The A mating type and blue light regulate all known differentiation processes in the basidiomycete Coprinus cinereus

Monokaryons of Coprinus cinereus constitutively form small spores (oidia) in the aerial mycelium. Some strains also produce large, inflated single cells (chlamydospores) at the agar/air interface, and hyphal aggregates (hyphal knots) that can develop into sclerotia. Monokaryons show various reactions upon transformation with heterologous A mating type genes. Production of oidia in such A-activated transformants is repressed in the dark and induced by blue light. Five of six monokaryons tested following transformation with A genes showed induced production of hyphal knots and sclerotia in the dark, and at least three strains showed enhanced chlamydospore production in the dark. Continuous incubation under blue light inhibited formation of hyphal knots, sclerotia and chlamydospores in both competent monokaryons and in A-activated transformants. On artificial medium and on a 12?h light/12?h dark regime, A-activated transformants of one distinct monokaryon (218) formed fruit-body primordia that were arrested in development before karyogamy. Our studies show that A mating type genes control all major differentiation processes in Coprinus, but whether developmental processes can proceed depends on the genetic background of the strain.     

The A mating type and blue light regulate all known differentiation processes in the basidiomycete Coprinus cinereus

Monokaryons of Coprinus cinereus constitutively form small spores (oidia) in the aerial mycelium. Some strains also produce large, inflated single cells (chlamydospores) at the agar/air interface, and hyphal aggregates (hyphal knots) that can develop into sclerotia. Monokaryons show various reactions upon transformation with heterologous A mating type genes. Production of oidia in such A-activated transformants is repressed in the dark and induced by blue light. Five of six monokaryons tested following transformation with A genes showed induced production of hyphal knots and sclerotia in the dark, and at least three strains showed enhanced chlamydospore production in the dark. Continuous incubation under blue light inhibited formation of hyphal knots, sclerotia and chlamydospores in both competent monokaryons and in A-activated transformants. On artificial medium and on a 12 h light/12 h dark regime, A-activated transformants of one distinct monokaryon (218) formed fruit-body primordia that were arrested in development before karyogamy. Our studies show that A mating type genes control all major differentiation processes in Coprinus, but whether developmental processes can proceed depends on the genetic background of the strain. Received: 11 May 1998 / Accepted: 15 July 1998     

Life history and developmental processes in the basidiomycete Coprinus cinereus.

Coprinus cinereus has two main types of mycelia, the asexual monokaryon and the sexual dikaryon, formed by fusion of compatible monokaryons. Syngamy (plasmogamy) and karyogamy are spatially and temporally separated, which is typical for basidiomycetous fungi. This property of the dikaryon enables an easy exchange of nuclear partners in further dikaryotic-monokaryotic and dikaryotic-dikaryotic mycelial fusions. Fruiting bodies normally develop on the dikaryon, and the cytological process of fruiting-body development has been described in its principles. Within the specialized basidia, present within the gills of the fruiting bodies, karyogamy occurs in a synchronized manner. It is directly followed by meiosis and by the production of the meiotic basidiospores. The synchrony of karyogamy and meiosis has made the fungus a classical object to study meiotic cytology and recombination. Several genes involved in these processes have been identified. Both monokaryons and dikaryons can form multicellular resting bodies (sclerotia) and different types of mitotic spores, the small uninucleate aerial oidia, and, within submerged mycelium, the large thick-walled chlamydospores. The decision about whether a structure will be formed is made on the basis of environmental signals (light, temperature, humidity, and nutrients). Of the intrinsic factors that control development, the products of the two mating type loci are most important. Mutant complementation and PCR approaches identified further genes which possibly link the two mating-type pathways with each other and with nutritional regulation, for example with the cAMP signaling pathway. Among genes specifically expressed within the fruiting body are those for two galectins, beta-galactoside binding lectins that probably act in hyphal aggregation. These genes serve as molecular markers to study development in wild-type and mutant strains. The isolation of genes for potential non-DNA methyltransferases, needed for tissue formation within the fruiting body, promises the discovery of new signaling pathways, possibly involving secondary fungal metabolites.     

Isolation of the <Emphasis Type="Italic">B</Emphasis> incompatibility factor mutants in <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

 B incompatibility factor mutants (Bmut) in Pleurotus ostreatus were recovered from common-B mating heterokaryons resulted from matings between wild-type monokaryons with different A but the same B factors (A1B2 and A2B2) after NTG mutagenesis. The mutant monokaryons such as A1B2mut and A2B2mut were observed to have regularly uninucleated hyphal cells and to be compatible with each other. Matings between A1B2mut and A2B2mut monokaryons produced stable heterokaryons (A1B2mut + A2B2mut) that had binucleated hyphal cells with true clamp connections and formed normal fruit-bodies. Mating tests using basidiospore progeny from each of these heterokaryons revealed the bipolar mating pattern. Genetic analysis suggested that the mutation of B factor in P. ostreatus might occur in the B incompatibility factor genes. Received: August 3, 2001 / Accepted: January 18, 2002     

Identification of new mutant alleles of <Emphasis Type="Italic">pcc1</Emphasis> in the homobasidiomycete <Emphasis Type="Italic">Coprinopsis cinerea</Emphasis>

It is known that a mutation in the pcc1 gene of the homobasidiomycete Coprinopsis cinerea leads to pseudoclamp development and fruit-body formation in a homokaryon without mating. In this study we characterize two strains that were reported previously to exhibit pseudoclamp development and fruiting without mating, together with six new mutants exhibiting the same phenotype. A frame-shift, nonsense, or intron splice site mutation was present within pcc1 in each of the eight mutants. The results suggest that the Pcc1 protein is a key element in a pathway(s) leading to pseudoclamp development and fruiting.     

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.     

Isolation and characterization of a sporeless mutant in <Emphasis Type="Italic">Pleurotus eryngii</Emphasis>

 A sporeless mutant dikaryon, completely defective in sporulation, was isolated from mycelial protoplasts of Pleurotus eryngii mutagenized by UV irradiation. Newly established dikaryons between one component monokaryon from the mutant, and 12 different wild type monokaryons from 3 other wild type dikaryons, all exhibited the sporeless phenotype, whereas those between the other monokaryon and the same wild type monokaryons all produced normal fruiting bodies. These results indicated that the sporeless mutation was induced in one of two nuclei of the mutant and was dominant. In the wild type basidia, the pattern of nuclear behavior during sporulation corresponded to the pattern C nuclear behavior as defined by Duncan and Galbraith. Cytological observation revealed that in the sporeless mutant meiosis was blocked at the meta-anaphase I in most basidia and hence basidiospores and sterigmata were not produced. Although fruiting bodies of the sporeless mutant showed a somewhat leaning growth, their gross morphology and its fruiting body productivity were comparable to that of the original wild type strain. Based on these results, it was considered that the sporeless mutant could serve as a potential material in breeding of sporeless P. eryngii commercial strains. Received: September 5, 2002 / Accepted: October 16, 2002 Acknowledgments We are grateful to Mrs. Motoe Masuda for her skillful technical assistance. Contribution no. 358 from the Tottori Mycological Institute Correspondence to:Y. Obatake     

Binding of calmodulin to Nuf1p is required for karyogamy in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The role of calmodulin (CaM) during mating in Saccharomyces cerevisiae was examined by using a set of Phe-to-Ala substitutions. We identified ten CaM mutants that exhibited significantly reduced mating efficiencies when crossed to a strain of the opposite mating type harboring the same CaM mutation. Most of the mating-defective CaM mutants were bilateral, i.e., they also exhibited mating defects, albeit minor ones, when crossed to the wild type. When strains carrying different bilateral CaM mutations were mated, the mating efficiencies recovered dramatically. We termed this phenomenon "intragenic mating complementation", and classified the mating-defective CaM mutations into two intragenic mating complementation groups. Two mutant alleles belonging to different groups showed minor defects in cell adhesion and cell fusion, but exhibited severe defects in karyogamy. CaM is known to bind to the essential spindle pole body component Nuf1p. This binding appears to be important for karyogamy because the nuf1 ^C911R mutation, which impairs CaM-Nuf1p binding, resulted in a severe defect in karyogamy. Indeed, the two mating-defective CaM mutations were found to compromise formation of the CaM/Nuf1p complex, and the mating defects of these two CaM mutants were suppressible by a dominant, CaM-independent, mutation in NUF1. Taken together, these results suggest that loss of CaM binding to Nuf1p causes a defect in karyogamy, thereby inhibiting productive mating.Communicated by C. P. Hollenberg     

Characteristic analysis of transformants in T-DNA mutation library of <Emphasis Type="Italic">Monascus ruber</Emphasis>

Monascus ruber, a red mold species, has been widely used in the fields of food and medicine. In this research, we transformed Monascus ruber spores using Agrobacterium tumefaciens as a tool for random insertional mutagenesis with the hygromycin phosphotransferase gene as the selected marker. Three types of mutants including citrinin-producing mutants, mutants with abnormal aerial hyphae and pigment change mutants were screened for molecular analysis. Southern blot analysis showed that more than 83.3% of transformants contained single T-DNA insertions. The genomic DNA segments of the transformants flanking the T-DNA could be amplified from their left borders with TAIL-PCR. Homologous comparison using the Blast tool showed that none of the isolated DNA sequences had any similarity to each other, suggesting that the T-DNA was randomly integrated into the fungal genome, which provided the hypothetical reason for the variant phenotypes of the transformants. The successful creation of transformants with a single T-DNA tag insertion may help us to clone functional genes related to the metabolism and differentiation of Monascus spp., which will greatly facilitate the molecular analysis of this important fungus and the improvement of strains at the genetic level.     

<Emphasis Type="Italic">Sporidiobolus johnsonii</Emphasis> and <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> revisited

The relationship between Sporidiobolus johnsonii and S. salmonicolor was investigated using rDNA sequence data. Two statistically well-supported clades were obtained. One clade included the type strain of S. johnsonii and the other included the type strain of S. salmonicolor. However, some mating strains of S. salmonicolor were found in the S. johnsonii group. These strains belonged to mating type A2 and were sexually compatible with mating type A1 strains from the S. salmonicolor group. DNA–DNA reassociation values were high within each clade and moderate between the two clades. In the re-investigation of teliospore germination, we observed that the basidia of S. salmonicolor were two-celled. In S. johnsonii, basidia were not formed and teliospore germination resulted in direct formation of yeast cells. We hypothesize that the S. johnsonii clade is becoming genetically isolated from the S. salmonicolor group and that a speciation process is presently going on. We suspect that the observed sexual compatibility between strains of the S. johnsonii and S. salmonicolor groups and the possible genetic flow between the two species has little biological relevance because distinct phenotypes have been fixed in the two taxa and intermediate (hybrid) sequences for LSU and ITS rDNAs have not been detected. An erratum to this article can be found at     

Diversity of <Emphasis Type="Italic">A</Emphasis> mating type in <Emphasis Type="Italic">Lentinula edodes</Emphasis> 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. Twelve of them commonly appeared, leaving 63 unique A mating type alleles. It was also revealed that only A few A mating type alleles such as A1, A4, A5, and A7 were prevalent in the cultivated strains, accounting for 62.5% of all A mating types. This implies preferred selection of certain A mating types in the process of strain development and suggests potential role of A mating genes in the expression of genes governing mushroom quality. Dominant expression of an A mating gene HD1 was observed from A1 mating locus, the most prevalent A allele, in A1-containing dikaryons. However, connections between HD1 expression and A1 preference in the cultivated strains remain to be verified. The A mating type was highly diverse in the wild strains. Thirty-six unique A alleles were discovered from relatively small and confined area of mountainous region in Korean peninsula. The number will further increase because no A allele has been recurrently observed in the wild strains and thus newly discovered strain will have good chances to contain new A allele. The high diversity in small area also suggests that the A mating locus has evolved rapidly and thus its diversity will further increase.     

Elucidation of <Emphasis Type="Italic">veA</Emphasis>-dependent genes associated with aflatoxin and sclerotial production in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> by functional genomics

The aflatoxin-producing fungi, Aspergillus flavus and A. parasiticus, form structures called sclerotia that allow for survival under adverse conditions. Deletion of the veA gene in A. flavus and A. parasiticus blocks production of aflatoxin as well as sclerotial formation. We used microarray technology to identify genes differentially expressed in wild-type veA and veA mutant strains that could be involved in aflatoxin production and sclerotial development in A. flavus. The DNA microarray analysis revealed 684 genes whose expression changed significantly over time; 136 of these were differentially expressed between the two strains including 27 genes that demonstrated a significant difference in expression both between strains and over time. A group of 115 genes showed greater expression in the wild-type than in the veA mutant strain. We identified a subgroup of veA-dependent genes that exhibited time-dependent expression profiles similar to those of known aflatoxin biosynthetic genes or that were candidates for involvement in sclerotial production in the wild type.     

Gas discharge plasmas are effective in inactivating <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Clostridium</Emphasis> spores

Bacterial spores are the most resistant form of life and have been a major threat to public health and food safety. Nonthermal atmospheric gas discharge plasma is a novel sterilization method that leaves no chemical residue. In our study, a helium radio-frequency cold plasma jet was used to examine its sporicidal effect on selected strains of Bacillus and Clostridium. The species tested included Bacillus subtilis, Bacillus stearothermophilus, Clostridium sporogenes, Clostridium perfringens, Clostridium difficile, and Clostridium botulinum type A and type E. The plasmas were effective in inactivating selected Bacillus and Clostridia spores with D values (decimal reduction time) ranging from 2 to 8 min. Among all spores tested, C. botulinum type A and C. sporogenes were significantly more resistant to plasma inactivation than other species. Observations by phase contrast microscopy showed that B. subtilis spores were severely damaged by plasmas and the majority of the treated spores were unable to initiate the germination process. There was no detectable fragmentation of the DNA when the spores were treated for up to 20 min. The release of dipicolinic acid was observed almost immediately after the plasma treatment, indicating the spore envelope damage could occur quickly resulting in dipicolinic acid release and the reduction of spore resistance.     

Analysis of <Emphasis Type="Italic">cry</Emphasis> Gene Profiles in <Emphasis Type="Italic">Bacillus Thuringiensis</Emphasis> Strains Isolated During Epizootics in <Emphasis Type="Italic">Cydia pomonella</Emphasis> L.

The crystal morphology and the profiles of genes encoding protein toxins (Cry and Cyt) were analyzed in 12 Bacillus thuringiensis strains isolated during epizootics in laboratory culture lines of Cydia pomonella, 2 isolates cultured from Leucoma salicis larvae, and 9 reference strains. Epizootic isolates produced crystals of the same bipyramidal shape; however, they revealed a variety of number and type of cry genes. Genes cry1I, cry2Ab, and cry9B were the most frequently observed in epizootic strains. Gene cry1I was noted in of 50% epizootic isolates. Eighty-three percent of them harbored gene cry2Ab. Gene cry9B was found for 42% of strains isolated during epizootics. Three isolates showed the largest number of cry genes and their variety; hence, they were chosen for the toxicity assay of their crystals and spores on C. pomonella larvae. One of them had approximately sixfold higher insecticidal activity than the reference strain B. thuringiensis subsp. kurstaki BTK STANDARD.     

Transformation of a filamentous fungus<Emphasis Type="Italic">Cryphonectria parasitica</Emphasis> using<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

AsAgrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast,Saccharomyces cerevisiae, a variety of fungi were subjected to theA. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. TheA. tumefaciens-mediated transformation of chestnut blight fungus,Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of theAspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants per 1×10⁶ conidia ofC. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon.     

<Emphasis Type="Italic">Sporidiobolus johnsonii</Emphasis> and <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> revisited

The relationship between Sporidiobolus johnsonii and S. salmonicolor was investigated using rDNA sequence data. Two statistically well-supported clades were obtained. One clade included the type strain of S. johnsonii and the other included the type strain of S. salmonicolor. However, some mating strains of S. salmonicolor were found in the S. johnsonii group. These strains belonged to mating type A2 and were sexually compatible with mating type A1 strains from the S. salmonicolor group. DNA–DNA reassociation values were high within each clade and moderate between the two clades. In the re-investigation of teliospore germination, we observed that the basidia of S. salmonicolor were two-celled. In S. johnsonii, basidia were not formed and teliospore germination resulted in direct formation of yeast cells. We hypothesize that the S. johnsonii clade is becoming genetically isolated from the S. salmonicolor group and that a speciation process is presently going on. We suspect that the observed sexual compatibility between strains of the S. johnsonii and S. salmonicolor groups and the possible genetic flow between the two species has little biological relevance because distinct phenotypes have been fixed in the two taxa and intermediate (hybrid) sequences for LSU and ITS rDNAs have not been detected.     

Morphological characteristics of sclerotia formed from hyphae of <Emphasis Type="Italic">Grifola umbellata</Emphasis> under artificial conditions

The morphological characteristics of sclerotia were induced in cultures of the fungus Grifola umbellata by introducing an unidentified companion fungus were studied by light microscope, scanning and transmission electron microscope (SEM and TEM). Light microscope and SEM investigations of developing sclerotia revealed that aerial mycelial hyphae diminished with age, and mature sclerotia had two tissue layers, the rind and medulla. The medulla was comprised of thin and thick-walled hyphae of varying diameter. The thick-walled cells always formed below the hyphal tips. Retraction of the cytoplasm was accompanied by the thickening of cell wall. There were crystalline initials in the newly formed sclerotium. Crystalline initials were always formed in the tip of medullary hyphae and were not of regular shape. A series of changes occurred in the cells in which the crystalline initials would be formed, such as enlargement of size, formation of one or several large vacuoles. Crystalline initials developed via amorphous materials in the cytoplasm deposited in the vacuoles. At last crystalline initials was released by degradation of the cell wall.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-<Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of<Emphasis Type="Italic"> Helminthosporium turcicum</Emphasis>, the maize leaf-blight fungus

Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome. Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium-tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) and the enhanced green fluorescent protein (EGFP) genes controlled by the gpd promoter from Agaricus bisporus and the CaMV 35S terminator. Agrobacterium-tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B. The presence of hph in the transformants was confirmed by PCR, and integration of the T-DNA at random sites in the genome was demonstrated by Southern blot analysis. Agrobacterium-tumefaciens-mediated transformation of Helminthosporium turcicum provides an opportunity for advancing studies of the molecular genetics of the fungus and of the molecular basis of its pathogenicity on maize.