RESUMO
Pleurotus ostreatus is a global mushroom crop with nutritional and medicinal benefits. However, the genetic basis of several commercial traits remains unknown. To address this, we analyzed the quantitative trait loci (QTLs) for two representative cultivars, "Heuktari" and "Miso," with apparently distinct alleles. A genetic map with 11 linkage groups was constructed, in which 27 QTLs were assigned to 14 traits. The explained phenotypic variations in QTLs ranged from 7.8% to 22.0%. Relatively high LOD values of 6.190 and 5.485 were estimated for the pinheading period and the number of valid stipes, respectively. Some QTL-derived molecular markers showed potential enhancement rates of selection precision in inbred lines, especially for cap shape (50%) and cap thickness (30%). Candidate genes were inferred from the QTL regions and validated using qRT-PCR, particularly for the cysteine and glutathione pathway, in relation to cap yellowness. The molecular markers in this study are expected to facilitate the breeding of the Heuktari and Miso lines and provide probes to identify related genes in P. ostreatus.
Assuntos
Pleurotus , Locos de Características Quantitativas , Pleurotus/genética , Marcadores Genéticos , Mapeamento Cromossômico , Ligação Genética , Fenótipo , AgriculturaRESUMO
Oyster mushrooms have a high biological efficiency and are easy to cultivate, which is why they are produced all over the world. Cap color is an important commercial trait for oyster mushrooms. Little is known about the genetic mechanism of the cap color trait in oyster mushrooms, which limits molecular breeding for the improvement of cap color-type cultivars. In this study, a 0.8-Mb major quantitative trait locus (QTL) region controlling cap color in the oyster mushroom Pleurotus cornucopiae was mapped on chromosome 7 through bulked-segregant analysis sequencing (BSA-seq) and extreme-phenotype genome-wide association studies (XP-GWAS). Candidate genes were further selected by comparative transcriptome analysis, and a tyrosinase gene (PcTYR) was identified as the highest-confidence candidate gene. Overexpression of PcTYR resulted in a significantly darker cap color, while the cap color of RNA interference (RNAi) strains for this gene was significantly lighter than that of the wild-type (WT) strains, suggesting that PcTYR plays an essential role in cap color formation. This is the first report about fine mapping and functional verification of a gene controlling cap color in oyster mushrooms. This will enhance our understanding of the genetic basis for cap color formation in oyster mushrooms and will facilitate molecular breeding for cap color. IMPORTANCE Oyster mushrooms are widely cultivated and consumed over the world for their easy cultivation and high biological efficiency (mushroom fresh weight/substrate dry weight × 100%). Fruiting bodies with dark caps are more and more popular according to consumer preferences, but dark varieties are rarely seen on the market. Little is known about the genetic mechanism of the cap color trait in oyster mushrooms, which limits molecular breeding for the improvement of cap color-type cultivars. A major QTL of cap color in oyster mushroom P. cornucopiae was fine mapped by using bulked-segregant analysis (BSA) and extreme-phenotype genome-wide association study (XP-GWAS) analysis. A candidate gene PcTYR coding tyrosinase was further identified with the help of comparative transcriptome analysis. qPCR analysis and genetic transformation tests proved that PcTYR played an essential role in cap color formation. This study will contribute to revealing the genetic mechanism of cap color formation in mushrooms, thereby facilitating molecular breeding for cap color trait.
Assuntos
Pleurotus , Estudo de Associação Genômica Ampla , Monofenol Mono-Oxigenase/genética , Pleurotus/genética , Locos de Características QuantitativasRESUMO
In heterothallic basidiomycete fungi, sexual compatibility is restricted by mating types, typically controlled by two loci: PR, encoding pheromone precursors and pheromone receptors, and HD, encoding two types of homeodomain transcription factors. We analysed the single mating-type locus of the commercial button mushroom variety, Agaricus bisporus var. bisporus, and of the related variety burnettii. We identified the location of the mating-type locus using genetic map and genome information, corresponding to the HD locus, the PR locus having lost its mating-type role. We found the mip1 and ß-fg genes flanking the HD genes as in several Agaricomycetes, two copies of the ß-fg gene, an additional HD2 copy in the reference genome of A. bisporus var. bisporus and an additional HD1 copy in the reference genome of A. bisporus var. burnettii. We detected a 140 kb-long inversion between mating types in an A. bisporus var. burnettii heterokaryon, trapping the HD genes, the mip1 gene and fragments of additional genes. The two varieties had islands of transposable elements at the mating-type locus, spanning 35 kb in the A. bisporus var. burnettii reference genome. Linkage analyses showed a region with low recombination in the mating-type locus region in the A. bisporus var. burnettii variety. We found high differentiation between ß-fg alleles in both varieties, indicating an ancient event of recombination suppression, followed more recently by a suppression of recombination at the mip1 gene through the inversion in A. bisporus var. burnettii and a suppression of recombination across whole chromosomes in A. bisporus var. bisporus, constituting stepwise recombination suppression as in many other mating-type chromosomes and sex chromosomes.
Assuntos
Agaricus/genética , Cromossomos/genética , Genes Fúngicos Tipo Acasalamento/genética , Agaricus/metabolismo , Alelos , Basidiomycota/genética , DNA Fúngico/genética , Ligação Genética/genética , Genoma Fúngico/genética , Recombinação Genética/genéticaRESUMO
Nonself recognition leading to somatic incompatibility (SI) is commonly used by mycologists to distinguish fungal individuals. Despite this, the process remains poorly understood in basidiomycetes as all current models of SI are based on genetic and molecular research in ascomycete fungi. Ascomycete fungi are mainly found in a monokaryotic stage, with a single type of haploid nuclei, and only briefly during mating do two genomes coexist in heterokaryotic cells. The sister phylum, Basidiomycota, differs in several relevant aspects. Basidiomycete fungi have an extended heterokaryotic stage, and SI is generally observed between heterokaryons instead of between homokaryons. Additionally, considerable nuclear migration occurs during a basidiomycete mating reaction, introducing a nucleus into a resident homokaryon with cytoplasmic mixing limited to the fused or neighboring cells. To accommodate these differences, we describe a basidiomycete model for nonself recognition using post-translational modification, based on a reader-writer system as found in other organisms. This post-translational modification combined with nuclear migration allows for the coexistence of two genomes in one individual while maintaining nonself recognition during all life stages. Somewhat surprisingly, this model predicts localized cell death during mating, which is consistent with previous observations but differs from the general assumptions of basidiomycete mating. This model will help guide future research into the mechanisms behind basidiomycete nonself recognition.
RESUMO
Pleurotus ostreatus, one of the most widely cultivated edible mushrooms, produces high numbers of spores causing severe respiratory health problems for people, clogging of filters and spoilage of produce. A non-sporulating commercial variety (SPOPPO) has been successfully introduced into the market in 2006. This variety was generated by introgression breeding of a natural mutation into a commercial variety. Our cytological studies revealed that meiosis in the natural and derived sporeless strains was blocked in metaphase I, apparently resulting in a loss of spore formation. The gene(s) underlying this phenotype were mapped to an 80 kb region strongly linked to sporelessness and identified by transformation of wild type genes of this region into a sporeless strain. Sporulation was restored by re-introduction of the DNA sequence encoding the P. ostreatus meiotic recombination gene MSH4 homolog (poMSH4). Subsequent molecular analysis showed that poMSH4 in the sporeless P. ostreatus was interrupted by a DNA fragment containing a region encoding a CxC5/CxC6 cysteine cluster associated with Copia-type retrotransposons. The block of meiosis in metaphase I by a poMSH4 null mutant suggests that this protein plays an essential role in both Class I and II crossovers in mushrooms, similar to animals (mice), but unlike in plants. MSH4 was previously shown to be a target for breeding of sporeless varieties in P. pulmonarius, and the null mutant of the MSH4 homolog of S. commune (scMSH4) confers an extremely low level of spore formation. We propose that MSH4 homologs are likely to be a breeding target for sporeless strains both within Pleurotus sp. and in other Agaricales.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Meiose , Pleurotus/fisiologia , Esporos Fúngicos/genética , Troca Genética , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Ligação Genética , Metáfase , Fenótipo , Pleurotus/genética , Retroelementos/genéticaRESUMO
BACKGROUND: Sexual development in Flammulina velutipes is controlled by two different mating type loci (HD and PR). The HD locus contains homeodomain (Hd) genes on two separate HD subloci: HD-a and HD-b. While the functionality of the HD-b sublocus has been largely confirmed, the status and content of the HD-a sublocus has remained unclear. METHODS: To examine the function of the HD-a sublocus, genome sequences of a series of F. velutipes strains were analyzed and tested through series of amplification by specific primer sets. Furthermore, activity of di-allelic HD-a locus was confirmed by crossing strains with different combinations of HD-a and HD-b subloci. RESULTS: Sublocus HD-b contained a large variety of fixed Hd1/Hd2 gene pairs, while the HD-a sublocus either contained a conserved Hd2 gene or, a newly discovered Hd1 gene that was also conserved. Identification of whole HD loci, that is, the contents of HD-a and HD-b subloci in a strain, revealed that strains with similar HD-b subloci could still form normal dikaryons if the two genes at the HD-a sublocus differed. At least di-allelic HD-a sublocus, is thus indicated to be actively involved in mating type compatibility. CONCLUSIONS: HD-a sublocus is active and di-allelic. Using the new information on the HD subloci, primers sets were developed that specifically amplify HD-a or HD-b subloci in the majority of F. velutipes strains. In this way, unknown HD mating types of F. velutipes can now be quickly identified, and HD mating type compatibility conferred by HD-a or HD-b can be confirmed by PCR.
RESUMO
Small GTPases play important roles in the growth, development and environmental responses of eukaryotes. Based on the genomic sequence of the straw mushroom Volvariella volvacea, 44 small GTPases were identified. A clustering analysis using human small GTPases as the references revealed that V. volvacea small GTPases can be grouped into five families: nine are in the Ras family, 10 are in the Rho family, 15 are in the Rab family, one is in the Ran family and nine are in the Arf family. The transcription of vvran1 was up-regulated upon hydrogen peroxide (H2O2) stress, and could be repressed by diphenyleneiodonium chloride (DPI), a NADPH oxidase-specific inhibitor. The number of vvran1 transcripts also increased upon cold stress. Diphenyleneiodonium chloride, but not the superoxide dismutase (SOD) inhibitor diethy dithiocarbamate (DDC), could suppress the up-regulation of vvran1 gene expression to cold stress. These results combined with the high correlations between gene expression and superoxide anion (O2(-)) generation indicated that vvran1 could be one of the candidate genes in the downstream of O2(-) mediated pathways that are generated by NADPH oxidase under low temperature and oxidative stresses.
Assuntos
Peróxido de Hidrogênio/farmacologia , Proteínas Monoméricas de Ligação ao GTP/genética , Estresse Fisiológico , Volvariella/enzimologia , Temperatura Baixa , Proteínas Fúngicas/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Humanos , Família Multigênica , Oniocompostos/farmacologia , Filogenia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Volvariella/genética , Proteína ran de Ligação ao GTP/genéticaRESUMO
The initiation of sexual development in the important edible and medicinal mushroom Flammulina velutipes is controlled by special genes at two different, independent, mating type (MAT) loci: HD and PR. We expanded our understanding of the F. velutipes mating type system by analyzing the MAT loci from a series of strains. The HD locus of F. velutipes houses homeodomain genes (Hd genes) on two separated locations: sublocus HD-a and HD-b. The HD-b subloci contained strain-specific Hd1/Hd2 gene pairs, and crosses between strains with different HD-b subloci indicated a role in mating. The function of the HD-a sublocus remained undecided. Many, but not all strains contained the same conserved Hd2 gene at the HD-a sublocus. The HD locus usually segregated as a whole, though we did detect one new HD locus with a HD-a sublocus from one parental strain, and a HD-b sublocus from the other. The PR locus of F. velutipes contained pheromone receptor (STE3) and pheromone precursor (Pp) genes at two locations, sublocus PR-a and PR-b. PR-a and PR-b both contained sets of strain-specific STE3 and Pp genes, indicating a role in mating. PR-a and PR-b cosegregated in our experiments. However, the identification of additional strains with identical PR-a, yet different PR-b subloci, demonstrated that PR subloci can recombine within the PR locus. In conclusion, at least three of the four MAT subloci seem to participate in mating, and new HD and PR loci can be generated through intralocus recombination in F. velutipes.
RESUMO
Volvariella volvacea is an important crop in Southeast Asia, but erratic fruiting presents a serious challenge for its production and breeding. Efforts to explain inconsistent fruiting have been complicated by the multinucleate nature, typical lack of clamp connections, and an incompletely identified sexual reproductive system. In this study, we addressed the life cycle of V. volvacea using whole genome sequencing, cloning of MAT loci, karyotyping of spores, and fruiting assays. Microscopy analysis of spores had previously indicated the possible coexistence of heterothallic and homothallic life cycles. Our analysis of the MAT loci showed that only MAT-A, and not MAT-B, controlled heterokaryotization. Thus, the heterothallic life cycle was bipolar. Karyotyping of single spore isolates (SSIs) using molecular markers supported the existence of heterokaryotic spores. However, most SSIs were clearly not heterokaryotic, yet contained structural variation (SV) markers relating to both alleles of both parents. Heterokaryons from crossed, self-sterile homokaryons could produce fruiting bodies, agreeing with bipolar heterothallism. Meanwhile, some SSIs with two different MAT-A loci also produced fruiting bodies, which supported secondary homothallism. Next, SSIs that clearly contained only one MAT-A locus (homothallism) were also able to fruit, demonstrating that self-fertile SSIs were not, per definition, secondary homothallic, and that a third life cycle or genetic mechanism must exist. Finally, recombination between SV markers was normal, yet 10 out of 24 SV markers showed 1:2 or 1:3 distributions in the spores, and large numbers of SSIs contained doubled SV markers. This indicated selfish genes, and possibly partial aneuploidy.
Assuntos
Carpóforos/genética , Genes Fúngicos Tipo Acasalamento , Variação Genética , Genoma Fúngico , Esporos Fúngicos/genética , Volvariella/genética , Sequência de Aminoácidos , Aneuploidia , Cruzamento , Mapeamento Cromossômico , Carpóforos/crescimento & desenvolvimento , Loci Gênicos , Marcadores Genéticos , Cariotipagem , Filogenia , Recombinação Genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Esporos Fúngicos/crescimento & desenvolvimento , Volvariella/classificação , Volvariella/crescimento & desenvolvimentoRESUMO
The edible white rot fungus Lentinula edodes possesses a variety of lignin degrading enzymes such as manganese peroxidases and laccases. Laccases belong to the multicopper oxidases, which have a wide range of catalytic activities including polyphenol degradation and synthesis, lignin degradation, and melanin formation. The exact number of laccases in L. edodes is unknown, as are their complete properties and biological functions. We analyzed the draft genome sequence of L. edodes D703PP-9 and identified 13 multicopper oxidase-encoding genes; 11 laccases in sensu stricto, of which three are new, and two ferroxidases. lcc8, a laccase previously reported in L. edodes, was not identified in D703PP-9 genome. Phylogenetic analysis showed that the 13 multicopper oxidases can be classified into laccase sensu stricto subfamily 1, laccase sensu stricto subfamily 2 and ferroxidases. From sequence similarities and expression patterns, laccase sensu stricto subfamily 1 can be divided into two subgroups. Laccase sensu stricto subfamily 1 group A members are mainly secreted from mycelia, while laccase sensu stricto subfamily 1 group B members are expressed mainly in fruiting bodies during growth or after harvesting but are lowly expressed in mycelia. Laccase sensu stricto subfamily 2 members are mainly expressed in mycelia, and two ferroxidases are mainly expressed in the fruiting body during growth or after harvesting, and are expressed at very low levels in mycelium. Our data suggests that L. edodes laccases in same group share expression patterns and would have common biological functions.
RESUMO
The edible mushroom Volvariella volvacea is an important crop in Southeast Asia and is predominantly harvested in the egg stage. One of the main factors that negatively affect its yield and value is the rapid transition from the egg to the elongation stage, which has a decreased commodity value and shelf life. To improve our understanding of the changes during stipe development and the transition from egg to elongation stage in particular, we analyzed gene transcription in stipe tissue of V. volvacea using 3'-tag based digital expression profiling. Stipe development turned out to be fairly complex with high numbers of expressed genes, and regulation of stage differences is mediated mainly by changes in expression levels of genes, rather than on/off modulation. Most explicit is the strong up-regulation of cell division from button to egg, and the very strong down-regulation hereof from egg to elongation, that continues in the maturation stage. Button and egg share cell division as means of growth, followed by a major developmental shift towards rapid stipe elongation based on cell extension as demonstrated by inactivation of cell division throughout elongation and maturation. Examination of regulatory genes up-regulated from egg to elongation identified three potential high upstream regulators for this switch. The new insights in stipe dynamics, together with a series of new target genes, will provide a sound base for further studies on the developmental mechanisms of mushroom stipes and the switch from egg to elongation in V. volvacea in particular.
Assuntos
Parede Celular/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Caules de Planta/crescimento & desenvolvimento , Volvariella/crescimento & desenvolvimento , Volvariella/genética , Caules de Planta/metabolismo , RNA Mensageiro/genética , RNA de Plantas/genética , RNA de Plantas/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The edible straw mushroom Volvariella volvacea is an important crop in South East Asia and is predominantly harvested in the egg stage. Rapid stipe elongation and cap expansion result in a swift transition from the egg to elongation and maturation stage, which are subjected to fast senescence and deterioration. In other mushrooms, ß-1,3-glucanases have been associated with degradation (softening) of the cell wall during stipe elongation and senescence. We present a new glycoside hydrolase family 55 (GH55) exo-ß-1,3-glucanase gene, exg2, and highly conserved deduced EXG2 protein. The 3D model and presumed catalytic residues of V. volvacea EXG2 are identical to Lentinula edodes EXG2 and Phanerochaete chrysosporium Lam55A, supporting similar enzymatic functions. In addition to previous association to stipe elongation and senescence, our data clearly indicates a role for cap (pileus) expansion. Digital gene expression, quantitative PCR and isobaric tags for relative and absolute quantification analysis showed low exg2 and EXG2 levels in primordia, button, egg and elongation stages and significantly increased levels in the maturation stage. Subsequent relative quantitative PCR analysis designated expression of exg2 to the stipe in the elongation stage and to the pileus and stipe in the maturation stage. EXG2 cell wall softening activity, close correlation of exg2 expression with the principal expanding mushroom tissues and a strong conservation of expression patterns and protein sequences in other mushrooms, make V. volvacea exg2 an important candidate for future studies on mechanisms of fruiting body expansion and senescence causing commodity value loss.
Assuntos
Carpóforos/enzimologia , Proteínas Fúngicas/genética , Glucana 1,3-beta-Glucosidase/genética , Volvariella/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Sequência Conservada , Carpóforos/genética , Carpóforos/crescimento & desenvolvimento , Proteínas Fúngicas/metabolismo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Glucana 1,3-beta-Glucosidase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Homologia Estrutural de Proteína , Transcrição Gênica , Volvariella/genética , Volvariella/crescimento & desenvolvimentoRESUMO
BACKGROUND: Mating-type loci of mushroom fungi contain master regulatory genes that control recognition between compatible nuclei, maintenance of compatible nuclei as heterokaryons, and fruiting body development. Regions near mating-type loci in fungi often show adapted recombination, facilitating the generation of novel mating types and reducing the production of self-compatible mating types. Compared to other fungi, mushroom fungi have complex mating-type systems, showing both loci with redundant function (subloci) and subloci with many alleles. The genomic organization of mating-type loci has been solved in very few mushroom species, which complicates proper interpretation of mating-type evolution and use of those genes in breeding programs. METHODOLOGY/PRINCIPAL FINDINGS: We report a complete genetic structure of the mating-type loci from the tetrapolar, edible mushroom Flammulina velutipes mating type A3B3. Two matB3 subloci, matB3a that contains a unique pheromone and matB3b, were mapped 177 Kb apart on scaffold 1. The matA locus of F. velutipes contains three homeodomain genes distributed over 73 Kb distant matA3a and matA3b subloci. The conserved matA region in Agaricales approaches 350 Kb and contains conserved recombination hotspots showing major rearrangements in F. velutipes and Schizophyllum commune. Important evolutionary differences were indicated; separation of the matA subloci in F. velutipes was diverged from the Coprinopsis cinerea arrangement via two large inversions whereas separation in S. commune emerged through transposition of gene clusters. CONCLUSIONS/SIGNIFICANCE: In our study we determined that the Agaricales have very large scale synteny at matA (â¼350 Kb) and that this synteny is maintained even when parts of this region are separated through chromosomal rearrangements. Four conserved recombination hotspots allow reshuffling of large fragments of this region. Next to this, it was revealed that large distance subloci can exist in matB as well. Finally, the genes that were linked to specific mating types will serve as molecular markers in breeding.
Assuntos
Flammulina/genética , Flammulina/fisiologia , Loci Gênicos/genética , Genômica , Sintenia/genética , Mapeamento Cromossômico , Proteínas Fúngicas/química , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Feromônios/genética , Filogenia , Polimorfismo Genético/genética , Estrutura Terciária de Proteína , Receptores de Feromônios/química , Receptores de Feromônios/classificação , Receptores de Feromônios/genética , Receptores de Feromônios/metabolismo , Reprodução/genéticaRESUMO
Mushroom-forming basidiomycetes colonize large areas in nature. Their hyphae are compartmentalized by perforated septa, which are usually covered by a septal pore cap (SPC). Here, we describe, for the first time, the composition and function of SPCs using the model system Schizophyllum commune. The SPC of S. commune was shown to consist of a proteinaceous matrix covered by a lipid membrane. The matrix was demonstrated to define the ultrastructure of the SPC and to consist of two main proteins, Spc14 and Spc33. Gene spc14 encodes a protein of 86 amino acids, which lacks known domain, signal or localization sequences. Gene spc33 encodes a 239 and a 340 amino acid variant. Both forms contain a predicted signal anchor that targets them to the ER. Immuno-localization showed the presence of Spc33 in the SPC but not in ER. From this and previous reports it is concluded that the SPC is derived from this organelle. Inactivation of spc33 resulted in loss of SPCs and the inability to close septa. The latter may well explain why vegetative growth and mushroom formation were severely reduced in strains in which spc33 was inactivated.
Assuntos
Proteínas Fúngicas/genética , Micélio/ultraestrutura , Schizophyllum/crescimento & desenvolvimento , Schizophyllum/genética , Sequência de Aminoácidos , DNA Fúngico/genética , Proteínas Fúngicas/metabolismo , Técnicas de Inativação de Genes , Genes Fúngicos , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Schizophyllum/metabolismo , Análise de Sequência de ProteínaRESUMO
BACKGROUND: Mycelia of higher fungi consist of interconnected hyphae that are compartmentalized by septa. These septa contain large pores that allow streaming of cytoplasm and even organelles. The cytoplasm of such mycelia is therefore considered to be continuous. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show by laser dissection that septa of Schizophyllum commune can be closed depending on the environmental conditions. The most apical septum of growing hyphae was open when this basidiomycete was grown in minimal medium with glucose as a carbon source. In contrast, the second and the third septum were closed in more than 50% and 90% of the cases, respectively. Interestingly, only 24 and 37% of these septa were closed when hyphae were growing in the absence of glucose. Whether a septum was open or closed also depended on physical conditions of the environment or the presence of toxic agents. The first septum closed when hyphae were exposed to high temperature, to hypertonic conditions, or to the antibiotic nourseothricin. In the case of high temperature, septa opened again when the mycelium was placed back to the normal growth temperature. CONCLUSIONS/SIGNIFICANCE: Taken together, it is concluded that the septal pores of S. commune are dynamic structures that open or close depending on the environmental conditions. Our findings imply that the cytoplasm in the mycelium of a higher fungus is not continuous per se.
Assuntos
Citoplasma/metabolismo , Hifas/fisiologia , Schizophyllum/metabolismo , Schizophyllum/fisiologia , Carbono/química , Técnicas de Cultura de Células/métodos , Meio Ambiente , Glucose/química , Glucose/metabolismo , Modelos Biológicos , Schizophyllum/genética , Temperatura , Fatores de TempoRESUMO
Phleomycin is mutagenic by introducing double-strand breaks in DNA. The ble gene of Streptoalloteychus hindustanus, which confers resistance to this substance, is widely used as a selection marker for transformation. Schizophyllum commune grows on 25 microg of phleomycin ml(-1) after introduction of a resistance cassette based on the ble gene. However, we here report that growth of resistant colonies on this concentration of phleomycin resulted in aberrant colony morphologies. Apparently, phleomycin was mutagenic despite acquired resistance. Therefore, a new selection system was developed based on resistance to the antibiotic nourseothricin. However, the transformation efficiency was tenfold lower than that obtained with phleomycin as a selection agent. This low transformation efficiency could be rescued by addition of a nonselective concentration of phleomycin during protoplast regeneration. This was accompanied by a higher incidence of single-copy integrations and with an increase of expression of key genes involved in double-strand break repair. Taken together, we conclude that the effect of a nonselective concentration of phleomycin strongly resembles the effect of restriction enzyme-mediated integration (REMI) but, unlike REMI, it does not depend on the presence of a target restriction site.
Assuntos
Mutagênicos/farmacologia , Fleomicinas/farmacologia , Recombinação Genética/efeitos dos fármacos , Schizophyllum/efeitos dos fármacos , Schizophyllum/fisiologia , Transformação Genética/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Enzimas Reparadoras do DNA/metabolismo , Schizophyllum/genéticaRESUMO
The hyphae of filamentous fungi are compartmentalized by septa that have a central pore. The fungal septa and septum-associated structures play an important role in maintaining cellular and intrahyphal homeostasis. The dolipore septa in the higher Basidiomycota (i.e., Agaricomycotina) are associated with septal pore caps. Although the ultrastructure of the septal pore caps has been studied extensively, neither the biochemical composition nor the function of these organelles is known. Here, we report the identification of the glycoprotein SPC18 that was found in the septal pore cap-enriched fraction of the basidiomycetous fungus Rhizoctonia solani. Based on its N-terminal sequence, the SPC18 gene was isolated. SPC18 encodes a protein of 158 amino acid residues, which contains a hydrophobic signal peptide for targeting to the endoplasmic reticulum and has an N-glycosylation motif. Immunolocalization showed that SPC18 is present in the septal pore caps. Surprisingly, we also observed SPC18 being localized in some plugs. The data reported here strongly support the hypothesis that septal pore caps are derived from endoplasmic reticulum and are involved in dolipore plugging and, thus, contribute to hyphal homeostasis in basidiomycetous fungi.
Assuntos
Proteínas Fúngicas/metabolismo , Glicoproteínas/metabolismo , Hifas/metabolismo , Rhizoctonia/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Glicoproteínas/química , Glicoproteínas/genética , Hifas/química , Hifas/genética , Hifas/ultraestrutura , Dados de Sequência Molecular , Transporte Proteico , Rhizoctonia/química , Rhizoctonia/genética , Rhizoctonia/ultraestruturaRESUMO
Septal pore caps occur in many filamentous basidiomycetes located at both sides of the dolipore septum and are at their base connected to the endoplasmic reticulum. The septal pore cap ultrastructure has been described extensively by the use of electron microscopy, but its composition and function are not yet known. To enable biochemical and functional analyses in the future, we here describe an enrichment method for perforate septal pore caps from Rhizoctonia solani. Our method is based on the combined use of French press and isopycnic centrifugation, using a discontinuous sucrose gradient followed by a treatment with Triton X-100. Enrichment was monitored by the use of scanning electron microscopy and transmission electron microscopy. Using the same isolation method, smaller septal pore caps were isolated from two other basidiomycetes as well. Furthermore, we showed pore-occluding material co-purified with the septal pore caps. This observation supports the hypothesis that septal pore caps play a key role in the plugging process of the septal pores in filamentous basidiomycetes.