurotus ostreatus strain PC9

The oyster mushroom Pleurotus ostreatus is a basidiomycete commonly found in the rotten wood and it is one of the most cultivated edible mushrooms globally. P. ostreatus is also a carnivorous fungus, which can paralyze and kill nematodes within minutes. However, the molecular mechanisms of the predator-prey interactions between P. ostreatus and nematodes remain unclear. PC9 and PC15 are two model strains of P. ostreatus and the genomes of both strains have been sequenced and deposited at the Joint Genome Institute (JGI). These two monokaryotic strains exhibit dramatic differences in growth, but because PC9 grows more robustly in laboratory conditions, it has become the strain of choice for many studies. Despite the fact that PC9 is the common strain for investigation, its genome is fragmentary and incomplete relative to that of PC15. To overcome this problem, we used PacBio long reads and Illumina sequencing to assemble and polish a more integrated genome for PC9. Our PC9 genome assembly, distributed across 17 scaffolds, is highly contiguous and includes six telomere-to-telomere scaffolds, dramatically improving the genome quality. We believe that our PC9 genome resource will be useful to the fungal research community investigating various aspects of P. ostreatus biology.

genomics↗

bioRxiv2020

Genomic analyses of two Italian oyster mushroom Pleurotus pulmonarius strains

Pleurotus mushrooms are among the most cultivated fungi in the world and are highly valuable for food, medicine and biotechnology industries. Furthermore, Pleurotus species are carnivorous fungi; they can rapidly paralyze and kill nematodes when nutrient-deprived. The predator-prey interactions between Pleurotus and nematodes are still widely unexplored. Moreover, the molecular mechanisms and the genes involved in the carnivorous behavior of Pleurotus mushrooms remain a mystery. We are attempting to understand the interactions between Pleurotus mushrooms and their nematode prey through genetic and genomic analyses. Two single spores (ss2 and ss5) isolated from a fruiting body of Pleurotus pulmonarius exhibited significant differences in growth and toxicity against nematodes. Thus, using PacBio long reads, we assembled and annotated two high-quality genomes for these two isolates of Pleurotus pulmonarius. Each of these assemblies contains 23 scaffolds, including 6 (ss2) and 8 (ss5) telomere-to-telomere scaffolds, and they are among the most complete assembled genomes of the Pleurotus species. Comparative analyses identified the genomic differences between the two P. pulmonarius strains. In sum, this work provides a genomic resource that will be invaluable for better understanding the Italian oyster mushroom P. pulmonarius.

genomics↗

bioRxiv2020

MicroRNA arm switching regulated by uridylation

Strand selection is a critical step in microRNA (miRNA) biogenesis. Although the dominant strand may alter depending on cellular contexts, the molecular mechanism and physiological significance of such alternative strand selection (or "arm switching") remain elusive. Here we find mir-324 as one of the strongly regulated miRNAs by arm switching, and identify terminal uridylyl transferases TUT4 and TUT7 as the key regulators. Uridylation of pre-mir-324 by TUT4/7 re-positions DICER on the pre-miRNA and shifts the cleavage site. This alternative processing produces a duplex with a different terminus, from which the 3' strand (3p) is selected instead of the 5' strand (5p). In glioblastoma, the TUT4/7 and 3p levels are upregulated while the 5p level is reduced. Manipulation of the strand ratio is sufficient to impair glioblastoma cell proliferation. This study uncovers a role of uridylation as a molecular switch in alternative strand selection and implicates its therapeutic potential.

molecular biology↗

bioRxiv2018

TGFam-Finder: An optimal solution for target-gene family annotation in eukaryotic genomes

Whole genome annotation errors that omit essential protein-coding genes hinder further research. We developed Target Gene Family Finder (TGFam-Finder), an optimal tool for structural annotation of protein-coding genes containing target domain(s) of interest in eukaryotic genomes. Large-scale re-annotation of 100 publicly available eukaryotic genomes led to the discovery of essential genes that were missed in previous annotations. An average of 117 (346%) and 148 (45%) additional FAR1 and NLR genes were newly identified in 50 plant genomes. Furthermore, 117 (47%) additional C2H2 zinc finger genes were detected in 50 animal genomes including human and mouse. Accuracy of the newly annotated genes was validated by RT-PCR and cDNA sequencing in human, mouse and rice. In the human genome, 26 newly annotated genes were identical with known functional genes. TGFam-Finder along with the new gene models provide an optimized platform for unbiased functional and comparative genomics and comprehensive evolutionary study in eukaryotes.

bioinformatics↗