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Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    Experimental demonstration of the benefits of somatic fusion and the consequences for allorecognition
    Bastiaans, E. ; Debets, A.J.M. ; Aanen, D.K. - \ 2015
    Evolution 69 (2015)4. - ISSN 0014-3820 - p. 1091 - 1099.
    vegetative incompatibility - neurospora-crassa - heterokaryon incompatibility - natural-populations - filamentous fungi - recognition - evolution - selection - genetics - cooperation
    Allorecognition, the ability to distinguish “self” from “nonself” based on allelic differences at allorecognition loci, is common in all domains of life. Allorecognition restricts the opportunities for social parasitism, and is therefore crucial for the evolution of cooperation. However, the maintenance of allorecognition diversity provides a paradox. If allorecognition is costly relative to cooperation, common alleles will be favored. Thus, the cost of allorecognition may reduce the genetic variation upon which allorecognition crucially relies, a prediction now known as “Crozier's paradox.” We establish the relative costs of allorecognition, and their consequences for the short-term evolution of recognition labels theoretically predicted by Crozier. We use fusion among colonies of the fungus Neurospora crassa, regulated by highly variable allorecognition genes, as an experimental model system. We demonstrate that fusion among colonies is mutually beneficial, relative to absence of fusion upon allorecognition. This benefit is due not only to absence of mutual antagonism, which occurs upon allorecognition, but also to an increase in colony size per se. We then experimentally demonstrate that the benefit of fusion selects against allorecognition diversity, as predicted by Crozier. We discuss what maintains allorecognition diversity
    Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants
    Muniz, C.R. ; Silva, C.F. da; Souza, M.T. ; Freire, F.C.O. ; Kema, G.H.J. ; Guedes, M.I.F. - \ 2014
    Genetics and Molecular Research 13 (2014)2. - ISSN 1676-5680 - p. 2906 - 2913.
    filamentous fungi - genetic-transformation - proteins - tool
    Lasiodiplodia theobromae is a major pathogen of many different crop cultures, including cashew nut plants. This paper describes an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for the successful delivery of T-DNA, transferring the genes of green fluorescent protein (gfp) and hygromycin B phosphotransferase (hph) to L. theobromae. When the fungal pycnidiospores were co-cultured with A. tumefaciens harboring the binary vector with hph-gfp gene, hygromycin-resistant fungus only developed with acetosyringone supplementation. The cashew plants inoculated with the fungus expressing GFP revealed characteristic pathogen colonization by epifluorescence microscopy. Intense and bright green hyphae were observed for transformants in all extensions of mycelium cultures. The penetration of parenchyma cells near to the inoculation site, beneath the epicuticle surface, was observed prior to 25 dpi. Penetration was followed by the development of hyphae within invaded host cells. These findings provide a rapid and reproducible ATMT method for L. theobromae transformation.
    Natural Variation of Heterokaryon Incompatibility Gene het-c in Podospora anserina Reveals Diversifying Selection
    Bastiaans, E. ; Debets, A.J.M. ; Aanen, D.K. ; Diepeningen, A.D. van; Saupe, S.J. ; Paoletti, M. - \ 2014
    Molecular Biology and Evolution 31 (2014)4. - ISSN 0737-4038 - p. 962 - 974.
    plant immune-system - glycolipid transfer protein - chestnut blight fungus - amino-acid sites - vegetative incompatibility - neurospora-crassa - cell-death - cryphonectria-parasitica - membrane interaction - filamentous fungi
    In filamentous fungi, allorecognition takes the form of heterokaryon incompatibility, a cell death reaction triggered when genetically distinct hyphae fuse. Heterokaryon incompatibility is controlled by specific loci termed het-loci. In this article, we analyzed the natural variation in one such fungal allorecognition determinant, the het-c heterokaryon incompatibility locus of the filamentous ascomycete Podospora anserina. The het-c locus determines an allogenic incompatibility reaction together with two unlinked loci termed het-d and het-e. Each het-c allele is incompatible with a specific subset of the het-d and het-e alleles. We analyzed variability at the het-c locus in a population of 110 individuals, and in additional isolates from various localities. We identified a total of 11 het-c alleles, which define 7 distinct incompatibility specificity classes in combination with the known het-d and het-e alleles. We found that the het-c allorecognition gene of P. anserina is under diversifying selection. We find a highly unequal allele distribution of het-c in the population, which contrasts with the more balanced distribution of functional groups of het-c based on their allorecognition function. One explanation for the observed het-c diversity in the population is its function in allorecognition. However, alleles that are most efficient in allorecognition are rare. An alternative and not exclusive explanation for the observed diversity is that het-c is involved in pathogen recognition. In Arabidopsis thaliana, a homolog of het-c is a pathogen effector target, supporting this hypothesis. We hypothesize that the het-c diversity in P. anserina results from both its functions in pathogen-defense, and allorecognition
    Selection against somatic parasitism can maintain allorecognition in fungi
    Czaran, T. ; Hoekstra, R.F. ; Aanen, D.K. - \ 2014
    Fungal Genetics and Biology 73 (2014). - ISSN 1087-1845 - p. 128 - 137.
    vegetative incompatibility - neurospora-crassa - kin discrimination - filamentous fungi - self-recognition - botryllus-schlosseri - marine-invertebrates - ascomycete fungi - cell parasitism - genetic-control
    Fusion between multicellular individuals is possible in many organisms with modular, indeterminate growth, such as marine invertebrates and fungi. Although fusion may provide various benefits, fusion usually is restricted to close relatives by allorecognition, also called heterokaryon or somatic incompatibility in fungi. A possible selective explanation for allorecognition is protection against somatic parasites. Such mutants contribute less to colony functions but more to reproduction. However, previous models testing this idea have failed to explain the high diversity of allorecognition alleles in nature. These models did not, however, consider the possible role of spatial structure. We model the joint evolution of allorecognition and somatic parasitism in a multicellular organism resembling an asexual ascomycete fungus in a spatially explicit simulation. In a 1000-by-1000 grid, neighbouring individuals can fuse, but only if they have the same allotype. Fusion with a parasitic individual decreases the total reproductive output of the fused individuals, but the parasite compensates for this individual-level fitness reduction by a disproportional share of the offspring. Allorecognition prevents the invasion of somatic parasites, and vice versa, mutation towards somatic parasitism provides the selective conditions for extensive allorecognition diversity. On the one hand, if allorecognition diversity did not build up fast enough, somatic parasites went to fixation; conversely, once parasites had gone to fixation no allorecognition diversity built up. On the other hand, the mere threat of parasitism could select for high allorecognition diversity, preventing invasion of somatic parasites. Moderate population viscosity combined with weak global dispersal was optimal for the joint evolution of allorecognition and protection against parasitism. Our results are consistent with the widespread occurrence of allorecognition in fungi and the low degree of somatic parasitism. We discuss the implications of our results for allorecognition in other organism groups.
    Overexpression of the Aspergillus niger GatA transporter leads to preferential use of D-galacturonic acid over D-xylose
    Sloothaak, J. ; Schilders, M. ; Schaap, P.J. ; Graaff, L.H. de - \ 2014
    AMB Express 4 (2014). - ISSN 2191-0855 - 9 p.
    filamentous fungi - expression - xlnr - degradation - identification - models - genes - crea - ph
    Pectin is a structural heteropolysaccharide of the primary cell walls of plants and as such is a significant fraction of agricultural waste residues that is currently insufficiently used. Its main component, D-galacturonic acid, is an attractive substrate for bioconversion. The complete metabolic pathway is present in the genome of Aspergillus niger, that is used in this study. The objective was to identify the D-galacturonic acid transporter in A. niger and to use this transporter to study D-galacturonic acid metabolism. We have functionally characterized the gene An14g04280 that encodes the D-galacturonic acid transporter in A. niger. In a mixed sugar fermentation it was found that the An14g04280 overexpression strain, in contrast to the parent control strain, has a preference for D-galacturonic acid over D-xylose as substrate. Overexpression of this transporter in A. niger resulted in a strong increase of D-galacturonic acid uptake and induction of the D-galacturonic acid reductase activity, suggesting a metabolite controlled regulation of the endogenous D-galacturonic acid catabolic pathway.
    Regular bottlenecks and restrictions to somatic fusion prevent the accumulation of mitochondrial defects in Neurospora
    Bastiaans, E. ; Aanen, D.K. ; Debets, A.J.M. ; Hoekstra, R.F. ; Lestrada, B. ; Maas, M.F.P.M. - \ 2014
    Philosophical Transactions of the Royal Society B. Biological sciences 369 (2014)1646. - ISSN 0962-8436
    vegetative incompatibility - filamentous fungi - dna mutations - hyphal fusion - evolution - populations - senescence - intermedia - selection - crassa
    The replication and segregation of multi-copy mitochondrial DNA (mtDNA) are not under strict control of the nuclear DNA. Within-cell selection may thus favour variants with an intracellular selective advantage but a detrimental effect on cell fitness. High relatedness among the mtDNA variants of an individual is predicted to disfavour such deleterious selfish genetic elements, but experimental evidence for this hypothesis is scarce. We studied the effect of mtDNA relatedness on the opportunities for suppressive mtDNA variants in the fungus Neurospora carrying the mitochondrial mutator plasmid pKALILO. During growth, this plasmid integrates into the mitochondrial genome, generating suppressive mtDNA variants. These mtDNA variants gradually replace the wild-type mtDNA, ultimately culminating in growth arrest and death. We show that regular sequestration of mtDNA variation is required for effective selection against suppressive mtDNA variants. First, bottlenecks in the number of mtDNA copies from which a 'Kalilo' culture started significantly increased the maximum lifespan and variation in lifespan among cultures. Second, restrictions to somatic fusion among fungal individuals, either by using anastomosis-deficient mutants or by generating allotype diversity, prevented the accumulation of suppressive mtDNA variants. We discuss the implications of these results for the somatic accumulation of mitochondrial defects during ageing
    Production of (+)-valencene in the mushroom-forming fungus S. commune
    Scholtmeijer, K. ; Beekwilder, J. ; Cankar, K. ; Wösten, H.A.B. ; Lugones, L.G. ; Bosch, H.J. - \ 2014
    Applied Microbiology and Biotechnology 98 (2014)11. - ISSN 0175-7598 - p. 5059 - 5068.
    basidiomycete schizophyllum-commune - sc3 hydrophobin gene - secondary metabolism - filamentous fungi - aerial hyphae - biosynthesis - mutation - protein - sesquiterpenes - microorganisms
    Production of commercially interesting sesquiterpenes was previously examined in plants and microorganisms such as Escherichia coli and Saccharomyces cerevisiae. We here investigate the potential of the mushroom Schizophyllum commune for the production of sesquiterpenes. Genomic analysis of S. commune revealed that the mevalonate pathway required for the synthesis of the farnesyl diphosphate substrate for sesquiterpene production is operational. Introduction of a valencene synthase gene resulted in production of the sesquiterpene (+)-valencene, both in mycelium and in fruiting bodies. Levels of (+)-valencene in culture media of strains containing a mutated RGS regulatory protein gene (thn) were increased fourfold compared to those in wild-type transformants. Up to 16 mg L-1 (+)-valencene was produced in these strains. In addition, the amount of (+)-valencene containing n-dodecane recovered from the culture medium increased sixfold to sevenfold in the thn mutant strains due to the absence of schizophyllan.
    Genome-wide analysis of pectate-induced gene expression in Botrytis cinerea: identification and functional analysis of putative D-galacturonic acid transporters
    Zhang, L. ; Hua, C. ; Stassen, J.H.M. ; Chatterjee, S. ; Cornelissen, M. ; Kan, J.A.L. van - \ 2014
    Fungal Genetics and Biology 72 (2014). - ISSN 1087-1845 - p. 182 - 191.
    wall degrading enzymes - saccharomyces-cerevisiae - filamentous fungi - endopolygalacturonase genes - colletotrichum fungi - hexose transporters - confocal microscopy - rna-seq - acid - virulence
    The fungal plant pathogen Botrytis cinerea produces a spectrum of cell wall degrading enzymes for the decomposition of host cell wall polysaccharides and the consumption of the monosaccharides that are released. Especially pectin is an abundant cell wall component, and the decomposition of pectin by B. cinerea has been extensively studied. An effective concerted action of the appropriate pectin depolymerising enzymes, monosaccharide transporters and catabolic enzymes is important for complete d-galacturonic acid utilization by B. cinerea. In this study, we performed RNA sequencing to compare genome-wide transcriptional profiles between B. cinerea cultures grown in media containing pectate or glucose as sole carbon source. Transcript levels of 32 genes that are induced by pectate were further examined in cultures grown on six different monosaccharides, by means of quantitative RT-PCR, leading to the identification of 8 genes that are exclusively induced by d-galacturonic acid. Among these, the hexose transporter encoding genes Bchxt15 and Bchxt19 were functionally characterised. The subcellular location was studied of BcHXT15-GFP and BcHXT19-GFP fusion proteins expressed under control of their native promoter, in a B. cinerea wild-type strain. Both genes are expressed during growth on d-galacturonic acid and the fusion proteins are localized in plasma membranes and intracellular vesicles. Target gene knockout analysis revealed that BcHXT15 contributes to d-galacturonic acid uptake at pH 5~5.6. The virulence of all B. cinerea hexose transporter mutants tested was unaltered on tomato and Nicotiana benthamiana leaves.
    Fragmentation of an aflatoxin-like gene cluster in a forest pathogen
    Bradshaw, R.E. ; Slot, J.C. ; Moore, G.G. ; Chettri, P. ; Wit, P.J.G.M. de; Ehrlich, K.C. ; Ganley, A.R.D. ; Olson, M.A. ; Rokas, A. ; Carbone, I. ; Cox, M.P. - \ 2013
    New Phytologist 198 (2013)2. - ISSN 0028-646X - p. 525 - 535.
    aspergillus-parasiticus - dothistroma-septosporum - phylogenetic analyses - biosynthetic-pathway - recombination events - secondary metabolism - functional-analysis - horizontal transfer - filamentous fungi - evolution
    Plant pathogens use a complex arsenal of weapons, such as toxic secondary metabolites, to invade and destroy their hosts. Knowledge of how secondary metabolite pathways evolved is central to understanding the evolution of host specificity. The secondary metabolite dothistromin is structurally similar to aflatoxins and is produced by the fungal pine pathogen Dothistroma septosporum. Our study focused on dothistromin genes, which are widely dispersed across one chromosome, to determine whether this unusual distributed arrangement evolved from an ancestral cluster. We combined comparative genomics and population genetics approaches to elucidate the origins of the dispersed arrangement of dothistromin genes over a broad evolutionary time-scale at the phylum, class and species levels. Orthologs of dothistromin genes were found in two major classes of fungi. Their organization is consistent with clustering of core pathway genes in a common ancestor, but with intermediate cluster fragmentation states in the Dothideomycetes fungi. Recombination hotspots in a D.septosporum population matched sites of gene acquisition and cluster fragmentation at higher evolutionary levels. The results suggest that fragmentation of a larger ancestral cluster gave rise to the arrangement seen in D.septosporum. We propose that cluster fragmentation may facilitate metabolic retooling and subsequent host adaptation of plant pathogens.
    Fungal fidelity: nuclear divorce from a dikaryon by mating or monokaryon regeneration
    Nieuwenhuis, B.P.S. ; Debets, A.J.M. ; Aanen, D.K. - \ 2013
    Fungal Biology 117 (2013)4. - ISSN 1878-6146 - p. 261 - 267.
    basidiomycete schizophyllum-commune - heterobasidion-annosum - homokaryotic matings - coprinus-cinereus - filamentous fungi - buller phenomenon - genomic conflict - pholiota-nameko - evolution - heterokaryons
    Basidiomycete fungi perform fertilizations by incorporation of nuclei into a monokaryotic mycelium to establish a dikaryon. The dikaryon cannot incorporate another type of nucleus, but can still act as a nucleus donor in a dikaryon–monokaryon (di–mon) mating, known as the Buller phenomenon. Previously, it has been observed that: (1) in a particular di–mon mating, one of the nuclear types of the dikaryon generally performs better as a donor than the other, and (2) when nuclei from a dikaryon are separated to form monokaryons again (dedikaryotisation), recovery of monokaryons of the two nuclear types is usually unequal. In this study, we investigated if these two observations of asymmetry are functionally related. We tested this hypothesis by performing both di–mon matings and dedikaryotisation of dikaryons derived from five different monokaryons. When a single mechanism controls both processes, the nucleus better at fertilizing a monokaryon in a Buller pairing should also be recovered upon dedikaryotisation with a higher frequency. The results showed a hierarchical structure for recovery among nuclei in dedikaryotisation, but this hierarchy did not correspond to the fertilization success during di–mon mating. These findings thus show that the mechanism causing asymmetric regeneration of nuclei, is most likely not the same as the mechanism responsible for increased chance of fertilization in di–mon matings. We discuss the complexity of the interactions that occur during di–mon matings with regards to the mating type loci
    Botrytis cinerea mutants deficient in D-galacturonic acid catabolism have a perturbed virulence on Nicotiana benthamiana and Arabidopsis, but not on tomato
    Zhang, L. ; Kan, J.A.L. van - \ 2013
    Molecular Plant Pathology 14 (2013)1. - ISSN 1464-6722 - p. 19 - 29.
    cell-wall polysaccharides - plant defensin gene - salicylic-acid - filamentous fungi - thaliana - pathogen - resistance - pathway - camalexin - biosynthesis
    d-Galacturonic acid is the most abundant monosaccharide component of pectic polysaccharides that comprise a significant part of most plant cell walls. Therefore, it is potentially an important nutritional factor for Botrytis cinerea when it grows in and t
    Dothistromin genes at multiple separate loci are regulated by AflR
    Chettri, P. ; Ehrlich, K.C. ; Cary, J.W. ; Collemare, J. ; Cox, M.P. ; Griffiths, S.A. ; Olson, M.A. ; Wit, P.J.G.M. de; Bradshaw, R.E. - \ 2013
    Fungal Genetics and Biology 51 (2013). - ISSN 1087-1845 - p. 12 - 20.
    aspergillus-parasiticus - aflatoxin biosynthesis - forest pathogen - filamentous fungi - secondary metabolism - cluster protein - pathway genes - needle blight - pini - expression
    In fungi, genes involved in the production of secondary metabolites are generally clustered at one location. There are some exceptions, such as genes required for synthesis of dothistromin, a toxin that is a chemical analog of the aflatoxin precursor versicolorin A and made by the pine needle pathogen Dothistroma septosporum. The availability of the D. septosporum genome sequence enabled identification of putative dothistromin genes, including an ortholog of the aflatoxin regulatory gene AflR, and revealed that most of the genes are spread over six separate regions (loci) on chromosome 12 (1.3 Mb). Here we show that levels of expression of the widely dispersed genes in D. septosporum are not correlated with gene location with respect to their distance from a telomere, but that AflR regulates them. The production of dothistromin by D. septosporum in which the AflR gene was knocked out (¿DsAflR) was drastically reduced, but still detectable. This is in contrast to orthologous ¿AflR mutants in Aspergillus species that lack any aflatoxin production. Expression patterns in ¿DsAflR mutants helped to predict the complete set of genes involved in dothistromin production. This included a short-chain aryl alcohol dehydrogenase (NorB), which is located on chromosome 11 rather than chromosome 12, but was 24-fold down regulated in ¿DsAflR. An orthologous set of dothistromin genes, organized in a similar fragmented cluster arrangement to that seen in D. septosporum, was found in the closely related tomato pathogen Cladosporium fulvum even though this species does not produce dothistromin. In C. fulvum, pseudogenization of key biosynthetic genes explains the lack of dothistromin production. The fragmented arrangement of dothistromin genes provides an example of coordinated control of a dispersed set of secondary metabolite genes; it also provides an example where loss of dothistromin production might have allowed adaptation to a new pathogenic lifestyle.
    Biocatalytic potential of laccase-like multicopper oxidases from Aspergillus niger
    Tamayo Ramos, J.A. ; Berkel, W.J.H. van; Graaff, L.H. de - \ 2012
    Microbial Cell Factories 11 (2012). - ISSN 1475-2859 - 11 p.
    malachite green - fungal laccases - yellow laccase - phanerochaete-chrysosporium - pleurotus-ostreatus - filamentous fungi - blue - degradation - expression - nidulans
    BACKGROUND: Laccase-like multicopper oxidases have been reported in several Aspergillus species but they remain uncharacterized. The biocatalytic potential of the Aspergillus niger fungal pigment multicopper oxidases McoA and McoB and ascomycete laccase McoG was investigated. RESULTS: The laccase-like multicopper oxidases McoA, McoB and McoG from the commonly used cell factory Aspergillus niger were homologously expressed, purified and analyzed for their biocatalytic potential. All three recombinant enzymes were monomers with apparent molecular masses ranging from 80 to 110 kDa. McoA and McoG resulted to be blue, whereas McoB was yellow. The newly obtained oxidases displayed strongly different activities towards aromatic compounds and synthetic dyes. McoB exhibited high catalytic efficiency with N,N-dimethyl-p-phenylenediamine (DMPPDA) and 2,2-azino-di(3-ethylbenzthiazoline) sulfonic acid (ABTS), and appeared to be a promising biocatalyst. Besides oxidizing a variety of phenolic compounds, McoB catalyzed successfully the decolorization and detoxification of the widely used textile dye malachite green. CONCLUSIONS: The A. niger McoA, McoB, and McoG enzymes showed clearly different catalytic properties. Yellow McoB showed broad substrate specificity, catalyzing the oxidation of several phenolic compounds commonly present in different industrial effluents. It also harbored high decolorization and detoxification activity with the synthetic dye malachite green, showing to have an interesting potential as a new industrial biocatalyst
    Correlated evolution of senescence and ephemeral substrate use in the Sordariomycetes
    Geydan, T.D. ; Debets, A.J.M. ; Verkleij, G.J. ; Diepeningen, A.D. van - \ 2012
    Molecular Ecology 21 (2012)11. - ISSN 0962-1083 - p. 2816 - 2828.
    podospora-anserina - metarhizium-anisopliae - colony sectorization - filamentous fungi - phylogenies - plasmids - genes - dna - populations - ascomycota
    Evolutionary theory predicts that senescence--a decline in reproduction and survival with increasing age--can evolve as a trade-off between investment in reproduction on one side and in somatic maintenance and repair on the other. The ecology of a species is crucial because it provides the external causes of death that determine the statistical limit to a species' lifespan. Filamentous fungi are generally believed to be nonsenescent, and there are indeed spectacular examples of very old fungal individuals in nature. However, some fungi utilize ephemeral resources, and therefore, senescence is expected to have evolved, like in the coprophilic Podospora anserina, the only well-studied filamentous fungus with intrinsic senescence. Here, we hypothesize that rapid senescence is more common in fungi than generally believed and that the phylogenetic distribution of senescence correlates with ecology. We collected lifespan data for a set of Sordariomycetes and constructed phylogenies based on several nuclear sequences. Several of the strains were from the CBS culture collection, originally isolated from various substrates, some of which ephemeral. In addition, we isolated new strains from short-lived substrates. Senescence was observed throughout the phylogeny. Correlation tests support the hypothesis that in the Sordariomycetes, senescence is a trait that has arisen in response to ephemeral substrates, and that it has evolved repeatedly and independently along the phylogeny
    The Aspergillus niger multicopper oxidase family: analysis and overexpression of laccase-like encoding genes
    Tamayo Ramos, J.A. ; Barends, S. ; Verhaert, R.M. ; Graaff, L.H. de - \ 2011
    Microbial Cell Factories 10 (2011). - ISSN 1475-2859 - p. 78 - 78.
    heterologous expression - recombinant enzyme - saccharomyces-cerevisiae - melanocarpus-albomyces - secreted proteins - filamentous fungi - multigene family - nidulans - fumigatus - sequence
    BACKGROUND: Many filamentous fungal genomes contain complex groups of multicopper oxidase (MCO) coding genes that makes them a good source for new laccases with potential biotechnological interest. A bioinformatics analysis of the Aspergillus niger ATCC 1015 genome resulted in the identification of thirteen MCO genes. Ten of them were cloned and homologously overexpressed. RESULTS: A bioinformatic analysis of the A. niger ATCC 1015 genome revealed the presence of 13 MCO genes belonging to three different subfamilies on the basis of their phylogenetic relationships: ascomycete laccases, fungal pigment MCOs and fungal ferroxidases. According to in silico amino acid sequence analysis, the putative genes encoding for functional extracellular laccases (mcoA, mcoB, mcoC, mcoD, mcoE, mcoF, mcoG, mcoI, mcoJ and mcoM) were placed under the control of the glaA promoter and overexpressed in A. niger N593. Enzyme activity plate assays with several common laccase substrates showed that all genes are actually expressed and code for active MCOs. Interestingly, expressed enzymes show different substrate specificities. In addition, optimization of fungal pigment MCOs extracellular production was investigated. The performance of the widely used glucoamylase signal sequence (ssGlaA) in McoA secretion was studied. Results obtained suggest that ssGlaA do not yield higher levels of secreted McoA when compared to its native secretion signal. Also, McoB synthesis was investigated using different nitrogen sources in minimal medium liquid cultures. Higher yields of extracellular McoB were achieved with (NH4)2 tartrate. CONCLUSIONS: Aspergillus niger is a good source of new laccases. The different substrate specificity observed in plate assays makes them interesting to be purified and biochemically compared. The homologous signal sequence of McoA has been shown to be a good choice for its extracellular overexpression. From the nitrogen sources tested (NH4)2 tartrate has been found to be the most appropriate for McoB production in A. niger
    The FRP1 F-box gene has different functions in sexuality, pathogenicity and metabolism in three fungal pathogens
    Jonkers, W. ; Kan, J.A.L. van; Tijm, P. ; Lee, Y.W. ; Tudzynski, P. ; Rep, M. ; Michielse, C.B. - \ 2011
    Molecular Plant Pathology 12 (2011)6. - ISSN 1464-6722 - p. 548 - 563.
    vascular wilt fungus - fusarium-oxysporum - botrytis-cinerea - gibberella-zeae - map kinase - reduced pathogenicity - filamentous fungi - protein-kinase - g-alpha - virulence
    Plant-pathogenic fungi employ a variety of infection strategies; as a result, fungi probably rely on different sets of proteins for successful infection. The F-box protein Frp1, only present in filamentous fungi belonging to the Sordariomycetes, Leotiomycetes and Dothideomycetes, is required for nonsugar carbon catabolism and pathogenicity in the root-infecting fungus Fusarium oxysporum. To assess the role of Frp1 in other plant-pathogenic fungi, FRP1 deletion mutants were generated in Fusarium graminearum and Botrytis cinerea, and their phenotypes were analysed. Deletion of FgFRP1 in F. graminearum led to impaired infection of barley roots, but not of aerial plant parts. Deletion of BcFRP1 in B. cinerea did not show any effect on pathogenicity. Sexual reproduction, however, was impaired in both F. graminearum and B. cinerea FRP1 deletion mutants. The mutants of all three fungi displayed different phenotypes when grown on an array of carbon sources. The F. oxysporum and B. cinerea deletion mutants showed opposite growth phenotypes on sugar and nonsugar carbon sources. Replacement of FoFRP1 in F. oxysporum with the B. cinerea BcFRP1 resulted in the restoration of pathogenicity, but also in a switch from impaired growth on nonsugar carbon sources to impaired growth on sugar carbon sources. This effect could be ascribed in part to the B. cinerea BcFRP1 promoter sequence. In conclusion, the function of the F-box protein Frp1, despite its high sequence conservation, is not conserved between different fungi, leading to differential requirements for pathogenicity and carbon source utilization.
    The D-galacturonic acid catabolic pathway in Botrytis cinerea
    Zhang, L. ; Thiewes, H. ; Kan, J.A.L. van - \ 2011
    Fungal Genetics and Biology 48 (2011)10. - ISSN 1087-1845 - p. 990 - 997.
    mold hypocrea-jecorina - aspergillus-nidulans - filamentous fungi - identification - pathogenesis - expression - virulence - aldolase - mutants - enzymes
    d-galacturonic acid is the most abundant component of pectin, one of the major polysaccharide constituents of plant cell walls. Galacturonic acid potentially is an important carbon source for microorganisms living on (decaying) plant material. A catabolic pathway was proposed in filamentous fungi, comprising three enzymatic steps, involving d-galacturonate reductase, l-galactonate dehydratase, and 2-keto-3-deoxy-l-galactonate aldolase. We describe the functional, biochemical and genetic characterization of the entire d-galacturonate-specific catabolic pathway in the plant pathogenic fungus Botrytis cinerea. The B. cinerea genome contains two non-homologous galacturonate reductase genes (Bcgar1 and Bcgar2), a galactonate dehydratase gene (Bclgd1), and a 2-keto-3-deoxy-l-galactonate aldolase gene (Bclga1). Their expression levels were highly induced in cultures containing GalA, pectate, or pectin as the sole carbon source. The four proteins were expressed in Escherichia coli and their enzymatic activity was characterized. Targeted gene replacement of all four genes in B. cinerea, either separately or in combinations, yielded mutants that were affected in growth on d-galacturonic acid, pectate, or pectin as the sole carbon source. In Aspergillus nidulans and A. niger, the first catabolic conversion only involves the Bcgar2 ortholog, while in Hypocrea jecorina, it only involves the Bcgar1 ortholog. In B. cinerea, however, BcGAR1 and BcGAR2 jointly contribute to the first step of the catabolic pathway, albeit to different extent. The virulence of all B. cinerea mutants in the d-galacturonic acid catabolic pathway on tomato leaves, apple fruit and bell peppers was unaltered.
    DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer
    Robideau, G.P. ; Cock, A.W.A.M. de; Coffey, M.D. ; Voglmayr, H. ; Brouwer, H. ; Bala, K. ; Chitty, D.W. ; Désaulniers, N. ; Eggertson, Q.A. ; Gachon, C.M.M. ; Hu, C.H. ; Küpper, F.C. ; Rintoul, T.L. ; Sarhan, E. ; Verstappen, E.C.P. ; Zhang, Y. ; Bonants, P.J.M. ; Ristaino, J.B. ; Lévesque, C.A. - \ 2011
    Molecular Ecology Resources 11 (2011)6. - ISSN 1755-098X - p. 1002 - 1011.
    multiple sequence alignment - phylogenetic-relationships - molecular phylogeny - ribosomal dna - genus phytophthora - filamentous fungi - lsu rdna - pythium - mitochondrial - identification
    Oomycete species occupymany different environments andmany ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes
    Culture collections, the new herbaria for fungal pathogens
    Abd-Elsalam, K.A. ; Yassin, M.A. ; Moslem, M.A. ; Bahkali, A.H. ; Wit, P.J.G.M. de; McKenzie, E.H.C. ; Stephenson, S.L. ; Cai, L. ; Hyde, K.D. - \ 2010
    Fungal Diversity 45 (2010)1. - ISSN 1560-2745 - p. 21 - 32.
    long-term preservation - sterile distilled water - anhydrous silica-gel - liquid-nitrogen - aspergillus-fumigatus - filamentous fungi - stock cultures - storage - viability - mycosphaerella
    This paper discusses the importance of culture collections in plant pathology and reviews the methods currently available to store cultures. The preservation and maintenance of plant pathogenic fungi in a viable yet stable state for long periods has always been important, because isolates of these fungi can serve as standards for identification of quarantine taxa. Such isolates are also important for testing disease resistance and for plant breeding programs. The increasing use of molecular sequences analysis in the systematics of plant pathogenic fungi has meant that maintaining fungi in culture collections has become essential. Herein we discuss trends in the identification of plant and post-harvest pathogens, using Aspergillus, Colletotrichum, Phyllosticta and Mycosphaerella and its anamorphs as examples. Herbarium specimens, although still a requirement of the Botanical Code when describing new species are, perhaps, less important in providing useful information when defining a pathogenic species. Many pathogen groups consist of complexes of species and morphology alone can no longer distinguish among species. However, ex-type living cultures are essential for identification and future species comparisons that incorporate the use of molecular techniques. As such, ex-type cultures of any new species of pathogen, or when new diseases are reported or studies involving pathogenic strains are published, cultures of the taxa studied should be deposited in widely available culture collections, preferably in at least three members of World Federation for Culture Collections. Methods for the storage of fungal cultures such as water preservation, freezing, mineral oil overlay, freeze drying and lyophilization are reviewed in this paper. The main objective of culture preservation is to maintain the vigor and genetic characteristics of a pure culture. Therefore, safe long-term preservation methods are required to ensure fungal survival and retention of any valuable characteristics. To minimize the risk of any morphological, physiological, or genetic changes, several different preservation conditions should be used whenever possible. The present review also describes a complete preservation methodology that can be used for plant pathogenic fungi.
    Identification of modules in Aspergillus niger by gene co-expression network analysis
    Berg, R.A. van den; Braaksma, M. ; Veen, D. van der; Werf, M.J. van der; Punt, P.J. ; Oost, J. van der; Graaff, L.H. de - \ 2010
    Fungal Genetics and Biology 47 (2010)6. - ISSN 1087-1845 - p. 539 - 550.
    filamentous fungi - regulatory networks - expression analysis - analysis reveals - microarray data - scale - yeast - cell - conservation - encyclopedia
    The fungus Aspergillus niger has been studied in considerable detail with respect to various industrial applications. Although its central metabolic pathways are established relatively well, the mechanisms that control the adaptation of its metabolism are understood rather poorly. In this study, clustering of co-expressed genes has been performed on the basis of DNA microarray data sets from two experimental approaches. In one approach, low amounts of inducer caused a relatively mild perturbation, while in the other approach the imposed environmental conditions including carbon source starvation caused severe perturbed stress. A set of conserved genes was used to construct gene co-expression networks for both the individual and combined data sets. Comparative analysis revealed the existence of modules, some of which are present in all three networks. In addition, experimental condition-specific modules were identified. Module-derived consensus expression profiles enabled the integration of all protein-coding A. niger genes to the co-expression analysis, including hypothetical and poorly conserved genes. Conserved sequence motifs were detected in the upstream region of genes that cluster in some modules, e.g., the binding site for the amino acid metabolism-related transcription factor CpcA as well as for the fatty acid metabolism-related transcription factors, FarA and FarB. Moreover, not previously described putative transcription factor binding sites were discovered for two modules: the motif 5'-CGACAA is overrepresented in the module containing genes encoding cytosolic ribosomal proteins, while the motif 5'-GGCCGCG is overrepresented in genes related to 'gene expression', such as RNA helicases and translation initiation factors
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