Publications by authors named "Theo A J van der Lee"

32 Publications

No to : Phylogenomic and Practical Reasons for Continued Inclusion of the Fusarium solani Species Complex in the Genus .

mSphere 2020 09 16;5(5). Epub 2020 Sep 16.

Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

This article is to alert medical mycologists and infectious disease specialists of recent name changes of medically important species of the filamentous mold species can cause localized and life-threating infections in humans. Of the 70 species that have been reported to cause infections, close to one-third are members of the species complex (FSSC), and they collectively account for approximately two-thirds of all reported infections. Many of these species were recently given scientific names for the first time by a research group in the Netherlands, but they were misplaced in the genus In this paper, we present genetic arguments that strongly support inclusion of the FSSC in There are potentially serious consequences associated with using the name for species because clinicians need to be aware that fusaria are broadly resistant to the spectrum of antifungals that are currently available.
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http://dx.doi.org/10.1128/mSphere.00810-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494836PMC
September 2020

Detecting Introgression Between Members of the and Species Complexes by Comparative Mitogenomics.

Front Microbiol 2020 3;11:1092. Epub 2020 Jun 3.

B.U. Biointeractions and Plant Health, Wageningen Plant Research, Wageningen University & Research, Wageningen, Netherlands.

The species complex (FFSC) and species complex (FOSC) are two related groups of plant pathogens causing a wide diversity of diseases in agricultural crops world wide. The aims of this study are (1) to clarify the phylogeny of the FFSC, (2) to identify potential deviation from tree-like evolution, (3) to explore the value of using mitogenomes for these kinds of analyses, and (4) to better understand mitogenome evolution. In total, we have sequenced 24 species from the FFSC and a representative set of recently analyzed FOSC strains was chosen, while was used as outgroup for the two species complexes. A species tree was constructed based on the concatenated alignment of seven nuclear genes and the mitogenome, which was contrasted to individual gene trees to identify potential conflicts. These comparisons indicated conflicts especially within the previously described African clade of the FFSC. Furthermore, the analysis of the mitogenomes revealed the presence of a variant of the large variable (LV) region in FFSC which was previously only reported for FOSC. The distribution of this variant and the results of sequence comparisons indicate horizontal genetic transfer between members of the two species complexes, most probably through introgression. In addition, a duplication of was found inside an intron of , which suggests that even highly conserved mitochondrial genes can have paralogs. Paralogization in turn may lead to inaccurate single gene phylogenies. In conclusion, mitochondrial genomes provide a robust basis for phylogeny. Comparative phylogenetic analysis indicated that gene flow among and between members of FFSC and FOSC has played an important role in the evolutionary history of these two groups. Since mitogenomes show greater levels of conservation and synteny than nuclear regions, they are more likely to be compatible for recombination than nuclear regions. Therefore, mitogenomes can be used as indicators to detect interspecies gene flow.
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http://dx.doi.org/10.3389/fmicb.2020.01092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7285627PMC
June 2020

Population Genomic Analysis Reveals a Highly Conserved Mitochondrial Genome in .

Front Microbiol 2020 5;11:839. Epub 2020 May 5.

State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agriculture Sciences, Beijing, China.

is one of the pivotal members of the species complex (FGSC) causing Fusarium head blight (FHB) on wheat, barley and rice in large parts of Asia. Besides resulting in yield losses, FHB also causes the accumulation of mycotoxins such as nivalenol (NIV) and deoxynivalenol (DON). The aim of this study was to conduct population studies on from Southern China through mitochondrial genome analyses. All strains were isolated from wheat or rice from several geographic areas in seven provinces in Southern China. Based on geographic location and host, 210 isolates were selected for next generation sequencing, and their mitogenomes were assembled by GRAbB and annotated to explore the mitochondrial genome variability of . The mitogenome proves extremely conserved and variation is mainly caused by absence/presence of introns harboring homing endonuclease genes. These variations could be utilized to develop molecular markers for track and trace of migrations within and between populations. This study illustrates how mitochondrial introns can be used as markers for population genetic analysis. SNP analysis demonstrate the occurrence of mitochondrial recombination in as was previously found for and implied for . Furthermore, varying degrees of genetic diversity and recombination showed a high association with different geographic regions as well as with cropping systems. The mitogenome of showed a much higher SNP diversity while the interspecies intron variation showed no evidence of gene flow between the two closely related and sexual compatible species.
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http://dx.doi.org/10.3389/fmicb.2020.00839DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7214670PMC
May 2020

The AvrSen1 Triggers a Hypersensitive Response in Potatoes While Natural Variants Evade Detection.

Mol Plant Microbe Interact 2019 Nov 17;32(11):1536-1546. Epub 2019 Sep 17.

Wageningen University and Research, Plant Breeding.

is an obligate biotrophic fungus of division Chytridiomycota. It causes potato wart disease, has a worldwide quarantine status and is included on the Health and Human Services and United States Department of Agriculture Select Agent list. isolates are grouped in pathotypes based on their ability to evade host resistance in a set of differential potato varieties. Thus far, 39 pathotypes are reported. A single dominant gene () governs pathotype 1 (D1) resistance and we anticipated that the underlying molecular model would involve a pathogen effector (AvrSen1) that is recognized by the host. The -specific secretome of 14 isolates representing six different pathotypes was screened for effectors specifically present in pathotype 1 (D1) isolates but absent in others. We identified a single candidate. Expression of this candidate in potato plants showed a specific hypersensitive response (HR), which cosegregated with the resistance in potato populations. No HR was obtained with truncated genes found in pathotypes that evaded recognition by . These findings established that our candidate gene was indeed The is a single-copy gene and encodes a 376-amino-acid protein without predicted function or functional domains, and is the first effector gene identified in Chytridiomycota, an extremely diverse yet underrepresented basal lineage of fungi.
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http://dx.doi.org/10.1094/MPMI-05-19-0138-RDOI Listing
November 2019

Comparative genomics of chytrid fungi reveal insights into the obligate biotrophic and pathogenic lifestyle of Synchytrium endobioticum.

Sci Rep 2019 06 17;9(1):8672. Epub 2019 Jun 17.

Wageningen University & Research, Droevendaalsesteeg 1, Plant Science Group, 6708PB, Wageningen, The Netherlands.

Synchytrium endobioticum is an obligate biotrophic soilborne Chytridiomycota (chytrid) species that causes potato wart disease, and represents the most basal lineage among the fungal plant pathogens. We have chosen a functional genomics approach exploiting knowledge acquired from other fungal taxa and compared this to several saprobic and pathogenic chytrid species. Observations linked to obligate biotrophy, genome plasticity and pathogenicity are reported. Essential purine pathway genes were found uniquely absent in S. endobioticum, suggesting that it relies on scavenging guanine from its host for survival. The small gene-dense and intron-rich chytrid genomes were not protected for genome duplications by repeat-induced point mutation. Both pathogenic chytrids Batrachochytrium dendrobatidis and S. endobioticum contained the largest amounts of repeats, and we identified S. endobioticum specific candidate effectors that are associated with repeat-rich regions. These candidate effectors share a highly conserved motif, and show isolate specific duplications. A reduced set of cell wall degrading enzymes, and LysM protein expansions were found in S. endobioticum, which may prevent triggering plant defense responses. Our study underlines the high diversity in chytrids compared to the well-studied Ascomycota and Basidiomycota, reflects characteristic biological differences between the phyla, and shows commonalities in genomic features among pathogenic fungi.
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http://dx.doi.org/10.1038/s41598-019-45128-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6572847PMC
June 2019

Development and Evaluation of a Triplex TaqMan Assay and Next-Generation Sequence Analysis for Improved Detection of Xylella in Plant Material.

Plant Dis 2019 Apr 18;103(4):645-655. Epub 2019 Feb 18.

Wageningen University and Research, Business Unit Biointeractions and Plant Health, 6700 AA Wageningen, The Netherlands.

Xylella fastidiosa is a heterogenous gram-negative bacterial plant pathogen with a wide host range covering over 300 plant species. Since 2013, in Europe, the presence of the pathogen is increasing in a part of the Mediterranean area, but it causes in particular severe disease problems in olive orchards in the Southern part of Italy. Various subspecies of the pathogen were also diagnosed in natural outbreaks and intercepted ornamental plants in Europe, among them Olea europaea, Coffea arabica, and Nerium oleander. The host range of the pathogen can vary, depending on the subspecies and even the strain. The availability of fast and reliable diagnostic tools is indispensable in management strategies to control diseases caused by X. fastidiosa. To improve the reliability of the TaqMan assay, currently widely used in surveys, a triplex TaqMan assay was developed in which two specific and sensitive TaqMan assays, previously designed for X. fastidiosa, were combined with an internal control. The triplex assay exhibited the same diagnostic sensitivity as the simplex assays. In addition, the usefulness of a metagenomic approach using next-generation sequencing (NGS) was demonstrated, in which total DNA extracted from plant material was sequenced. DNA extracts from plant material free of X. fastidiosa, from artificially inoculated hosts plants or from naturally infected plants sampled in France, Spain, and Italy, or intercepted in Austria and the Netherlands, were analyzed for the presence of X. fastidiosa using the metagenomic approach. In all samples, even in samples with a low infection level, but not in the pathogen-free samples, DNA reads were detected specific for X. fastidiosa. In most cases, the pathogen could be identified to the subspecies level, and for one sample even the whole genome could be assembled and the sequence type could be determined. All results of NGS-analyzed samples were confirmed with the triplex TaqMan polymerase chain reaction and loop-mediated isothermal amplification.
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http://dx.doi.org/10.1094/PDIS-08-18-1433-REDOI Listing
April 2019

The linear mitochondrial genome of the quarantine chytrid Synchytrium endobioticum; insights into the evolution and recent history of an obligate biotrophic plant pathogen.

BMC Evol Biol 2018 09 10;18(1):136. Epub 2018 Sep 10.

Wageningen UR, Droevendaalsesteeg 1, Biointeractions and Plant Health & Plant Breeding, 6708, PB, Wageningen, The Netherlands.

Background: Chytridiomycota species (chytrids) belong to a basal lineage in the fungal kingdom. Inhabiting terrestrial and aquatic environments, most are free-living saprophytes but several species cause important diseases: e.g. Batrachochytrium dendrobatidis, responsible for worldwide amphibian decline; and Synchytrium endobioticum, causing potato wart disease. S. endobioticum has an obligate biotrophic lifestyle and isolates can be further characterized as pathotypes based on their virulence on a differential set of potato cultivars. Quarantine measures have been implemented globally to control the disease and prevent its spread. We used a comparative approach using chytrid mitogenomes to determine taxonomical relationships and to gain insights into the evolution and recent history of introductions of this plant pathogen.

Results: We assembled and annotated the complete mitochondrial genome of 30 S. endobioticum isolates and generated mitochondrial genomes for five additional chytrid species. The mitochondrial genome of S. endobioticum is linear with terminal inverted repeats which was validated by tailing and PCR amplifying the telomeric ends. Surprisingly, no conservation in organisation and orientation of mitochondrial genes was observed among the Chytridiomycota except for S. endobioticum and its sister species Synchytrium microbalum. However, the mitochondrial genome of S. microbalum is circular and comprises only a third of the 72.9 Kbp found for S. endobioticum suggesting recent linearization and expansion. Four mitochondrial lineages were identified in the S. endobioticum mitochondrial genomes. Several pathotypes occur in different lineages, suggesting that these have emerged independently. In addition, variations for polymorphic sites in the mitochondrial genome of individual isolates were observed demonstrating that S. endobioticum isolates represent a community of different genotypes. Such communities were shown to be complex and stable over time, but we also demonstrate that the use of semi-resistant potato cultivars triggers a rapid shift in the mitochondrial haplotype associated with increased virulence.

Conclusions: Mitochondrial genomic variation shows that S. endobioticum has been introduced into Europe multiple times, that several pathotypes emerged multiple times, and that isolates represent communities of different genotypes. Our study represents the most comprehensive dataset of chytrid mitogenomes, which provides new insights into the extraordinary dynamics and evolution of mitochondrial genomes involving linearization, expansion and reshuffling.
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http://dx.doi.org/10.1186/s12862-018-1246-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6131824PMC
September 2018

Evolution and Diversity of Biosynthetic Gene Clusters in .

Front Microbiol 2018 5;9:1158. Epub 2018 Jun 5.

Bioinformatics Group, Wageningen University, Wageningen, Netherlands.

Plant pathogenic fungi in the genus cause severe damage to crops, resulting in great financial losses and health hazards. Specialized metabolites synthesized by these fungi are known to play key roles in the infection process, and to provide survival advantages inside and outside the host. However, systematic studies of the evolution of specialized metabolite-coding potential across have been scarce. Here, we apply a combination of bioinformatic approaches to identify biosynthetic gene clusters (BGCs) across publicly available genomes from , to group them into annotated families and to study gain/loss events of BGC families throughout the history of the genus. Comparison with MIBiG reference BGCs allowed assignment of 29 gene cluster families (GCFs) to pathways responsible for the production of known compounds, while for 57 GCFs, the molecular products remain unknown. Comparative analysis of BGC repertoires using ancestral state reconstruction raised several new hypotheses on how BGCs contribute to pathogenicity or host specificity, sometimes surprisingly so: for example, a gene cluster for the biosynthesis of hexadehydro-astechrome was identified in the genome of the biocontrol strain Fo47, while being absent in that of the tomato pathogen f.sp. . Several BGCs were also identified on supernumerary chromosomes; heterologous expression of genes for three terpene synthases encoded on the supernumerary chromosome and subsequent GC/MS analysis showed that these genes are functional and encode enzymes that each are able to synthesize koraiol; this observed functional redundancy supports the hypothesis that localization of copies of BGCs on supernumerary chromosomes provides freedom for evolutionary innovations to occur, while the original function remains conserved. Altogether, this systematic overview of biosynthetic diversity in paves the way for targeted natural product discovery based on automated identification of species-specific pathways as well as for connecting species ecology to the taxonomic distributions of BGCs.
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http://dx.doi.org/10.3389/fmicb.2018.01158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5996196PMC
June 2018

The Histone Acetyltransferases Are Important for Morphogenesis, DON Biosynthesis, and Pathogenicity.

Front Microbiol 2018 26;9:654. Epub 2018 Apr 26.

State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.

Post-translational modifications of chromatin structure by histone acetyltransferase (HATs) play a central role in the regulation of gene expression and various biological processes in eukaryotes. Although HAT genes have been studied in many fungi, few of them have been functionally characterized. In this study, we identified and characterized four putative HATs () in the plant pathogenic ascomycete , the causal agent of head blight of wheat and barley. We replaced the genes and all mutant strains showed reduced growth of . The Δ and Δ mutant increased sensitivity to oxidative and osmotic stresses. Additionally, Δ showed reduced conidia sporulation and perithecium formation. Mutant Δ was unable to generate any conidia and lost its ability to form perithecia. Our data showed also that and are pathogenicity factors required for infecting wheat heads as well as tomato fruits. Importantly, almost no Deoxynivalenol (DON) was produced either in Δ or Δ mutants, which was consistent with a significant downregulation of TRI genes expression. Furthermore, we discovered for the first time that is indispensable for the acetylation of histone site H3K4, while is essential for the acetylation of H3K9, H3K18, and H3K27. H3K14 can be completely acetylated when and were both present. The RNA-seq analyses of the two mutant strains provide insight into their functions in development and metabolism. Results from this study clarify the functional divergence of HATs in , and may provide novel targeted strategies to control secondary metabolite expression and infections of .
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http://dx.doi.org/10.3389/fmicb.2018.00654DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932188PMC
April 2018

Stress and sexual reproduction affect the dynamics of the wheat pathogen effector AvrStb6 and strobilurin resistance.

Nat Genet 2018 03 12;50(3):375-380. Epub 2018 Feb 12.

Laboratory of Phytopathology, Wageningen University and Research, Wageningen, the Netherlands.

Host resistance and fungicide treatments are cornerstones of plant-disease control. Here, we show that these treatments allow sex and modulate parenthood in the fungal wheat pathogen Zymoseptoria tritici. We demonstrate that the Z. tritici-wheat interaction complies with the gene-for-gene model by identifying the effector AvrStb6, which is recognized by the wheat resistance protein Stb6. Recognition triggers host resistance, thus implying removal of avirulent strains from pathogen populations. However, Z. tritici crosses on wheat show that sex occurs even with an avirulent parent, and avirulence alleles are thereby retained in subsequent populations. Crossing fungicide-sensitive and fungicide-resistant isolates under fungicide pressure results in a rapid increase in resistance-allele frequency. Isolates under selection always act as male donors, and thus disease control modulates parenthood. Modeling these observations for agricultural and natural environments reveals extended durability of host resistance and rapid emergence of fungicide resistance. Therefore, fungal sex has major implications for disease control.
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http://dx.doi.org/10.1038/s41588-018-0052-9DOI Listing
March 2018

Mitochondrial genomes reveal recombination in the presumed asexual Fusarium oxysporum species complex.

BMC Genomics 2017 Sep 18;18(1):735. Epub 2017 Sep 18.

Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, Utrecht, 3584CT, The Netherlands.

Background: The Fusarium oxysporum species complex (FOSC) contains several phylogenetic lineages. Phylogenetic studies identified two to three major clades within the FOSC. The mitochondrial sequences are highly informative phylogenetic markers, but have been mostly neglected due to technical difficulties.

Results: A total of 61 complete mitogenomes of FOSC strains were de novo assembled and annotated. Length variations and intron patterns support the separation of three phylogenetic species. The variable region of the mitogenome that is typical for the genus Fusarium shows two new variants in the FOSC. The variant typical for Fusarium is found in members of all three clades, while variant 2 is found in clades 2 and 3 and variant 3 only in clade 2. The extended set of loci analyzed using a new implementation of the genealogical concordance species recognition method support the identification of three phylogenetic species within the FOSC. Comparative analysis of the mitogenomes in the FOSC revealed ongoing mitochondrial recombination within, but not between phylogenetic species.

Conclusions: The recombination indicates the presence of a parasexual cycle in F. oxysporum. The obstacles hindering the usage of the mitogenomes are resolved by using next generation sequencing and selective genome assemblers, such as GRAbB. Complete mitogenome sequences offer a stable basis and reference point for phylogenetic and population genetic studies.
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http://dx.doi.org/10.1186/s12864-017-4116-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5604515PMC
September 2017

A single-nucleotide-polymorphism-based genotyping assay for simultaneous detection of different carbendazim-resistant genotypes in the species complex.

PeerJ 2016 25;4:e2609. Epub 2016 Oct 25.

Institute of Plant Protection, Chinese Academy of Agricultural Sciences , Beijing , China.

The occurrence resistance to methyl benzimidazole carbamates (MBC)-fungicides in the species complex (FGSC) is becoming a serious problem in the control of Fusarium head blight in China. The resistance is caused by point mutations in the gene. So far, five resistant genotypes (F167Y, E198Q, E198L, E198K and F200Y) have been reported in the field. To establish a high-throughput method for rapid detection of all the five mutations simultaneously, an efficient single-nucleotide-polymorphism-based genotyping method was developed based on the Luminex xMAP system. One pair of amplification primers and five allele specific primer extension probes were designed and optimized to specially distinguish the different genotypes within one single reaction. This method has good extensibility and can be combined with previous reported probes to form a highly integrated tool for species, trichothecene chemotype and MBC resistance detection. Using this method, carbendazim resistant FGSC isolates from Jiangsu, Anhui and Sichuan Province in China were identified. High and moderate frequencies of resistance were observed in Jiangsu and Anhui Province, respectively. Carbendazim resistance in is only observed in the 3ADON genotype. Overall, our method proved to be useful for early detection of MBC resistance in the field and the result aids in the choice of fungicide type.
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http://dx.doi.org/10.7717/peerj.2609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5088611PMC
October 2016

Combating a Global Threat to a Clonal Crop: Banana Black Sigatoka Pathogen Pseudocercospora fijiensis (Synonym Mycosphaerella fijiensis) Genomes Reveal Clues for Disease Control.

PLoS Genet 2016 08 11;12(8):e1005876. Epub 2016 Aug 11.

Plant Research International, Wageningen University and Research, Wageningen, The Netherlands.

Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana breeding programs.
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http://dx.doi.org/10.1371/journal.pgen.1005876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4981457PMC
August 2016

GRAbB: Selective Assembly of Genomic Regions, a New Niche for Genomic Research.

PLoS Comput Biol 2016 06 16;12(6):e1004753. Epub 2016 Jun 16.

CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands.

GRAbB (Genomic Region Assembly by Baiting) is a new program that is dedicated to assemble specific genomic regions from NGS data. This approach is especially useful when dealing with multi copy regions, such as mitochondrial genome and the rDNA repeat region, parts of the genome that are often neglected or poorly assembled, although they contain interesting information from phylogenetic or epidemiologic perspectives, but also single copy regions can be assembled. The program is capable of targeting multiple regions within a single run. Furthermore, GRAbB can be used to extract specific loci from NGS data, based on homology, like sequences that are used for barcoding. To make the assembly specific, a known part of the region, such as the sequence of a PCR amplicon or a homologous sequence from a related species must be specified. By assembling only the region of interest, the assembly process is computationally much less demanding and may lead to assemblies of better quality. In this study the different applications and functionalities of the program are demonstrated such as: exhaustive assembly (rDNA region and mitochondrial genome), extracting homologous regions or genes (IGS, RPB1, RPB2 and TEF1a), as well as extracting multiple regions within a single run. The program is also compared with MITObim, which is meant for the exhaustive assembly of a single target based on a similar query sequence. GRAbB is shown to be more efficient than MITObim in terms of speed, memory and disk usage. The other functionalities (handling multiple targets simultaneously and extracting homologous regions) of the new program are not matched by other programs. The program is available with explanatory documentation at https://github.com/b-brankovics/grabb. GRAbB has been tested on Ubuntu (12.04 and 14.04), Fedora (23), CentOS (7.1.1503) and Mac OS X (10.7). Furthermore, GRAbB is available as a docker repository: brankovics/grabb (https://hub.docker.com/r/brankovics/grabb/).
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http://dx.doi.org/10.1371/journal.pcbi.1004753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4911045PMC
June 2016

A gapless genome sequence of the fungus Botrytis cinerea.

Mol Plant Pathol 2017 01 9;18(1):75-89. Epub 2016 Jun 9.

Syngenta Crop Protection Münchwilen AG, Crop Protection Research, CH-4332, Stein, Switzerland.

Following earlier incomplete and fragmented versions of a genome sequence for the grey mould Botrytis cinerea, a gapless, near-finished genome sequence for B. cinerea strain B05.10 is reported. The assembly comprised 18 chromosomes and was confirmed by an optical map and a genetic map based on approximately 75 000 single nucleotide polymorphism (SNP) markers. All chromosomes contained fully assembled centromeric regions, and 10 chromosomes had telomeres on both ends. The genetic map consisted of 4153 cM and a comparison of the genetic distances with the physical distances identified 40 recombination hotspots. The linkage map also identified two mutations, located in the previously described genes Bos1 and BcsdhB, that conferred resistance to the fungicides boscalid and iprodione. The genome was predicted to encode 11 701 proteins. RNAseq data from >20 different samples were used to validate and improve gene models. Manual curation of chromosome 1 revealed interesting features, such as the occurrence of a dicistronic transcript and fully overlapping genes in opposite orientations, as well as many spliced antisense transcripts. Manual curation also revealed that the untranslated regions (UTRs) of genes can be complex and long, with many UTRs exceeding lengths of 1 kb and possessing multiple introns. Community annotation is in progress.
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http://dx.doi.org/10.1111/mpp.12384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638203PMC
January 2017

Development of Polymorphic Microsatellite Loci for Potato Wart from Next-Generation Sequence Data.

Phytopathology 2016 Jun 8;106(6):636-44. Epub 2016 Apr 8.

First and seventh authors: Canadian Food Inspection Agency, 3851 Fallowfield Road, Ottawa, ON K2H 8P9, Canada; second and third authors: Wageningen University and Research Center, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands; fourth and fifth authors: Canadian Food Inspection Agency, 93 Mount Edward Road, Charlottetown, PEI C1A 5T1, Canada; and sixth author: Agriculture and Agri-Food Canada, 960 Carling Ave, Ottawa, ON K1A 0C6, Canada.

Synchytrium endobioticum is the fungal agent causing potato wart disease. Because of its severity and persistence, quarantine measures are enforced worldwide to avoid the spread of this disease. Molecular markers exist for species-specific detection of this pathogen, yet markers to study the intraspecific genetic diversity of S. endobioticum were not available. Whole-genome sequence data from Dutch pathotype 1 isolate MB42 of S. endobioticum were mined for perfect microsatellite motifs. Of the 62 selected microsatellites, 21 could be amplified successfully and displayed moderate levels of polymorphism in 22 S. endobioticum isolates from different countries. Nineteen multilocus genotypes were observed, with only three isolates from Canada displaying identical profiles. The majority of isolates from Canada clustered genetically. In contrast, most isolates collected in Europe show no genetic clustering associated with their geographic origin. S. endobioticum isolates with the same pathotype displayed highly variable genotypes and none of the microsatellite markers correlated with a specific pathotype. The markers developed in this study can be used to assess intraspecific genetic diversity of S. endobioticum and allow track and trace of genotypes that will generate a better understanding of the migration and spread of this important fungal pathogen and support management of this disease.
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http://dx.doi.org/10.1094/PHYTO-12-15-0317-RDOI Listing
June 2016

Computational strategies for genome-based natural product discovery and engineering in fungi.

Fungal Genet Biol 2016 Apr 13;89:29-36. Epub 2016 Jan 13.

Bioinformatics Group, Wageningen University, Wageningen, The Netherlands. Electronic address:

Fungal natural products possess biological activities that are of great value to medicine, agriculture and manufacturing. Recent metagenomic studies accentuate the vastness of fungal taxonomic diversity, and the accompanying specialized metabolic diversity offers a great and still largely untapped resource for natural product discovery. Although fungal natural products show an impressive variation in chemical structures and biological activities, their biosynthetic pathways share a number of key characteristics. First, genes encoding successive steps of a biosynthetic pathway tend to be located adjacently on the chromosome in biosynthetic gene clusters (BGCs). Second, these BGCs are often are located on specific regions of the genome and show a discontinuous distribution among evolutionarily related species and isolates. Third, the same enzyme (super)families are often involved in the production of widely different compounds. Fourth, genes that function in the same pathway are often co-regulated, and therefore co-expressed across various growth conditions. In this mini-review, we describe how these partly interlinked characteristics can be exploited to computationally identify BGCs in fungal genomes and to connect them to their products. Particular attention will be given to novel algorithms to identify unusual classes of BGCs, as well as integrative pan-genomic approaches that use a combination of genomic and metabolomic data for parallelized natural product discovery across multiple strains. Such novel technologies will not only expedite the natural product discovery process, but will also allow the assembly of a high-quality toolbox for the re-design or even de novo design of biosynthetic pathways using synthetic biology approaches.
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http://dx.doi.org/10.1016/j.fgb.2016.01.006DOI Listing
April 2016

Diagnosis of Infections: Approaches to Identification by the Clinical Mycology Laboratory.

Curr Fungal Infect Rep 2015;9(3):135-143

Plant Research International Wageningen UR, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands.

Infections caused by the genus have emerged over the past decades and range from onychomycosis and keratitis in healthy individuals to deep and disseminated infections with high mortality rates in immune-compromised patients. As antifungal susceptibility can differ between the different species, identification at species level is recommended. Several clinical observations as hyaline hyphae in tissue, necrotic lesions in the skin and positive blood tests with fungal growth or presence of fungal cell wall components may be the first hints for fusariosis. Many laboratories rely on morphological identification, but especially multi-locus sequencing proves better to discriminate among members of the species complexes involved in human infection. DNA-based diagnostic tools have best discriminatory power when based on translation elongation factor 1-α or the RNA polymerase II second largest subunit. However, assays based on the detection of other fusarial cell compounds such as peptides and cell wall components may also be used for identification. The purpose of this review is to provide an overview and a comparison of the different tools currently available for the diagnosis of fusariosis.
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http://dx.doi.org/10.1007/s12281-015-0225-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537702PMC
January 2015

FPLC and liquid-chromatography mass spectrometry identify candidate necrosis-inducing proteins from culture filtrates of the fungal wheat pathogen Zymoseptoria tritici.

Fungal Genet Biol 2015 Jun;79:54-62

Wageningen University & Research Center, Plant Research International, 6708 PB Wageningen, The Netherlands. Electronic address:

Culture filtrates (CFs) of the fungal wheat pathogen Zymoseptoria tritici were assayed for necrosis-inducing activity after infiltration in leaves of various wheat cultivars. Active fractions were partially purified and characterized. The necrosis-inducing factors in CFs are proteinaceous, heat stable and their necrosis-inducing activity is temperature and light dependent. The in planta activity of CFs was tested by a time series of proteinase K (PK) co-infiltrations, which was unable to affect activity 30min after CF infiltrations. This suggests that the necrosis inducing proteins (NIPs) are either absent from the apoplast and likely actively transported into mesophyll cells or protected from the protease by association with a receptor. Alternatively, plant cell death signaling pathways might be fully engaged during the first 30min and cannot be reversed even after PK treatment. Further fractionation of the CFs with the highest necrosis-inducing activity involved fast performance liquid chromatography, SDS-PAGE and mass spectrometry. This revealed that most of the proteins present in the fractions have not been described before. The two most prominent ZtNIP encoding candidates were heterologously expressed in Pichia pastoris and subsequent infiltration assays showed their differential activity in a range of wheat cultivars.
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http://dx.doi.org/10.1016/j.fgb.2015.03.015DOI Listing
June 2015

Proteome catalog of Zymoseptoria tritici captured during pathogenesis in wheat.

Fungal Genet Biol 2015 Jun;79:42-53

Wageningen University & Research Center, Plant Research International, 6708PB Wageningen, The Netherlands. Electronic address:

Zymoseptoria tritici is an economically important pathogen of wheat. However, the molecular basis of pathogenicity on wheat is still poorly understood. Here, we present a global survey of the proteins secreted by this fungus in the apoplast of resistant (cv. Shafir) and susceptible (cv. Obelisk) wheat cultivars after inoculation with reference Z. tritici strain IPO323. The fungal proteins present in apoplastic fluids were analyzed by gel electrophoresis and by data-independent acquisition liquid chromatography/mass spectrometry (LC/MS(E)) combined with data-dependent acquisition LC-MS/MS. Subsequent mapping mass spectrometry-derived peptide sequence data against the genome sequence of strain IPO323 identified 665 peptides in the MS(E) and 93 in the LC-MS/MS mode that matched to 85 proteins. The identified fungal proteins, including cell-wall degrading enzymes and proteases, might function in pathogenicity, but the functions of many remain unknown. Most fungal proteins accumulated in cv. Obelisk at the onset of necrotrophy. This inventory provides an excellent basis for future detailed studies on the role of these genes and their encoded proteins during pathogenesis in wheat.
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http://dx.doi.org/10.1016/j.fgb.2015.04.018DOI Listing
June 2015

Effector discovery in the fungal wheat pathogen Zymoseptoria tritici.

Mol Plant Pathol 2015 Dec 28;16(9):931-45. Epub 2015 Apr 28.

Wageningen University and Research Centre, Plant Research International, Droevendaalsesteeg 1, 6708, PB, Wageningen, The Netherlands.

Fungal plant pathogens, such as Zymoseptoria tritici (formerly known as Mycosphaerella graminicola), secrete repertoires of effectors to facilitate infection or trigger host defence mechanisms. The discovery and functional characterization of effectors provides valuable knowledge that can contribute to the design of new and effective disease management strategies. Here, we combined bioinformatics approaches with expression profiling during pathogenesis to identify candidate effectors of Z. tritici. In addition, a genetic approach was conducted to map quantitative trait loci (QTLs) carrying putative effectors, enabling the validation of both complementary strategies for effector discovery. In planta expression profiling revealed that candidate effectors were up-regulated in successive waves corresponding to consecutive stages of pathogenesis, contrary to candidates identified by QTL mapping that were, overall, expressed at low levels. Functional analyses of two top candidate effectors (SSP15 and SSP18) showed their dispensability for Z. tritici pathogenesis. These analyses reveal that generally adopted criteria, such as protein size, cysteine residues and expression during pathogenesis, may preclude an unbiased effector discovery. Indeed, genetic mapping of genomic regions involved in specificity render alternative effector candidates that do not match the aforementioned criteria, but should nevertheless be considered as promising new leads for effectors that are crucial for the Z. tritici-wheat pathosystem.
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http://dx.doi.org/10.1111/mpp.12251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638447PMC
December 2015

Distribution of anticoagulant rodenticide resistance in Rattus norvegicus in the Netherlands according to Vkorc1 mutations.

Pest Manag Sci 2014 Nov 21;70(11):1761-6. Epub 2014 May 21.

Wageningen UR Livestock Research, Lelystad, The Netherlands.

Background: Rodenticide resistance to anticoagulants in Rattus norvegicus will lead to increased difficulties in combating these pest animals. Here, the authors present the results of a survey in the Netherlands where tissue samples and droppings were tested using a newly developed TaqMan PCR test for genotypic variation at codon 139 in the Vkorc1 gene associated with anticoagulant rodenticide resistance. Test results are linked to results of a questionnaire that was conducted among pest controllers.

Results: Genetic mutations at codon 139 of the Vkorc1 gene in R. norvegicus can be encountered in many parts of the Netherlands. In 34/61 rat tails, a genotype was found that is linked to anticoagulant rodenticide resistance (56%). In droppings, 42/169 samples (25%) showed a resistance-mediating genotype. In addition, indications of a clear genetic substructure in the Netherlands were found. In some regions, only resistance-mediating genotypes were found, corroborating results from the questionnaire in which pest controllers indicated they suspected resistance to anticoagulant rodenticides.

Conclusion: This is the first study to demonstrate the presence of multiple genetic mutations at codon 139 of the Vkorc1 gene in R. norvegicus in the Netherlands. As rodenticides should keep their efficacy because they are a last resort in rodent management, more studies are urgently needed that link specific genetic mutations to the efficacy of active substances.
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http://dx.doi.org/10.1002/ps.3809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4238836PMC
November 2014

Rapid identification of Fusarium graminearum species complex using Rolling Circle Amplification (RCA).

J Microbiol Methods 2012 Apr 3;89(1):63-70. Epub 2012 Feb 3.

Department of Plant Pathology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran.

Rolling Circle Amplification (RCA) of DNA is a sensitive and cost effective method for the rapid identification of pathogenic fungi without the need for sequencing. Amplification products can be visualized on 1% agarose gel to verify the specificity of probe-template binding or directly by adding fluorescent dyes. Fusarium Head Blight (FHB) is currently the world's largest threat to the production of cereal crops with the production of a range of mycotoxins as an additional risk. We designed sets of RCA padlock probes based on polymorphisms in the elongation factor 1-α (EF-1α) gene to detect the dominant FHB species, comprising lineages of the Fusarium graminearum species complex (FGSC). The method also enabled the identification of species of the Fusarium oxysporum (FOSC), the Fusarium incarnatum-equiseti (FIESC), and the Fusarium tricinctum (FTSC) species complexes, and used strains from the CBS culture collection as reference. Subsequently probes were applied to characterize isolates from wheat and wild grasses, and inoculated wheat kernels. The RCA assays successfully amplified DNA of the target fungi, both in environmental samples and in the contaminated wheat samples, while no cross reactivity was observed with uncontaminated wheat or related Fusarium species. As RCA does not require expensive instrumentation, the technique has a good potential for local and point of care screening for toxigenic Fusarium species in cereals.
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http://dx.doi.org/10.1016/j.mimet.2012.01.017DOI Listing
April 2012

New broad-spectrum resistance to septoria tritici blotch derived from synthetic hexaploid wheat.

Theor Appl Genet 2012 Jan 13;124(1):125-42. Epub 2011 Sep 13.

Plant Research International, Biointeractions and Plant Health, P.O. Box 16, 6700 AA, Wageningen, The Netherlands.

Septoria tritici blotch (STB), caused by the ascomycete Mycosphaerella graminicola, is one of the most devastating foliar diseases of wheat. We screened five synthetic hexaploid wheats (SHs), 13 wheat varieties that represent the differential set of cultivars and two susceptible checks with a global set of 20 isolates and discovered exceptionally broad STB resistance in SHs. Subsequent development and analyses of recombinant inbred lines (RILs) from a cross between the SH M3 and the highly susceptible bread wheat cv. Kulm revealed two novel resistance loci on chromosomes 3D and 5A. The 3D resistance was expressed in the seedling and adult plant stages, and it controlled necrosis (N) and pycnidia (P) development as well as the latency periods of these parameters. This locus, which is closely linked to the microsatellite marker Xgwm494, was tentatively designated Stb16q and explained from 41 to 71% of the phenotypic variation at seedling stage and 28-31% in mature plants. The resistance locus on chromosome 5A was specifically expressed in the adult plant stage, associated with SSR marker Xhbg247, explained 12-32% of the variation in disease, was designated Stb17, and is the first unambiguously identified and named QTL for adult plant resistance to M. graminicola. Our results confirm that common wheat progenitors might be a rich source of new Stb resistance genes/QTLs that can be deployed in commercial breeding programs.
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http://dx.doi.org/10.1007/s00122-011-1692-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249545PMC
January 2012

Finished genome of the fungal wheat pathogen Mycosphaerella graminicola reveals dispensome structure, chromosome plasticity, and stealth pathogenesis.

PLoS Genet 2011 Jun 9;7(6):e1002070. Epub 2011 Jun 9.

USDA-Agricultural Research Service, Purdue University, West Lafayette, Indiana, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pgen.1002070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3111534PMC
June 2011

Genetic analysis of resistance to septoria tritici blotch in the French winter wheat cultivars Balance and Apache.

Theor Appl Genet 2011 Sep 8;123(5):741-54. Epub 2011 Jun 8.

Plant Research International, Biointeractions and Plant Health, P.O. Box 16, 6700 AA, Wageningen, The Netherlands.

The ascomycete Mycosphaerella graminicola is the causal agent of septoria tritici blotch (STB), one of the most destructive foliar diseases of bread and durum wheat globally, particularly in temperate humid areas. A screening of the French bread wheat cultivars Apache and Balance with 30 M. graminicola isolates revealed a pattern of resistant responses that suggested the presence of new genes for STB resistance. Quantitative trait loci (QTL) analysis of a doubled haploid (DH) population with five M. graminicola isolates in the seedling stage identified four QTLs on chromosomes 3AS, 1BS, 6DS and 7DS, and occasionally on 7DL. The QTL on chromosome 6DS flanked by SSR markers Xgpw5176 and Xgpw3087 is a novel QTL that now can be designated as Stb18. The QTLs on chromosomes 3AS and 1BS most likely represent Stb6 and Stb11, respectively, and the QTLs on chromosome 7DS are most probably identical with Stb4 and Stb5. However, the QTL identified on chromosome 7DL is expected to be a new Stb gene that still needs further characterization. Multiple isolates were used and show that not all isolates identify all QTLs, which clearly demonstrates the specificity in the M. graminicola-wheat pathosystem. QTL analyses were performed with various disease parameters. The development of asexual fructifications (pycnidia) in the characteristic necrotic blotches of STB, designated as parameter P, identified the maximum number of QTLs. All other parameters identified fewer but not different QTLs. The segregation of multiple QTLs in the Apache/Balance DH population enabled the identification of DH lines with single QTLs and multiple QTL combinations. Analyses of the marker data of these DH lines clearly demonstrated the positive effect of pyramiding QTLs to broaden resistance spectra as well as epistatic and additive interactions between these QTLs. Phenotyping of the Apache/Balance DH population in the field confirmed the presence of the QTLs that were identified in the seedling stage, but Stb18 was inconsistently expressed and might be particularly effective in young plants. In contrast, an additional QTL for STB resistance was identified on chromosome 2DS that is exclusively and consistently expressed in mature plants over locations and time, but it was also strongly related with earliness, tallness as well as resistance to Fusarium head blight. Although to date no Stb gene has been reported on chromosome 2D, the data provide evidence that this QTL is only indirectly related to STB resistance. This study shows that detailed genetic analysis of contemporary commercial bread wheat cultivars can unveil novel Stb genes that can be readily applied in marker-assisted breeding programs.
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http://dx.doi.org/10.1007/s00122-011-1623-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3155673PMC
September 2011

Meiosis drives extraordinary genome plasticity in the haploid fungal plant pathogen Mycosphaerella graminicola.

PLoS One 2009 Jun 10;4(6):e5863. Epub 2009 Jun 10.

Plant Research International BV, Wageningen University and Research Centre, Wageningen, The Netherlands.

Meiosis in the haploid plant-pathogenic fungus Mycosphaerella graminicola results in eight ascospores due to a mitotic division following the two meiotic divisions. The transient diploid phase allows for recombination among homologous chromosomes. However, some chromosomes of M. graminicola lack homologs and do not pair during meiosis. Because these chromosomes are not present universally in the genome of the organism they can be considered to be dispensable. To analyze the meiotic transmission of unequal chromosome numbers, two segregating populations were generated by crossing genetically unrelated parent isolates originating from Algeria and The Netherlands that had pathogenicity towards durum or bread wheat, respectively. Detailed genetic analyses of these progenies using high-density mapping (1793 DArT, 258 AFLP and 25 SSR markers) and graphical genotyping revealed that M. graminicola has up to eight dispensable chromosomes, the highest number reported in filamentous fungi. These chromosomes vary from 0.39 to 0.77 Mb in size, and represent up to 38% of the chromosomal complement. Chromosome numbers among progeny isolates varied widely, with some progeny missing up to three chromosomes, while other strains were disomic for one or more chromosomes. Between 15-20% of the progeny isolates lacked one or more chromosomes that were present in both parents. The two high-density maps showed no recombination of dispensable chromosomes and hence, their meiotic processing may require distributive disjunction, a phenomenon that is rarely observed in fungi. The maps also enabled the identification of individual twin isolates from a single ascus that shared the same missing or doubled chromosomes indicating that the chromosomal polymorphisms were mitotically stable and originated from nondisjunction during the second division and, less frequently, during the first division of fungal meiosis. High genome plasticity could be among the strategies enabling this versatile pathogen to quickly overcome adverse biotic and abiotic conditions in wheat fields.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0005863PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2689623PMC
June 2009

G(alpha) and Gbeta proteins regulate the cyclic AMP pathway that is required for development and pathogenicity of the phytopathogen Mycosphaerella graminicola.

Eukaryot Cell 2009 Jul 1;8(7):1001-13. Epub 2009 May 1.

Plant Research International BV, Wageningen University and Research Centre, 6700 AA Wageningen, The Netherlands.

We identified and functionally characterized genes encoding three Galpha proteins and one Gbeta protein in the dimorphic fungal wheat pathogen Mycosphaerella graminicola, which we designated MgGpa1, MgGpa2, MgGpa3, and MgGpb1, respectively. Sequence comparisons and phylogenetic analyses showed that MgGPA1 and MgGPA3 are most related to the mammalian Galpha(i) and Galpha(s) families, respectively, whereas MgGPA2 is not related to either of these families. On potato dextrose agar (PDA) and in yeast glucose broth (YGB), MgGpa1 mutants produced significantly longer spores than those of the wild type (WT), and these developed into unique fluffy mycelia in the latter medium, indicating that this gene negatively controls filamentation. MgGpa3 mutants showed more pronounced yeast-like growth accompanied with hampered filamentation and secreted a dark-brown pigment into YGB. Germ tubes emerging from spores of MgGpb1 mutants were wavy on water agar and showed a nested type of growth on PDA that was due to hampered filamentation, numerous cell fusions, and increased anastomosis. Intracellular cyclic AMP (cAMP) levels of MgGpb1 and MgGpa3 mutants were decreased, indicating that both genes positively regulate the cAMP pathway, which was confirmed because the WT phenotype was restored by adding cAMP to these mutant cultures. The cAMP levels in MgGpa1 mutants and the WT were not significantly different, suggesting that this gene might be dispensable for cAMP regulation. In planta assays showed that mutants of MgGpa1, MgGpa3, and MgGpb1 are strongly reduced in pathogenicity. We concluded that the heterotrimeric G proteins encoded by MgGpa3 and MgGpb1 regulate the cAMP pathway that is required for development and pathogenicity in M. graminicola.
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http://dx.doi.org/10.1128/EC.00258-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708459PMC
July 2009

The het-c heterokaryon incompatibility gene in Aspergillus niger.

Mycol Res 2009 Feb 26;113(Pt 2):222-9. Epub 2008 Oct 26.

Laboratory of Genetics, Department of Plant Sciences, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands.

Heterokaryon incompatibility among Aspergillus niger strains is a widespread phenomenon that is observed as the inability to form stable heterokaryons. The genetic basis of heterokaryon incompatibility reactions is well established in some sexual filamentous fungi but largely unknown in presumed asexual species, such as A. niger. To test whether the genes that determine heterokaryon incompatibility in Neurospora crassa, such as het-c, vib-1 and pin-c, have a similar function in A. niger, we performed a short in silico search for homologues of these genes in the A. niger and several related genomes. For het-c, pin-c and vib-1 we did indeed identify putative orthologues. We then screened a genetically diverse worldwide collection of incompatible black Aspergilli for polymorphisms in the het-c orthologue. No size variation was observed in the variable het-c indel region that determines the specificity in N. crassa. Sequence comparison showed only minor variation in the number of glutamine coding triplets. However, introduction of one of the three N. crassa alleles (het-c2) in A. niger by transformation resulted in an abortive phenotype, reminiscent of the heterokaryon incompatibility in N. crassa. We conclude that although the genes required are present and the het-c homologue could potentially function as a heterokaryon incompatibility gene, het-c has no direct function in heterokaryon incompatibility in A. niger because the necessary allelic variation is absent.
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http://dx.doi.org/10.1016/j.mycres.2008.10.003DOI Listing
February 2009

Large-scale gene discovery in the septoria tritici blotch fungus Mycosphaerella graminicola with a focus on in planta expression.

Mol Plant Microbe Interact 2008 Sep;21(9):1249-60

Plant Research International B.V., Wageningen, The Netherlands.

The foliar disease septoria tritici blotch, caused by the fungus Mycosphaerella graminicola, is currently the most important wheat disease in Europe. Gene expression was examined under highly different conditions, using 10 expressed sequence tag libraries generated from M. graminicola isolate IPO323 using seven in vitro and three in planta growth conditions. To identify fungal clones in the interaction libraries, we developed a selection method based on hybridization with the entire genomic DNA of M. graminicola, to selectively enrich these libraries for fungal genes. Assembly of the 27,007 expressed sequence tags resulted in 9,190 unigenes, representing 5.2 Mb of the estimated 39-Mb genome size of M. graminicola. All libraries contributed significantly to the number of unigenes, especially the in planta libraries representing different stages of pathogenesis, which covered 15% of the library-specific unigenes. Even under presymptomatic conditions (5 days postinoculation), when fungal biomass is less than 5%, this method enabled us to efficiently capture fungal genes expressed during pathogenesis. Many of these genes were uniquely expressed in planta, indicating that in planta gene expression significantly differed from in vitro expression. Examples of gene discovery included a number of cell wall-degrading enzymes, a broad set of genes involved in signal transduction (n=11) and a range of ATP-binding cassette (n=20) and major facilitator superfamily transporter genes (n=12) potentially involved in protection against antifungal compounds or the secretion of pathogenicity factors. In addition, evidence is provided for a mycovirus in M. graminicola that is highly expressed under various stress conditions, in particular, under nitrogen starvation. Our analyses provide a unique window on in vitro and in planta gene expression of M. graminicola.
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http://dx.doi.org/10.1094/MPMI-21-9-1249DOI Listing
September 2008