Monokariotic fruiting body and clamp cell formation in Mycoleptodonoides aitchisonii (Bunaharitake)

The ability to produce monokaryotic fruiting bodies and clamp cells in culture was examined in monokaryotic strain isolated from several dikaryotic parental strains of the edible mushroom, Mycoleptodonoides aitchisonii (Bunaharitake). We describe a single dikaryotic M. aitchisonii strain, TUFC50005, and 20 monokaryons derived from it, which exhibited a wide spectrum of monokaryotic fruiting types. Most strains formed primordia, or young fruiting body-like structures, but only one of the monokaryons, strain TUFC50005-4, formed a fruiting body, even though it had only one nucleus and produced only two spores after meiosis. We demonstrated that dikariotization was not required for clamp cell formation, fruiting body formation, or meiosis, in this mushroom.     

Dikaryotic fruiting body development in a single dikaryon of <Emphasis Type="Italic">Agrocybe aegerita</Emphasis> and the spectrum of monokaryotic fruiting types in its monokaryotic progeny

Using monokaryotic offspring from several dikaryotic parental strains, the phenomenon of monokaryotic fruiting has been previously analysed in the commercially cultivated high-quality edible mushroom Agrocybe aegerita, revealing a variety of monokaryotic fruiting types. Here, we report a single dikaryotic A. aegerita strain, A. aegerita AAE-3, and 40 monokaryons derived from it, which exhibit a wide spectrum of monokaryotic fruiting types, including a rare, previously unknown type. Advantageously, the selected parental strain A. aegerita AAE-3 completes its life cycle within three weeks by the formation of dikaryotic fruiting bodies of typical agaric morphology on malt extract agar plates. In order to morphologically compare normal dikaryotic fruiting to monokaryotic fruiting, histology was performed from all dikaryotic fruiting body development stages and all fruiting types of monokaryotic origin. No clamp connections or dikaryotic hyphae were observed within the plectenchyma of monokaryotic fruiting stages. Among the monokaryotic fruiting types of the A. aegerita AAE-3-derived monokaryons, we also characterised the rare ‘stipe type’ here described as ‘elongated initials type’ as no differentiation into a future cap and stipe was seen. The two mating-compatible monokaryotic strains representing the extremes of the fruiting type spectrum observed, A. aegerita AAE-3-13 (‘mycelium type’) and A. aegerita AAE-3-32 (‘abortive?+?true homokaryotic fruiting fruiter type, AHF?+?THF fruiter type’), were also found to readily produce oidia (arthrospores). In order to obtain a set of mating-compatible monokaryons covering the whole observed spectrum of monokaryotic fruiting, the two monokaryons A. aegerita AAE-3-40 (‘initials type’) and A. aegerita AAE-3-37 (‘elongated initials type’) have been selected for their mating compatibility with A. aegerita AAE-3-32 and A. aegerita AAE-3-13, respectively. Together with the parental dikaryotic strain A. aegerita AAE-3, this set of standard monokaryons could prove useful for studies exploring the factors regulating monokaryotic fruiting in comparison to dikaryotic mushroom formation.     

长根小奥德蘑双孢菌株与四孢菌株核相变化的比较

用双苯并咪唑（Hoechst 33258）染色法分别对长根小奥德蘑Oudemansiella radicata双孢菌株和四孢菌株的菌丝、子实体、担孢子进行染色观察,结果表明：双孢长根小奥德蘑菌丝细胞多为单核,无锁状联合;原担子中单核进行一次有丝分裂形成两个横向或纵向排列的子核,这2个子核分别进入2个担孢子中,留下无核的空担子;成熟担孢子具有一个核。四孢长根小奥德蘑菌丝细胞大多数为双核,具有锁状联合;进入原担子中的两个单倍性细胞核先发生核配,形成一个二倍性的核,再经过减数分裂形成四个染色体减半的单倍性子核,     

Purification and Identification of the Fruiting-Inducing Substances in Coprinus macrorhizus

Substances which are effective in inducing fruiting bodies in monokaryotic mycelia of the fis(+) strain of Coprinus macrorhizus were purified and characterized. The active components of fruiting-inducing substances were identified as adenosine-3'-monophosphate, adenosine 3',5'-cyclic monophosphate (cyclic AMP), and a protein which is bound with the cyclic AMP. Cyclic AMP was synthesized from adenine within mycelia of the mutant strains which form monokaryotic fruiting bodies without the addition of fruiting-inducing substances, but not in those of the strains which do not form monokaryotic fruiting bodies. The proteins which bind with cyclic AMP were detected in crude extracts of mycelia of those strains which form monokaryotic fruiting bodies and of the dikaryon, but not in those of the strains which do not form monokaryotic fruiting bodies.     

Map-based cloning of genes encoding key enzymes for pigment synthesis in Auricularia cornea

Color is an important quality attribute of fungi, and a useful marker for classification, genetic, and molecular research. However, there is much debate over which enzymes play key regulatory roles in pigment synthesis pathways among different fungi and even within the same species. Auricularia cornea is the most widely cultivated mushroom in the genus Auricularia; 1.834 million tons of this mushroom were produced in 2016 in China. Thus, systematic studies on its color inheritance and the genes encoding key enzymes for pigment synthesis have high scientific and economic value. In this study, the white strain ACW001 and the purple strain ACP004 of A. cornea were used as dikaryotic parents. Selfing populations of ACW001 and ACP004 were constructed with their monokaryotic strains. The fruiting body color of the two populations was consistent with that of their parents, confirming that the two parents were color homozygotes. All strains in the hybrid population of the two parents produced purple fruiting bodies. A robust hybrid strain (ACW001-33×ACP004-33) was selected from the hybrid population, and 87 monokaryotic strains of ACW001-33×ACP004-33 were obtained as a mapping population. Finally, a testcross population was constructed by crossing the mapping population with the test strain ACW001-9. The color genotype of each monokaryotic strain in the mapping population was identified by a fruiting test. The genomes of the two monokaryotic strains ACW001-33 and ACP004-33 were sequenced, and then simple sequence repeat (SSR) and sequence-related amplified polymorphism (SRAP) molecular marker primers were developed. Then, 88 pairs of primers that could distinguish the genotypes of the mapping population were used to construct a genetic linkage map. The genetic linkage map consisted of 12 linkage groups (LGs) spanning 1315.2 cM. The color control locus was preliminarily located at 24.5 cM of the 11th LG. Fine-mapping primers were designed based on sequence differences between ACW001-33 and ACP004-33 in the primary location region. Four color control candidate genes were located in an 8.2-kb region of ACW001-33_contig733 and a 9.2-kb region of ACP004-33_contig802. Homologous alignment and prediction of conserved domain analyses indicated that two of the color control candidate genes encoded proteins with unknown function, and the other two, ACP004_g11815 and ACP004_g11816, encoded glutamyl aminotransferases. These two genes were consecutively arranged on ACP004-33_contig802, and were likely to encode key enzymes in the γ-glutamine-4-hydroxy-benzoate (GHB) pigment synthesis pathway. Primers were designed from the flanking sequences of the two genes and used to analyze the testcross population. Products were amplified only from the 30 testcross strains with purple fruiting bodies, confirming the accuracy of the localization results. We discuss the deficiencies and advantages of map-based cloning in fungi vs. plants, and summarize the steps and requirements of the map-based cloning method for fungi. This study has provided novel ideas and methods for locating functional genes in fungi.     

Basidiospore formation in monokaryotic fruiting bodies of a mutant strain of Coprinus macrorhizus

The process of basidiospore formation in a mutant strain Fis^c of Coprinus macrorhizus, a heterothallic species of Basidiomycete, which forms monokaryotic fruiting bodies was examined. A single nucleus in a young basidium divided mitotically and two daughter nuclei were fused subsequently. The fused nucleus then divided meiotically forming four basidiospores on a basidium. The typical chromosome behaviours in the first meiotic prophase were observed. Synaptonemal complexes were observed in a basidium at the first meiotic prophase. A continuous illumination of fruiting bodies was effective to arrest meiosis in monokaryotic fruiting bodies at the particular stage of meiotic division.     

Quantitative Trait Loci Controlling Vegetative Growth Rate in the Edible Basidiomycete Pleurotus ostreatus

Mycelium growth rate is a quantitative characteristic that exhibits continuous variation. This trait has applied interest, as growth rate is correlated with production yield and increased advantage against competitors. In this work, we studied growth rate variation in the edible basidiomycete Pleurotus ostreatus growing as monokaryotic or dikaryotic mycelium on Eger medium or on wheat straw. Our analysis resulted in identification of several genomic regions (quantitative trait loci [QTLs]) involved in the control of growth rate that can be mapped on the genetic linkage map of this fungus. In some cases monokaryotic and dikaryotic QTLs clustered at the same map position, indicating that there are principal genomic areas responsible for growth rate control. The availability of this linkage map of growth rate QTLs can help in the design of rational strain breeding programs based on genomic information.     

Identification and Functional Analysis of Pheromone and Receptor Genes in the B3 Mating Locus of Pleurotus eryngii

Pleurotus eryngii has recently become a major cultivated mushroom; it uses tetrapolar heterothallism as a part of its reproductive process. Sexual development progresses only when the A and B mating types are compatible. Such mating incompatibility occasionally limits the efficiency of breeding programs in which crossing within loci-shared strains or backcrossing strategies are employed. Therefore, understanding the mating system in edible mushroom fungi will help provide a short cut in the development of new strains. We isolated and identified pheromone and receptor genes in the B3 locus of P. eryngii and performed a functional analysis of the genes in the mating process by transformation. A genomic DNA library was constructed to map the entire mating-type locus. The B3 locus was found to contain four pheromone precursor genes and four receptor genes. Remarkably, receptor PESTE3.3.1 has just 34 amino acid residues in its C-terminal cytoplasmic region; therefore, it seems likely to be a receptor-like gene. Real-time quantitative RT-PCR (real-time qRT-PCR) revealed that most pheromone and receptor genes showed significantly higher expression in monokaryotic cells than dikaryotic cells. The pheromone genes PEphb3.1 and PEphb3.3 and the receptor gene PESTE3.3.1 were transformed into P5 (A3B4). The transformants were mated with a tester strain (A4B4), and the progeny showed clamp connections and a normal fruiting body, which indicates the proposed role of these genes in mating and fruiting processes. This result also confirms that PESTE3.3.1 is a receptor gene. In this study, we identified pheromone and receptor genes in the B3 locus of P. eryngii and found that some of those genes appear to play a role in the mating and fruiting processes. These results might help elucidate the mechanism of fruiting differentiation and improve breeding efficiency.     

Chemical and genetical control of induction of monokaryotic fruiting bodies in Coprinus macrorhizus

Summary Fruiting-inducing substance (FIS), which is effective to induce fruiting bodies in monokaryotic mycelia of a strain of the Basidiomycete, Coprinus macrorhizus, has been detected in cell-free extracts of fruiting bodies and dikaryotic mycelia of C. macrorhizus itself and fruiting bodies of several other Basidiomycete species. Inducibility by FIS was controlled by genetic and environmental factors. Constitutive mutants which form monokaryotic fruiting bodies without addition of FIS were isolated. Fruiting bodies and monokaryotic mycelia of these mutants also contained FIS.FIS was stable to heat, acid and alkaline hydrolysis, and several enzymes degradating proteins and nucleic acids. Two fractions of FIS were obtained after Sephadex G-25 chromatography. One of them contained protein and the other appears to have similar chemical nature as adenosine-3, 5-monophosphate or adenosine-3-monophosphate which were active to induce monokaryotic fruiting.     

Self-fructification associated with genetic instability in Coprinus radiatus

In the basidiomycete Coprinus radiatus, crosses gave rise with a high frequency to 2 types of self-fructifying progeny. The first type showed monokaryotic fruiting due to the occurrence of a mutation associated with the B1 incompatibility factor. The second type showed fruiting due to the presence of the 2 parental genomes in the original spore. Mutability and abnormal development of the life cycle are responsible for self-fructification. These features occurred in progeny of related strains harbouring a genetic instability located at the Nic-2 locus. High mutability, disturbance of the development of the life cycle and genetic instability are traits which resemble hybrid dysgenesis in Drosophila and meiotic dysgenesis in Phycomyces.     

Quantitative trait loci controlling vegetative growth rate in the edible basidiomycete Pleurotus ostreatus

Mycelium growth rate is a quantitative characteristic that exhibits continuous variation. This trait has applied interest, as growth rate is correlated with production yield and increased advantage against competitors. In this work, we studied growth rate variation in the edible basidiomycete Pleurotus ostreatus growing as monokaryotic or dikaryotic mycelium on Eger medium or on wheat straw. Our analysis resulted in identification of several genomic regions (quantitative trait loci [QTLs]) involved in the control of growth rate that can be mapped on the genetic linkage map of this fungus. In some cases monokaryotic and dikaryotic QTLs clustered at the same map position, indicating that there are principal genomic areas responsible for growth rate control. The availability of this linkage map of growth rate QTLs can help in the design of rational strain breeding programs based on genomic information.     

蛹虫草表型多态性对子实体产生及虫草菌素的影响

为选育高产子实体和虫草菌素的蛹虫草菌株,从野生蛹虫草中分离单孢子并进行分型,之后进行产子实体实验;同时对蛹虫草子实体进行了产孢结构的观察,并用高效液相色谱仪检测了子实体和培养基中的虫草菌素。结果表明,菌落背面颜色为橙铬色的菌株容易产生子实体;出发(原代)菌株所产子实体在形态上更接近野生蛹虫草,而角变株的子实体畸形;出发菌株产子实体能力要比该菌株未发生角变部分分离株和角变部分分离株都强。在虫草菌素的产量上也以出发菌株为高,表明表型多态性对蛹虫草产子实体和虫草菌素有很大的影响。这对于优势菌种的筛选和生产实践有借鉴意义。     

Entamoeba histolytica, E. invadens, and E. moshkovskii: fluctuations of the DNA content of axenic trophozoites.

DNA content was determined by means of diphenylamine reaction in trophozoites of exponentially growing, axenized Entamoeba histolytica (strains HK-9:NIH, HM-2:IMSS, and HM-3:IMSS), E. invadens (strain PZ), and E. moshkovskii (strain FIC). DNA content was variable in all strains. Variations generally, but not always, occurred within a range characteristic of each species. Average DNA content in strains analyzed was in decreasing order: E. histolytica > E. invadens > E. moshkovskii. Two types of variation were clearly seen in E. histolytica: (i) In one strain (HM-2) the initial content was higher, but, after subculturing it for 6 months (24 passages), the amount of DNA decreased almost four times and became similar to that of the other strains; (ii) a clonal derivative of HK-9 had a small but significant increase and less dispersion in DNA content than the parental strain. The proportion of trophozoites with more than one nucleus was variable; average DNA content per nucleus was slightly smaller than that per trophozoite. We believe that small variations in DNA content may be due to (i) slight changes in ploidy, (ii) genomic heterogeneity, or (iii) differences in the degree of synchrony of the cultures. Large differences may be caused mainly by large changes in ploidy.     

The G-protein beta subunit GPB1 is required for mating and haploid fruiting in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle. The gene encoding a heterotrimeric G-protein beta subunit, GPB1, was cloned and disrupted. gpb1 mutant strains are sterile, indicating a role for this gene in mating. GPB1 plays an active role in mediating responses to pheromones in early mating steps (conjugation tube formation and cell fusion) and signals via a mitogen-activated protein (MAP) kinase cascade in both MATalpha and MATa cells. The functions of GPB1 are distinct from those of the Galpha protein GPA1, which functions in a nutrient-sensing cyclic AMP (cAMP) pathway required for mating, virulence factor induction, and virulence. gpb1 mutant strains are also defective in monokaryotic fruiting in response to nitrogen starvation. We show that MATa cells stimulate monokaryotic fruiting of MATalpha cells, possibly in response to mating pheromone, which may serve to disperse cells and spores to locate mating partners. In summary, the Gbeta subunit GPB1 and the Galpha subunit GPA1 function in distinct signaling pathways: one (GPB1) senses pheromones and regulates mating and haploid fruiting via a MAP kinase cascade, and the other (GPA1) senses nutrients and regulates mating, virulence factors, and pathogenicity via a cAMP cascade.     

Mating type and mating strategies in Neurospora

In the heterothallic species Neurospora crassa, strains of opposite mating type, A and a, must interact to give the series of events resulting in fruiting body formation, meiosis, and the generation of dormant ascospores. The mating type of a strain is specified by the DNA sequence it carries in the mating type region; strains that are otherwise isogenic can mate and produce ascospores. The DNA of the A and a regions have completely dissimilar sequences. Probing DNA from strains of each mating type with labelled sequences from the A and the a regions has shown that, unlike in Saccharomyces cerevisiae, only a single copy of a mating type sequence is present in a haploid genome. The failure to switch is explainable by the physical absence of DNA sequences characteristic of the opposite mating type. While the mating type sequences must be of the opposite kind for mating to occur in the sexual cycle, two strains of opposite mating type cannot form a stable heterokaryon during vegetative growth; instead, they fuse abortively to give a heterokaryon incompatibility reaction, which results in death of the cells along the fusion line. The DNA sequences responsible for this reaction are coextensive with those sequences in the A and a regions which are necessary to initiate fruiting body formation. The genus Neurospora also includes homothallic species--ones in which a single haploid nucleus carries all the information necessary to form fruiting bodies, undergo meiosis, and produce new haploid spores. One such species, N. terricola, contains one copy each of the A and the a sequences within each haploid genome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insertional mutagenesis combined with an inducible filamentation phenotype reveals a conserved STE50 homologue in Cryptococcus neoformans that is required for monokaryotic fruiting and sexual reproduction

Cryptococcus neoformans typically grows in a yeast-like morphology; however, under specific conditions the fungus can produce hyphae that are either dikaryotic or monokaryotic. In this study, we developed a simple method for inducing robust monokaryotic fruiting and combined the assay with Agrobacterium tumefaciens insertional mutagenesis to screen for hyphal mutants. A C. neoformans homologue of the Saccharomyces cerevisiae STE50 gene was identified and characterized. STE50 was found to be required for sexual reproduction and monokaryotic fruiting. Ste50p has conserved SAM and RA domains, as well as two SH3 domains specific to basidiomycetous Ste50 proteins. Analysis of protein-protein interaction showed that Ste50p can interact with Ste11p and Ste20p, and epistasis experiments placed STE50 between STE20 and STE11. Genetic analysis of the role of STE50 in sexual reproduction showed that it was required for all steps, from response to pheromone to production of hyphae. Analysis of the effect of individual Ste50p domains on sexual reproduction and monokaryotic fruiting revealed domain-specific effects for both processes. This study revealed that the C. neoformans STE50 gene has both conserved and novel functions during sexual reproduction and monokaryotic fruiting, and these functions are domain-dependent.     

Genomic comparison of Salmonella typhimurium DT104 with non-DT104 strains

DT104 emerged as a new branch of Salmonella typhimurium with resistance to multiple antimicrobials. To reveal some general genomic features of DT104 for clues of evolutionary events possibly associated with the emergence of this relatively new type of this pathogen, we mapped 11 independent DT104 strains and compared them with non-DT104 S. typhimurium strains. We found that all 11 DT104 strains contained three insertions absent in non-DT104 strains, i.e., the previously reported ST104, ST104B and ST64B. However, SGI-1, a genomic island known to be responsible for DT104 multidrug resistance, was not present in all DT104 strains examined in this study: one DT104 strain did not contain SGI-1 but carried a 144 kb plasmid, suggesting possible evolutionary relationships between the two DNA elements in the development of antimicrobial resistance.     

Relationship between fruiting body development and extracellular laccase production in the edible mushroom Flammulina velutipes

The biochemical mechanism underlying the development of fruiting bodies in Flammulina velutipes, an edible mushroom, was investigated using the YBLB colorimetric assay to distinguish between the normal strain (FVN-1) and the degenerate strain (FVD-1). In this assay, the color of the YBLB medium (blue-green) inoculated with FVN-1 exhibiting normal fruiting body development changed to yellow, while the color of the medium inoculated with FVD-1 changed to blue. In this study, we found that this color difference originated from extracellular laccase produced by FVN-1. Moreover, FVN-1 exhibited considerably higher extracellular laccase activity than FVD-1, under conditions facilitating fruiting body formation. Overall, these findings suggest that extracellular laccase is involved in the fruiting body development process in F. velutipes.