ronald de vries | Utrecht University (original) (raw)

Papers by ronald de vries

Research paper thumbnail of The genome sequence of the model ascomycete fungus Podospora anserina

Genome Biology, 2008

The dung-inhabiting ascomycete fungus Podospora anserina is a model used to study various aspects... more The dung-inhabiting ascomycete fungus Podospora anserina is a model used to study various aspects of eukaryotic and fungal biology, such as ageing, prions and sexual development.

Research paper thumbnail of The effect of hematocrit on bioanalysis of DBS: results from the EBF DBS-microsampling consortium

Bioanalysis, 2013

The European Bioanalysis Forum dried blood spots (DBS)/microsampling consortium is reporting back... more The European Bioanalysis Forum dried blood spots (DBS)/microsampling consortium is reporting back from the experiments they performed on further documenting the potential hurdles of the DBS technology. This paper is focused on the impact of hematocrit changes on DBS analyses. The hematocrit can have an effect on the size of the blood spot, on spot homogeneity and on extraction recovery in a compound-dependent manner. The extraction recovery can change upon aging in an hematocrit-dependent way. Different card materials can give different outcomes. The results from the conducted experiments show that the issues of DBS in regulated bioanalysis are real and that the technology will need improvements to be ready for use as a general tool for regulated bioanalysis.

Research paper thumbnail of The Aspergillus nigerd-xylulose kinase gene is co-expressed with genes encoding arabinan degrading enzymes, and is essential for growth on d-xylose and l-arabinose: Aspergillus niger xylulose kinase

European Journal of Biochemistry, 2001

Research paper thumbnail of aguA, the Gene Encoding an Extracellular a-Glucuronidase from Aspergillus tubingensis, Is Specifically Induced on Xylose and Not on Glucuronic Acid

An extracellular a-glucuronidase was purified and characterized from a commercial Aspergillus pre... more An extracellular a-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrom- etry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged

Research paper thumbnail of Update of the EBF recommendation for the use of DBS in regulated bioanalysis integrating the conclusions from the EBF DBS-microsampling consortium

Bioanalysis, 2013

The European Bioanalysis Forum dried blood spots/microsampling consortium is reporting back from ... more The European Bioanalysis Forum dried blood spots/microsampling consortium is reporting back from the experiments they performed on further documenting the potential hurdles of the DBS technology. Their experiments focused on the impact of hematocrit changes, IS addition, spot homogeneity, aging of spots and stability of fresh blood and cards. Results from these experiments demonstrate that the issues of DBS in regulated bioanalysis are real and that the technology will need additional improvements to be ready for use as a general tool for regulated bioanalysis. In addition, results on fresh blood and card stability were shared at international meetings and will be reported at a later date.

Research paper thumbnail of In-depth study of homogeneity in DBS using two different techniques: results from the EBF DBS-microsampling consortium

Bioanalysis, 2013

At the start of their work, the European Bioanalysis Forum dried blood spots microsampling consor... more At the start of their work, the European Bioanalysis Forum dried blood spots microsampling consortium did not form a dedicated team to investigate the spot homogeneity. However, two teams performed experiments that produced results relating to sample homogeneity. The data, which were produced via two different approaches (a radiolabeled and a nonradiolabeled approach), are highly complementary and demonstrate clear effects on sample inhomogeneity due to the substrate type, compound and hematocrit levels. The results demonstrate that sample inhomogeneity is a significant hurdle to the use of dried blood spots for regulated bioanalysis that should be investigated further in the method establishment phase if the whole spot is not sampled.

Research paper thumbnail of Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire

Genome Biology, 2010

Pythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on ... more Pythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species. The P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions, although, surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host-specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome, including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report of these in a genome outside the metazoans. Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage-specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae.

Research paper thumbnail of Genomic Analysis of the Necrotrophic Fungal Pathogens Sclerotinia sclerotiorum and Botrytis cinerea

PLoS Genetics, 2011

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic f... more Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to ,1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.

Research paper thumbnail of The α-glucuronidase Agu1 from Schizophyllum commune is a member of a novel glycoside hydrolase family (GH115)

Applied Microbiology and Biotechnology, 2011

Schizophyllum commune produces an α-glucuronidase that is active on polymeric xylan, while the as... more Schizophyllum commune produces an α-glucuronidase that is active on polymeric xylan, while the ascomycete α-glucuronidases are only active on xylan oligomers. In this study, we have identified the gene (agu1) encoding this enzyme and confirmed the functionality by overexpression of the gene in S. commune and degradation of aldopentauronic acids, (MeGlcA)3-Xyl4, in the cultivation medium of the transformants. Expression analysis

Research paper thumbnail of Cloning and characterization of Aspergillus niger genes encoding an ������-galactosidase and a ������-mannosidase involved in galactomannan degradation: ������Galactosidase and ������-mannosidase from A.���������niger

European Journal of Biochemistry - EUR J BIOCHEM, 2001

Alpha-galactosidase (EC 3.2.1.22) and beta-mannosidase (EC 3.2.1.25) participate in the hydrolysi... more Alpha-galactosidase (EC 3.2.1.22) and beta-mannosidase (EC 3.2.1.25) participate in the hydrolysis of complex plant saccharides such as galacto(gluco)mannans. Here we report on the cloning and characterization of genes encoding an alpha-galactosidase (AglC) and a beta-mannosidase (MndA) from Aspergillus niger. The aglC and mndA genes code for 747 and 931 amino acids, respectively, including the eukaryotic signal sequences. The predicted isoelectric points of AglC and MndA are 4.56 and 5.17, and the calculated molecular masses are 79.674 and 102.335 kDa, respectively. Both AglC and MndA contain several putative N-glycosylation sites. AglC was assigned to family 36 of the glycosyl hydrolases and MndA was assigned to family 2. The expression patterns of aglC and mndA and two other genes encoding A. niger alpha-galactosidases (aglA and aglB) during cultivation on galactomannan were studied by Northern analysis. A comparison of gene expression on monosaccharides in the A. niger wild-type and a CreA mutant strain showed that the carbon catabolite repressor protein CreA has a strong influence on aglA, but not on aglB, aglC or mndA. AglC and MndA were purified from constructed overexpression strains of A. niger, and the combined action of these enzymes degraded a galactomanno-oligosaccharide into galactose and mannose. The possible roles of AglC and MndA in galactomannan hydrolysis is discussed.

Research paper thumbnail of Vaccination with a soluble recombinant hemagglutinin trimer protects pigs against a challenge with pandemic (H1N1) 2009 influenza virus

Research paper thumbnail of Secondary metabolite profiling, growth profiles and other tools for species recognition and important Aspergillus mycotoxins

Studies in Mycology, 2007

Species in the genus Aspergillus have been classified primarily based on morphological features. ... more Species in the genus Aspergillus have been classified primarily based on morphological features. Sequencing of house-hold genes has also been used in Aspergillus taxonomy and phylogeny, while extrolites and physiological features have been used less frequently. Three independent ways of classifying and identifying aspergilli appear to be applicable: Morphology combined with physiology and nutritional features, secondary metabolite profiling and DNA sequencing. These three ways of identifying Aspergillus species often point to the same species. This consensus approach can be used initially, but if consensus is achieved it is recommended to combine at least two of these independent ways of characterising aspergilli in a polyphasic taxonomy. The chemical combination of secondary metabolites and DNA sequence features has not been explored in taxonomy yet, however. Examples of these different taxonomic approaches will be given for Aspergillus section Nigri.

Research paper thumbnail of CreA modulates the XlnR-induced expression on xylose of Aspergillus niger genes involved in xylan degradation

Research in Microbiology, 1999

The expression of the feruloyl esterase gene faeA, the alpha-glucuronidase gene aguA, the endoxyl... more The expression of the feruloyl esterase gene faeA, the alpha-glucuronidase gene aguA, the endoxylanase gene xlnB, and the beta-xylosidase gene xlnD from Aspergillus niger on xylose was studied in a wild-type strain and in a CreA mutant. A decrease in expression of all four genes was observed with increasing xylose concentrations in the wild-type strain, whereas expression levels in the CreA mutant were not influenced. The results in the wild type indicated that xylose concentrations higher than 1 mM resulted in repression of the expression of the xylanolytic genes tested mediated by the carbon catabolite repressor protein CreA. On xylose, the expression levels of the xylanolytic genes were therefore not only determined by induction via XlnR, but also by repression via CreA. The genes tested were not influenced to the same extent by XlnR or CreA, resulting in specific expression levels and patterns for each individual gene.

Research paper thumbnail of Finished Genome of the Fungal Wheat Pathogen Mycosphaerella graminicola Reveals Dispensome Structure, Chromosome Plasticity, and Stealth Pathogenesis

PLoS Genetics, 2011

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes s... more The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed ''mesosynteny'' is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors. (GHJK); IVGrigoriev@lbl.gov (IVG) . These authors contributed equally to this work.

Research paper thumbnail of Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

Nature Biotechnology, 2007

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the... more The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.

Research paper thumbnail of Genome sequence of the model mushroom Schizophyllum commune

Nature Biotechnology, 2010

Much remains to be learned about the biology of mushroom-forming fungi, which are an important so... more Much remains to be learned about the biology of mushroom-forming fungi, which are an important source of food, secondary metabolites and industrial enzymes. The wood-degrading fungus Schizophyllum commune is both a genetically tractable model for studying mushroom development and a likely source of enzymes capable of efficient degradation of lignocellulosic biomass. Comparative analyses of its 38.5-megabase genome, which encodes 13,210 predicted genes, reveal the species's unique wood-degrading machinery. One-third of the 471 genes predicted to encode transcription factors are differentially expressed during sexual development of S. commune. Whereas inactivation of one of these, fst4, prevented mushroom formation, inactivation of another, fst3, resulted in more, albeit smaller, mushrooms than in the wild-type fungus. Antisense transcripts may also have a role in the formation of fruiting bodies. Better insight into the mechanisms underlying mushroom formation should affect commercial production of mushrooms and their industrial use for producing enzymes and pharmaceuticals.

Research paper thumbnail of Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis

Research paper thumbnail of Isolation and characterization of two specific regulatory Aspergillus niger mutants shows antagonistic regulation of arabinan and xylan metabolism

Microbiology, 2003

This paper describes two Aspergillus niger mutants (araA and araB) specifically disturbed in the ... more This paper describes two Aspergillus niger mutants (araA and araB) specifically disturbed in the regulation of the arabinanase system in response to the presence of L-arabinose. Expression of the three known L-arabinose-induced arabinanolytic genes, abfA, abfB and abnA, was substantially decreased or absent in the araA and araB strains compared to the wild-type when incubated in the presence of L-arabinose or L-arabitol. In addition, the intracellular activities of L-arabitol dehydrogenase and L-arabinose reductase, involved in L-arabinose catabolism, were decreased in the araA and araB strains. Finally, the data show that the gene encoding D-xylulose kinase, xkiA, is also under control of the arabinanolytic regulatory system. L-Arabitol, most likely the true inducer of the arabinanolytic and L-arabinose catabolic genes, accumulated to a high intracellular concentration in the araA and araB mutants. This indicates that the decrease of expression of the arabinanolytic genes was not due to lack of inducer accumulation. Therefore, it is proposed that the araA and araB mutations are localized in positive-acting components of the regulatory system involved in the expression of the arabinanase-encoding genes and the genes encoding the L-arabinose catabolic pathway.

Research paper thumbnail of Technical advance in fungal biotechnology: development of a miniaturized culture method and an automated high-throughput screening

Letters in Applied Microbiology, 2009

Aims: The goal of the study was to develop a reliable, reproducible and rapid method of culture i... more Aims: The goal of the study was to develop a reliable, reproducible and rapid method of culture in order to screen a large number of fungal transformants. Methods and Results: The method is based upon miniaturized cell cultures and automated expression screening in microwell plates. For the method development, 50 recombinant Aspergillus vadensis clones producing feruloyl esterase B (FaeB) from Aspergillus niger were screened in 6 days. Then a panel of clones showing various behaviours was checked in flasks in order to demonstrate the reproducibility of the method. Using this method, a transformant of A. vadensis producing 1AE2 g l )1 of FaeB was selected (12-fold more than the A. niger overproducing strain). Conclusions: This miniaturized culture method allows to obtain reliable and reproducible results. The procedure has the advantages of being efficient, timesaving and more efficient than conventional in-flask culture screening as it can screen 800 clones per day after a culture of 3 days. Significance and Impact of the Study: This method could be applied to any other fungal strain culture, enzyme activity or biodiversity screening.

Research paper thumbnail of Recombinant Soluble, Multimeric HA and NA Exhibit Distinctive Types of Protection against Pandemic Swine-Origin 2009 A(H1N1) Influenza Virus Infection in Ferrets

Journal of Virology, 2010

once again emphasizes the strong need for effective vaccines that can be developed rapidly and ap... more once again emphasizes the strong need for effective vaccines that can be developed rapidly and applied safely. With this aim, we produced soluble, multimeric forms of the 2009 A(H1N1) HA (sHA 3 ) and NA (sNA 4 ) surface glycoproteins using a virus-free mammalian expression system and evaluated their efficacy as vaccines in ferrets. Immunization twice with 3.75-g doses of these antigens elicited strong antibody responses, which were adjuvant dependent. Interestingly, coadministration of both antigens strongly enhanced the HA-specific but not the NA-specific responses. Distinct patterns of protection were observed upon challenge inoculation with the homologous H1N1 virus. Whereas vaccination with sHA 3 dramatically reduced virus replication (e.g., by lowering pulmonary titers by about 5 log 10 units), immunization with sNA 4 markedly decreased the clinical effects of infection, such as body weight loss and lung pathology. Clearly, optimal protection was achieved by the combination of the two antigens. Our observations demonstrate the great vaccine potential of multimeric HA and NA ectodomains, as these can be easily, rapidly, flexibly, and safely produced in high quantities. In particular, our study underscores the underrated importance of NA in influenza vaccination, which we found to profoundly and specifically contribute to protection by HA. Its inclusion in a vaccine is likely to reduce the HA dose required and to broaden the protective immunity.

Research paper thumbnail of The genome sequence of the model ascomycete fungus Podospora anserina

Genome Biology, 2008

The dung-inhabiting ascomycete fungus Podospora anserina is a model used to study various aspects... more The dung-inhabiting ascomycete fungus Podospora anserina is a model used to study various aspects of eukaryotic and fungal biology, such as ageing, prions and sexual development.

Research paper thumbnail of The effect of hematocrit on bioanalysis of DBS: results from the EBF DBS-microsampling consortium

Bioanalysis, 2013

The European Bioanalysis Forum dried blood spots (DBS)/microsampling consortium is reporting back... more The European Bioanalysis Forum dried blood spots (DBS)/microsampling consortium is reporting back from the experiments they performed on further documenting the potential hurdles of the DBS technology. This paper is focused on the impact of hematocrit changes on DBS analyses. The hematocrit can have an effect on the size of the blood spot, on spot homogeneity and on extraction recovery in a compound-dependent manner. The extraction recovery can change upon aging in an hematocrit-dependent way. Different card materials can give different outcomes. The results from the conducted experiments show that the issues of DBS in regulated bioanalysis are real and that the technology will need improvements to be ready for use as a general tool for regulated bioanalysis.

Research paper thumbnail of The Aspergillus nigerd-xylulose kinase gene is co-expressed with genes encoding arabinan degrading enzymes, and is essential for growth on d-xylose and l-arabinose: Aspergillus niger xylulose kinase

European Journal of Biochemistry, 2001

Research paper thumbnail of aguA, the Gene Encoding an Extracellular a-Glucuronidase from Aspergillus tubingensis, Is Specifically Induced on Xylose and Not on Glucuronic Acid

An extracellular a-glucuronidase was purified and characterized from a commercial Aspergillus pre... more An extracellular a-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrom- etry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged

Research paper thumbnail of Update of the EBF recommendation for the use of DBS in regulated bioanalysis integrating the conclusions from the EBF DBS-microsampling consortium

Bioanalysis, 2013

The European Bioanalysis Forum dried blood spots/microsampling consortium is reporting back from ... more The European Bioanalysis Forum dried blood spots/microsampling consortium is reporting back from the experiments they performed on further documenting the potential hurdles of the DBS technology. Their experiments focused on the impact of hematocrit changes, IS addition, spot homogeneity, aging of spots and stability of fresh blood and cards. Results from these experiments demonstrate that the issues of DBS in regulated bioanalysis are real and that the technology will need additional improvements to be ready for use as a general tool for regulated bioanalysis. In addition, results on fresh blood and card stability were shared at international meetings and will be reported at a later date.

Research paper thumbnail of In-depth study of homogeneity in DBS using two different techniques: results from the EBF DBS-microsampling consortium

Bioanalysis, 2013

At the start of their work, the European Bioanalysis Forum dried blood spots microsampling consor... more At the start of their work, the European Bioanalysis Forum dried blood spots microsampling consortium did not form a dedicated team to investigate the spot homogeneity. However, two teams performed experiments that produced results relating to sample homogeneity. The data, which were produced via two different approaches (a radiolabeled and a nonradiolabeled approach), are highly complementary and demonstrate clear effects on sample inhomogeneity due to the substrate type, compound and hematocrit levels. The results demonstrate that sample inhomogeneity is a significant hurdle to the use of dried blood spots for regulated bioanalysis that should be investigated further in the method establishment phase if the whole spot is not sampled.

Research paper thumbnail of Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire

Genome Biology, 2010

Pythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on ... more Pythium ultimum is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species. The P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions, although, surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host-specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome, including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report of these in a genome outside the metazoans. Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage-specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae.

Research paper thumbnail of Genomic Analysis of the Necrotrophic Fungal Pathogens Sclerotinia sclerotiorum and Botrytis cinerea

PLoS Genetics, 2011

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic f... more Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to ,1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.

Research paper thumbnail of The α-glucuronidase Agu1 from Schizophyllum commune is a member of a novel glycoside hydrolase family (GH115)

Applied Microbiology and Biotechnology, 2011

Schizophyllum commune produces an α-glucuronidase that is active on polymeric xylan, while the as... more Schizophyllum commune produces an α-glucuronidase that is active on polymeric xylan, while the ascomycete α-glucuronidases are only active on xylan oligomers. In this study, we have identified the gene (agu1) encoding this enzyme and confirmed the functionality by overexpression of the gene in S. commune and degradation of aldopentauronic acids, (MeGlcA)3-Xyl4, in the cultivation medium of the transformants. Expression analysis

Research paper thumbnail of Cloning and characterization of Aspergillus niger genes encoding an ������-galactosidase and a ������-mannosidase involved in galactomannan degradation: ������Galactosidase and ������-mannosidase from A.���������niger

European Journal of Biochemistry - EUR J BIOCHEM, 2001

Alpha-galactosidase (EC 3.2.1.22) and beta-mannosidase (EC 3.2.1.25) participate in the hydrolysi... more Alpha-galactosidase (EC 3.2.1.22) and beta-mannosidase (EC 3.2.1.25) participate in the hydrolysis of complex plant saccharides such as galacto(gluco)mannans. Here we report on the cloning and characterization of genes encoding an alpha-galactosidase (AglC) and a beta-mannosidase (MndA) from Aspergillus niger. The aglC and mndA genes code for 747 and 931 amino acids, respectively, including the eukaryotic signal sequences. The predicted isoelectric points of AglC and MndA are 4.56 and 5.17, and the calculated molecular masses are 79.674 and 102.335 kDa, respectively. Both AglC and MndA contain several putative N-glycosylation sites. AglC was assigned to family 36 of the glycosyl hydrolases and MndA was assigned to family 2. The expression patterns of aglC and mndA and two other genes encoding A. niger alpha-galactosidases (aglA and aglB) during cultivation on galactomannan were studied by Northern analysis. A comparison of gene expression on monosaccharides in the A. niger wild-type and a CreA mutant strain showed that the carbon catabolite repressor protein CreA has a strong influence on aglA, but not on aglB, aglC or mndA. AglC and MndA were purified from constructed overexpression strains of A. niger, and the combined action of these enzymes degraded a galactomanno-oligosaccharide into galactose and mannose. The possible roles of AglC and MndA in galactomannan hydrolysis is discussed.

Research paper thumbnail of Vaccination with a soluble recombinant hemagglutinin trimer protects pigs against a challenge with pandemic (H1N1) 2009 influenza virus

Research paper thumbnail of Secondary metabolite profiling, growth profiles and other tools for species recognition and important Aspergillus mycotoxins

Studies in Mycology, 2007

Species in the genus Aspergillus have been classified primarily based on morphological features. ... more Species in the genus Aspergillus have been classified primarily based on morphological features. Sequencing of house-hold genes has also been used in Aspergillus taxonomy and phylogeny, while extrolites and physiological features have been used less frequently. Three independent ways of classifying and identifying aspergilli appear to be applicable: Morphology combined with physiology and nutritional features, secondary metabolite profiling and DNA sequencing. These three ways of identifying Aspergillus species often point to the same species. This consensus approach can be used initially, but if consensus is achieved it is recommended to combine at least two of these independent ways of characterising aspergilli in a polyphasic taxonomy. The chemical combination of secondary metabolites and DNA sequence features has not been explored in taxonomy yet, however. Examples of these different taxonomic approaches will be given for Aspergillus section Nigri.

Research paper thumbnail of CreA modulates the XlnR-induced expression on xylose of Aspergillus niger genes involved in xylan degradation

Research in Microbiology, 1999

The expression of the feruloyl esterase gene faeA, the alpha-glucuronidase gene aguA, the endoxyl... more The expression of the feruloyl esterase gene faeA, the alpha-glucuronidase gene aguA, the endoxylanase gene xlnB, and the beta-xylosidase gene xlnD from Aspergillus niger on xylose was studied in a wild-type strain and in a CreA mutant. A decrease in expression of all four genes was observed with increasing xylose concentrations in the wild-type strain, whereas expression levels in the CreA mutant were not influenced. The results in the wild type indicated that xylose concentrations higher than 1 mM resulted in repression of the expression of the xylanolytic genes tested mediated by the carbon catabolite repressor protein CreA. On xylose, the expression levels of the xylanolytic genes were therefore not only determined by induction via XlnR, but also by repression via CreA. The genes tested were not influenced to the same extent by XlnR or CreA, resulting in specific expression levels and patterns for each individual gene.

Research paper thumbnail of Finished Genome of the Fungal Wheat Pathogen Mycosphaerella graminicola Reveals Dispensome Structure, Chromosome Plasticity, and Stealth Pathogenesis

PLoS Genetics, 2011

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes s... more The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed ''mesosynteny'' is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors. (GHJK); IVGrigoriev@lbl.gov (IVG) . These authors contributed equally to this work.

Research paper thumbnail of Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

Nature Biotechnology, 2007

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the... more The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.

Research paper thumbnail of Genome sequence of the model mushroom Schizophyllum commune

Nature Biotechnology, 2010

Much remains to be learned about the biology of mushroom-forming fungi, which are an important so... more Much remains to be learned about the biology of mushroom-forming fungi, which are an important source of food, secondary metabolites and industrial enzymes. The wood-degrading fungus Schizophyllum commune is both a genetically tractable model for studying mushroom development and a likely source of enzymes capable of efficient degradation of lignocellulosic biomass. Comparative analyses of its 38.5-megabase genome, which encodes 13,210 predicted genes, reveal the species's unique wood-degrading machinery. One-third of the 471 genes predicted to encode transcription factors are differentially expressed during sexual development of S. commune. Whereas inactivation of one of these, fst4, prevented mushroom formation, inactivation of another, fst3, resulted in more, albeit smaller, mushrooms than in the wild-type fungus. Antisense transcripts may also have a role in the formation of fruiting bodies. Better insight into the mechanisms underlying mushroom formation should affect commercial production of mushrooms and their industrial use for producing enzymes and pharmaceuticals.

Research paper thumbnail of Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis

Research paper thumbnail of Isolation and characterization of two specific regulatory Aspergillus niger mutants shows antagonistic regulation of arabinan and xylan metabolism

Microbiology, 2003

This paper describes two Aspergillus niger mutants (araA and araB) specifically disturbed in the ... more This paper describes two Aspergillus niger mutants (araA and araB) specifically disturbed in the regulation of the arabinanase system in response to the presence of L-arabinose. Expression of the three known L-arabinose-induced arabinanolytic genes, abfA, abfB and abnA, was substantially decreased or absent in the araA and araB strains compared to the wild-type when incubated in the presence of L-arabinose or L-arabitol. In addition, the intracellular activities of L-arabitol dehydrogenase and L-arabinose reductase, involved in L-arabinose catabolism, were decreased in the araA and araB strains. Finally, the data show that the gene encoding D-xylulose kinase, xkiA, is also under control of the arabinanolytic regulatory system. L-Arabitol, most likely the true inducer of the arabinanolytic and L-arabinose catabolic genes, accumulated to a high intracellular concentration in the araA and araB mutants. This indicates that the decrease of expression of the arabinanolytic genes was not due to lack of inducer accumulation. Therefore, it is proposed that the araA and araB mutations are localized in positive-acting components of the regulatory system involved in the expression of the arabinanase-encoding genes and the genes encoding the L-arabinose catabolic pathway.

Research paper thumbnail of Technical advance in fungal biotechnology: development of a miniaturized culture method and an automated high-throughput screening

Letters in Applied Microbiology, 2009

Aims: The goal of the study was to develop a reliable, reproducible and rapid method of culture i... more Aims: The goal of the study was to develop a reliable, reproducible and rapid method of culture in order to screen a large number of fungal transformants. Methods and Results: The method is based upon miniaturized cell cultures and automated expression screening in microwell plates. For the method development, 50 recombinant Aspergillus vadensis clones producing feruloyl esterase B (FaeB) from Aspergillus niger were screened in 6 days. Then a panel of clones showing various behaviours was checked in flasks in order to demonstrate the reproducibility of the method. Using this method, a transformant of A. vadensis producing 1AE2 g l )1 of FaeB was selected (12-fold more than the A. niger overproducing strain). Conclusions: This miniaturized culture method allows to obtain reliable and reproducible results. The procedure has the advantages of being efficient, timesaving and more efficient than conventional in-flask culture screening as it can screen 800 clones per day after a culture of 3 days. Significance and Impact of the Study: This method could be applied to any other fungal strain culture, enzyme activity or biodiversity screening.

Research paper thumbnail of Recombinant Soluble, Multimeric HA and NA Exhibit Distinctive Types of Protection against Pandemic Swine-Origin 2009 A(H1N1) Influenza Virus Infection in Ferrets

Journal of Virology, 2010

once again emphasizes the strong need for effective vaccines that can be developed rapidly and ap... more once again emphasizes the strong need for effective vaccines that can be developed rapidly and applied safely. With this aim, we produced soluble, multimeric forms of the 2009 A(H1N1) HA (sHA 3 ) and NA (sNA 4 ) surface glycoproteins using a virus-free mammalian expression system and evaluated their efficacy as vaccines in ferrets. Immunization twice with 3.75-g doses of these antigens elicited strong antibody responses, which were adjuvant dependent. Interestingly, coadministration of both antigens strongly enhanced the HA-specific but not the NA-specific responses. Distinct patterns of protection were observed upon challenge inoculation with the homologous H1N1 virus. Whereas vaccination with sHA 3 dramatically reduced virus replication (e.g., by lowering pulmonary titers by about 5 log 10 units), immunization with sNA 4 markedly decreased the clinical effects of infection, such as body weight loss and lung pathology. Clearly, optimal protection was achieved by the combination of the two antigens. Our observations demonstrate the great vaccine potential of multimeric HA and NA ectodomains, as these can be easily, rapidly, flexibly, and safely produced in high quantities. In particular, our study underscores the underrated importance of NA in influenza vaccination, which we found to profoundly and specifically contribute to protection by HA. Its inclusion in a vaccine is likely to reduce the HA dose required and to broaden the protective immunity.