Multinucleate Spores Research Articles

High Environmentally Induced Plasticity in Spore Size and Numbers of Nuclei per Spore in Physarum albescens (Myxomycetes)

Spore size enables dispersal in plasmodial slime molds (Myxomycetes) and is an important taxonomic character. We recorded size and the number of nuclei per spore for 39 specimens (colonies of 50-1000 sporocarps) of the nivicolous myxomycete Physarum albescens, a morphologically defined taxon with several biological species. For each colony, three sporocarps were analyzed from the same spore mount under brightfield and DAPI-fluorescence, recording ca. 14,000 spores per item. Diagrams for spore size distribution showed narrow peaks of mostly uninucleate spores. Size was highly variable within morphospecies (10.6-13.5µm, 11-13%), biospecies (3-13%), even within spatially separated colonies of one clone (ca. 8%); but fairly constant for a colony (mean variation 0.4µm, ca. 1.5%). ANOVA explains most of this variation by the factor locality (within all colonies: 32.7%; within a region: 21.4%), less by biospecies (13.5%), whereas the contribution of intra-colony variation was negligible (<0.1%). Two rare aberrations occur: 1) multinucleate spores and 2) oversized spores with a double or triple volume of normal spores. Both are not related to each other or limited to certain biospecies. Spore size shows high phenotypic plasticity, but the low variation within a colony points to a strong genetic background.

Nuclear conditions of basidiospores and hyphal cells in the edible mushroom Oudemansiella aparlosarca.

Oudemansiella aparlosarca is an edible mushroom possessing medicinal and health benefits. Although there are studies on the cultivation of O. aparlosarca, only a few studies have focused on its genetics and life cycle. Therefore, the main objective of this study was to identify the nuclear conditions of basidiospores and homokaryotic and heterokaryotic hyphal cells and to determine the influence of different nuclear conditions on basidiospore diameter in O. aparlosarca. Two parental strains: strain‐55 and strain‐81 were used. Staining of basidiospores and hyphal cells in the apical region was performed. We observed the following nuclear conditions: non‐nucleate, mononucleate, binucleate, and multinucleate. In both parental strains, binucleate spores were predominant, while the number of non‐nucleate spores was the lowest. The diameter of non‐nucleate spores was the smallest, being 11.52 µm and 12.15 µm in parental strain‐81 and strain‐55, respectively, while multinucleate spores had the largest diameter, being 14.78 µm in both parental strains. Both homokaryotic and heterokaryotic strains were identified in isolated single spores from parental strains. Binucleate cells were majorly present in heterokaryotic hyphal cells, and multinucleate cells were predominant in homokaryotic hyphal cells. We conclude that O. aparlosarca contains homokaryotic and heterokaryotic basidiospores, which indicates an amphithallic life cycle. The observed binucleate spores might be the result of post‐meiotic mitosis.

Efficient genome editing in filamentous fungi via an improved CRISPR-Cas9 ribonucleoprotein method facilitated by chemical reagents.

DNA double-strand break (DSB) repair induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing. Direct genome editing via Cas9-CRISPR gRNA (guide RNA) ribonucleoprotein (RNP) complexes assembled invitro has also been successful in some fungi. However, the efficiency of direct RNP transformation into fungal protoplasts is currently too low. Here, we report an optimized genome editing approach for filamentous fungi based on RNPs facilitated by adding chemical reagents. We increased the transformation efficiency of RNPs significantly by adding Triton X-100 and prolonging the incubation time, and the editing efficiency reached 100% in Trichoderma reesei and Cordyceps militaris. The optimized RNP-based method also achieved efficient (56.52%) homologous recombination integration with short homology arms (20 bp) and gene disruption (7.37%) that excludes any foreign DNA (selection marker) in T. reesei. In particular, after adding reagents related to mitosis and cell division, the further optimized protocol showed an increased ratio of edited homokaryotic transformants (from 0% to 40.0% for inositol and 71.43% for benomyl) from Aspergillus oryzae, which contains multinucleate spores and protoplasts. Furthermore, the multi-target engineering efficiency of the optimized RNP transformation method was similar to those of methods based on invivo expression of Cas9. This newly established genome editing system based on RNPs may be widely applicable to construction of genome-edited fungi for the food and medical industries, and has good prospects for commercialization.

Variation in allele frequencies at the bg112 locus reveals unequal inheritance of nuclei in a dikaryotic isolate of the fungus Rhizophagus irregularis

The genetic state of the arbuscular mycorrhizal fungus species Rhizophagus irregularis differs among isolates, including both homokaryotic and dikaryotic isolates. Via the production of multi-nucleate axexual spores, siblings of dikaryotic isolates may inherit unequal frequencies of nucleotypes. Using bg112, a microsatellite marker, previous studies revealed that lines deriving from single spores of the dikaryotic R. irregularis isolate C3 differed in their proportions of different alleles. A genomic study of single nuclei of R. irregularis, however, suggested that this marker was a multi-copy locus and that therefore it was inappropriate to study the inheritance of nuclei in dikaryotic isolates. In this study, we first analysed whole genome data of several R. irregularis isolates and demonstrated that bg112 is indeed a single copy locus in these genomes. Thus, the bg112 locus is a suitable marker to study the relative frequency of nucleotypes in R. irregularis. Second, by using amplicon sequencing, we confirmed the existence of one allele of bg112 in two homokaryotic isolates (DAOM197198 and C2) and two alleles in the dikaryotic isolate (C3). Finally, we found that the relative proportions of two bg112 alleles differed significantly among dikaryotic single-spore lines derived from isolate C3, indicating that genetically different nucleotypes are inherited unequally in this dikaryotic R. irregularis isolate.

Rapid mitochondrial genome evolution through invasion of mobile elements in two closely related species of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are common and important plant symbionts. They have coenocytic hyphae and form multinucleated spores. The nuclear genome of AMF is polymorphic and its organization is not well understood, which makes the development of reliable molecular markers challenging. In stark contrast, their mitochondrial genome (mtDNA) is homogeneous. To assess the intra- and inter-specific mitochondrial variability in closely related Glomus species, we performed 454 sequencing on total genomic DNA of Glomus sp. isolate DAOM-229456 and we compared its mtDNA with two G. irregulare isolates. We found that the mtDNA of Glomus sp. is homogeneous, identical in gene order and, with respect to the sequences of coding regions, almost identical to G. irregulare. However, certain genomic regions vary substantially, due to insertions/deletions of elements such as introns, mitochondrial plasmid-like DNA polymerase genes and mobile open reading frames. We found no evidence of mitochondrial or cytoplasmic plasmids in Glomus species, and mobile ORFs in Glomus are responsible for the formation of four gene hybrids in atp6, atp9, cox2, and nad3, which are most probably the result of horizontal gene transfer and are expressed at the mRNA level. We found evidence for substantial sequence variation in defined regions of mtDNA, even among closely related isolates with otherwise identical coding gene sequences. This variation makes it possible to design reliable intra- and inter-specific markers.

Significant genetic and phenotypic changes arising from clonal growth of a single spore of an arbuscular mycorrhizal fungus over multiple generations

Arbuscular mycorrhizal fungi (AMF) are highly successful plant symbionts. They reproduce clonally producing multinucleate spores. It has been suggested that some AMF harbor genetically different nuclei. However, recent advances in sequencing the Glomus irregulare genome have indicated very low within-fungus polymorphism. We tested the null hypothesis that, with no genetic differences among nuclei, no significant genetic or phenotypic variation would occur among clonal single spore lines generated from one initial AMF spore. Furthermore, no additional variation would be expected in the following generations of single spore lines. Genetic diversity contained in one initial spore repeatedly gave rise to genetically different variants of the fungus with novel phenotypes. The genetic changes represented quantitative changes in allele frequencies, most probably as a result of changes in the frequency of genetic variation partitioned on different nuclei. The genetic and phenotypic variation is remarkable, given that it arose repeatedly from one clonal individual. Our results highlight the dynamic nature of AMF genetics. Even though within-fungus genetic variation is low, some is probably partitioned among nuclei and potentially causes changes in the phenotype. Our results are important for understanding AMF genetics, as well as for researchers and biotechnologists hoping to use AMF genetic diversity for the improvement of AMF inoculum.

RIBOSOMAL RNA GENE DIVERSITY, EFFECTIVE POPULATION SIZE, AND EVOLUTIONARY LONGEVITY IN ASEXUAL GLOMEROMYCOTA

Arbuscular mycorrhizal fungi (phylum Glomeromycota) are among the oldest and most successful symbionts of land plants. With no evidence of sexual reproduction, their evolutionary success is inconsistent with the prediction that asexual taxa are vulnerable to extinction due to accumulation of deleterious mutations. To explore why Glomeromycota defy this prediction, we studied ribosomal RNA (rRNA) gene evolution in the Claroideoglomus lineage and estimated effective population size, N(e) , in C. etunicatum. We found that rRNA genes of these fungi exhibit unusual and complex patterns of molecular evolution. In C. etunicatum, these patterns can be collectively explained by an unexpectedly large N(e) combined with imperfect genome-wide and population-level rRNA gene repeat homogenization. The mutations accumulated in rRNA gene sequences indicate that natural selection is effective at purging deleterious mutations in the Claroideoglomus lineage, which is also consistent with the large N(e) of C. etunicatum. We propose that in the near absence of recombination, asexual reproduction involving massively multinucleate spores typical for Glomeromycota is responsible for the improved efficacy of selection relative to drift. We postulate that large effective population sizes contribute to the evolutionary longevity of Glomeromycota.

Searching for clues of sexual reproduction in the genomes of arbuscular mycorrhizal fungi

Abstract Arbuscular mycorrhizal fungi (AMF) represent an ecologically relevant and evolutionarily intriguing group of land plant symbionts, which produce multinucleated spores and hyphae that are currently thought to have propagated clonally for over 500 million years. This long-term absence of sex in AMF is a puzzling evolutionary feature that has sparked scientific interest for some time, but a provoking explanation for their successful evolutionary history in the absence of an obvious sexual cycle is that these organisms may have cryptic sex, or a parasexual life cycle, allowing them to recombine alleles and compensate for deleterious mutations. Interestingly, the recent acquisition of large sequence data from many AMF species can finally allow this hypothesis to be tested more extensively. In this perspective, we highlight emerging evidence based on sequence data for the potential of AMF to have sexual reproduction, and propose a number of routes that could be taken to further explore the presence (or absence thereof) of sex in this poorly studied, yet highly relevant, fungal group.

Spore development and nuclear inheritance in arbuscular mycorrhizal fungi.

BackgroundA conventional tenet of classical genetics is that progeny inherit half their genome from each parent in sexual reproduction instead of the complete genome transferred to each daughter during asexual reproduction. The transmission of hereditary characteristics from parents to their offspring is therefore predictable, although several exceptions are known. Heredity in microorganisms, however, can be very complex, and even unknown as is the case for coenocytic organisms such as Arbuscular Mycorrhizal Fungi (AMF). This group of fungi are plant-root symbionts, ubiquitous in most ecosystems, which reproduce asexually via multinucleate spores for which sexuality has not yet been observed.ResultsWe examined the number of nuclei per spore of four AMF taxa using high Z-resolution live confocal microscopy and found that the number of nuclei was correlated with spore diameter. We show that AMF have the ability, through the establishment of new symbioses, to pass hundreds of nuclei to subsequent generations of multinucleated spores. More importantly, we observed surprising heterogeneity in the number of nuclei among sister spores and show that massive nuclear migration and mitosis are the mechanisms by which AMF spores are formed. We followed spore development of Glomus irregulare from hyphal swelling to spore maturity and found that the spores reached mature size within 30 to 60 days, and that the number of nuclei per spores increased over time.ConclusionsWe conclude that the spores used for dispersal of AMF contain nuclei with two origins, those that migrate into the spore and those that arise by mitosis in the spore. Therefore, these spores do not represent a stage in the life cycle with a single nucleus, raising the possibility that AMF, unlike all other known eukaryotic organisms, lack the genetic bottleneck of a single-nucleus stage.

Conserved Meiotic Machinery in Glomus spp., a Putatively Ancient Asexual Fungal Lineage

Arbuscular mycorrhizal fungi (AMF) represent an ecologically important and evolutionarily intriguing group of symbionts of land plants, currently thought to have propagated clonally for over 500 Myr. AMF produce multinucleate spores and may exchange nuclei through anastomosis, but meiosis has never been observed in this group. A provocative alternative for their successful and long asexual evolutionary history is that these organisms may have cryptic sex, allowing them to recombine alleles and compensate for deleterious mutations. This is partly supported by reports of recombination among some of their natural populations. We explored this hypothesis by searching for some of the primary tools for a sustainable sexual cycle—the genes whose products are required for proper completion of meiotic recombination in yeast—in the genomes of four AMF and compared them with homologs of representative ascomycete, basidiomycete, chytridiomycete, and zygomycete fungi. Our investigation used molecular and bioinformatic tools to identify homologs of 51 meiotic genes, including seven meiosis-specific genes and other “core meiotic genes” conserved in the genomes of the AMF Glomus diaphanum (MUCL 43196), Glomus irregulare (DAOM-197198), Glomus clarum (DAOM 234281), and Glomus cerebriforme (DAOM 227022). Homology of AMF meiosis-specific genes was verified by phylogenetic analyses with representative fungi, animals (Mus, Hydra), and a choanoflagellate (Monosiga). Together, these results indicate that these supposedly ancient asexual fungi may be capable of undergoing a conventional meiosis; a hypothesis that is consistent with previous reports of recombination within and across some of their populations.

Ras GTPase-Activating Protein Regulation of Actin Cytoskeleton and Hyphal Polarity in Aspergillus nidulans

Aspergillus nidulans gapA1, a mutation leading to compact, fluffy colonies and delayed polarity establishment, maps to a gene encoding a Ras GTPase-activating protein. Domain organization and phylogenetic analyses strongly indicate that GapA regulates one or more "true" Ras proteins. A gapADelta strain is viable. gapA colonies are more compact than gapA1 colonies and show reduced conidiation. gapADelta strains have abnormal conidiophores, characterized by the absence of one of the two layers of sterigmata seen in the wild type. gapA transcript levels are very low in conidia but increase during germination and reach their maximum at a time coincident with germ tube emergence. Elevated levels persist in hyphae. In germinating conidiospores, gapADelta disrupts the normal coupling of isotropic growth, polarity establishment, and mitosis, resulting in a highly heterogeneous cell population, including malformed germlings and a class of giant cells with no germ tubes and a multitude of nuclei. Unlike wild-type conidia, gapADelta conidia germinate without a carbon source. Giant multinucleated spores and carbon source-independent germination have been reported in strains carrying a rasA dominant active allele, indicating that GapA downregulates RasA. gapADelta cells show a polarity maintenance defect characterized by apical swelling and subapical branching. The strongly polarized wild-type F-actin distribution is lost in gapADelta cells. As GapA-green fluorescent protein shows cortical localization with strong predominance at the hyphal tips, we propose that GapA-mediated downregulation of Ras signaling at the plasma membrane of these tips is involved in the polarization of the actin cytoskeleton that is required for hyphal growth and, possibly, for asexual morphogenesis.

The morphology of Ichthyophonus sp. in their mugilid hosts (Pisces: Teleostei) and following cultivation in vitro. A light and electron microscopy study.

The morphology of Ichthyophonus sp., a parasite of Mugil capito and Liza saliens, was investigated by light and transmission electron microscopy. The most frequent stage found in the fish hosts was the multinucleate spore, though germinating stages, hyphae, and endospores were also found. Different development patterns were observed in the media assayed for in vitro culture. Optimal growth and development were obtained in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum at pH 7. Ultrastructural features of multinucleate spores, both in the fish host and in culture, were a fibrillar thick wall and an electron-lucent matrix, with large glycogen granules, some electron-dense bodies, large vacuoles, lipid inclusions, and endoplasmic reticulum mainly appearing among the nuclei. Mitochondria with scarce tubulovesicular cristae were observed in the different stages, mainly near the wall and the germinating sites. Condensed heterochromatin was rarely seen. A nucleus-associated organelle (NAO) was frequently observed, and dictyosome cisternae and vesicles appeared in its vicinity.

Morphology of Ichthyophonus hoferi assessed by light and scanning electron microscopy

Abstract. The morphology of Ichthyophonus hoferi in vitro at pH 3.5 and 7.0 is described using light and scanning electron microscopy. Only vegetative growth was observed. At pH 3.5, hyphal growth was seen. The hyphae of I. hoferi are characterized by evacuated hyphal walls with the cytoplasm migrating to the apex and no septation. In contrast, the growth at pH 7.0 is mainly seen as spherical bodies which vary in size and are uni‐ to multinucleate. Amoeboid bodies showing slow movements were observed within 3–6 h of transfer to pH 7.0. We propose a life‐cycle involving the germination of thick‐walled multinucleate spores in the fish stomach as a response to the low pH. The hyphae then penetrate the digestive tract and rupture when they reach a blood vessel (neutral pH), whereby uni‐ and binucleate bodies and/or amoeboid bodies are released. The small cells are transported in the blood vessels and spread in the organs richly supplied with blood (heart, kidney, spleen, liver and muscle tissue) where they grow to form multinucleate spores.

Laccaria fraterna, a common ectomycorrhizal fungus with mono- and bi-sporic basidia and multinucleate spores: comparison with the quadristerigmate, binucleate spored L. laccata and the hypogeous relative Hydnangium carneum

Examination of Laccaria collections from Western Australia revealed that the most common species L. fraterna had basidia which were monosporic, bisporic or very occasionally trisporic, in contrast to L. laccata which was quadrisporic. For L. fraterna, all post-meiotic nuclei migrated into the spores leaving basidia anucleate. Spores of monosterigmate basidia had four nuclei, and bisterigmate basidia usually had two. Post-meiotic mitosis occurred after spores were delimited and prior to morphological maturation. Mature spores had up to eight nuclei. That most spores had compatible mating-type nuclei was indicated by most single germinated spores having clamped mycelium with dikaryotic cells. In Hydnangium carneum, a hypogeous relative of Laccaria, post-meiotic mitosis occurred in the basidium and two nuclei migrated into each of the four spores. Migration patterns indicated that some spores had compatible mating-type nuclei, as in L. fraterna. Allocation of compatible mating types to spores partly explains why L. fraterna ‘weed-like’ behaviour and is a pioneer in plantations of eucalypts on former farmland or rehabilitated mine sites. Mycorrhizas of these fungi have been synthesized aseptically with several species of Eucalyptus.

Causes of cell death following exposure to different fungicides and heat

The lethal effects of several agricultural fungicides, the mutagen ICR-170, and heat have been studied in multinucleated spores from heterokaryotic mycelia of Phycomyces blakesleeanus. The fungicides captafol, captan, Manzate (maneb), Benlate (benomyl), and ZZ-melprex (dodine) induce exclusively cytoplasmic lethality. The mutagen ICR-170 causes three different kinds of lethal events: mitotic, recessive, and cytoplasmic. Heat exposure of dormant spores results in only cytoplasmic lethal damage. Germination increases heat sensitivity, particularly that of nuclei; while most of the lethality in germinating spores is still cytoplasmic, there is a definite contribution of mitotic lethality, i.e., events blocking the division of the affected nuclei.

Further studies on sexuality in azygosporic strains of Mucor hiemalis

A study of azygosporic strains of Mucor hiemalis suggested that the strains were heterozygous for mating-type. The length of uninucleate sporangiospores from most azygosporic strains was 1·3–1·6 times the length of spores from tester strains. Sporangiospores from azygosporic strains were predominantly uninucleate but more than 35 % of the spores of the tester strains were multinucleate. It is possible, however, to find strains derived from azygosporic cultures which are normal in appearance and which have predominantly long uninucleate spores, as well as azygosporic strains which have relatively short uninucleate spores and a relatively high percentage of multinucleate spores. Azygosporic strains have been resynthesized by germinating zygospores derived from a cross of two normal strains of opposite mating-type, both of which were obtained as single vegetative spore isolates of a single azygosporic strain.

Irregular microspore formation inDatura innoxia and its relevance to anther culture

Meiotic irregularities in pollen mother cells ofDatura innoxia lead to the formation of diploid (uninucleate) spores, and to multinucleate spores containing 2, 3 or 4 haploid nuclei. At the free microspore stage, irregular spores form three distinct size-classes having mean diameters of 46 (class II), 52 (III) and 64 (IV) ⧎m, compared with 36 Μm (I) for regular haploid spores. At the beginning of flowering, irregular spores account for about 0.8% (400–450) of the total pollen per anther, but the frequency increases with plant age, and towards the end of flowering, it may be as high as 8%. Irregular spores give rise either to non-haploid pollen grains (2 n, 3 n, 4 n) consisting of one generative and one vegetative nucleus, or to multinucleate pollen grains containing several generative and vegetative nuclei which may be of the same or of different ploidy.

Karyology of conidiogenesis and endoconidium germination in Thielaviopsis basicola

Cytological studies on Thielaviopsis basicola revealed that other than a low frequency of multinucleate endoconidia in some isolates the uninucleate condition prevailed. Of the six isolates that were studied, three possessed some multinucleate spores. The nuclei in a multinucleate endoconidium were all derived by successive mitotic divisions of the single generative nucleus in the conidiophore. The nuclei in the multinucleate endoconidium divided independently and segregated into uninucleate and (or) multinucleate components during the course of endoconidial germination and subsequent hyphal development. The implications of the results are discussed with regard to the occurrence of cultural variability in this fungus.

Genetic classification of the lethal effects of various agents on heterokaryotic spores of phycomyces

The lethal effects of X and UV radiations, N-methyl- N′-nitro- N-nitrosoguanidine and heat have been investigated in multinucleate spores from heterokaryotic mycelia of Phycomyces blakesleeanus. According to their relation to the nuclei, 4 types of lethal events have been considered: mitotic (the affected nucleus cannot undergo unlimited mitoses); recessive (the affected nucleus can divide forever, but the cell dies if the affected nucleus is the only one able to divide forever); dominant (when a nucleus is affected, the cell dies irrespective of the condition of other nuclei); and cytoplasmic (the nuclei are not affected, but the cells dies). A theoretical prediction of the genetic changes produced by these events on the spore population has been compared to actual determinations of the proportions of heterokaryons among the survivors and the presence of recessive lethals in surviving heterokaryons. X-rays cause principally mitotic lethals, and, somewhat less frequently, recessive lethals. UV light produces mitotic and recessive lethals at similar rates; photoreactivation reverses them about equally and simulates a dose reduction. The lethal action of nitrosoguanidine depends on the pH and the chemical composition of the buffer; as extreme cases, in pH 7 citrate-phosphate buffer it consists mainly of equal proportions of mitotic and recessive events, but in pH 5 Tris-malate buffer cytoplasmic and recessive events seem predominant. Heat (55°) causes only, or nearly only, cytoplasmic lethality. Functionally uninucleate cells, important in the isolation of recessive mutants, are obtained best with X-rays. After treatments with UV radiation or nitrosoguanidine in pH 7 citrate-phosphate buffer leading to survival of one-tenth of the spores, about one-third of the survivors are functionally uninucleate.

The numbers of nuclei in conidia of aspergilli

Examinations to determine the number of conidial nuclei characteristic of the majority of species of Aspergillus have been made. The results are given in Table I. Stained preparations of about sixty species were examined: approximately two-thirds have uni-nucleate and one-third multi-nucleate conidia. The distribution of uni- and multi-nucleate species among the groups of the genus is not haphazard, but is generally what might be expected with genetically related species. There is a close relationship between average spore size and number of nuclei: uni-nucleate spores rarely exceed 4μ in diameter, multi-nucleate spores are rarely less than 5μ. The stained preparations mentioned above also gave information as to the source of nuclei in sterigmata and conidia; single-spore isolations from heterokaryotic heads of A. carbonarius and mutant, confirmed the microscopic evidence that in this species, the nuclei of a single conidium are normally derived ultimately from more than one nucleus in the vesicle.