Monokaryotic Pleurotus sapidus Strains with Intraspecific Variability of an Alkene Cleaving DyP-Type Peroxidase Activity as a Result of Gene Mutation and Differential Gene Expression.

The basidiomycete Pleurotus sapidus produced a dye-decolorizing peroxidase (PsaPOX) with alkene cleavage activity, implying potential as a biocatalyst for the fragrance and flavor industry. To increase the activity, a daughter-generation of 101 basidiospore-derived monokaryons (MK) was used. After a pre-selection according to the growth rate, the activity analysis revealed a stable intraspecific variability of the strains regarding peroxidase and alkene cleavage activity of PsaPOX. Ten monokaryons reached activities up to 2.6-fold higher than the dikaryon, with MK16 showing the highest activity. Analysis of the PsaPOX gene identified three different enzyme variants. These were co-responsible for the observed differences in activities between strains as verified by heterologous expression in Komagataella phaffii. The mutation S371H in enzyme variant PsaPOX_high caused an activity increase alongside a higher protein stability, while the eleven mutations in variant PsaPOX_low resulted in an activity decrease, which was partially based on a shift of the pH optimum from 3.5 to 3.0. Transcriptional analysis revealed the increased expression of PsaPOX in MK16 as reason for the higher PsaPOX activity in comparison to other strains producing the same PsaPOX variant. Thus, different expression profiles, as well as enzyme variants, were identified as crucial factors for the intraspecific variability of the PsaPOX activity in the monokaryons.

Agrobacterium-mediated insertional mutagenesis in the mycorrhizal fungus Laccaria bicolor.

Agrobacterium-mediated gene transfer (AMT) is extensively employed as a tool in fungal functional genomics and accordingly, in previous studies we used AMT on a dikaryotic strain of the ectomycorrhizal basidiomycete Laccaria bicolor. The interest in this fungus derives from its capacity to establish a symbiosis with tree roots, thereby playing a major role in nutrient cycling of forest ecosystems. The ectomycorrhizal symbiosis is a highly complex interaction involving many genes from both partners. To advance in the functional characterization of fungal genes, AMT was used on a monokaryotic L. bicolor. A collection of over 1200 transgenic strains was produced, of which 200 randomly selected strains were analyzed for their genomic T-DNA insertion patterns. By means of insertional mutagenesis, a number of transgenic strains were obtained displaying differential growth features. Moreover, mating with a compatible strain resulted in dikaryons that retained altered phenotypic features of the transgenic monokaryon. The analysis of the T-DNA integration pattern revealed mostly similar results to those reported in earlier studies, confirming the usefulness of AMT on different genetic backgrounds of L. bicolor. Taken together, our studies display the great versatility and potentiality of AMT as a tool for the genetic characterization of L. bicolor.

Morphological and molecular characterization of yellow oyster mushroom, Pleurotus citrinopileatus, hybrids obtained by interspecies mating.

Pleurotus citrinopileatus (yellow oyster mushroom) has an attractive shape and yellow colour but the fragile texture complicates packaging, and its strong aroma is unappealing to consumers. This study aimed to improve the characteristics and yield of P. citrinopileatus by interspecies mating between monokaryotic cultures of P. citrinopileatus and P. pulmonarius. Ten monokaryon cultures of the parental lines were crossed in all combinations to obtain hybrids. Eleven compatible mating pairs were obtained and cultivated to observe their sporophore morphology and yield. The selected hybrid, i.e. P1xC9, was beige in colour while hybrid P3xC8 was yellow in colour. Their sporophores had less offensive aroma, improved texture and higher yield. The DNA sequences of these hybrids were found to be in the same clade as the P. citrinopileatus parent with a bootstrap value of 99%. High bootstrap values indicate high genetic homology between hybrids and the P. citrinopileatus parent. The biological efficiencies of these hybrids P1xC9 (70.97%) and P3xC8 (52.14%) were also higher than the P. citrinopileatus parent (35.63%). Interspecies hybrids obtained by this mating technique can lead to better strains of mushrooms for genetic improvement of the Pleurotus species.

Pathogenicity of monokaryotic and dikaryotic mycelia of Ganoderma boninense revealed via LC-MS-based metabolomics.

This study compared the pathogenicity of monokaryotic (monokaryon) and dikaryotic (dikaryon) mycelia of the oil palm pathogen Ganoderma boninense via metabolomics approach. Ethyl acetate crude extracts of monokaryon and dikaryon were analysed by liquid chromatography quadrupole/time-of-flight-mass spectrometry (LC-Q/TOF-MS) coupled with multivariate data analysis using MetaboAnalyst. The mummichog algorithm was also used to identify the functional activities of monokaryon and dikaryon without a priori identification of all their secondary metabolites. Results revealed that monokaryon produced lesser fungal metabolites than dikaryon, suggesting that monokaryon had a lower possibility of inducing plant infection. These findings were further supported by the identified functional activities. Monokaryon exhibits tyrosine, phenylalanine, and tryptophan metabolism, which are important for fungal growth and development and to produce toxin precursors. In contrast, dikaryon exhibits the metabolism of cysteine and methionine, arginine and proline, and phenylalanine, which are important for fungal growth, development, virulence, and pathogenicity. As such, monokaryon is rendered non-pathogenic as it produces growth metabolites and toxin precursors, whereas dikaryon is pathogenic as it produces metabolites that are involved in fungal growth and pathogenicity. The LC-MS-based metabolomics approach contributes significantly to our understanding of the pathogenesis of Ganoderma boninense, which is essential for disease management in oil palm plantations.

Whole Genome Sequence of an Edible Mushroom Oudemansiella raphanipes (Changgengu).

Oudemansiella raphanipes, considered as a well-known culinary edible mushroom with a high content of natural bioactive substances, is widely cultivated in China with the commercial name Changgengu. However, due to the lack of genomic data, molecular and genetic study on O. raphanipes is rare. To obtain a comprehensive overview of genetic characteristics and enhance the value of O. raphanipes, two mating-compatible monokaryons isolated from the dikaryon were applied for de novo genome sequencing and assembly using Nanopore and /or Illumina sequencing platforms. One of the monokaryons, O. raphanipes CGG-A-s1, was annotated with 21,308 protein-coding genes, of which 56 were predicted to be involved in the biosynthesis of secondary metabolites such as terpene, type I PKS, NRPS, and siderophore. Phylogenetic and comparative analysis of multiple fungi genomes revealed a close evolutionary relationship between O. raphanipes and Mucidula mucid based on single-copy orthologous protein genes. Significant collinearity was detected between O. raphanipes and Flammulina velutipes on the synteny of inter-species genomes. 664 CAZyme genes in CGG-A-s1 were identified with GHs and AAs families significantly elevated when compared with the other 25 sequenced fungi, indicating a strong wood degradation ability. Furthermore, the mating type locus analysis revealed that CGG-A-s1 and CGG-A-s2 were conserved in the gene organization of the mating A locus but various in that of the mating B locus. The genome resource of O. raphanipes will provide new insights into its development of genetic studies and commercial production of high-quality varieties.

Comprehensive Genetic Analysis of Monokaryon and Dikaryon Populations Provides Insight Into Cross-Breeding of Flammulina filiformis.

Flammulina filiformis, as one of the most popular edible fungi in East Asia, is produced in an industrialized and standardized way. However, its monotonous variety and product convergence have seriously restricted the development of the industry. In this study, 11 cultivated strains and 13 wild strains of F. filiformis were collected from multiple regions of China and Japan and were performed genome sequencing. Together with genome data of six strains previously released, in total 23 dikaryons (formed by two monokaryons mating, can making fruiting body), 35 monokaryons (formed by protoplast-regenerating of dikaryon and isolating) were used for genetic diversity and population structure analysis based on the high-throughput genotyping. Firstly, a set of SNP markers with intrapopulation polymorphism including 849,987 bi-allelic SNPs were developed and basically covered all of 11 chromosomes with a high distribution density of 24.16 SNP markers per kb. The cultivated dikaryotic strains were divided into three subgroups, and their breeding history was made inferences, which is consistent with the available pedigree records. The wild dikaryotic strains were divided into two subgroups and showed varied contributions of genetic components with high genetic diversity. All the investigated dikaryons have a symmetric distribution pattern with their two constituent monokaryons in principal component analysis. Finally, we summarized the pedigree relationship diagram of F. filiformis main strains including six modules, and the genotypes of hybrids can be directly phased by the known parental allele according to it. This study provides a method to distinguish two sets of monokaryon haplotypes, and several valuable genetic resources of wild F. filiformis, and an effective strategy for guiding F. filiformis breeding based on the population structure and pedigree relationship in future.

A Resequencing-Based Ultradense Genetic Map of Hericium erinaceus for Anchoring Genome Sequences and Identifying Genetic Loci Associated With Monokaryon Growth.

Hericium erinaceus has attracted tremendous interest owing to its compelling health-promoting properties. However, breeding of elite cultivars of H. erinaceus is hindered by the lack of a genetic and molecular toolbox. Here, we performed resequencing analysis of 127 F1 single-spore isolates and constructed the first high-resolution genetic map of H. erinaceus. With the use of recombination bins as markers, an ultradense genetic map consisting of 1,174 bins (including 37,082 single-nucleotide polymorphisms) was generated. This newly developed genetic map covered 1,096.5 cM, with an average bin spacing of 0.95 cM. High collinearity between genetic map and H. erinaceus genome assembly was revealed by aligning scaffolds to this genetic map using bin markers as anchors. The application of this newly developed genetic map in quantitative trait locus (QTL) mapping was also elucidated, and four QTLs for monokaryon growth were recovered. One QTL, mgr1, which contributes 12.1% of growth variations, was located near the mating type A (MAT-A) loci. Overall, this newly constructed high-resolution genetic map (or bin map) could be used as reference in future genetic, genomic, and breeding studies on H. erinaceus.

Biorefining via solid-state fermentation of rice and sunflower by-products employing novel monosporic strains from Pleurotus sapidus.

Pleurotus sapidus monokaryotic strains (Mk) were screened as a novel source of mycelia to valorize rice straw (RS), rice husks (RH) and sunflower seed hulls (SSH) into value-added products through solid-state fermentation (SSF). P. sapidus Dk3174 basidiospores were cultured in the presence of Remazol Brillant Blue R for strain selection, revealing the ligninolytic ability of emerging colonies. Further screening demonstrated the intraspecific variability in dye degradation and enzyme production of 63 strains. Growth rate, biomass and enzyme production in plates containing RS, RH or SSH pointed at MkP6 as a suitable strain for pilot-scale SSF. MkP6 presented a similar laccase profile as the parental Dk3174, being greater in pasteurized substrates (300-1200 U/Kg) than in sterilized substrates (30-250 U/Kg). Peroxidase represented 25% of the total ligninolytic activity measured. The SSH fermented biomass with MkP6 obtained good yields of nanocellulose (67%) and the saccharide release for ethanol production increased by 3-4 times.

Functional diversity in Dichomitus squalens monokaryons.

Dichomitussqualens is a white-rot fungus that colonizes and grows mainly on softwood and is commonly found in the northern parts of Europe, North America, and Asia. We analyzed the genetic and physiological diversity of eight D. squalens monokaryons derived from a single dikaryon. In addition, an unrelated dikaryon and a newly established dikaryon from two of the studied monokaryons were included. Both growth and lignocellulose acting enzyme profiles were highly variable between the studied monokaryotic and dikaryotic strains, demonstrating a high level of diversity within the species.

Development of Novel Polymorphic EST-SSR Markers in Bailinggu (Pleurotus tuoliensis) for Crossbreeding.

Identification of monokaryons and their mating types and discrimination of hybrid offspring are key steps for the crossbreeding of Pleurotus tuoliensis (Bailinggu). However, conventional crossbreeding methods are troublesome and time consuming. Using RNA-seq technology, we developed new expressed sequence tag-simple sequence repeat (EST-SSR) markers for Bailinggu to easily and rapidly identify monokaryons and their mating types, genetic diversity and hybrid offspring. We identified 1110 potential EST-based SSR loci from a newly-sequenced Bailinggu transcriptome and then randomly selected 100 EST-SSRs for further validation. Results showed that 39, 43 and 34 novel EST-SSR markers successfully identified monokaryons from their parent dikaryons, differentiated two different mating types and discriminated F1 and F2 hybrid offspring, respectively. Furthermore, a total of 86 alleles were detected in 37 monokaryons using 18 highly informative EST-SSRs. The observed number of alleles per locus ranged from three to seven. Cluster analysis revealed that these monokaryons have a relatively high level of genetic diversity. Transfer rates of the EST-SSRs in the monokaryons of closely-related species Pleurotuseryngii var. ferulae and Pleurotus ostreatus were 72% and 64%, respectively. Therefore, our study provides new SSR markers and an efficient method to enhance the crossbreeding of Bailinggu and closely-related species.

Genome-wide gene expression patterns in dikaryon of the basidiomycete fungus Pleurotus ostreatus.

Dikarya is a subkingdom of fungi that includes Ascomycota and Basidiomycota. The gene expression patterns of dikaryon are poorly understood. In this study, we bred a dikaryon DK13×3 by mating monokaryons MK13 and MK3, which were from the basidiospores of Pleurotus ostreatus TD300. Using RNA-Seq, we obtained the transcriptomes of the three strains. We found that the total transcript numbers in the transcriptomes of the three strains were all more than ten thousand, and the expression profile in DK13×3 was more similar to MK13 than MK3. However, the genes involved in macromolecule utilization, cellular material synthesis, stress-resistance and signal transduction were much more up-regulated in the dikaryon than its constituent monokaryons. All possible modes of differential gene expression, when compared to constituent monokaryons, including the presence/absence variation, and additivity/nonadditivity gene expression in the dikaryon may contribute to heterosis. By sequencing the urease gene poure sequences and mRNA sequences, we identified the monoallelic expression of the poure gene in the dikaryon, and its transcript was from the parental monokaryon MK13. Furthermore, we discovered RNA editing in the poure gene mRNA of the three strains. These results suggest that the gene expression patterns in dikaryons should be similar to that of diploids during vegetative growth.

Callose deposition during the interaction between cowpea (Vigna unguiculata) and the monokaryotic stage of the cowpea rust fungus (Uromyces vignae).

The interaction between the race 1 of the cowpea rust fungus (Uromyces vignae Barclay) and the resistant cowpea (Vigna unguiculata (L.) Walp.) ev. Queen Anne is characterized by the deposition of callose around intracellular fungal structures. Ultrastructural examination of the early stages of infection by basidiospores of the fungus revealed two types of deposits induced by intracellular hyphae: a non-callose collar at the penetration site and a callose encasement around the invasion hypha. The callose encasement was developed from the site where the fungus encountered the inside of the plant cell wall and was separated from the fungus by the extrahyphal membrane and an extension of plant plasma membrane. The incidence of encasements was reduced in plants treated with inhibitors of transcription (actinomycin D), protein synthesis (cycloheximide), protein glycosylation (tunicamycin) and microfilament polymerization (cytochalasin E). Inhibitors of Golgi-associated vesicle transfer (monensin, brefeldin A) and anti-microtubule agents (colchicine, oryzalin) had no effect. When the fungus was killed by heat treatment in either the resistant or the susceptible cultivar, callose was deposited at various locations along the fungus, mostly in the extrahyphal matrix. The data suggest that unlike the extrahaustorial membrane surrounding D-haustoria of this fungus, the extrahyphal membrane is capable of generating callose. Since this process does not normally occur in the susceptible or the resistant cv. even when callose is deposited in the latter by regions of the plasma membrane not associated with the fungus, we conclude that the deposition of callose by the extrahyphal membrane is inhibited by the living fungus.

Genetic variation of nitrate reductase activity in mono- and dikaryotic populations of the ectomycorrhizal fungus, Hebeloma cylindrosporum Romagnési.

Intraspecific variability in the activity of nitrate reductase (NR) has been studied in the ectomycorrhizal fungus Hebeloma cylindrosporum Romagnési at the interstrain and intrastrain levels, the latter within a population of 11 wild dikaryotic strains collected in'Les Landes'(SW France) at four locations less than 100 km away from one another. An attempt was made to determine whether variability within the monokaryotic and dikaryotic progeny of the HC1 fruiting strain could be used as a basis for an improvement programme involving breeding between selected monokaryons. The NR activity of the wild strains ranged from 201 to 700 nmol NO2 synthesized h-1 mg -1 fungal protein whilst that of 20 sib-monokaryons (5 per mating type) of the HCl strain varied from 51 to 510 nmol NO2 synthesized h -1 mg-1 fungal protein. Fifty controlled dikaryotic myeclia were obtained from all the compatible fusions with these monokaryons. In these, variation of NR activity was of the same order of magnitude as that recorded at the interstrain level, ranging from 72 to 689 nmol NO2 synthesized h -1 rag -1 fungal protein. Analysis of the components of the variation of NR activity in these controlled dikaryons demonstrated that the additive component of this variation accounted for less than 1 % of the total observed variation. The NR activity of any one controlled dikaryon could not therefore be predicted from the activity of its parental monokaryons However 14 of the 50 controlled dikaryons e8hibited an NR activity higher than that of the HCl parental dikaryon. These results indicate that breeding with sib-monokaryons can be used as a basis for an improvement programme of NR activity in this ectomycorrhizal Basidiomycete.

Crosses between monokaryons of Pleurotus sapidus or Pleurotus florida show an improved biotransformation of (+)-valencene to (+)-nootkatone.

Several hundred monokaryotic and new dikaryotic strains derived thereof were established from (+)-valencene tolerant Pleurotus species. When grouped according to their growth rate on agar plates and compared to the parental of Pleurotus sapidus 69, the slowly growing monokaryons converted (+)-valencene more efficiently to the grapefruit flavour compound (+)-nootkatone. The fast growing monokaryons and the slow×slow and the fast×fast dikaryotic crosses showed similar or inferior yields. Some slow×fast dikaryons, however, exceeded the biotransformation capability of the parental dikaryon significantly. The activity of the responsible enzyme, lipoxygenase, showed a weak correlation with the yields of (+)-nootkatone indicating that the determination of enzyme activity using the primary substrate linoleic acid may be misleading in predicting the biotransformation efficiency. This exploratory study indicated that a classical genetics approach resulted in altered and partly improved terpene transformation capability (plus 60%) and lipoxygenase activity of the strains.

Genome-wide gene expression patterns in dikaryon of the basidiomycete fungus Pleurotus ostreatus

Abstract Dikarya is a subkingdom of fungi that includes Ascomycota and Basidiomycota. The gene expression patterns of dikaryon are poorly understood. In this study, we bred a dikaryon DK13 × 3 by mating monokaryons MK13 and MK3, which were from the basidiospores of Pleurotus ostreatus TD300. Using RNA-Seq, we obtained the transcriptomes of the three strains. We found that the total transcript numbers in the transcriptomes of the three strains were all more than ten thousand, and the expression profile in DK13 × 3 was more similar to MK13 than MK3. However, the genes involved in macromolecule utilization, cellular material synthesis, stress-resistance and signal transduction were much more up-regulated in the dikaryon than its constituent monokaryons. All possible modes of differential gene expression, when compared to constituent monokaryons, including the presence/absence variation, and additivity/nonadditivity gene expression in the dikaryon may contribute to heterosis. By sequencing the urease gene poure sequences and mRNA sequences, we identified the monoallelic expression of the poure gene in the dikaryon, and its transcript was from the parental monokaryon MK13. Furthermore, we discovered RNA editing in the poure gene mRNA of the three strains. These results suggest that the gene expression patterns in dikaryons should be similar to that of diploids during vegetative growth.