RESUMO
Tilletia indica Mitra causes Karnal bunt (KB) in wheat by pathogenic dikaryophase. The present study is the first to provide the draft genomes of the dikaryon (PSWKBGD-3) and its two monosporidial lines (PSWKBGH-1 and 2) using Illumina and PacBio reads, their annotation and the comparative analyses among the three genomes by extracting polymorphic SSR markers. The trancriptome from infected wheat grains of the susceptible wheat cultivar WL711 at 24 h, 48h, and 7d after inoculation of PSWKBGH-1, 2 and PSWKBGD-3 were also isolated. Further, two transcriptome analyses were performed utilizing T. indica transcriptome to extract dikaryon genes responsible for pathogenesis, and wheat transcriptome to extract wheat genes affected by dikaryon involved in plant-pathogen interaction during progression of KB in wheat. A total of 54, 529, and 87 genes at 24hai, 48hai, and 7dai, respectively were upregulated in dikaryon stage while 21, 35, and 134 genes of T. indica at 24hai, 48hai, and 7dai, respectively, were activated only in dikaryon stage. While, a total of 23, 17, and 52 wheat genes at 24hai, 48hai, and 7dai, respectively were upregulated due to the presence of dikaryon stage only. The results obtained during this study have been compiled in a web resource called TiGeR ( http://backlin.cabgrid.res.in/tiger/ ), which is the first genomic resource for T. indica cataloguing genes, genomic and polymorphic SSRs of the three T. indica lines, wheat and T. indica DEGs as well as wheat genes affected by T. indica dikaryon along with the pathogenecity related proteins of T. indica dikaryon during incidence of KB at different time points. The present study would be helpful to understand the role of dikaryon in plant-pathogen interaction during progression of KB, which would be helpful to manage KB in wheat, and to develop KB-resistant wheat varieties.
Assuntos
Basidiomycota , Doenças das Plantas , Transcriptoma , Triticum , Triticum/genética , Triticum/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Basidiomycota/patogenicidade , Basidiomycota/fisiologia , Perfilação da Expressão Gênica , Genoma Fúngico , Interações Hospedeiro-Patógeno/genéticaRESUMO
Cell-to-cell fusion is a fundamental biological process across the tree of life. In filamentous fungi, somatic fusion (or anastomosis) is required for the normal development of their syncytial hyphal networks, and it can initiate non-sexual genetic exchange processes, such as horizontal genetic transfer and the parasexual cycle. Although these could be important drivers of the evolution of asexual fungi, this remains a largely unexplored possibility due to the lack of suitable resources for their study in these puzzling organisms. We thus aimed at the characterization of cell fusion in the important asexual fungus Verticillium dahliae via Conidial Anastomosis Tubes (CATs), which can be useful for the analysis of parasexuality. We optimized appropriate procedures for their highly reproducible quantification and live-cell imaging, which were used to characterize their physiology and cell biology, and to start elucidating their underlying genetic machinery. Formation of CATs was shown to depend on growth conditions and require functional Fus3 and Slt2 MAP kinases, as well as the NADPH oxidase NoxA, whereas the GPCR Ste2 and the mating-type protein MAT1-2-1 were dispensable. We show that nuclei and other organelles can migrate through CATs, which often leads to the formation of transient dikaryons. Their nuclei have possible windows of opportunity for genetic interaction before degradation of one by a presumably homeostatic mechanism. We establish here CAT-mediated fusion in V. dahliae as an experimentally convenient system for the cytological analysis of fungal non-sexual genetic interactions. We expect that it will facilitate the dissection of sexual alternatives in asexual fungi.
Assuntos
Acremonium/genética , Proteínas Fúngicas/genética , Reprodução Assexuada/genética , Esporos Fúngicos/genética , Acremonium/patogenicidade , Ascomicetos/genética , Ascomicetos/patogenicidade , Núcleo Celular/genética , Transferência Genética Horizontal/genética , Genes Fúngicos Tipo Acasalamento/genética , Hifas/genética , Hifas/crescimento & desenvolvimento , Proteínas Quinases Ativadas por Mitógeno/genética , NADPH Oxidases/genética , Proteínas de Saccharomyces cerevisiae/genética , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
The basidiomycete Pleurotus sapidus produced a dye-decolorizing peroxidase (PsaPOX) with alkene cleavage activity, implying potential as a biocatalyst for the fragrance and flavor industry. To increase the activity, a daughter-generation of 101 basidiospore-derived monokaryons (MK) was used. After a pre-selection according to the growth rate, the activity analysis revealed a stable intraspecific variability of the strains regarding peroxidase and alkene cleavage activity of PsaPOX. Ten monokaryons reached activities up to 2.6-fold higher than the dikaryon, with MK16 showing the highest activity. Analysis of the PsaPOX gene identified three different enzyme variants. These were co-responsible for the observed differences in activities between strains as verified by heterologous expression in Komagataella phaffii. The mutation S371H in enzyme variant PsaPOX_high caused an activity increase alongside a higher protein stability, while the eleven mutations in variant PsaPOX_low resulted in an activity decrease, which was partially based on a shift of the pH optimum from 3.5 to 3.0. Transcriptional analysis revealed the increased expression of PsaPOX in MK16 as reason for the higher PsaPOX activity in comparison to other strains producing the same PsaPOX variant. Thus, different expression profiles, as well as enzyme variants, were identified as crucial factors for the intraspecific variability of the PsaPOX activity in the monokaryons.
Assuntos
Alcenos/metabolismo , Corantes/metabolismo , Proteínas Fúngicas/metabolismo , Peroxidase/metabolismo , Pleurotus/metabolismo , Biotransformação , Proteínas Fúngicas/genética , Modelos Moleculares , Mutação , Peroxidase/genética , Pleurotus/enzimologia , Pleurotus/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TranscriptomaRESUMO
A barrier to cost-efficient biomanufacturing is the instability of engineered genetic elements, such as plasmids. Instability can also manifest at the whole-genome level, when fungal dikaryons revert to parental species due to nuclear segregation during cell division. Here, we show that by encapsulating Saccharomyces cerevisiae-Pichia stipitis dikaryons in an alginate matrix, we can limit cell division and preserve their expanded metabolic capabilities. As a proxy to cellulosic ethanol production, we tested the capacity of such cells to carry out ethanologenic fermentation of glucose and xylose, examining substrate use, ploidy, and cell viability in relation to planktonic fusants, as well as in relation to planktonic and encapsulated cell cultures consisting of mixtures of these species. Glucose and xylose consumption and ethanol production by encapsulated dikaryons were greater than planktonic controls. Simultaneous co-fermentation did not occur; rather the order and kinetics of glucose and xylose catabolism by encapsulated dikaryons were similar to cultures where the two species were encapsulated together. Over repeated cycles of fed-batch culture, encapsulated S. cerevisiae-P. stipitis fusants exhibited a dramatic increase in genomic stability, relative to planktonic fusants. Encapsulation also increased the stability of antibiotic-resistance plasmids used to mark each species and preserved a fixed ratio of S. cerevisiae to P. stipitis cells in mixed cultures. Our data demonstrate how encapsulating cells in an extracellular matrix restricts cell division and, thereby, preserves the stability and biological activity of entities ranging from genomes to plasmids to mixed populations, each of which can be essential to cost-efficient biomanufacturing.
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Alginatos/química , Células Imobilizadas/citologia , Protoplastos/citologia , Saccharomyces cerevisiae/citologia , Saccharomycetales/citologia , Materiais Biocompatíveis/química , Divisão Celular , Células Imobilizadas/metabolismo , Protoplastos/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismoRESUMO
Ustilago maydis causes common smut disease in maize. Although pathogenic diploid strains of the fungus have been known for many years, the normal life cycle was thought to involve an extended dikaryotic stage, with nuclear fusion occurring in immature teliospores. However, microscopic examination of both living and fixed tumor material showed that nuclei fuse long before sporulation begins and that tumors are filled with uninucleate cells undergoing mitosis. Quantification of DNA in the nuclei confirmed these observations. Additionally, fungal cells from tumor material placed on nutrient agar produced colonies of diploid budding cells. Time-lapse observations showed that at least some of these colonies arose from thin-walled fungal cells rather than from immature spores. Ultrastructural examination of developing teliospores from tumors confirmed that they were uninucleate. Condensed chromatin and other structures characteristic of nuclei in prophase I of meiosis were observed. These observations support revising the U. maydis life cycle to include a diploid mitotic stage that corresponds with rapid tumor enlargement and conversion of plant to fungal biomass. Because mitotic division of diploid nuclei is so unusual as a life cycle feature in the fungi, it will be interesting to explore the consequences of its presence in U. maydis.
Assuntos
Diploide , Mitose , Ustilago/citologia , Ustilago/crescimento & desenvolvimento , Microscopia , Doenças das Plantas/microbiologia , Ustilago/genética , Zea mays/microbiologiaRESUMO
The uracil auxotrophic monokaryotic strain 423-9 of Lentinula edodes was crossed with nine monokaryons (cro2-2-9, W66-1, xd2-3-2, QingKe 20A, 241-1-1, 9015-1, L66-2, 241-1-2, and Qing 23A) derived from wild type strains of L. edodes. Nine dikaryotic hybrids were established from these crosses. These hybrids were fruited and 496 single spore isolates were obtained. Among these single spore isolates, 166 were identified as monokaryons under a microscope. We screened these monokaryons on selective medium and obtained 19 uracil auxotrophic monokaryons. By using the Monkaryon-monkaryon crossing method among the uracil auxotrophic monokaryons, 56 uracil auxotrophic dikaryotic strains were established on selective medium. These dikaryotic strains were unable to grow on minimal medium without uracil and exhibited slow growth rates on PDA plates compared to the wild type strain. The uracil auxotrophic dikaryotic strains also showed more vigorous growth on sawdust cultivation medium containing uracil than that without uracil. The fruiting tests showed that they formed normal fruiting bodies on the sawdust medium containing uracil. The results show that the uracil auxotrophic dikaryotic strain of L. edodes could be produced by mating, and will provide a valuable resource for future genetic studies and for spawn protection and identification.
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Pleurotus citrinopileatus (yellow oyster mushroom) has an attractive shape and yellow colour but the fragile texture complicates packaging, and its strong aroma is unappealing to consumers. This study aimed to improve the characteristics and yield of P. citrinopileatus by interspecies mating between monokaryotic cultures of P. citrinopileatus and P. pulmonarius. Ten monokaryon cultures of the parental lines were crossed in all combinations to obtain hybrids. Eleven compatible mating pairs were obtained and cultivated to observe their sporophore morphology and yield. The selected hybrid, i.e. P1xC9, was beige in colour while hybrid P3xC8 was yellow in colour. Their sporophores had less offensive aroma, improved texture and higher yield. The DNA sequences of these hybrids were found to be in the same clade as the P. citrinopileatus parent with a bootstrap value of 99%. High bootstrap values indicate high genetic homology between hybrids and the P. citrinopileatus parent. The biological efficiencies of these hybrids P1xC9 (70.97%) and P3xC8 (52.14%) were also higher than the P. citrinopileatus parent (35.63%). Interspecies hybrids obtained by this mating technique can lead to better strains of mushrooms for genetic improvement of the Pleurotus species.
Assuntos
Hibridização Genética , Pleurotus/classificação , Pleurotus/genética , Sequência de Bases , Cruzamentos Genéticos , DNA Fúngico/genética , Transferência Genética Horizontal , Genes Fúngicos , Genes de RNAr , Filogenia , Pleurotus/citologia , Pleurotus/isolamento & purificação , Homologia de SequênciaRESUMO
We test our "one-log-one-genet" sampling method for the Hawaiian mushroom Rhodocollybia laulaha that posits all R. laulaha mushrooms collected from a single log represent a single genet. We also examine the potential expansion of single genets beyond the confines of one log and the temporal persistence of genets in nature. Finally, we estimate error rates in AFLP scoring. To our knowledge, this is one of few examinations of naturally occurring fungal genets in the tropics and a novel report of AFLP error rates in fungi. Forty-six mushrooms from seven logs were genotyped with the IGS1 locus, two microsatellite loci and 184 AFLP loci from three primer pair combinations. One hundred fifty-three mushroom collections representing the geographic range of R. laulaha were genotyped with the IGS1 and microsatellite loci. The probabilities of two genets sharing identical multilocus genotypes by chance (without actually being the same genet) were calculated for each genotype recovered. The data suggest that R. laulaha mushrooms from one log typically represent one genet, that genets might expand beyond the confines of a single log and that a single genet may persist in a collecting site for as much as 13 y. We offer initial evidence to support the "one-log-one genet" sampling method and the idea that R. laulaha vegetative expansion and persistence in nature might be common. In addition, we caution against exclusive use of AFLP loci for identifying fungal genets due to relatively high error rates in scoring.
Assuntos
Agaricales/genética , Agaricales/isolamento & purificação , Alelos , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Primers do DNA/genética , DNA Fúngico/genética , Loci Gênicos/genética , Genética Populacional , Genótipo , Geografia , Havaí , Madeira/microbiologiaRESUMO
This study compared the pathogenicity of monokaryotic (monokaryon) and dikaryotic (dikaryon) mycelia of the oil palm pathogen Ganoderma boninense via metabolomics approach. Ethyl acetate crude extracts of monokaryon and dikaryon were analysed by liquid chromatography quadrupole/time-of-flight-mass spectrometry (LC-Q/TOF-MS) coupled with multivariate data analysis using MetaboAnalyst. The mummichog algorithm was also used to identify the functional activities of monokaryon and dikaryon without a priori identification of all their secondary metabolites. Results revealed that monokaryon produced lesser fungal metabolites than dikaryon, suggesting that monokaryon had a lower possibility of inducing plant infection. These findings were further supported by the identified functional activities. Monokaryon exhibits tyrosine, phenylalanine, and tryptophan metabolism, which are important for fungal growth and development and to produce toxin precursors. In contrast, dikaryon exhibits the metabolism of cysteine and methionine, arginine and proline, and phenylalanine, which are important for fungal growth, development, virulence, and pathogenicity. As such, monokaryon is rendered non-pathogenic as it produces growth metabolites and toxin precursors, whereas dikaryon is pathogenic as it produces metabolites that are involved in fungal growth and pathogenicity. The LC-MS-based metabolomics approach contributes significantly to our understanding of the pathogenesis of Ganoderma boninense, which is essential for disease management in oil palm plantations.
Assuntos
Ganoderma , Espectrometria de Massa com Cromatografia Líquida , Espectrometria de Massas em Tandem , Virulência , Cromatografia Líquida , FenilalaninaRESUMO
This paper addresses the stability of mycelial growth in fungi and differences between ascomycetes and basidiomycetes. Starting with general evolutionary theories of multicellularity and the role of sex, we then discuss individuality in fungi. Recent research has demonstrated the deleterious consequences of nucleus-level selection in fungal mycelia, favoring cheaters with a nucleus-level benefit during spore formation but a negative effect on mycelium-level fitness. Cheaters appear to generally be loss-of-fusion (LOF) mutants, with a higher propensity to form aerial hyphae developing into asexual spores. Since LOF mutants rely on heterokaryosis with wild-type nuclei, we argue that regular single-spore bottlenecks can efficiently select against such cheater mutants. We then zoom in on ecological differences between ascomycetes being typically fast-growing but short-lived with frequent asexual-spore bottlenecks and basidiomycetes being generally slow-growing but long-lived and usually without asexual-spore bottlenecks. We argue that these life history differences have coevolved with stricter nuclear quality checks in basidiomycetes. Specifically, we propose a new function for clamp connections, structures formed during the sexual stage in ascomycetes and basidiomycetes but during somatic growth only in basidiomycete dikaryons. During dikaryon cell division, the two haploid nuclei temporarily enter a monokaryotic phase, by alternatingly entering a retrograde-growing clamp cell, which subsequently fuses with the subapical cell to recover the dikaryotic cell. We hypothesize that clamp connections act as screening devices for nuclear quality, with both nuclei continuously testing each other for fusion ability, a test that LOF mutants will fail. By linking differences in longevity of the mycelial phase to ecology and stringency of nuclear quality checks, we propose that mycelia have a constant and low lifetime cheating risk, irrespective of their size and longevity.
Assuntos
Hifas , Micélio , Hifas/genética , FungosRESUMO
In this review, I explore the pervasive but underappreciated role of local adaptation in fungi. It has been difficult historically to study local adaptation in fungi because of the limited understanding of fungal species and their traits, but new hope has been offered with technological advances in sequencing. The filamentous nature of fungi invalidates some assumptions made in evolution because of their ability to exist as multinucleate entities with genetically different nuclei sharing the same cytoplasm. Many insights on local adaptation have come from studying fungi, and much of the empirical evidence gathered about local adaptation in the context of host-pathogen interactions comes from studying fungal virulence genes, drug resistance, and environmental adaptation. Together, these insights paint a picture of the variety of processes involved in fungal local adaptation and their connections to the unusual cell biology of Fungi (multinucleate, filamentous habit), but there is much that remains unknown, with major gaps in our knowledge of fungal species, their phenotypes, and the ways by which they adapt to local conditions.
Assuntos
Adaptação Fisiológica , Interações Hospedeiro-Patógeno , Adaptação Fisiológica/genética , Virulência/genética , Fenótipo , Fungos/genéticaRESUMO
Lentinula edodes is a tetrapolar basidiomycete with two haploid nuclei in each cell during most of their life cycle. Understanding the two haploid nuclei genome structures and their interactions on growth and fruiting body development has significant practical implications, especially for commercial cultivars. In this study, we isolated and assembled the two haploid genomes from a commercial strain of L. edodes using Illumina, HiFi, and Hi-C technologies. The total genome lengths were 50.93 Mb and 49.80 Mb for the two monokaryons SP3 and SP30, respectively, with each assembled into 10 chromosomes with 99.63% and 98.91% anchoring rates, respectively, for contigs more than 100 Kb. Genome comparisons suggest that two haploid nuclei likely derived from distinct genetic ancestries, with ~30% of their genomes being unique or non-syntenic. Consistent with a tetrapolar mating system, the two mating-type loci A (matA) and B (matB) of L. edodes were found located on two different chromosomes. However, we identified a new but incomplete homeodomain (HD) sublocus at ~2.8 Mb from matA in both monokaryons. Our study provides a solid foundation for investigating the relationships among cultivars and between cultivars and wild strains and for studying how two genetically divergent nuclei coordinate to regulate fruiting body formation in L. edodes.
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Flammulina filiformis, as one of the most popular edible fungi in East Asia, is produced in an industrialized and standardized way. However, its monotonous variety and product convergence have seriously restricted the development of the industry. In this study, 11 cultivated strains and 13 wild strains of F. filiformis were collected from multiple regions of China and Japan and were performed genome sequencing. Together with genome data of six strains previously released, in total 23 dikaryons (formed by two monokaryons mating, can making fruiting body), 35 monokaryons (formed by protoplast-regenerating of dikaryon and isolating) were used for genetic diversity and population structure analysis based on the high-throughput genotyping. Firstly, a set of SNP markers with intrapopulation polymorphism including 849,987 bi-allelic SNPs were developed and basically covered all of 11 chromosomes with a high distribution density of 24.16 SNP markers per kb. The cultivated dikaryotic strains were divided into three subgroups, and their breeding history was made inferences, which is consistent with the available pedigree records. The wild dikaryotic strains were divided into two subgroups and showed varied contributions of genetic components with high genetic diversity. All the investigated dikaryons have a symmetric distribution pattern with their two constituent monokaryons in principal component analysis. Finally, we summarized the pedigree relationship diagram of F. filiformis main strains including six modules, and the genotypes of hybrids can be directly phased by the known parental allele according to it. This study provides a method to distinguish two sets of monokaryon haplotypes, and several valuable genetic resources of wild F. filiformis, and an effective strategy for guiding F. filiformis breeding based on the population structure and pedigree relationship in future.
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During sexual development the human fungal pathogen Cryptococcus neoformans undergoes a developmental transition from yeast-form growth to filamentous growth. This transition requires cellular restructuring to form a filamentous dikaryon. Dikaryotic growth also requires tightly controlled nuclear migration to ensure faithful replication and dissemination of genetic material to spore progeny. Although the gross morphological changes that take place during dikaryotic growth are largely known, the molecular underpinnings that control this process are uncharacterized. Here we identify and characterize a C. neoformans homolog of the Saccharomyces cerevisiae BIM1 gene, and establish the importance of BIM1 for proper filamentous growth of C. neoformans. Deletion of BIM1 leads to truncated sexual development filaments, a severe defect in diploid formation, and a block in monokaryotic fruiting. Our findings lead to a model consistent with a critical role for BIM1 in both filament integrity and nuclear congression that is mediated through the microtubule cytoskeleton.
Assuntos
Cryptococcus neoformans/fisiologia , Proteínas Fúngicas/fisiologia , Proteínas dos Microtúbulos/fisiologia , Citoesqueleto/metabolismo , Proteínas Fúngicas/genética , Deleção de Genes , Humanos , Proteínas dos Microtúbulos/deficiênciaRESUMO
Arbuscular mycorrhizal fungi (AMF) are plant root symbionts that continuously carry thousands of nuclei in their spores and hyphae. This unique cellular biology raises fundamental questions regarding their nuclear dynamics. This review aims to address these by synthesizing current knowledge of nuclear content and behavior in these ubiquitous soil fungi. Overall, we find that that nuclear counts, as well as the nuclei shape and organization, vary drastically both within and among species in this group. By comparing these features with those of other fungi, we highlight unique aspects of the AMF nuclear biology that require further attention. The potential implications of the observed nuclear variability for the biology and evolution of these widespread plant symbionts are discussed.
Assuntos
Micorrizas , Núcleo Celular , Fungos , Raízes de Plantas , Plantas , Solo , Microbiologia do Solo , SimbioseRESUMO
Stripe rust of wheat, caused by the obligate biotrophic fungus Puccinia striiformis f.sp. tritici, is a major threat to wheat production worldwide with an estimated yearly loss of US $1 billion. The recent advances in long-read sequencing technologies and tailored-assembly algorithms enabled us to disentangle the two haploid genomes of Pst. This provides us with haplotype-specific information at a whole-genome level. Exploiting this novel information, we perform whole-genome comparative genomics of two P. striiformis f.sp. tritici isolates with contrasting life histories. We compare one isolate of the old European lineage (PstS0), which has been asexual for over 50 years, and a Warrior isolate (PstS7 lineage) from a novel incursion into Europe in 2011 from a sexual population in the Himalayan region. This comparison provides evidence that long-term asexual evolution leads to genome expansion, accumulation of transposable elements, and increased heterozygosity at the single nucleotide, structural, and allele levels. At the whole-genome level, candidate effectors are not compartmentalized and do not exhibit reduced levels of synteny. Yet we were able to identify two subsets of candidate effector populations. About 70% of candidate effectors are invariant between the two isolates, whereas 30% are hypervariable. The latter might be involved in host adaptation on wheat and explain the different phenotypes of the two isolates. Overall, this detailed comparative analysis of two haplotype-aware assemblies of P. striiformis f.sp. tritici is the first step in understanding the evolution of dikaryotic rust fungi at a whole-genome level.
Assuntos
Evolução Molecular , Genoma Fúngico , Haplótipos , Doenças das Plantas/genética , Puccinia/genética , Puccinia/patogenicidade , Triticum/microbiologia , Proteínas Fúngicas/genética , Fenótipo , Doenças das Plantas/microbiologiaRESUMO
A long-standing biological question is how evolution has shaped the genomic architecture of dikaryotic fungi. To answer this, high-quality genomic resources that enable haplotype comparisons are essential. Short-read genome assemblies for dikaryotic fungi are highly fragmented and lack haplotype-specific information due to the high heterozygosity and repeat content of these genomes. Here, we present a diploid-aware assembly of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici based on long reads using the FALCON-Unzip assembler. Transcriptome sequencing data sets were used to infer high-quality gene models and identify virulence genes involved in plant infection referred to as effectors. This represents the most complete Puccinia striiformis f. sp. tritici genome assembly to date (83 Mb, 156 contigs, N50 of 1.5 Mb) and provides phased haplotype information for over 92% of the genome. Comparisons of the phase blocks revealed high interhaplotype diversity of over 6%. More than 25% of all genes lack a clear allelic counterpart. When we investigated genome features that potentially promote the rapid evolution of virulence, we found that candidate effector genes are spatially associated with conserved genes commonly found in basidiomycetes. Yet, candidate effectors that lack an allelic counterpart are more distant from conserved genes than allelic candidate effectors and are less likely to be evolutionarily conserved within the P. striiformis species complex and Pucciniales In summary, this haplotype-phased assembly enabled us to discover novel genome features of a dikaryotic plant-pathogenic fungus previously hidden in collapsed and fragmented genome assemblies.IMPORTANCE Current representations of eukaryotic microbial genomes are haploid, hiding the genomic diversity intrinsic to diploid and polyploid life forms. This hidden diversity contributes to the organism's evolutionary potential and ability to adapt to stress conditions. Yet, it is challenging to provide haplotype-specific information at a whole-genome level. Here, we take advantage of long-read DNA sequencing technology and a tailored-assembly algorithm to disentangle the two haploid genomes of a dikaryotic pathogenic wheat rust fungus. The two genomes display high levels of nucleotide and structural variations, which lead to allelic variation and the presence of genes lacking allelic counterparts. Nonallelic candidate effector genes, which likely encode important pathogenicity factors, display distinct genome localization patterns and are less likely to be evolutionary conserved than those which are present as allelic pairs. This genomic diversity may promote rapid host adaptation and/or be related to the age of the sequenced isolate since last meiosis.
Assuntos
Basidiomycota/genética , Variação Genética , Genoma Fúngico , Haplótipos , Basidiomycota/isolamento & purificação , Doenças das Plantas/microbiologia , Triticum/microbiologia , Fatores de Virulência/genéticaRESUMO
Intraspecific variability in the activity of nitrate reductase (NR) has been studied in the ectomycorrhizal fungus Hebeloma cylindrosporum Romagnési at the interstrain and intrastrain levels, the latter within a population of 11 wild dikaryotic strains collected in'Les Landes'(SW France) at four locations less than 100 km away from one another. An attempt was made to determine whether variability within the monokaryotic and dikaryotic progeny of the HC1 fruiting strain could be used as a basis for an improvement programme involving breeding between selected monokaryons. The NR activity of the wild strains ranged from 201 to 700 nmol NO2 synthesized h-1 mg -1 fungal protein whilst that of 20 sib-monokaryons (5 per mating type) of the HCl strain varied from 51 to 510 nmol NO2 synthesized h -1 mg-1 fungal protein. Fifty controlled dikaryotic myeclia were obtained from all the compatible fusions with these monokaryons. In these, variation of NR activity was of the same order of magnitude as that recorded at the interstrain level, ranging from 72 to 689 nmol NO2 synthesized h -1 rag -1 fungal protein. Analysis of the components of the variation of NR activity in these controlled dikaryons demonstrated that the additive component of this variation accounted for less than 1 % of the total observed variation. The NR activity of any one controlled dikaryon could not therefore be predicted from the activity of its parental monokaryons However 14 of the 50 controlled dikaryons e8hibited an NR activity higher than that of the HCl parental dikaryon. These results indicate that breeding with sib-monokaryons can be used as a basis for an improvement programme of NR activity in this ectomycorrhizal Basidiomycete.
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To determine mechanisms of assembly of ciliary dyneins, we focused on the Chlamydomonas inner dynein arm, I1 dynein, also known as dynein f. I1 dynein assembles in the cytoplasm as a 20S complex similar to the 20S I1 dynein complex isolated from the axoneme. The intermediate chain subunit, IC140 (IDA7), and heavy chains (IDA1, IDA2) are required for 20S I1 dynein preassembly in the cytoplasm. Unlike I1 dynein derived from the axoneme, the cytoplasmic 20S I1 complex will not rebind I1-deficient axonemes in vitro. To test the hypothesis that I1 dynein is transported to the distal tip of the cilia for assembly in the axoneme, we performed cytoplasmic complementation in dikaryons formed between wild-type and I1 dynein mutant cells. Rescue of I1 dynein assembly in mutant cilia occurred first at the distal tip and then proceeded toward the proximal axoneme. Notably, in contrast to other combinations, I1 dynein assembly was significantly delayed in dikaryons formed between ida7 and ida3. Furthermore, rescue of I1 dynein assembly required new protein synthesis in the ida7 × ida3 dikaryons. On the basis of the additional observations, we postulate that IDA3 is required for 20S I1 dynein transport. Cytoplasmic complementation in dikaryons using the conditional kinesin-2 mutant, fla10-1 revealed that transport of I1 dynein is dependent on kinesin-2 activity. Thus, I1 dynein complex assembly depends upon IFT for transport to the ciliary distal tip prior to docking in the axoneme.
Assuntos
Axonema/metabolismo , Chlamydomonas/metabolismo , Cílios/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Transporte Biológico , Cinesinas/metabolismo , Modelos Biológicos , Mutação , Proteínas de Plantas/metabolismo , Biossíntese de ProteínasRESUMO
The fungal fruiting body or mushroom is a multicellular structure essential for sexual reproduction. It is composed of dikaryotic cells that contain one haploid nucleus from each mating partner sharing the same cytoplasm without undergoing nuclear fusion. In the mushroom, the pileus bears the hymenium, a layer of cells that includes the specialized basidia in which nuclear fusion, meiosis, and sporulation occur. Coprinopsis cinerea is a well-known model fungus used to study developmental processes associated with the formation of the fruiting body. Here we describe that knocking down the expression of Atr1 and Chk1, two kinases shown to be involved in the response to DNA damage in a number of eukaryotic organisms, dramatically impairs the ability to develop fruiting bodies in C. cinerea, as well as other developmental decisions such as sclerotia formation. These developmental defects correlated with the impairment in silenced strains to sustain an appropriated dikaryotic cell cycle. Dikaryotic cells in which chk1 or atr1 genes were silenced displayed a higher level of asynchronous mitosis and as a consequence aberrant cells carrying an unbalanced dose of nuclei. Since fruiting body initiation is dependent on the balanced mating-type regulator doses present in the dikaryon, we believe that the observed developmental defects were a consequence of the impaired cell cycle in the dikaryon. Our results suggest a connection between the DNA damage response cascade, cell cycle regulation, and developmental processes in this fungus.