Effect of a Mating Type Gene Editing in Using RNP/Nanoparticle Complex.

Gene editing using CRISPR/Cas9 is an innovative tool for developing new mushroom strains, offering a promising alternative to traditional breeding methods that are time-consuming and labor-intensive. However, plasmid-based gene editing presents several challenges, including the need for selecting appropriate promoters for Cas9 expression, optimizing codons for the Cas9 gene, the unintended insertion of fragmented plasmid DNA into genomic DNA (gDNA), and regulatory concerns related to genetically modified organisms (GMOs). To address these issues, we utilized a Ribonucleoprotein (RNP) complex consisting of Cas9 and gRNA for gene editing to modify the A mating-type gene of .

The Cas9-gRNA ribonucleoprotein complex-mediated editing of pyrG in Ganoderma lucidum and unexpected insertion of contaminated DNA fragments.

Gene editing is a promising alternative to traditional breeding for the generation of new mushroom strains. However, the current approach frequently uses Cas9-plasmid DNA to facilitate mushroom gene editing, which can leave residual foreign DNA in the chromosomal DNA raising concerns regarding genetically modified organisms. In this study, we successfully edited pyrG of Ganoderma lucidum using a preassembled Cas9-gRNA ribonucleoprotein complex, which primarily induced a double-strand break (DSB) at the fourth position prior to the protospacer adjacent motif.

SNP-Based Genetic Linkage Map and Quantitative Trait Locus Mapping Associated with the Agronomically Important Traits of .

White strains of are more difficult to cultivate than are brown strains; therefore, new white strain breeding strategies are required. Accordingly, we constructed the genetic map of with 1996 SNP markers on 11 linkage groups (LGs) spanning 1380.49 cM.

Growth Characteristics of Polyporales Mushrooms for the Mycelial Mat Formation.

Mushroom strains of Polyporales from the genera , , , , and 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, 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.

Generation of Iron-Independent Siderophore-Producing through the Constitutive Expression of .

secretes siderophore to uptake environmental iron. Siderophore secretion in was enabled only in the iron-free minimal medium due to iron repression of , a transcriptional activator of siderophore biosynthetic genes. Aiming to produce siderophore using conventional iron-containing complex media, we constructed a recombinant strain of that escapes gene repression.

Investigation of Mating Pheromone-Pheromone Receptor Specificity in .

The mating-type locus of , a representative edible mushroom, is highly complex because of allelic variations in the mating pheromone receptors (s) and the mating pheromones (s) in both the and subloci. The complexity of the mating-type locus, five subloci with five alleles of and nine and three subloci with 3 alleles of and five s, has led us to investigate the specificity of the PHB-RCB interaction because the interaction plays a key role in non-self-recognition. In this study, the specificities of PHBs to RCB1-2 and RCB1-4 from the sublocus and RCB2-1 from the sublocus were investigated using recombinant yeast strains generated by replacing , an endogenous yeast mating pheromone receptor, with the s.

Variable Number Tandem Repeats in the Mitochondrial DNA of .

Variable number tandem repeats (VNTRs) in mitochondrial DNA (mtDNA) of are of interest for their role in mtDNA variation and their application as genetic marker. Sequence analysis of three mtDNAs revealed the presence of VNTRs of two categories. Type I VNTRs consist of two types of repeat units in a symmetric distribution, whereas Type II VNTRs contain tandemly arrayed repeats of 7- or 17-bp DNA sequences.

Activation of the Mating Pheromone Response Pathway of by Synthetic Pheromones.

Pheromone (PHB)-receptor (RCB) interaction in the mating pheromone response pathway of was investigated using synthetic PHBs. Functionality of the C-terminally carboxymethylated synthetic PHBs was demonstrated by concentration-dependent induction of a mating-related gene () expression and by pseudoclamp formation in a monokaryotic strain S1-11 of . Treatment with synthetic PHBs activated the expression of homeodomain genes (s) residing in the mating type locus, and of -regulated genes, including , , and , as well as genes in the mating type locus, including pheromone () and receptor () genes.

Diversity of A mating type in Lentinula edodes and mating type preference in the cultivated strains.

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.

Nucleus-Selective Expression of Laccase Genes in the Dikaryotic Strain of .

In mating of , 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 , suggesting an expression bias in the allelic genes of the two nuclei.

Differential Expression of Laccase Genes in Pleurotus ostreatus and Biochemical Characterization of Laccase Isozymes Produced in Pichia pastoris.

In this study, transcriptome analysis of twelve laccase genes in Pleurotus ostreatus revealed that their expression was differentially regulated at different developmental stages. Lacc5 and Lacc12 were specifically expressed in fruiting bodies and primordia, respectively, whereas Lacc6 was expressed at all developmental stages. Lacc1 and Lacc3 were specific to the mycelial stage in solid medium.