Variable number tandem repeats (VNTRs) in mitochondrial DNA (mtDNA) of Lentinula edodes are of interest for their role in mtDNA variation and their application as genetic marker. Sequence analysis of three L. edodes 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. The number of repeat units was variable depending on the mtDNA of different strains. Using the variations in VNTRs as a mitochondrial marker and the A mating type as a nuclear type marker, we demonstrated that one of the two nuclei in the donor dikaryon preferentially enters into the monokaryotic cytoplasm to establish a new dikaryon which still retains the mitochondria of the monokaryon in the individual mating. Interestingly, we found 6 VNTRs with newly added repeat units from the 22 mates, indicating that elongation of VNTRs occurs during replication of mtDNA. This, together with comparative analysis of the repeating pattern, enables us to propose a mechanistic model that explains the elongation of Type I VNTRs through reciprocal incorporation of basic repeat units, 5’-TCCCTTTAGGG-3’ and its complementary sequence (5’-CCCTAAAGGGA-3’).

Agaricus bisporus secretes siderophore to uptake environmental iron. Siderophore secretion in A. bisporus was enabled only in the iron-free minimal medium due to iron repression of hapX, a transcriptional activator of siderophore biosynthetic genes. Aiming to produce siderophore using conventional iron-containing complex media, we constructed a recombinant strain of A. bisporus that escapes hapX gene repression. For this, the A. bisporushapX gene was inserted next to the glyceraldehyde 3-phosphate dehydrogenase promoter (pGPD) in a binary vector, pBGgHg, for the constitutive expression of hapX. Transformants of A. bisporus were generated using the binary vector through Agrobacterium tumefaciens-mediated transformation. PCR and Northern blot analyses of the chromosomal DNA of the transformants confirmed the successful integration of pGPD-hapX at different locations with different copy numbers. The stable integration of pGPD-hapX was supported by PCR analysis of chromosomal DNA obtained from the 20 passages of the transformant. The transformants constitutively over-expressed hapX by 3- to 5-fold and sidD, a key gene in the siderophore biosynthetic pathway, by 1.5- to 4-fold in mRNA levels compared to the wild-type strain (without Fe³⁺), regardless of the presence of iron. Lastly, HPLC analysis of the culture supernatants grown in minimal medium with or without Fe³⁺ ions presented a peak corresponding to iron-chelating siderophore at a retention time of 5.12 min. The siderophore concentrations of the transformant T2 in the culture supernatant were 9.3-fold (−Fe³⁺) and 8-fold (+Fe³⁺) higher than that of the wild-type A. bisporus grown without Fe³⁺ ions, while no siderophore was detected in the wild-type supernatant grown with Fe³⁺. The results described here demonstrate the iron-independent production of siderophore by a recombinant strain of A. bisporus, suggesting a new application for mushrooms through molecular biological manipulation.

White strains of Hypsizygus marmoreus are more difficult to cultivate than are brown strains; therefore, new white strain breeding strategies are required. Accordingly, we constructed the genetic map of H. marmoreus with 1996 SNP markers on 11 linkage groups (LGs) spanning 1380.49 cM. Prior to analysis, 82 backcrossed strains (HM8 lines) were generated by mating between KMCC03106-31 and the progenies of the F₁ hybrid (Hami-18 × KMCC03106-93). Using HM8, the first 23 quantitative trait loci (QTLs) of yield-related traits were detected with high limit of detection (LOD) scores (1.98–9.86). The length, thickness, and hardness of the stipe were colocated on LG 1. Especially, length of stipe and thickness of stipe were highly correlated given that the correlation coefficients were negative (−0.39, p value ≤ .01). And a typical biomodal distribution was observed for lightness of the pileus and the lightness of the pileus trait belonged to the LG 8, as did traits of earliness and mycelial growth in potato dextrose agar (PDA) medium. Therefore, results for color traits can be suggested that color is controlled by a multi-gene of one locus. The yield trait was highly negatively correlated with the traits for thickness of the stipe (−0.45, p value ≤ .01). Based on additive effects, the white strain was confirmed as recessive; however, traits of mycelial growth, lightness, and quality were inherited by backcrossed HM8 lines. This new genetic map, finely mapped QTLs, and the strong selection markers could be used in molecular breeding of H. marmoreus.

The B mating-type locus of Lentinula edodes, a representative edible mushroom, is highly complex because of allelic variations in the mating pheromone receptors (RCBs) and the mating pheromones (PHBs) in both the Bα and Bβ subloci. The complexity of the B mating-type locus, five Bα subloci with five alleles of RCB1 and nine PHBs and three Bβ subloci with 3 alleles of RCB2 and five PHBs, 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 Bα sublocus and RCB2-1 from the Bb sublocus were investigated using recombinant yeast strains generated by replacing STE2, an endogenous yeast mating pheromone receptor, with the L. edodes RCBs. Fourteen synthetic PHBs with C-terminal carboxymethylation but without farnesylation were added to the recombinant yeast cells and the PHB–RCB interaction was monitored by the expression of the FUS1 gene—a downstream gene of the yeast mating signal pathway. RCB1-2 (Bα2) was activated by PHB1 (4.3-fold) and PHB2 (2.1-fold) from the Bα1 sublocus and RCB1-4 (Bα4) was activated by PHB5 (3.0-fold) and PHB6 (2.7-fold) from the Bα2 sublocus and PHB13 (3.0-fold) from the Bα5 sublocus. In particular, PHB3 from Bβ2 and PHB9 from Bβ3 showed strong activation of RCB2-1 of the Bβ1 sublocus by 59-fold. The RCB–PHB interactions were confirmed in the monokaryotic S1–10 strain of L. edodes by showing increased expression of clp1, a downstream gene of the mating signal pathway and the occurrence of clamp connections after the treatment of PHBs. These results indicate that a single PHB can interact with a non-self RCB in a sublocus-specific manner for the activation of the mating pheromone signal pathways in L. edodes.

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 Lentinula edodes. To overcome the challenges posed by the large size of the Cas9 protein, which limits its penetration through the protoplast membrane, and the susceptibility of sgRNA to degradation, we developed a nanoparticle complex using calcium phosphate and polyacrylic acid. This approach significantly improved gene editing efficiency. Consequently, we successfully edited the mating-controlling genes hd1 and hd2 in L. edodes and examined the effects of their disruption on mating. Disruption of the hd1 gene, which is known to influence mycelial growth, did not significantly affect growth or mating. In contrast, editing the hd2 gene disrupted mating with compatible partners, highlighting its critical role in the mating process. The RNP-based transformation technology presented here offers significant advancement over traditional plasmid-based methods, enhancing the efficiency of targeted gene modification while avoiding the insertion of foreign genetic material, thereby mitigating GMO-related regulatory concerns.