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. Of the 66 edited transformants, 42 had deletions ranging from a single base to large deletions of up to 796 bp, with 30 being a single base deletion. Interestingly, the remaining 24 contained inserted sequences with variable sizes at the DSB site that originated from the fragmented host mitochondrial DNA, E. coli chromosomal DNA, and the Cas9 expression vector DNA. The latter two were thought to be contaminated DNAs that were not removed during the purification process of the Cas9 protein. Despite this unexpected finding, the study demonstrated that editing G. lucidum genes using the Cas9-gRNA complex is achievable with comparable efficiency to the plasmid-mediated editing system.

Structural Analysis of the A Mating Type Locus and Development of the Mating Type Marker of Agaricus bisporus var. bisporus.

Karyotyping in Agaricus bisporus is crucial for both the isolation of homokaryotic strains and the confirmation of dikaryon establishment. For the verification of the karyotype, the A mating type loci of two homokaryotic strains, H39 and H97, were analyzed through comparative sequence analysis. The two loci showed major differences in two sequence regions designated as Region 1 and Region 2. H97 had a putative DNA transposon in Region 1 that had target site duplications (TSDs), terminal inverted repeats (TIRs), and a loop sequence, in contrast to H39, which only had the insertional target sequence. Homologous sequences of the transposon were discovered in the two different chromosomes of H97 and in one of H39, all of which have different TSDs but share high sequence homology in TIR. Region 2 shared three consensus sequences between H97 and H39. However, it was only from H97 that a large insertional sequence of unknown origin was discovered between the first and second consensus sequences. The difference in length in Region 1, employed for the verification of the A mating type, resulted in the successful verification of mating types in the heterokaryotic and homokaryotic strains. This length difference enables the discrimination between homo- and heterokaryotic spores by PCR. The present study suggests that the A mating type locus in A. bisporus H97 has evolved through transposon insertion, allowing the discrimination of the mating type, and thus the nuclear type, between A. bisporus H97 and H39.

Mitochondrial Effects on the Physiological Characteristics of Lentinula edodes.

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.