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Strain degeneration has been defined as a decrease or loss in the yield of important commercial traits resulting from subsequent culture, which ultimately leads to Reactive Oxygen Species (ROS) production. Pleurotus ostreatus is a lignin-producing nematophagous edible mushroom. Mycelia for mushroom production are usually maintained in subsequent culture in solid media and frequently show symptoms of strain degeneration. The dikaryotic strain P. ostreatus (DkN001) has been used in our lab as a model organism for different purposes. Hence, different tools have been developed to uncover genetic and molecular aspects of this fungus. In this work, strain degeneration was studied in a full-sib monokaryotic progeny of the DkN001 strain with fast (F) and slow (S) growth rates by using different experimental approaches (light microscopy, malondialdehyde levels, whole-genome transcriptome analysis, and chitosan effect on monokaryotic mycelia). The results obtained showed that: (i) strain degeneration in P. ostreatus is linked to oxidative stress, (ii) the oxidative stress response in monokaryons is genotype dependent, (iii) stress and detoxifying genes are highly expressed in S monokaryons with symptoms of strain degeneration, (iv) chitosan addition to F and S monokaryons uncovered the constitutive expression of both oxidative stress and cellular detoxifying genes in S monokaryon strains which suggest their adaptation to oxidative stress, and (v) the overexpression of the cell wall genes, Uap1 and Cda1, in S monokaryons with strain degeneration phenotype indicates cell wall reshaping and the activation of High Osmolarity Glycerol (HOG) and Cell Wall Integrity (CWI) pathways. These results could constitute a hallmark for mushroom producers to distinguish strain degeneration in commercial mushrooms. This research was funded by Research Projects RTI2018-099371-B-I00 (MCIU, AEI, FEDER/UE) and AGL2015-66833-R (MINECO) of the Spanish National Research Programme, H2020 MUSA 727624 (EU), and by funds of the Public University of Navarre (UPNA).

Lentinula edodes or better known as Shiitake mushroom contains β-1,3;1,6-glucan as part of the cell wall components and studies have shown that it has the ability to enhance the immune system and work as anticancer. The aim of this study is to create potential strain of L. edodes with the high content of β-1,3;1,6-glucan, which has the potential for biotechnological purposes. L. edodes spores were irradiated with gamma ray (Ɣ-rays) and incubated, which formed monokaryon mycelium (MM). Compatible mating of mutated monokaryon formed mutant line dikaryon mycelium (MLDM) that were selected for genotypic and phenotypic comparison with the wild-type line dikaryon mycelium (WLDM). The concentration of β-1,3;1,6-glucan were measured using a commercial β-Glucan Megazyme Assay Kit (Yeast & Mushroom). Three MLDM, (A37, A26, and C07) 38.8, 36.0 and 34.5 %w/w of 100 mg respectively, significantly produced higher amount of β-1,3;1,6-glucan in comparison with WLDM (20.2 %w/w of 100 mg).

The interaction between the race 1 of the cowpea rust fungus (Uromyces vignae Barclay) and the resistant cowpea (Vigna unguiculata (L.) Walp.) cv. 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.

The secretome complexity and lignocellulose degrading capacity of Pleurotus ostreatus monokaryons mkPC9 and mkPC15 and mated dikaryon dkN001 were studied in submerged liquid cultures containing wood, glucose, and wood plus glucose as carbon sources. The study revealed that this white-rot basidiomycete attacks all the components of the plant cell wall. P. ostreatus secretes a variety of glycoside hydrolases, carbohydrate esterases, and polysaccharide lyases, especially when wood is the only carbon source. The presence of wood increased the secretome complexity, whereas glucose diminished the secretion of enzymes involved in cellulose, hemicellulose and pectin degradation. In contrast, the presence of glucose did not influence the secretion of redox enzymes or proteases, which shows the specificity of glucose on the secretion of cellulolytic enzymes. The comparison of the secretomes of monokaryons and dikaryons reveals that secretome complexity is unrelated to the nuclear composition of the strain. Grants numbers AGL2014-55971-R (AEI, FEDER, UE) and RTI2018-099371-B-I00 (MCIU/AEI/FEDER, UE) of the Spanish National Research Plan, and PRO-UPNA18 from the Public University of Navarre have funded this research. Proteomics work in Goettingen was supported within the framework of a Common Lower Saxony-Israel-Project (ZN2043) by the Ministry of Science and Culture of Lower Saxony granted to UK and AM. MA appreciates a grant for research exchanges in Goettingen and a postdoctoral grant by Universidad Pública de Navarra. Grants numbers AGL2014-55971-R (AEI, FEDER, UE) and RTI2018-099371-B-I00 (MCIU/AEI/FEDER, UE) of the Spanish National Research Plan, and PRO-UPNA18 from the Public University of Navarre have funded this research. Proteomics work in Goettingen was supported within the framework of a Common Lower Saxony-Israel-Project (ZN2043) by the Ministry of Science and Culture of Lower Saxony granted to UK and AM. MA appreciates a grant for research exchanges in Goettingen and a postdoctoral grant by Universidad Pública de Navarra. Some of these experiments also featured in the thesis of Manuel Alfaro.

We introduced a site-directed mutation in the sdi1 gene and used it as a selective marker for the polyethylene glycol-mediated transformation of Pleurotus eryngii monokaryon protoplasts. The transformants displayed obvious and stable resistance to the fungicide carboxin indicating that the mutant Pesdi1 gene is an efficient selective marker.

Entsprechend dem ökonomischen Konzept „Reduzierung, Wiederverwendung und Recycling“ stellen präkursorenreiche Nebenströme der Citrus-verarbeitenden Industrie ein ideales Ausgangsmaterial für die Produktion und industrielle Anwendung von wertgesteigerten Verbindungen dar. Citrus-Nebenströme enthalten eine vergleichsweise große Menge an Monoterpenen, einschließlich (R)-Limonen und β-Myrcen. Pilze der Abteilung Basidiomycota sind in der Lage, diese Terpene in attraktive aromaaktive Substanzen zu transformieren. Zum Beispiel konnte für β-Myrcen eine Biotransformation durch den Austernseitling Pleurotus ostreatus zu dem holzig, citrusartig riechenden Aromastoff Perillen gezeigt werden. Fil: Bürger, F.. No especifíca; Fil: Omarini, Alejandra Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencias de la Tierra y Ambientales de La Pampa. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Instituto de Ciencias de la Tierra y Ambientales de La Pampa; Argentina Fil: Krahe, N. K.. No especifíca; Fil: Fraatz, M. A.. No especifíca; Fil: Berger, R. G.. No especifíca; Fil: Zorn, H.. Universität Gießen; Alemania

The aim of this study was to breed G . lucidum strains simultaneously rich in polysaccharides and triterpenes by mating of basidiospore monokaryons of two commercial cultivars, G . lucidum JC and AL. By incubation of freshly collected basidiospores, 31 monokaryons were isolated (19 from strain JC, and 12 from strain AC), and showed significant differences in mycelial growth and contents of polysaccharide and triterpene. Mating between the two strains yielded 83 instances of sexual compatibility. Of the hybrids isolated, sixteen ones that showed moderate and fast substrate colonization were evaluated in terms of fruiting. All the tested hybrids produced fruiting bodies but varied considerably in time to first harvest, productivity, yield distribution, and pileus shape and color. Significant differences in polysaccharide content (from 6.79 to 18.21 mg/g dry weight) and triterpene content (from 5.90 to 10.87 mg/g dry weight) were also observed among the hybrids. Notably, high concentrations of polysaccharides (16.63 mg/g dry weight) and triterpenes (10.50 mg/g dry weight) were both present in hybrid H-23, with satisfactory biological efficiency (19.41%). An antagonism test and random amplified polymorphic DNA (RAPD) analysis indicated that H-23 is a new hybrid strain. This hybrid may be suitable for use in commercial production of G . lucidum for manufacture of more effective health-promoting and medicinal products. To our knowledge, the present study is the first report on the development of new G . lucidum strains via mating by means of basidiospore-derived monokaryons.

Suppression subtractive hybridization (SSH) was employed to investigate bioluminescence in Panellus stipticus (Bull.) P. Karst. by detecting proteins differentially expressed in bioluminescent and luminescent strains. Comparisons of luminescent and non-luminescent monokaryon cultures of North American strains revealed differences in transcript levels of proteins responsible for post-translational modification (PTM) of enzymes. A similar comparison of a luminescent strain of P. stipticus from North America with a non-luminescent European strain revealed the presence of extracellular manganese superoxide dismutase (MnSOD) in the luminescent form, in addition to proteins involved in PTM. The application of MnSOD-specific inhibitors to luminescent mycelium resulted in the rapid loss of luminescence. The relevance to luminescence of proteins involved in PTM is discussed, together with a possible role for MnSOD that considers the potential for SODs to form stable complexes with catechols revealed in previously published research. In light of the recent discovery that hispidine may be the precursor of fungal luciferin, we consider a hypothetical mechanism for fungal luminescence in which the ο-hydroquinone moiety of a hispidine derivative ligates with the extracellular form of MnSOD producing a semiquinone-radical complex, with the resultant semiquinonato complex potentially reacting with molecular oxygen or other reactive oxygen species to produce sufficiently excited intermediates to emit light on relaxation.

‘Baekjung’ adaptable to high temperature was made by crossing between monokaryon derived from selfing of brown strain and monokaryon derived from Korea white strain. In the condition that temperature is maintained at 10oC without low temperature of 4oC suppressing treatment and wrapping during cultivation period, it showed good productivity than Uri1ho(control). The optimum temperature of mycelial growth was 30oC and that of fruiting body initiation and development were 14oC and 7oC, respectively. The days for the fruiting and yield were 7 days and 277±11.2 g per 1,100 ml bottle, respectively. This variety needed high concentration of carbon dioxide up to 4,000 ppm for the good quality.

Frontiers in microbiology 9, 2545 (2018). doi:10.3389/fmicb.2018.02545, Published by Frontiers Media, Lausanne

D. squalens, a white-rot fungus that efficiently degrades lignocellulose in nature, can be used in various biotechnological applications and has several strains with sequenced and annotated genomes. Here we present a method for the transformation of this basidiomycete fungus, using a recently introduced commercial ascomycete protoplasting enzyme cocktail, Protoplast F. In protoplasting of D. squalens mycelia, Protoplast F outperformed two other cocktails while releasing similar amounts of protoplasts to a third cocktail. The protoplasts released using Protoplast F had a regeneration rate of 12.5% (±6 SE). Using Protoplast F, the D. squalens monokaryon CBS464.89 was conferred with resistance to the antibiotics hygromycin and G418 via polyethylene glycol mediated protoplast transformation with resistance cassettes expressing the hygromycin phosphotransferase (hph) and neomycin phosphotransferase (nptII) genes, respectively. The hph gene was expressed in D. squalens using heterologous promoters from genes encoding β-tubulin or glyceraldehyde 3-phosphate dehydrogenase. A Southern blot confirmed integration of a resistance cassette into the D. squalens genome. An average of six transformants (±2 SE) were obtained when at least several million protoplasts were used (a transformation efficiency of 0.8 (±0.3 SE) transformants per μg DNA). Transformation of D. squalens demonstrates the suitability of the Protoplast F cocktail for basidiomycete transformation and furthermore can facilitate understanding of basidiomycete gene function and development of improved strains for biotechnological applications.

Clitopilus passeckerianus is the fungal species responsible for the production of pleuromutilin, a diterpene antibiotic that is gaining in commercial interest. Production of the antibiotic is constrained by the low titers typically obtained from isolates. We therefore set out to investigate the possibility of using classical breeding techniques coupled with genetic manipulation as a means to develop such fungi. We show that the original production strain of C. passeckerianus is able to fruit under laboratory conditions, giving viable haploid meiotic basidiospores. The derived progeny displayed the typical physiological and genetic characteristics of a tetrapolar mating system. The monokaryon haploids produced pleuromutilin and haploid lines were amenable to genetic manipulation. Together this shows that the basic requirements for a classical breeding approach are present and the tools required to undertake directed genetic engineering on haploid strains are available, demonstrating that strain improvement may be feasible in this fungus.

Pleurotus eryngii (DC.) Quel. is widely used for bioconverting lignocellulosic byproducts into biofuel and value added products. Sequencing and annotating the genome of a monokaryon strain P. eryngii 183 allows us to gain a better understanding of carbohydrate-active enzymes (CAZymes) and oxidoreductases for degradation of lignocellulose in white-rot fungi. The genomic data provides insights into genomic basis of degradation mechanisms of lignin and cellulose and may pave new avenues for lignocellulose bioconversion.

To examine the potential of a method of double-stranded (ds) RNA infection to Helicobasidium mompa, the transmissibility of dsRNA between strains of this fungus was investigated. Strain V70 was used as a dsRNA donor. The dsRNA recipients were six strains that were mycelially incompatible with V70, plus two monokaryotic strains. Random amplified polymorphic DNA analysis suggested that the mycelially incompatible strains were genetically different mutually; however, the analysis also suggested that V70 was genetically homogeneous with the two monokaryotic strains. When V70 was paired with either of the mycelially incompatible strains, the dsRNAs did not transmit to the recipients at all. When V70 was paired with the two monokaryotic strains, the dsRNAs were transmitted to the monokaryotic strains. The two monokaryotic strains, which had acquired dsRNAs from V70 in the previous experiment, were used as new dsRNA donors in a next experiment so that we could investigate dsRNA transmission from these monokaryotic strains to the six strains used in the previous experiment. One of the monokaryotic strains permitted dsRNA transmission to two of the six recipients. We conclude that we can infect genetically different strains of H. mompa with dsRNA using the monokaryotic strains.

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. Fil: Omarini, Alejandra Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Leibniz Universität Hannover. Hannover; Alemania Fil: Plagemann, Ina. Leibniz Universität Hannover. Hannover; Alemania Fil: Schimanski, Silke. Leibniz Universität Hannover. Hannover; Alemania Fil: Krings, Ulrich. Leibniz Universität Hannover. Hannover; Alemania Fil: Berger, Ralf G.. Leibniz Universität Hannover. Hannover; Alemania

Patterns of protein expression in the monokaryons and the derived dikaryon of the basidiomycete Flammulina velutipes were investigated using two-dimensional polyacrylamide gel electrophoresis and silver staining. Over 300 spots were detected on each gel, and 19 proteins were expressed in the dikaryon before fruiting treatment but not expressed in the monokaryon before fruiting treatment. On the other hand, 9 proteins were expressed in the monokaryon specifically. Newly expressed proteins increased after fruiting treatment (at 7 d and 14 d), but decreased after fruit bodies were formed (at 21 d). The number of newly expressed proteins after the fruiting treatment was similar in the monokaryon. Proteins expressed characteristically were classified into 7 types. The highest number of proteins (13 proteins) were specifically expressed in the dikaryon at 14 d when the fruit-bodies were formed. Twenty-seven proteins were expressed specifically in the fruit body. Fruit body specific proteins Pf1 and Pf3 were sequenced. This revealed Pf1 and Pf3 had amino acid sequences that were similar to each other, as well as a high similarity to the deduced amino acid sequence of the FDS gene isolated from F. velutipes.

Monokaryotic Pycnoporus cinnabarinus strains were obtained from the dikaryotic strain I-938. One of these, designated MK18, consistently produced high laccase activity. In cultures of MK18 and I-938 where ferulic acid was added as laccase inducer, laccase activity was enhanced about 2.5-fold reaching 3400 U/l for the MK18 strain. Laccase was purified to homogeneity and under the selected growth conditions, only one isoform of the enzyme was produced. The N-terminal sequence was similar to the amino terminal sequence of laccase II from Trametes versicolor. The enzyme was stable at 60 ∘C for more than 1 h.

Compatible monokaryotic strains of Agaricus bisporus ATCC 36975 and 36976 and the resulting dikaryon of their mating were grown in a liquid medium, and their respective cell walls were prepared. Significant differences were not found in the gross chemical composition of the three hyphal types. However, the neutral carbohydrate composition of the complete walls and their fractions was found to be somewhat different in each strain. More consistent differences were encountered in the chemical structure of the distinct polysaccharidic wall fractions in the three types of organisms. Some of these structural wall differences can be considered as characteristic markers for differentiating the mono- and dikaryotic types of A. bisporus.

The rust fungi appear to have evolved a sophisticated complex of molecular interactions with their host plants that govern both plant resistance and susceptibility. It is suggested that many of these interactions relate to the maintenance and effective exploitation of biotrophy, and that host specificity and the obligacy of parasitism are a consequence of the resulting interactive molecular control of plant and fungal activities. For the dikaryon, plant signals are required for locating stomata and the formation of infection structures, haustorial mother cells, and haustoria. Host susceptibility to both the monokaryon and the dikaryon appears to involve the suppression of defensive secretory processes, the induction of cellular alterations in invaded cells, and, for the dikaryon at least, changes in nutrient translocation. Parasite-specific resistance involves cultivar-specific fungal signals (elicitors) of defense responses such as cell death and callose deposition. The nature of, and evidence for, the signals involved in these interactions are reviewed. Key words: biotrophy, elicitors, rust fungi, signal exchange.

summary The ectomycorrhizal activity (expressed as the number of mycorrhizas per plant) of indole-3-acetic acid (IAA) overproducer mutants of Hebeloma cylindruspurum Rumagnesi and of their mono- and dikaryotic progenies has been studied in an attempt to clarify the role of fungal IAA in the establishment of ectomycorrhizal symbiosis, The original mutants obtained from the hi monokaryon produced a higher number of mycorrhizas than did the wild type. The study of the mycorrhizal activity of synthesized dikaryons heterozygous for the FIR (fluoroindoleresistant) mutations, which are responsible for IAA overproduction, showed that these mutations are recessive. The vitro fruiting of a dikaryon heterozygous for a FIR mutation allowed the isolation of IAA-overproducing sib-monokaryons. Their average mycorrhizal activity was higher than that of non-overproducers. indicating that, although the mycorrhizal activity of sib-monokaryons is under polygenic control, high IAA production gives an advantage in mycorrhiza formation. This was confirmed by the higher number of mycorrhizas formed by synthesized dikaryons homozygous for a FIR mutation than by wild type mycelia. The mycorrhizas formed by the mono- or dikaryotic wild types were characterized by a uniseriate Hartig net, whereas it was pluriseriate in the case of mycorrhizas formed by IAA overproducer mutants. The mutants stimulated the growth of the host plants to the same extent as did the wild types, indicating that growth stimulation is not a direct consequence of fungal auxin production. The usefulness of these mutants as a model to study the role of fungal IAA in mycorrhiza formation is discussed.

Rapid cell death induced in the resistant cowpea cultivar, Dixie Cream, by the monokaryotic stage of race 1 of the cowpea rust fungus was delayed by treatments with Superoxide dismutase, mannitol, catalase and the lipoxygenase inhibitor salicyl hydroxamic acid. These treatments also delayed cell death caused by a cultivar-specific elicitor of necrosis present in intercellular washing fluids from monokaryon-infectcd susceptible plants. The data suggest that active oxygen species, hydrogen peroxide and lipid peroxidation may be involved in both elicitor- and fungal-induced cell death, and in both cases, treatments were effective when applied close to the onset of the first cytological signs of necrosis. Pre-inoculation treatment of cv. Dixie Cream with heat, or prenecrosis treatments with the protein synthesis inhibitor blasticidin S or abscisic acid, also delayed fungal-induced cell death, but heat was the only treatment of all those tested that resulted in a slight increase in fungal growth. Cell death was also delayed by damaging or killing the fungus at various stages before the normal onset of necrosis suggesting the necessity for sustained fungal metabolism. All of these data, and the time-course of elicitor-induced necrosis, are consistent with the hypothesis that the elicitor is the primary cause of cell death in this incompatible plant-fungal interaction. However, the data also suggest that cell death may not be the primary cause of the cessation of fungal growth.

Production of extracellular β-1, 3-glucanase activity by a monokaryotic Schizophyllum commune strain was monitored and results indicated that the β-glucanase activity consisted of an endo- β-1, 3-glucanase activity, besides a negligible amount of β-1, 6-glucanase and β-glucosidase activity. Unlike the β-1, 3-glucanase production of the dikaryotic parent strain S. commune ATCC 38548, the β-1, 3-glucanase formation of the monokaryon was not regulated by catabolite repression. The endo- β-1, 3-glucanase of the monokaryon was purified from the culture filtrate by lyophilization, anion exchange chromatography on Mono Q, and gel filtration on Sephacryl S-100. It appeared homogeneous on SDS-PAGE with a molecular mass of 35.5 kDa and the isoelectric point was 3.95. The enzyme was only active toward glucans containing β-1, 3-linkages, including lichenan, a β-1, 3-1, 4-D-glucan. It attacked laminarin in an endo-like fashion to form laminaribiose, laminaritriose, and high oligosaccharides. While the extracellular β-glucanases from the dikaryotic S. commune ATCC 38548 degraded significant amounts of schizophyllan, the endo- β-1, 3-glucanase from the monokaryon showed greatly reduced activity toward this high molecular mass β-1, 3-/β-1, 6-glucan. The Km of the endoglucanase, using laminarin as substrate, was 0.28 mg/mL. Optimal pH and temperature were 5.5 and 50 °C, respectively. The enzyme was stable between pH 5.5 and 7.0 and at temperatures below 50 °C. The enzyme was completely inhibited by 1 mM Hg2+. Growth of the monokaryotic S. commune strain was not affected by its constitutive endo- β-1, 3-glucanase formation.Key words: endo- β-1, 3-glucanase, Schizophyllum commune, monokaryon, constitutive endo- β-1, 3-glucanase formation.

summary IAA-overproducer mutants of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum Romagnesi were isolated in an attempt to clarify the role of fungal IAA in ectomycorrhiza formation. By contrast to the wild type, mutants are able to metabolize endogenous Trp to IAA. They were isolated using a two-step method. One hundred and forty-four mutants with altered regulation of the Trp branch of the shikimate pathway were isolated from two compatible monokaryons by selecting for resistance to the indole analogue, 5-fluoroindole. These mutants are Trp-overproducers and most of them accumulate free Trp. Seventeen mutants able to synthesize IAA in the absence of exogenous precursor (tryptophan or intermediates of the tryptophan biosynthetic pathway) were identified by screening for the ability to metabolize endogenous Trp into IAA. By studying the activity of key enzymes of the Trp biosynthetic pathway (i.e. anthranilate synthase and Trp synthase), it was evident that different mechanisms led to IAA-overproduction. A correlation was detected between the level of intracellular Trp accumulation and the ability of the mutants to metabolize endogenous Trp to IAA. This indicates that, irrespective of the mutation, intracellular Trp accumulation is a prerequisite for IAA overproduction. A positive correlation was also detected between, on the one hand, resistance to fluoroindole and, on the other hand, intracellular Trp accumulation and, subsequently, IAA-overproduction. Thus, resistance to high fluoroindole concentrations appeared to provide a method for the direct selection of IAA-overproducer mutants. In order to be able to compare these mutants with the wild parental dikaryon and to perform genetic analyses, the mutants were fused with a compatible wild monokaryon. The resulting dikaryons were able to form mycorrhizas with the normal host plant, Pinus pinaster, and to fruit under controlled culture conditions, thus allowing stabilization and purification of the mutations. IAA-overproducer monokaryons were detected within the progeny of all the mutants. They are now being used to study the role of fungal IAA in ectomycorrhizas.

We have examined the kinetic relationship between the rate of entry into the S phase in human diploid fibroblast-like (HDFL) monokaryon cells and (1) the concentration of DNA polymerase α activity and (2) the cell volume. In the former studies, a first-order dependence between the rate of entry into the S phase and the concentration of DNA polymerase α activity was observed, consistent with the enzyme, or a coregulated factor, being rate limiting for this metabolic process. Examination of the nature of the dependence of the rate of entry into the S phase upon cell volume revealed a more complex relationship. The results obtained in studies with synchronized cultures are consistent with the presence of two to three rate-limiting reactants when cell volume is the independent variable. Studies with asynchronous HDFL cell cultures revealed that the smallest cells in the G1 population, presumably the early G1 cells, enter the S phase at an increasing rate as a function of cell volume up to a certain size, beyond which the cells enter at a decreasing rate similar to that observed in the studies with the synchronized cultures. Similar studies examining the relationship between cell volume and the rate of entry into S phase in three established immortal cell lines revealed positive correlation between the rate of entry into S phase and cell volume throughout the size range of the G1 population. This latter observation suggests that the factors involved in the initiation of the S phase may be present in concentrations that are not rate limiting in immortal cell lines.

Coprinus congregatus has a membrane-associated laccase which is not secreted into the culture media. A mutant monokaryon has been generated by UV irradiation which shows slower radial growth than the wild-type (72.7%) but which secretes laccase. The laccase of the mutant was compared with that of wild-type by native PAGE analysis, and showed identical mobility.

Sc3p is the major hydrophobin produced and excreted by the wood-rotting fungus Schizophyllum commune when grown as a monokaryon in culture. In addition to Sc3p, several lower molecular weight proteins of unknown function and a high molecular weight polysaccharide, schizophyllan, are present in this fungal culture medium. Previous studies in our laboratory indicated that schizophyllan and hydrophobin, as produced in the culture supernatant, work in concert to ultimately produce stable films on hydrophilic substrates. We report a new procedure for isolation of hydrophobin that does not require treatment with strong organic acids. This procedure allows a systematic evaluation of the importance of the individual hydrophobin and schizophyllan components in solution stabilization and film formation from a conformational state more representative of that found during fungal production. Experimental results indicate that schizophyllan stabilizes small hydrophobin multimers in aqueous solution. In addition, schizophyllan is necessary for subsequent hydrophobin assembly into films on hydrophilic surfaces. By contrast, hydrophobin alone is sufficient for thin film assembly onto a hydrophobic surface; however, hydrophobin aggregation at high protein concentration in the absence of schizophyllan may limit the efficiency of film formation. A model has been proposed in which the polysaccharide acts as a hydrophilic stabilizer of hydrophobin in solution preventing self-aggregation. The schizophyllan-stabilized complex may also allow conformational mobility for lateral reorganization during deposition and film coalescence.

Our examination of the cytological characteristics of the vegetative incompatibility reaction in a filamentous basidiomycete, Helicobasidium monpa, by analyzing the fluorescence emitted by ethidium bromide and acridine orange stained nuclei is described. Hyphal anastomoses between strains belonging to different mycelium compatibility groups (MCG) were observed with cell death in fused hyphae, whose nuclei were intensified by ethidium bromide. In contrast, the nuclei in a living cell were not intensified by staining with ethidium bromide, but were intensified by staining with acridine orange. These results indicate that in H. monpa, ethidium bromide staining is a useful method for detecting dead cells. We also examined the relationships between the alternation of ploidy and hyphal anastomosis formation using the newly developed method on filamentous fungi. The tetraploid monokaryon strain derived from the original dikaryon strain by continuous subculture could not be fused to any wild type strains, but the original dikaryon strain could be fused without cell death to only the same MCG strain. In contrast, the haploid dikaryon strain derived from the original monokaryon strain fuses to several strains belonging to different MCGs without cell death. These results suggested that the cellular ploidy of this fungus is closely related to its mating system and, H. monpa may be a self-fertilizing fungus.

A comparison of Pycnoporus cinnabarinus strains for laccase production was carried out. A dikaryotic strain, I-937 strain, producing a high level of laccase (9500 U l(-1)) was selected. The study of the life cycle in vitro of this dikaryotic strain led to isolation of monokaryons. Forty-eight monokaryotic strains were isolated and screened for laccase production. One of these strains, ss3, produced a higher level of laccase than the parental strain I-937. The maximum production reached 29000 U l(-1) in medium supplemented with ferulic acid.

The ability of 10 dikaryotic and 20 monokaryotic strains of Trametes ( Coriolus ) versicolor to bleach and delignify hardwood and softwood kraft pulps was assessed. A dikaryon (52P) and two of its mating-compatible monokaryons (52J and 52D) derived via protoplasting were compared. All three regularly bleached hardwood kraft pulp more than 20 brightness points (International Standards Organization) in 5 days and softwood kraft pulp the same amount in 12 days. Delignification (kappa number reduction) by the dikaryon and the monokaryons was similar, but the growth of the monokaryons was slower. Insoluble dark pigments were commonly found in the mycelium, medium, and pulp of the dikaryon only. Laccase and manganese peroxidase (MnP) but not lignin peroxidase activities were secreted during bleaching by all three strains. Their laccase and MnP isozyme patterns were compared on native gels. No segregation of isozyme bands between the monokaryons was found. Hardwood kraft pulp appeared to adsorb several laccase isozyme bands. One MnP isozyme (pI, 3.2) was secreted in the presence of pulp by all three strains, but a second (pI, 4.9) was produced only by 52P. A lower level of soluble MnP activity in one monokaryon (52D) was associated with reduced bleaching ability and a lower level of methanol production. Since monokaryon 52J bleached pulp better than its parent dikaryon 52P, especially per unit of biomass, this genetically simpler monokaryon will be the preferred subject for further genetic manipulation and improvement of fungal pulp biological bleaching.

The relationship between fungal nuclear condition and growth of the two parasitic stages of the cowpea rust fungus was studied by light microscopy in living and fixed material. Uninucleate teliospores of the cowpea rust fungus germinated to form a promycelium that produced four basidiospores, each containing two nuclei as the result of a nuclear division during nuclear migration into the developing spore. Basidiospore-derived hyphae within an epidermal cell of a susceptible host cultivar rapidly became monokaryotic by (i) synchronous mitosis of the two basidiospore nuclei after they had migrated into the invasion hypha and the latter had grown to about 40 μm; (ii) the subsequent immediate division by septa of the invasion hypha into one binucleate and two uninucleate cells; and (iii) the disappearance of one of the nuclei in the binucleate cell. However, in the same host cultivar, the intercellular infection hyphae, secondary hyphae, and first two haustorial mother cells (HMCs) derived from dikaryotic urediospores of the same fungus had variable nuclear numbers due to nuclear degradation and asynchronous nuclear divisions. Nevertheless, by 3 days after inoculation, all subsequently formed intercellular hyphae, HMCs, and haustoria were binucleate. In the three resistant cultivars tested, the presence or absence of nuclear division in the basidiospore-derived, intracellular invasion hyphae depended on the degree of maximum fungal growth; growth inhibition was related to plant cell death in two of the cultivars and to callose encasement in cultivar Queen Anne. In urediospore-derived infections of these same cultivars, the intercellular infection hyphae and HMCs developed normally, but nuclear migration into the haustorium was reduced, often before callose encasement of the haustorium in Queen Anne. The data suggest that in Queen Anne, fungal encasement plays a greater role in resistance to the basidiospore-derived than to the urediospore-derived stage of infection.

Teliospores of Tilletia caries separated from contaminating micro-organisms by centrifugation in sucrose gradients yielded, in axenic culture, mycelia which became dominated by a monokaryon if cultures were incubated at 22 °C, or stable dikaryons if incubated at 15°. Dikaryotic mycelia occasionally produced teliospores.

The RNA/protein ratios in two co-isogenic monokaryons ofSchizophyllum commune, growing in shaken cultures, were similar (about 5.2). However, dikaryotic hyphae on average contained only 1.3 times as much RNA and protein as did monokaryotic hyphae. The hyphal compartment volume of the dikaryon was also found to be 1.3 times larger than that of the monokaryons. This difference resulted from an increase in hyphal diameter rather than an increase in cell length. Autoradiographic estimation of the rate of RNA synthesis showed that the individual nuclei of the dikaryon synthesized RNA at a rate which was about 28% less than that of the monokaryotic nuclei. This largely explained the higherRNA/DNA ratio observed in the monokaryons compared with the dikaryon. The partner nuclei in dikaryotic cells contribute equally to RNA synthesis.

Total polysomes, including free and membrane-bound polysomes, were isolated from the monokaryotic and dikaryotic mycelial cell types of the basidiomycete Schizophyllum commune grown under submerged conditions. Sucrose gradient centrifugation showed that these isolated polysomes had a size distribution as expected for polysomes functioning in vivo and thus contained intact mRNA. RNA preparations extracted from the polysomes were used to analyze the mRNA sets in the monokaryon and dikaryon. Saturation hybridization of single-copy DNA with a vast excess of polysomal RNA and cell-free translation of polysomal RNA in a wheat germ system followed by two-dimensional gel electrophoresis of the products did not reveal significant differences between the mRNA sets in both mycelial cell types. Moreover, it was found that the sequence complexities and coding properties of polysomal RNA and total (nuclear and cytoplasmic) RNA from S. commune are not detectably different. From these results and those obtained in previous studies it was concluded that the differentiation between monokaryotic and dikaryotic mycelial cell types as controlled by A and B incompatibility genes involves differential modification of polypeptides during or after mRNA translation.

Extracts of the mycelium of Coprinus lagopua (sensu Buller) contain two glutamate dehydro genases with different optimum pH values. One is assayed with nicotinamide adenine dinucleotide (NAD-GDH) and the other with nicotinamide adenine dinucleotide phosphate (NADP-GDH). Changes in specific activity of the enzymes were investigated during the growth of both a monokaryon (H9) and a dikaryon (H9 x TO) in different media and after the transfer of mycelium from one growth medium into another. In the latter case the magnitude of the changes in enzyme activity could be altered by modification of either the carbon or the nitrogen source in the transfer medium. It is concluded from the results obtained that neither glutamate nor the ammonium ion seems to regulate directly the synthesis of either enzyme. However, some of the results are in accordance with the view that a product of glucose metabolism represses the synthesis of the NAD-GDH and derepresses or induces that of the NADP-GDH and evidence that this regulator is 2-oxoglutarate was obtained. It is also concluded that the complete system of regulation must involve more than one molecule.

Proteins and isozymes of several different classes of enzymes in partially purified protein extracts of monokaryons, dikaryons, and monokaryon mixtures of a bipolar Coprinus sp. were separated on polyacrylamide gels by slab electrophoresis. Differences in protein and isozyme spectra were correlated with the operation of the incompatibility factors and with the results of Wang and Raper on Schizophyllum . It was concluded that the shift from monokaryon to dikaryon mediated a major change in the nature, quantity, or distribution of the proteins of this Coprinus sp.

An alpha-D-glucan was isolated from a monokaryon of Polyporus circinatus; it has been characterized as a highly branched glycogn.

The possible involvement of the glutamine synthetase/glutamate synthase pathway in ammonia assimilation is reported in a monokaryon of Coprinus cinereus.

Background Two reference strains have been sequenced from the mushroom Coprinopsis cinerea, monokaryon Okayama 7/#130 (OK130) and the self-compatible homokaryon AmutBmut. An adenine-auxotrophy in OK130 (ade8-1) and a para-aminobenzoic acid (PABA)-auxotrophy in AmutBmut (pab1-1) offer selection markers for transformations. Of these two strains, homokaryon AmutBmut had been transformed before to PABA-prototrophy and with the bacterial hygromycin resistance marker hph, respectively. Results Gene ade8 encodes a bifunctional enzyme with an N-terminal glycinamide ribonucleotide synthase (GARS) and a C-terminal aminoimidazole ribonucleotide synthase (AIRS) domain required for steps 2 and 5 in the de novo biosynthesis of purines, respectively. In OK130, a missense mutation in ade8-1 rendered residue N231 for ribose recognition by the A loop of the GARS domain into D231. The new ade8+ vector pCcAde8 complements the auxotrophy of OK130 in transformations. Transformation rates with pCcAde8 in single-vector and co-transformations with ade8+-selection were similarly high, unlike for trp1+ plasmids which exhibit suicidal feedback-effects in single-vector transformations with complementation of tryptophan synthase defects. As various other plasmids, unselected pCcAde8 helped in co-transformations of trp1 strains with a trp1+-selection vector to overcome suicidal effects by transferred trp1+. Co-transformation rates of pCcAde8 in OK130 under adenine selection with nuclear integration of unselected DNA were as high as 80% of clones. Co-transformation rates of expressed genes reached 26–42% for various laccase genes and up to 67% with lcc9 silencing vectors. The bacterial gene hph can also be used as another, albeit less efficient, selection marker for OK130 transformants, but with similarly high co-transformation rates. We further show that the pab1-1 defect in AmutBmut is due to a missense mutation which changed the conserved PIKGT motif for chorismate binding in the C-terminal PabB domain to PIEGT in the mutated 4-amino-4-deoxychorismate synthase. Conclusions ade8-1 and pab1-1 auxotrophic defects in C. cinerea reference strains OK130 and AmutBmut for complementation in transformation are described. pCcAde8 is a new transformation vector useful for selection in single and co-transformations of the sequenced monokaryon OK130 which was transformed for the first time. The bacterial gene hph can also be used as an additional selection marker in OK130, making in combination with ade8+ successive rounds of transformation possible.

A monokaryotic strain of Puccinia graminis f.sp. tritici , having uredospores with an unusually large nucleus, was obtained in axenic culture from a normal parent. By studying uredospores formed on wheat the characteristics of these two strains were compared. The uredospore nucleus of the monokaryon and the diploid fusion nucleus of the parental teleutospores both had a nuclear volume double that of the parental uredospores. Most of the uredospores of the monokaryon were uninucleate and were the same size as the binucleate uredospores of the parent. Abnormally large uredospores occurred infrequently in samples from the monokaryon and these had two nuclei both the size of the nucleus of a uninucleate uredospore. The results indicate that the monokaryotic strain is a diploid form of P. graminis f.sp. tritici and not haploid as formerly believed. A formaldehyde-alkaline ethanol-acetic acid orcein technique is described for staining nuclei in mature uredospores.

The intercellular and intracellular structures of Puccinia poarum on the pycnial-aecial host, Tussilago farfara , and the uredial-telial host, Poa pratensis , were compared by transmission and scanning electron microscopy. The intercellular growth of the monokaryon on Tussilago is denser and more extensive than that of the dikaryon in Poa leaf tissue. Although ultra-structurally similar, monokaryotic hyphae differ from dikaryotic hyphae in the intracellular structures which they bear and in their interaction with host walls, often growing embedded in wall material which may project into host cells. The relatively undifferentiated intracellular hyphae of the monokaryon differ from the haustoria of the dikaryotic growth stages of P. poarum in their irregular growth, septation, lack of a neck-band or markedly constricted neck, the deposition of host wall-like material in the external matrix bounded by the invaginated host plasmalemma and in the association of callose reactions with intracellular hyphae and adjacent parts of host walls. Whereas penetration by monokaryotic hyphae involves ingrowth of host wall layers, entry by haustoria induces little disturbance of the host wall but is characterized by a thickening of the haustorium mother cell wall at its point of contact with the host cell. The extra-haustorial matrix contains electron dense fibrils of similar staining reaction to the fungal wall but lacks any material resembling the host cell wall. The significance of the differences between the monokaryotic and dikaryotic development of this rust fungus is discussed.