Control of fungal development: I. The effects of two regulatory genes on growth in Schizophyllum commune

In Schizophyllum the various events comprising the transition from the homokaryotic to the dikaryotic stage of the life cycle are triggered by two sets of developmental regulatory genes known as the A and B incompatability factors. This paper defines the effects of these genes on growth of surface colonies by establishing the growth curves of dikaryons, comon-A heterokaryons, and strains that morphologically mimic dikaryons or heterokaryons because of constitutive mutations in A and/or B.Like homokaryons, all these developmental stages and genetic mimics have triphasic growth curves with definite exponential phases. Growth curves of dikaryons and common-A heterokaryons are indistinguishable from those of their corresponding genetic mimics. However, the growth rate during exponential phase and the timing of the entire curve are dependent upon developmental type, with the consequence that colonies of different developmental stages harvested either at equal times or at equal weights are not necessarily in the same phase of colonial growth. Data presented allow choice of harvesting times such that colonies of the different developmental types will be within the same growth phase.Biochemical differences between homokaryons and dikaryons must be due to differentiation since the two stages have virtually the same growth rate.     

Isolation and characterization of compatible diploids of Schizophyllum commune.

Summary Common-AB diploids with several heterozygous biochemical markers were mated with appropriately marked haploid strains of S. commune in an effort to obtain compatible, common-A, and common-B diploid progeny with biochemical markers identical to those of the common-AB parent. The spores from these crosses were germinated on minimal medium. Five compatible diploids, but no common-A or common-B diploids, marked as desired, were isolated by this method. Two possessed some dikaryotic cells and two had many dikaryotic cells. One of the latter was shown to have peculiar behaviour associated with one of its B mating-type factors.     

PHASE-MICROSCOPIC OBSERVATIONS OF FUSIONS OF NUCLEI IN SOMATIC CELLS OF A HETEROKARYON OF SCHIZOPHYLLUM COMMUNE

Pairs of nuclei in apical cells of a common-B heterokaryon (A41 B41 + A2 B41) of Schizophyllum commune fused prior to the nuclear division. It is assumed that each fusion was followed by a reduction division. Five consecutive fusions and divisions in a single hypha were photographed in situ with a phase microscope. These fusions and reduction divisions are possibly related to the genetically determined phenomenon of somatic recombination as a part of the parasexual cycle in fungi.     

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     

EVIDENCES OF COMMON-AB HETEROKARYOSIS IN SCHIZOPHYLLUM COMMUNE

Since preliminary experimental evidence for the existence of common-AB heterokaryons in Schizophyllum commune was inconclusive, experiments were performed to test for and to characterize these heterokaryons. Prototrophic mycelia regularly result from the growing together of pairs of mutant strains of identical mating type which carry non-allelic nutritional deficiencies. Since crossfeeding through a dialyzing Cellophane membrane does not occur, the prototrophic common-AB mycelia apparently have both parental nuclear types within a common cytoplasm. Hyphal tips isolated from the peripheries of these prototrophic mycelia usually are not prototrophic. The distributions of the parental nuclear types in the subcultured prototrophic mycelia, as revealed by grid sampling, are irregular in pattern and extent; these patterns probably reflect the nuclear ratios in the transfer inocula, as well as the distributions of nuclear types in various parts of the inocula. Since a common-AB prototroph typically consists of regions containing a single nuclear type as well as regions containing both nuclear types, marked fluctuations of the estimated nuclear ratio occur upon subculture, and many small transfer-inocula are not prototrophic as they contain only a single auxotrophic nuclear type. The patterns of nuclear distributions in prototrophic common-AB mycelia are probably maintained by the restriction of nuclear migration.     

Selectively recombining B incompatibility factors of Schizophyllum commune

Summary Analysis of genetic crosses among strains of Schizophyllum commune carrying recombining B factors has revealed that not all heteroallelic pairs of B factors are able to recombine with each other. This suppression of recombination is highly specific and appears to be determined by the B factors themselves.     

THE KINETICS OF INITIAL NUCLEAR EXCHANGE IN COMPATIBLE AND NONCOMPATIBLE MATINGS OF SCHIZOPHYLLUM COMMUNE

The effect of mating-type factors on exchange of nuclei between mycelial fragments mated in liquid culture was studied. Evidence for exchange of nuclei was based on three criteria for detecting nuclei from both mating partners in mycelial fragments of the developing dikaryon or hetero-karyon. The kinetics of nuclear exchange were shown to be relatively independent of the method used. It was shown that the kinetics of nuclear exchange in the first 96 hr are different in the compatible (A41 B41 × A42 B42), common-A (A42 B42 × A42 B41), common-B (A41 B42 × A42 B42), and common-AB (A41 B41 × A41 B41) matings. In all four types of matings, the percentage of fragments possessing both types of nuclei 12-24 hr after mating is nearly equal. After this time, significant differences appear in the patterns for compatible vs. the three non-compatible matings and the common-A vs. the common-B and common-AB matings. The percentage of mycelial fragments possessing both types of nuclei throughout the 96-hr test period is similar for both the common-B and common-AB matings. The kinetics of nuclear exchange were shown to be independent of the particular mating-type alleles and nutritional markers used. When the efficiency of nuclear exchange in complete and minimal media was compared, it was shown that nuclear exchange occurred more rapidly and synchronously in minimal medium. This difference is not due to growth differences in the two media. These data indicate that the earliest mating interactions, i.e., hyphal anastomosis and nuclear exchange, are independent of the mating-type factors but that subsequent events are determined by these genes.     

DNA amplification polymorphisms of the cultivated mushroom Agaricus bisporus.

Single 10-bp primers were used to generate random amplified polymorphic DNA (RAPD) markers from commercial and wild strains of the cultivated mushroom Agaricus bisporus via the polymerase chain reaction. Of 20 primers tested, 19 amplified A. bisporus DNA, each producing 5 to 15 scorable markers ranging from 0.5 to 3.0 kbp. RAPD markers identified seven distinct genotypes among eight heterokaryotic strains; two of the commercial strains were shown to be related to each other through single-spore descent. Homokaryons recovered from protoplast regenerants of heterokaryotic strains carried a subset of the RAPD markers found in the heterokaryon, and both of the haploid nuclei from two heterokaryons were distinguishable. RAPD markers also served to verify the creation of a hybrid heterokaryon and to analyze meiotic progeny from this new strain: most of the basidiospores displayed RAPD fingerprints identical to that of the parental heterokaryon, although a few selected slow growers were homoallelic at a number of loci that were heteroallelic in the parent, suggesting that they represented rare homokaryotic basidiospores; crossover events between a RAPD marker locus and its respective centromere appeared to be infrequent. These results demonstrate that RAPD markers provide an efficient alternative for strain fingerprinting and a versatile tool for genetic studies and manipulations of A. bisporus.     

A distinctive phenotype in the common-<Emphasis Type="Italic">A</Emphasis> heterokaryon of <Emphasis Type="Italic">Coprinus cinereus</Emphasis>

The homobasidiomycete Coprinus cinereus, unlike Schizophyllum commune, is not known to exhibit an obvious heterokaryotic phenotype in common-A matings. In the present study we found that progeny isolated from a fruit-body collected in the field exhibit a distinctive mycelial development in common-A matings. Genetic analysis suggested that the common-A heterokaryotic phenotype is brought about by a nuclear factor(s) other than the mating type genes. Received: March 30, 2001 / Accepted: October 1, 2001     

Allelic Diversity at the het-c Locus in Neurospora tetrasperma Confirms Outcrossing in Nature and Reveals an Evolutionary Dilemma for Pseudohomothallic Ascomycetes

Vegetative cells of the filamentous ascomycete Neurospora tetrasperma are typically heterokaryotic, possessing haploid nuclei of both A and a mating types. As a consequence, N. tetrasperma is self-fertile. This life cycle, referred to as pseudohomothallism, clearly derives from true heterothallism of the type exhibited by related species such as N. crassa. Occasional homokaryotic, single-mating-type (heterothallic) isolates occur; in the laboratory, such strains can be outcrossed. The potential for outcrossing in N. tetrasperma raises the question of how this organism avoids heterokaryon incompatibility. Heterokaryon incompatability in vegetatively growing fungi is controlled by multiple loci. Two strains must be identical at each het locus (11 in N. crassa) to form a stable heterokaryon. Prior to the present survey, it seemed plausible that N. tetrasperma avoids heterokaryon incompatibility by maintaining compatible allele combinations through continual selfing. A survey of het-c variation among wild-type isolates in this study demonstrated that N. tetrasperma outcrosses in nature and that such matings can result in incompatible combinations of het-c alleles. Whereas individual wild-type isolates are invariably homoallelic for het-c, closely related strains may possess functionally different het-c alleles, which predate the origin of N. tetrasperma. Therefore, pseudohomothallic ascomycetes such as N. tetrasperma face an apparent evolutionary dilemma: the benefits of outcrossing must be balanced against the fact that matings can produce unstable heterokaryons and disrupt the pseudohomothallic life cycle. Received: 22 October 1999 / Accepted: 7 September 2000     

DNA amplification polymorphisms of the cultivated mushroom Agaricus bisporus.

Single 10-bp primers were used to generate random amplified polymorphic DNA (RAPD) markers from commercial and wild strains of the cultivated mushroom Agaricus bisporus via the polymerase chain reaction. Of 20 primers tested, 19 amplified A. bisporus DNA, each producing 5 to 15 scorable markers ranging from 0.5 to 3.0 kbp. RAPD markers identified seven distinct genotypes among eight heterokaryotic strains; two of the commercial strains were shown to be related to each other through single-spore descent. Homokaryons recovered from protoplast regenerants of heterokaryotic strains carried a subset of the RAPD markers found in the heterokaryon, and both of the haploid nuclei from two heterokaryons were distinguishable. RAPD markers also served to verify the creation of a hybrid heterokaryon and to analyze meiotic progeny from this new strain: most of the basidiospores displayed RAPD fingerprints identical to that of the parental heterokaryon, although a few selected slow growers were homoallelic at a number of loci that were heteroallelic in the parent, suggesting that they represented rare homokaryotic basidiospores; crossover events between a RAPD marker locus and its respective centromere appeared to be infrequent. These results demonstrate that RAPD markers provide an efficient alternative for strain fingerprinting and a versatile tool for genetic studies and manipulations of A. bisporus.     

Parasexual analysis of common-A crosses in the basidiomycete Agrocybe aegerita

Summary Crosses were performed between homokaryons of Agrocybe aegerita having the same allele at the A incompatibility gene but different B alleles. Heterokaryotic mycelia originating from crosses between two complementary auxotrophs were characterized by their instability on complete medium and extensive anastomosis between hyphae. Diploid mycelia were selected by plating oidia recovered from these heterokaryons onto minimal medium. These mycelia were characterized by the production of larger oidia than those of homokaryons, the release of a brown pigment when growing on complete medium and extensive hyphal anastomoses. Diploids retained the two B incompatibility functions of their homokaryotic parents and gave rise to a diploid/haploid dikaryon when crossed with a compatible homokaryon. Nearly 1% of the oidia recovered from heterokaryons were diploid. These nuclear fusion frequencies as well as the production of brown pigments enabled the identification of diploid strains on complete medium. In this way, crosses between wild prototrophic strains were successfully performed. Somatic recombination was induced following the treatment of diploid mycelia with haploidizing compounds. Selection based on the inability of mycelia to produce the brown pigments on complete medium led to selection of strains homoallelic at the B locus.     

Enrichment for Nicotiana heterokaryons after protoplast fusion and subsequent growth in agarose microdrops

Protoplasts isolated from Nicotiana tabacum L. leaves and Nicotiana suaveolens Lehm. cell suspensions have been fused with polyethylene glycol (PEG). Enrichment for heterokaryons was based on a Percoll flotation protocol which allowed a preparation with 50% heterokaryons to be obtained. The heterokaryons developed into calli whose hybrid nature was shown by polyacrylamide gel electrophoresis of esterase isoenzymes. Sensitivity of the mesophyll protoplasts to PEG and different buoyant densities of the heterokaryon and cell-suspension protoplasts contribute to the enrichment. The 50%-fusion figure following purification is an improvement on standard PEG procedures.Heterokaryons obtained were embedded in 20l drops of agarose and placed in a liquid nurse culture that allows optimum growth of the heterokaryons and maintains a physical boundary between the heterokaryons and the nurse culture. Once colonies develop, the agarose microdrop is removed from the nurse culture and placed on shoot-induction medium. Agarose microdrops containing the heterokaryons can be readily removed at any stage and processed for electron microscopy to follow the early stages of colony development.The procedures we have utilised provide a robust physical selection method that allows the total variation from a heterokaryon population to be expressed.Abbreviations BAP N⁶-benzylaminopurine - BM basal medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthalene acetic acid - PEG polyethylene glycol - PKM modified Kao (1977) medium for protoplast culture     

Fumonisin B1 production and vegetative compatibility of strains from Gibberella fujikuroi mating population “A” (Fusarium moniliforme)

We examined 25 strains of Fusarium moniliforme from eight states known to be associated with equine leukoencephalomalacia, a disease caused by the mycotoxin fumonisin B₁. We determined the mating population, mating type, and vegetative compatibility group to which each of these strains belonged. All 25 strains were in the A mating population; 12 were A⁺ and 13 were A^–. Seventeen of the 25 strains were female fertile; these strains also averaged higher levels of fumonisin B₁ production than did the strains that were female sterile. Nitrate non-utilizing (nit) mutants were generated in all 25 strains and each strain was assigned to a unique vegetative compatibility group based on the inability of the derived nit mutants to form a prototrophic heterokaryon with complementary nit mutants derived from any of the other strains examined. From these data, we concluded that the production of fumonisin B₁ is a general characteristic of strains from the A mating population of Gibberella fujikuroi associated with equine leukoencephalomalacia, since all 25 of the isolates that we examined were genetically distinct individuals.     

Suppressive effect of protease inhibitors on heterokaryons containing chick erythrocyte nuclei

Nuclei of active cells (HeLa, mouse fibroblasts) partnered with chick erythrocyte nuclei in heterokaryons are suppressed, as judged by a decreased rate of ³H-uridine incorporation and diminished nuclear binding of ³H-actinomycin D. The extent to which active partner nuclei are suppressed, the extent to which erythrocyte nuclei are reactivated, and the degree of sensitivity of heterokaryons towards certain inhibitors of proteolytic enzymes, all correlate strongly with the ratios of erythrocyte nuclei to active nuclei. Thus, reactivation of individual erythrocyte nuclei is reduced progressively and active nuclei are suppressed progressively as the ratio of erythrocyte nuclei per active nucleus in heterokaryons increases. This erythrocyte nuclear-dose dependent suppression is markedly amplified when heterokaryons are grown in the presence of protease inhibitors. The protease inhibitors found to affect heterokaryons are low molecular weight (<400) inhibitors of trypsin-like enzymes: -1-tosylamide-2-leucyl chloromethyl ketone (TLCK), N-α-tosyl- -arginine methyl ester (TAME) and N-benzoyl- -arginine amide (BAA). They affect heterokaryons at concentrations comparable to the minimal concentrations at which they inhibit trypsin. Nonfused HeLa cells, mouse fibroblasts, or their homokaryons are refractory to protease inhibitors at these concentrations.Reactivation of chick erythrocyte nuclei in a heterokaryon may involve release of suppressors ordinarily confined to the erythrocyte nucleus, with subsequent redistribution of suppressor among all the nuclei of the heterokaryon. Under these circumstances the state of nuclear activity will depend on the quantity of suppressor per individual nucleus; within the erythrocyte nucleus the suppressors will decrease its rate of reactivation, when they migrate into an active nucleus they will suppress it. These suppressors, either in transit between the nuclei, or within the nuclei, may be hydrolysed by intracellular proteases.     

Use of Cobalt as a Mitochondrial Vital Stain to Study Cytoplasmic Exchange in Matings of the Basidiomycete Schizophyllum commune

Differential labeling of mates, based on the selective uptake of cobalt by mitochondria of one of the partners, has been used to determine visually by means of phase-contrast microscopy whether transfer of mitochondria occurs after hyphal fusion. Compatible and incompatible matings of the tetrapolar basidiomycete, Schizophyllum commune, were studied. Transfer was detectable in common-A, common-AB, and fully compatible matings. It was not detectable in common-B matings     

Interspecific competitive ability of homokaryotic and heterokaryotic wood decay basidiomycetes

The role of the homokaryotic life stage in the dynamics of fungal communities is relatively unknown. However, homokaryons are thought to be only a temporary stage and are therefore not generally used in ecological experiments with fungi. In this study, the relative competitive ability and growth rates of homokaryons and heterokaryons of wood decay fungi were tested to assess the potential role of homokaryons in community dynamics. A homokaryon and a heterokaryon of each of four species (Aleurodiscus lividocoeruleus, Peniophora sp. 1, Peniophora sp. 2 and Pereniporia medulla‐panis) were assessed for their competitive abilities on an agar medium. The relationship between nuclear status and competitive ability varied between species. The homokaryon of Peniophora sp. 2 was competitively superior to its heterokaryon, whereas the homokaryon of Peniophora sp. 1 was inferior to its heterokaryon. A hierarchy of competitive abilities of each isolate revealed that Pereniporia medulla‐panis homokaryon = P. medulla‐panis heterokaryon > Peniophora sp. 1 heterokaryon > Peniophora sp. 2 homokaryon > Peniophora sp. 2 heterokaryon > A. lividocoeruleus heterokaryon = A. lividocoeruleus homokaryon. This experiment indicates that homokaryons as well as heterokaryons have the potential to influence community structure through competitive effects.     

The Use of Duplication-Generating Rearrangements for Studying Heterokaryon Incompatibility Genes in Neurospora

Heterokaryon (vegetative) incompatibility, governing the fusion of somatic hyphal filaments to form stable heterokaryons, is of interest because of its widespread occurrence in fungi and its bearing on cellular recognition. Conventional investigations of the genetic basis of heterokaryon incompatibility in N. crassa are difficult because in commonly used stocks differences are present at several het loci, all with similar incompatibility phenotypes. This difficulty is overcome by using duplications (partial diploids) that are unlikely to contain more than one het locus. A phenotypically expressed incompatibility reaction occurs when unlike het alleles are present within the same somatic nucleus, and this parallels the heterokaryon incompatibility reaction that occurs when unlike alleles in different haploid nuclei are introduced into the same somatic hypha by mycelial fusion.—Nontandem duplications were used to confirm that the incompatibility reactions in heterokaryons and in duplications are alternate expressions of the same genes. This was demonstrated for three loci which had previously been established by conventional heterokaryon tests—het-e, het-c and mt. These were each obtained in duplications as recombinant meiotic segregants from crosses heterozygous for duplication-generating chromosome rearrangements. The particular method of producing the duplications is irrelevant so long as the incompatibility alleles are heterozygous.—The duplication technique has made it possible to determine easily the het-e and het-c genotypes of numerous laboratory and wild strains of unknown constitution. In laboratory strains both loci are represented simply by two alleles. Analysis of het-c is more complicated in some wild strains, where differences have been demonstrated at one or more additional het loci within the duplication used and multiple allelism is also possible.—The results show that the duplication method can be used to identify and map additional vegetative incompatibility loci, without the necessity of heterokaryon tests.     

Ultraviolet-inactivation of conidia from heterokaryons of Neurospora crassa containing uv-sensitive mutations.

The effect of three UV-sensitive mutations of Neurospora crassa, upr-I, uvs-4 and uvs-6, on the ultraviolet-inactivation of conidia from two-component heterokaryons was investigated. In two-component heterokaryons with wild-type sensitivity to radiation inactivation, all three conidial fractions exhibited similar ultraviolet-inactivation curves. Each UV-sensitive mutation studied uniquely modified the ultraviolet-inactivation curves of conidia from two-component heterokaryons. In heterokaryons heterokaryotic for upr-I, the upr-I mutation was recessive and the repair function determined by the wild type allele was functional to some degree in homokaryotic upr-I conidia. All three conidial fractions of heterokaryons containing upr-I in both components showed increased sensitivity to ultraviolet light. The uvs-4 mutation was recessive and resulted in conidia with increased UV-sensitivity only when included in both components of a heterokaryon. Homokaryotic uvs-4 conidia, which arose from heterokaryons containing both uvs-4 and wild-type components, exhibited wild-type survival. Therefore, as with upr-I, there was a carryover the repair capability to conidia which were genetically UV-sensitive. The uvs-6 mutation, when included in one component of a two-component heterokaryon, resulted in increased UV-sensitivity of both heterokaryotic and homokaryotic uvs-6 conidia. When both components contained uvs-6, the UV-sensitivity of all three conidial fractions was increased and all showed similar inactivation curves. Thus, as with upr-I and uvs-4, there was a carryover of the wild-type repair capability to genetically uvs-6 conidia. Heterokaryon tests for complementation between two non-allelic UV-sensitive mutations showed that in heterokaryotic conidia, complete complementation occurred between upr-I and uvs-4.     

COBALT AS A MITOCHONDRIAL DENSITY MARKER IN A STUDY OF CYTOPLASMIC EXCHANGE DURING MATING OF SCHIZOPHYLLUM COMMUNE

A slow in vivo uptake of cobalt from a growth medium resulted in an increase in density of mitochondria of Schizophyllum commune. Differential labeling of donor and resident mycelia, and subsequent analysis of resident mycelia surrounding donor implants, detected cobalt-dense mitochondria and demonstrated exchange of mitochondria after hyphal fusion. Transfer of mitochondria occurred in fully compatible, common-A, and common-AB matings, but was not detected in common-B matings of the tetrapolar Basidiomycete S. commune.