Update on Nox function, site of action and regulation in Botrytis cinerea

Additional file 1: Table S1. Strains used in this work

BcAtf1, a global regulator, controls various differentiation processes and phytotoxin production in Botrytis cinerea

Atf1-homologous basic region leucine zipper (bZIP) transcription factors are known to act downstream of the stress-activated mitogen-activated protein kinase (SAPK) cascade in mammals, as well as in several fungi; they regulate the transcription of genes involved in the general stress response. Functional analyses of BcAtf1 in Botrytis cinerea show that it is also connected to the SAPK BcSak1, as it shares several stress response target genes. However, Dbcatf1 mutants are not hypersensitive to osmotic or oxidative stress, as are Dbcsak1 mutants. Both BcSak1 and BcAtf1 are regulators of differentiation, but their roles in these processes are almost inverse as, in contrast with Dbcsak1, Dbcatf1 mutants are significantly impaired in conidia production and do not differentiate any sclerotia. They show extremely vigorous growth in axenic culture, with a thick layer of aerial hyphae and a marked increase in colonization efficiency on different host plants and tissues. In addition, the sensitivity to cell wall-interfering agents is increased strongly. Microarray analyses demonstrate that the loss of BcAtf1 leads to extensive transcriptional changes: apart from stress response genes, the expression of a broad set of genes, probably involved in primary metabolism, cell wall synthesis and development, is affected by BcAtf1. Unexpectedly, BcAtf1 also controls secondary metabolism: the mutant contains significantly elevated levels of phytotoxins. These data indicate that BcAtf1 controls a diversity of cellular processes and has broad regulatory functions

Natural Variation in the VELVET Gene bcvel1 Affects Virulence and Light-Dependent Differentiation in Botrytis cinerea

Botrytis cinerea is an aggressive plant pathogen causing gray mold disease on various plant species. In this study, we identified the genetic origin for significantly differing phenotypes of the two sequenced B. cinerea isolates, B05.10 and T4, with regard to light-dependent differentiation, oxalic acid (OA) formation and virulence. By conducting a map-based cloning approach we identified a single nucleotide polymorphism (SNP) in an open reading frame encoding a VELVET gene (bcvel1). The SNP in isolate T4 results in a truncated protein that is predominantly found in the cytosol in contrast to the fulllength protein of isolate B05.10 that accumulates in the nuclei. Deletion of the full-length gene in B05.10 resulted in the T4 phenotype, namely light-independent conidiation, loss of sclerotial development and oxalic acid production, and reduced virulence on several host plants. These findings indicate that the identified SNP represents a loss-of-function mutation of bcvel1. In accordance, the expression of the B05.10 copy in T4 rescued the wild-type/B05.10 phenotype. BcVEL1 is crucial for full virulence as deletion mutants are significantly hampered in killing and decomposing plant tissues. However, the production of the two best known secondary metabolites, the phytotoxins botcinic acid and botrydial, are not affected by the deletion of bcvel1 indicating that other factors are responsible for reduced virulence. Genome-wide expression analyses of B05.10- and Dbcvel1-infected plant material revealed a number of genes differentially expressed in the mutant: while several protease- encoding genes are under-expressed in Dbcvel1 compared to the wild type, the group of over-expressed genes is enriched for genes encoding sugar, amino acid and ammonium transporters and glycoside hydrolases reflecting the response of Dbcvel1 mutants to nutrient starvation conditions

The NADPH Oxidase Complexes in Botrytis cinerea: Evidence for a Close Association with the ER and the Tetraspanin Pls1

NADPH oxidases (Nox) are major enzymatic systems that generate reactive-oxygen species (ROS) in multicellular eukaryotes. In several fungi they have been shown to be involved in sexual differentiation and pathogenicity. However, in contrast to the well characterized mammalian systems, basic information on the composition, recruitment, and localization of fungal Nox complexes and on the molecular mechanisms of their cellular effects are still lacking. Here we give a detailed analysis of components of the Nox complexes in the gray mold fungus Botrytis cinerea. It had previously been shown that the two catalytic transmembrane subunits BcNoxA and B are important for development of sclerotia and for full virulence, with BcNoxA being involved in spreading of lesions and BcNoxB in penetration; BcNoxR functions as a regulator of both subunits. Here we present evidence (using for the first time a functional GFP fusion able to complement the ¿bcnoxA mutant) that BcNoxA localizes mainly to the ER and at the plasma membrane; BcNoxB shows a similar localization pattern, while the regulator BcNoxR is found in vesicles throughout the hyphae and at the hyphal tip. To identify possible interaction partners, which could be involved in the localization or recruitment of the Nox complexes, we functionally characterized the tetraspanin Pls1, a transmembrane protein, which had been suggested to be a NoxB-interacting partner in the saprophyte Podospora anserina. Knock-out experiments and GFP fusions substantiate a link between BcNoxB and BcPls1 because both deletion mutants have overlapping phenotypes (especially a defect in penetration), and the proteins show a similar localization pattern (ER). However, in contrast to the corresponding protein in P. anserina BcPls1 is important for female fertility, but not for ascospore germination

The Sfp-Type 4′-Phosphopantetheinyl Transferase Ppt1 of Fusarium fujikuroi Controls Development, Secondary Metabolism and Pathogenicity

The heterothallic ascomycete Fusarium fujikuroi is a notorious rice pathogen causing super-elongation of plants due to the production of terpene-derived gibberellic acids (GAs) that function as natural plant hormones. Additionally, F. fujikuroi is able to produce a variety of polyketide- and non-ribosomal peptide-derived metabolites such as bikaverins, fusarubins and fusarins as well as metabolites from yet unidentified biosynthetic pathways, e.g. moniliformin. The key enzymes needed for their production belong to the family of polyketide synthases (PKSs) and non-ribosomal peptide synthases (NRPSs) that are generally known to be post-translationally modified by a Sfp-type 4′phosphopantetheinyl transferase (PPTase). In this study we provide evidence that the F. fujikuroi Sfp-type PPTase FfPpt1 is essentially involved in lysine biosynthesis and production of bikaverins, fusarubins and fusarins, but not moniliformin as shown by analytical methods. Concomitantly, targeted Ffppt1 deletion mutants reveal an enhancement of terpene-derived metabolites like GAs and volatile substances such as α-acorenol. Pathogenicity assays on rice roots using fluorescent labeled wild-type and Ffppt1 mutant strains indicate that lysine biosynthesis and iron acquisition but not PKS and NRPS metabolism is essential for establishment of primary infections of F. fujikuroi. Additionally, FfPpt1 is involved in conidiation and sexual mating recognition possibly by activating PKS- and/or NRPS-derived metabolites that could act as diffusible signals. Furthermore, the effect on iron acquisition of Ffppt1 mutants led us to identify a previously uncharacterized putative third reductive iron uptake system (FfFtr3/FfFet3) that is closely related to the FtrA/FetC system of A. fumigatus. Functional characterization provides evidence that both proteins are involved in iron acquisition and are liable to transcriptional repression of the homolog of the Aspergillus GATA-type transcription factor SreA under iron-replete conditions. Targeted deletion of the first Fusarium homolog of this GATA-type transcription factor-encoding gene, Ffsre1, strongly indicates its involvement in regulation of iron homeostasis and oxidative stress resistance

Genome analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

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 t

Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

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.Fil: Ten Have, Arjen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Amselem, Joelle. Institut National de la Recherche Agronomique; FranciaFil: Cuomo, Christina A.. Broad Institute of MIT and Harvard; Estados UnidosFil: Jan, A. L. van Kan. Wageningen University; Países BajosFil: Viaud, Muriel. Institut National de la Recherche Agronomique; FranciaFil: Benito, Ernesto P.. Universidad de Salamanca; EspañaFil: Couloux, Arnaud. Centre National de Séquençage. Genoscope; FranciaFil: Coutinho, Pedro M.. Centre National de la Recherche Scientifique; FranciaFil: Vries, Ronald P. de. Microbiology and Kluyver Centre for Genomics of Industrial Fermentations; Países Bajos. Fungal Biodiversity Centre; Países BajosFil: Dyer, Paul S.. The University Of Nottingham; Reino UnidoFil: Fillinger, Sabine. Institut National de la Recherche Agronomique; FranciaFil: Fournier, Elisabeth. Institut National de la Recherche Agronomique; Francia. Centre de coopération internationale en recherche agronomique pour le développement; FranciaFil: Gout, Lilian. Institut National de la Recherche Agronomique; FranciaFil: Hahn, Matthias. University Of Kaiserlautern; AlemaniaFil: Kohn, Linda. University Of Toronto; CanadáFil: Lapalu, Nicolas. Institut National de la Recherche Agronomique; FranciaFil: Plummer, Kim M.. la Trobe University; AustraliaFil: Pradier, Jean-Marc. Institut National de la Recherche Agronomique; FranciaFil: Quévillon, Emmanuel. Institut National de la Recherche Agronomique; Francia. Centre National de la Recherche Scientifique; FranciaFil: Sharon, Amir. Tel Aviv University. Department of Molecular Biology and Ecology of Plants; IsraelFil: Simon, Adeline. Institut National de la Recherche Agronomique; FranciaFil: Tudzynski, Bettina. Institut für Biologie und Biotechnologie der Pflanzen; AlemaniaFil: Tudzynski, Paul. Institut für Biologie und Biotechnologie der Pflanzen; AlemaniaFil: Wincker, Patrick. Centre National de Séquençage. Genoscope; FranciaFil: Andrew, Marion. University Of Toronto; CanadáFil: Anthouard, Véronique. Centre National de Séquençage. Genoscope; FranciaFil: Beever, Ross E.. Landcare Research; Nueva ZelandaFil: Beffa, Rolland. Centre National de la Recherche Scientifique; FranciaFil: Benoit, Isabelle . Microbiology and Kluyver Centre for Genomics of Industrial Fermentations; Países BajosFil: Bouzid, Ourdia. Microbiology and Kluyver Centre for Genomics of Industrial Fermentations; Países Bajo

The identification of QTL controlling ergot sclerotia size in hexaploid wheat implicates a role for the Rht dwarfing alleles

The fungal pathogen Claviceps purpurea infects ovaries of a broad range of temperate grasses and cereals, including hexaploid wheat, causing a disease commonly known as ergot. Sclerotia produced in place of seed carry a cocktail of harmful alkaloid compounds that result in a range of symptoms in humans and animals, causing ergotism. Following a field assessment of C. purpurea infection in winter wheat, two varieties ‘Robigus’ and ‘Solstice’ were selected which consistently produced the largest differential effect on ergot sclerotia weights. They were crossed to produce a doubled haploid mapping population, and a marker map, consisting of 714 genetic loci and a total length of 2895 cM was produced. Four ergot reducing QTL were identified using both sclerotia weight and size as phenotypic parameters; QCp.niab.2A and QCp.niab.4B being detected in the wheat variety ‘Robigus’, and QCp.niab.6A and QCp.niab.4D in the variety ‘Solstice’. The ergot resistance QTL QCp.niab.4B and QCp.niab.4D peaks mapped to the same markers as the known reduced height (Rht) loci on chromosomes 4B and 4D, Rht-B1 and Rht-D1, respectively. In both cases, the reduction in sclerotia weight and size was associated with the semi-dwarfing alleles, Rht-B1b from ‘Robigus’ and Rht-D1b from ‘Solstice’. Two-dimensional, two-QTL scans identified significant additive interactions between QTL QCp.niab.4B and QCp.niab.4D, and between QCp.niab.2A and QCp.niab.4B when looking at sclerotia size, but not between QCp.niab.2A and QCp.niab.4D. The two plant height QTL, QPh.niab.4B and QPh.niab.4D, which mapped to the same locations as QCp.niab.4B and QCp.niab.4D, also displayed significant genetic interactions

Evidence for inter-specific recombination among the mitochondrial genomes of Fusarium species in the Gibberella fujikuroi complex

BACKGROUND: The availability of mitochondrial genomes has allowed for the resolution of numerous questions regarding the evolutionary history of fungi and other eukaryotes. In the Gibberella fujikuroi species complex, the exact relationships among the so-called “African”, “Asian” and “American” Clades remain largely unresolved, irrespective of the markers employed. In this study, we considered the feasibility of using mitochondrial genes to infer the phylogenetic relationships among Fusarium species in this complex. The mitochondrial genomes of representatives of the three Clades (Fusarium circinatum, F. verticillioides and F. fujikuroi) were characterized and we determined whether or not the mitochondrial genomes of these fungi have value in resolving the higher level evolutionary relationships in the complex. RESULTS: Overall, the mitochondrial genomes of the three species displayed a high degree of synteny, with all the genes (protein coding genes, unique ORFs, ribosomal RNA and tRNA genes) in identical order and orientation, as well as introns that share similar positions within genes. The intergenic regions and introns generally contributed significantly to the size differences and diversity observed among these genomes. Phylogenetic analysis of the concatenated protein-coding dataset separated members of the Gibberella fujikuroi complex from other Fusarium species and suggested that F. fujikuroi (“Asian” Clade) is basal in the complex. However, individual mitochondrial gene trees were largely incongruent with one another and with the concatenated gene tree, because six distinct phylogenetic trees were recovered from the various single gene datasets. CONCLUSION: The mitochondrial genomes of Fusarium species in the Gibberella fujikuroi complex are remarkably similar to those of the previously characterized Fusarium species and Sordariomycetes. Despite apparently representing a single replicative unit, all of the genes encoded on the mitochondrial genomes of these fungi do not share the same evolutionary history. This incongruence could be due to biased selection on some genes or recombination among mitochondrial genomes. The results thus suggest that the use of individual mitochondrial genes for phylogenetic inference could mask the true relationships between species in this complex.This work is based on the research supported in part by a number of grants from the South African National Department of Science and Technology (DST), National Research Foundation (NRF), Technology and Human Resources of Industry Programme (THRIP) (includes Grant specific unique reference number (UID) 83924).http://www.biomedcentral.com/1471-2164/14/605am201