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Transitioning from spores to hyphae is pivotal to host invasion by the plant pathogenic fungus Zymoseptoria tritici. This dimorphic switch can be initiated by high temperature in vitro (~27 °C); however, such a condition may induce cellular heat stress, questioning its relevance to field infections. Here, we study the regulation of the dimorphic switch by temperature and other factors. Climate data from wheat-growing areas indicate that the pathogen sporadically experiences high temperatures such as 27 °C during summer months. However, using a fluorescent dimorphic switch reporter (FDR1) in four wild-type strains, we show that dimorphic switching already initiates at 15–18 °C, and is enhanced by wheat leaf surface compounds. Transcriptomics reveals 1261 genes that are up- or down-regulated in hyphae of all strains. These pan-strain core dimorphism genes (PCDGs) encode known effectors, dimorphism and transcription factors, and light-responsive proteins (velvet factors, opsins, putative blue light receptors). An FDR1-based genetic screen reveals a crucial role for the white-collar complex (WCC) in dimorphism and virulence, mediated by control of PCDG expression. Thus, WCC integrates light with biotic and abiotic cues to orchestrate Z. tritici infection.

28 p.-12 fig., Intracellular traffic in Aspergillus nidulans hyphae must cope with the challenges that the high rates of apical extension (1μm/min) and the long intracellular distances (>100 μm) impose. Understanding the ways in which the hyphal tip cell coordinates traffic to meet these challenges is of basic importance, but is also of considerable applied interest, as fungal invasiveness of animals and plants depends critically upon maintaining these high rates of growth. Rapid apical extension requires localization of cell-wall-modifying enzymes to hyphal tips. By combining genetic blocks in different trafficking steps with multidimensional epifluorescence microscopy and quantitative image analyses we demonstrate that polarization of the essential chitin-synthase ChsB occurs by indirect endocytic recycling, involving delivery/exocytosis to apices followed by internalization by the sub-apical endocytic collar of actin patches and subsequent trafficking to TGN cisternae, where it accumulates for ~1 min before being re-delivered to the apex by a RAB11/TRAPPII-dependent pathway. Accordingly, ChsB is stranded at the TGN by Sec7 inactivation but re-polarizes to the apical dome if the block is bypassed by a mutation in geaAgea1 that restores growth in the absence of Sec7. That polarization is independent of RAB5, that ChsB predominates at apex-proximal cisternae, and that upon dynein impairment ChsB is stalled at the tips in an aggregated endosome indicate that endocytosed ChsB traffics to the TGN via sorting endosomes functionally located upstream of the RAB5 domain and that this step requires dynein-mediated basipetal transport. It also requires RAB6 and its effector GARP (Vps51/Vps52/Vps53/Vps54), whose composition we determined by MS/MS following affinity chromatography purification. Ablation of any GARP component diverts ChsB to vacuoles and impairs growth and morphology markedly, emphasizing the important physiological role played by this pathway that, we propose, is central to the hyphal mode of growth., This work was funded by the Agencia Estatal de Investigación, Ministerio de Economía, Industria y Competitividad, Spain (http://www.idi.mineco.gob.es/portal/site/MICINN/?lang_choosen=en) through Grant BIO2015-65090-R to MAP and by the Consejería de Educación, Comunidad de Madrid (http://www.madrid.org) through Grant S2010/BMD-2414 to MAP.

International audience; In bacteria, more than half of the genes in the genome are organized in operons. In contrast, in eukaryotes, functionally related genes are usually dispersed across the genome. There are, however, numerous examples of functional clusters of nonhomologous genes for metabolic pathways in fungi and plants. Despite superficial similarities with operons (physical clustering, coordinate regulation), these clusters have not usually originated by horizontal gene transfer from bacteria, and (unlike operons) the genes are typically transcribed separately rather than as a single polycistronic message. This clustering phenomenon raises intriguing questions about the origins of clustered metabolic pathways in eukaryotes and the significance of clustering for pathway function. Here we review metabolic gene clusters from fungi and plants, highlight commonalities and differences, and consider how these clusters form and are regulated. We also identify opportunities for future research in the areas of large-scale genomics, synthetic biology, and experimental evolution.

Allorecognition, the ability to distinguish “self” from “nonself” based on allelic differences at allorecognition loci, is common in all domains of life. Allorecognition restricts the opportunities for social parasitism, and is therefore crucial for the evolution of cooperation. However, the maintenance of allorecognition diversity provides a paradox. If allorecognition is costly relative to cooperation, common alleles will be favored. Thus, the cost of allorecognition may reduce the genetic variation upon which allorecognition crucially relies, a prediction now known as “Crozier's paradox.” We establish the relative costs of allorecognition, and their consequences for the short-term evolution of recognition labels theoretically predicted by Crozier. We use fusion among colonies of the fungus Neurospora crassa, regulated by highly variable allorecognition genes, as an experimental model system. We demonstrate that fusion among colonies is mutually beneficial, relative to absence of fusion upon allorecognition. This benefit is due not only to absence of mutual antagonism, which occurs upon allorecognition, but also to an increase in colony size per se. We then experimentally demonstrate that the benefit of fusion selects against allorecognition diversity, as predicted by Crozier. We discuss what maintains allorecognition diversity

Background The Aspergillus niger genome contains a large repertoire of genes encoding carbohydrate active enzymes (CAZymes) that are targeted to plant polysaccharide degradation enabling A. niger to grow on a wide range of plant biomass substrates. Which genes need to be activated in certain environmental conditions depends on the composition of the available substrate. Previous studies have demonstrated the involvement of a number of transcriptional regulators in plant biomass degradation and have identified sets of target genes for each regulator. In this study, a broad transcriptional analysis was performed of the A. niger genes encoding (putative) plant polysaccharide degrading enzymes. Microarray data focusing on the initial response of A. niger to the presence of plant biomass related carbon sources were analyzed of a wild-type strain N402 that was grown on a large range of carbon sources and of the regulatory mutant strains ΔxlnR, ΔaraR, ΔamyR, ΔrhaR and ΔgalX that were grown on their specific inducing compounds. Results The cluster analysis of the expression data revealed several groups of co-regulated genes, which goes beyond the traditionally described co-regulated gene sets. Additional putative target genes of the selected regulators were identified, based on their expression profile. Notably, in several cases the expression profile puts questions on the function assignment of uncharacterized genes that was based on homology searches, highlighting the need for more extensive biochemical studies into the substrate specificity of enzymes encoded by these non-characterized genes. The data also revealed sets of genes that were upregulated in the regulatory mutants, suggesting interaction between the regulatory systems and a therefore even more complex overall regulatory network than has been reported so far. Conclusions Expression profiling on a large number of substrates provides better insight in the complex regulatory systems that drive the conversion of plant biomass by fungi. In addition, the data provides additional evidence in favor of and against the similarity-based functions assigned to uncharacterized genes. Electronic supplementary material The online version of this article (10.1186/s12864-017-4164-x) contains supplementary material, which is available to authorized users.

Accurate estimates of gamete and offspring dispersal range are required for the understanding and prediction of spatial population dynamics and species persistence. Little is known about gamete dispersal in fungi, especially in lichen-forming ascomycetes. Here, we estimate the dispersal functions of clonal propagules, gametes and ascospores of the epiphytic lichen Lobaria pulmonaria. We use hierarchical Bayesian parentage analysis, which integrates genetic and ecological information from multiannual colonization and dispersal source data collected in a large, oldgrowth forest landscape. The effective dispersal range of gametes is several hundred metres to kilometres from potential paternal individuals. By contrast, clonal propagules disperse only tens of metres, and ascospores disperse over several thousand metres. Our study reveals the dispersal distances of individual reproductive units; clonal propagules, gametes and ascospores, which is of great importance for a thorough understanding of the spatial dynamics of ascomycetes. Sexual reproduction occurs between distant individuals. However, whereas gametes and ascospores disperse over long distances, the overall rate of colonization of trees is low. Hence, establishment is the limiting factor for the colonization of new host trees by the lichen in old- growth landscapes.

Light is perceived and transduced by fungi, where it modulates processes as diverse as growth and morphogenesis, sexual development and secondary metabolism. A special case in point is that of fungi with a subterranean, light-shielded habitat such as Tuber spp. Using as reference the genome sequence of the black truffle Tuber melanosporum, we used bioinformatic prediction tools and expression data to gain insight on the photoreceptor systems of this hypogeous ectomycorrhizal fungus. These include a chromophore-less opsin, a putative red-light-sensing phytochrome not expressed at detectable levels in any of the examined lifecycle stages, and a nearly canonical two-component (WC-1/WC-2) photoreceptor system similar to the Neurospora white collar complex (WCC). Multiple evidence, including expression at relatively high levels in all lifecycle stages except for fruiting-bodies and the results of heterologous functional complementation experiments conducted in Neurospora, suggests that the Tuber WCC is likely functional and capable of responding to blue-light. The other putative T. melanosporum photoreceptor components, especially the chromophore-less opsin and the likely non-functional phytochrome, may instead represent signatures of adaptation to a hypogeous (light-shielded) lifestyle.

The discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) has revealed new territory for chemical and biochemical analysis. These unique mononuclear copper enzymes are abundant, suggesting functional diversity beyond their established roles in the depolymerization of biomass polysaccharides. At the same time basic biochemical methods for characterizing LPMOs, such as activity assays are not well developed. Here we describe a method for quantification of C1-oxidized chitooligosaccharides (aldonic acids), and hence LPMO activity. The method was used to quantify the activity of a four-domain LPMO from Vibrio cholerae, GbpA, which is a virulence factor with no obvious role in biomass processing. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The Cf-5 gene of tomato confers resistance to strains of the fungal pathogen Cladosporium fulvum carrying the avirulence gene Avr5. Although Cf-5 has been cloned, Avr5 has remained elusive. We report the cloning of Avr5 using a combined bioinformatic and transcriptome sequencing approach. RNA-Seq was performed on the sequenced race 0 strain (0WU; carrying Avr5), as well as a race 5 strain (IPO 1979; lacking a functional Avr5 gene) during infection of susceptible tomato. Forty-four in planta–induced C. fulvum candidate effector (CfCE) genes of 0WU were identified that putatively encode a secreted, small cysteine-rich protein. An expressed transcript sequence comparison between strains revealed two polymorphic CfCE genes in IPO 1979. One of these conferred avirulence to IPO 1979 on Cf-5 tomato following complementation with the corresponding 0WU allele, confirming identification of Avr5. Complementation also led to increased fungal biomass during infection of susceptible tomato, signifying a role for Avr5 in virulence. Seven of eight race 5 strains investigated escape Cf-5-mediated resistance through deletion of the Avr5 gene. Avr5 is heavily flanked by repetitive elements, suggesting that repeat instability, in combination with Cf-5-mediated selection pressure, has led to the emergence of race 5 strains deleted for the Avr5 gene.

The actin cytoskeleton is a dynamic but well organized intracellular framework that is essential for proper functioning of eukaryotic cells. Here, we use the actin binding peptide Lifeact to investigate the in vivo actin cytoskeleton dynamics in the oomycete plant pathogen Phytophthora infestans. Lifeact-eGFP labelled thick and thin actin bundles and actin filament plaques allowing visualization of actin dynamics. All actin structures in the hyphae were cortically localized. In growing hyphae actin filament cables were axially oriented in the sub-apical region whereas in the extreme apex in growing hyphae, waves of fine F-actin polymerization were observed. Upon growth termination, actin filament plaques appeared in the hyphal tip. The distance between a hyphal tip and the first actin filament plaque correlated strongly with hyphal growth velocity. The actin filament plaques were nearly immobile with average lifetimes exceeding one hour, relatively long when compared to the lifetime of actin patches known in other eukaryotes. Plaque assembly required ~30 seconds while disassembly was accomplished in ~10 sec. Remarkably, plaque disassembly was not accompanied with internalization and the formation of endocytic vesicles. These findings suggest that the functions of actin plaques in oomycetes differ from those of actin patches present in other organisms.

A genome-wide inventory of proteins involved in cell wall synthesis and remodeling has been obtained by taking advantage of the recently released genome sequence of the ectomycorrhizal Tuber melanosporum black truffle. Genes that encode cell wall biosynthetic enzymes, enzymes involved in cell wall polysaccharide synthesis or modification, GPI-anchored proteins and other cell wall proteins were identified in the black truffle genome. As a second step, array data were validated and the symbiotic stage was chosen as the main focus. Quantitative RT-PCR experiments were performed on 29 selected genes to verify their expression during ectomycorrhizal formation. The results confirmed the array data, and this suggests that cell wall-related genes are required for morphogenetic transition from mycelium growth to the ectomycorrhizal branched hyphae. Labeling experiments were also performed on T. melanosporum mycelium and ectomycorrhizae to localize cell wall components.

Prions are infectious proteins that cause fatal diseases in mammals. Prions have also been found in fungi, but studies on their role in nature are scarce. The proposed biological function of fungal prions is debated and varies from detrimental to benign or even beneficial. [Het-s] is a prion of the fungus Podospora anserina. The het-s locus exists as two antagonistic alleles that constitute an allorecognition system: the het-s allele encoding the protein variant capable of prion formation and the het-S allele encoding a protein variant that cannot form a prion. We document here that het-s alleles, capable of prion formation, are nearly twice as frequent as het-S alleles in a natural population of 112 individuals. Then, we report a 92% prevalence of [Het-s] prion infection among the het-s isolates and find evidence of the role of the [Het-s]/het-S allorecognition system on the incidence of infection by a deleterious senescence plasmid. We explain the het-s/het-S allele ratios by the existence of two selective forces operating at different levels. We propose that during the somatic stage, the role of [Het-s]/HET-S in allorecognition leads to frequency-dependent selection for which an equilibrated frequency would be optimal. However, in the sexual cycle, the [Het-s] prion causes meiotic drive favoring the het-s allele. Our findings indicate that [Het-s] is a selected and, therefore, widespread prion whose activity as selfish genetic element is counteracted by balancing selection for allorecognition polymorphism

Peroxisomes are ubiquitous organelles characterized by a protein-rich matrix surrounded by a single membrane. In filamentous fungi, peroxisomes are crucial for the primary metabolism of several unusual carbon sources used for growth (e. g. fatty acids), but increasing evidence is presented that emphasize the crucial role of these organelles in the formation of a variety of secondary metabolites. In filamentous fungi, peroxisomes also play a role in development and differentiation whereas specialized peroxisomes, the Woronin bodies, play a structural role in plugging septal pores. The biogenesis of peroxisomes in filamentous fungi involves the function of conserved PEX genes, as well as genes that are unique for these organisms. Peroxisomes are also subject to autophagic degradation, a process that involves ATG genes. The interplay between organelle biogenesis and degradation may serve a quality control function, thereby allowing a continuous rejuvenation of the organelle population in the cells.

Kinetics of extracellular protease (ECP) production has typically been studied for processes that involve protease as a product. We argue that ECP is equally important in fermentations where protease is not a product of interest. Industrial fermentations typically use complex nitrogen substrates, which are proteolytically hydrolyzed to amino acids (AA) by ECP before assimilation. However, high AA concentrations may lead to nitrogen catabolite repression (NCR) of the products such as antibiotics. Thus, ECP plays a crucial role in managing the nitrogen substrate supply thereby affecting the antibiotic productivity. Here, we have studied the induction of ECP and its effect on the antibiotic productivity for a rifamycin B overproducer strain Amycolatopsis meditterranei S699. This organism produces ECP at the level of 14 U mL(-1) in complex media, which is sufficient for hydrolysis of proteins in the media but low compared to other ECP overproducers. We find ECP secretion to be repressed by ammonia, AA, and under conditions that support high growth rate. We propose a structured kinetic model which accounts for the kinetics of ECP secretion, amino acid availability, growth, and antibiotic production. In addition to the quantity, the timing of ECP induction was critical in achieving higher rifamycin productivity. We artificially created conditions that led to delayed protease secretion, which in turn led to premature termination of batch and lower productivity. The predictive value of the model can be useful in better management of the available nitrogen supply, minimization of NCR, and in the monitoring of fermentation batches.

Zn2Cys6 transcription factors are unique to fungi and have been reported to be involved in different regulatory functions. Here, we characterized EBR1 (enhanced branching 1), a novel Zn2Cys6 transcription factor of Fusarium graminearum. Knocking out EBR1 in F. graminearum PH-1 caused reduction of both radial growth and virulence. The conidia of knock-out strain PH-1Δebr1 germinated faster than those of wild-type PH-1, but the conidiation of the mutant was significantly reduced. Detailed analysis showed that the reduced radial growth might be due to reduced apical dominance of the hyphal tip, leading to increased hyphal branching. Inoculation assays on wheat heads with a green fluorescent protein (GFP)-labeled PH-1Δebr1 mutant showed that it was unable to penetrate the rachis of the spikelets. Protein fusion with GFP showed that EBR1 is localized in the nucleus of both conidia and hyphae. Knocking out the orthologous gene FOXG_05408 in F. oxysporum f. sp. lycopersici caused a much weaker phenotype than the PH-1Δebr1 mutant, which may be due to the presence of multiple orthologous genes in this fungus. Transformation of FOXG_05408 into PH-1Δebr1 restored the mutant phenotype. Similar to EBR1, FOXG_05408 is localized in the nucleus of F. oxysporum f. sp. lycopersici. Possible functions of EBR1 and its relation with other fungal transcription factors are discussed.

Interactions between bacteria and fungi cover a wide range of incentives, mechanisms and outcomes. The genus Collimonas consists of soil bacteria that are known for their antifungal activity and ability to grow at the expense of living fungi. In non-contact confrontation assays with the fungus Aspergillus niger, Collimonas fungivorans showed accumulation of biomass concomitant with inhibition of hyphal spread. Through microarray analysis of bacterial and fungal mRNA from the confrontation arena, we gained new insights into the mechanisms underlying the fungistatic effect and mycophagous phenotype of collimonads. Collimonas responded to the fungus by activating genes for the utilization of fungal-derived compounds and for production of a putative antifungal compound. In A. niger, differentially expressed genes included those involved in lipid and cell wall metabolism and cell defense, which correlated well with the hyphal deformations that were observed microscopically. Transcriptional profiles revealed distress in both partners: downregulation of ribosomal proteins and upregulation of mobile genetic elements in the bacteria and expression of endoplasmic reticulum stress and conidia-related genes in the fungus. Both partners experienced nitrogen shortage in each other’s presence. Overall, our results indicate that the Collimonas/ Aspergillus interaction is a complex interplay between trophism, antibiosis and competition for nutrients.

Experiments that systematically varied T, τ, and photoperiod in Neurospora crassa revealed that the traditional nonparametric and parametric approaches could not explain entrainment for all of the tested conditions. The authors have developed a new approach to understanding entrainment that incorporates several features of the old paradigms but allows exploration of the underlying mechanisms in synchronized clocks, making extrapolations from constant conditions to entrained state unnecessary. It is based on a circadian integrated response characteristic (CIRC) that makes no assumptions about how entrainment occurs (by phase shifts or velocity changes). All it presumes is that, during entrainment, the clock’s cycle length must match that of the zeitgeber. With the help of the CIRC, entrainment to all zeitgeber conditions can be modeled by changing 3 parameters: the CIRC’s shape and asymmetry and an assumed internal cycle length (τ under entrainment: τ E) that the clock adopts under stable entrainment to produce a specific phase relationship to the zeitgeber (τE is reflected in a period aftereffect when clocks are released to constant conditions). The few parameters of the CIRC make it highly amenable to modeling. Here, the authors describe the results of modeling Neurospora’s circadian surface and show that the new approach can explain and unify all results of the circadian surface. The qualities of the CIRC are highly systematic for the respective entrainment condition and show that τE is an important variable in the entrainment process. The results also show that the wild-type strain is excellently tuned for entrainment under the natural 24-h cycle despite its shorter period (~22 h) in constant darkness. Experiments measuring aftereffects support the prediction that τE plays an important role in entrainment.

The widespread potential for somatic fusion among different conspecific multicellular individuals suggests that such fusion is adaptive. However, because recognition of non-kin (allorecognition) usually leads to a rejection response, successful somatic fusion is limited to close kin. This is consistent with kin-selection theory, which predicts that the potential cost of fusion and the potential for somatic parasitism decrease with increasing relatedness. Paradoxically, however, Crozier found that, in the short term, positive-frequency-dependent selection eliminates the required genetic polymorphism at allorecognition loci. The 'Crozier paradox' may be solved if allorecognition is based on extrinsically balanced polymorphisms, for example at immune loci. Alternatively, the assumption of most models that self fusion is mutually beneficial is wrong. If fusion is on average harmful, selection will promote unconditional rejection. However, we propose that fusion within individuals is beneficial, selecting for the ability to fuse, but fusion between individuals on average costly, selecting for non-self recognition (rather than non-kin recognition). We discuss experimental data on fungi that are consistent with this hypothesis.

Plant pathogens secrete effector molecules that contribute to the establishment of disease in their plant hosts. The identification of cellular cues that regulate effector gene expression is an important aspect of understanding the infection process. Nutritional status in the cell has been postulated to be a cue for effector gene expression. Several studies have shown the induction of the same effector genes during growth under nitrogen-starved conditions in vitro as during growth in planta, suggesting that a nitrogen-poor environment exists during colonization. As a consequence, it has been proposed that growth in nitrogen-starved media mimics the environment that a pathogen encounters during growth in planta. Although this relationship has been implied in several studies, there is little known regarding available nitrogen during colonization for fungal pathogens. This review focuses on the effect of nitrogen starvation on fungal effector gene expression and examines the role of fungal nitrogen regulatory genes to help gain an understanding of whether nitrogen availability within the host is an important factor in disease development.

The projection structures of complex I and the I+III2 supercomplex from the C-4 plant Zea mays were determined by electron microscopy and single particle image analysis to a resolution of up to 11 angstrom. Maize complex I has a typical L-shape. Additionally, it has a large hydrophilic, extra-domain attached to the centre of the membrane arm on its matrix-exposed side, which previously was described for Arabidopsis and which was reported to include carbonic anhydrase subunits. A comparison with the X-ray structure of homotrimeric gamma-carbonic anhydrase from the archaebacterium Methanosarcina thermophila indicates that this domain is also composed of a trimer. Mass spectrometry analyses allowed to identify two different carbonic anhydrase isoforms, suggesting that the gamma-carbonic anhydrase domain of maize complex I most likely is a heterotrimer. Statistical analysis indicates that the maize complex I structure is heterogeneous: a less-abundant "type II" particle has a 15 angstrom shorter membrane arm and an additional small protrusion on the intermembrane-side of the membrane arm if compared to the more abundant "type P particle. The I+III2 supercomplex was found to be a rigid structure which did riot break down into subcomplexes at the interface between the hydrophilic and the hydrophobic arms of complex I. The complex I moiety of the supercomplex appears to be only of "type I". This would mean that the "type II" particles are not involved in the supercomplex formation and, hence, could have a different physiological role. (C) 2007 Elsevier B.V. All rights reserved.

Mycotoxin producing capability greatly varies within species. In theory, the major source of this variability is meiotic recombination. However, a number of important toxigenic species have no known sexual stage and, therefore, the origin of the intraspecific diversity in these fungi is poorly understood. Mating in sexually reproducing Ascomycetes is controlled by MAT genes, but fungi with no known sexual stage also may have fully functional, constitutively transcribed mating type genes. The MAT genes, MAT1-1-1 and MAT1-2-1 encode putative transcription factors which, besides regulating pheromone and pheromone receptor genes, may affect other genes not involved directly in the mating process. By comparing the transcript profiles of a ΔMAT1-2-1 knock-out mutant and the wild type of Fusarium verticillioides , more than 200 ESTs, either down- or up-regulated in the mutant, were identified. Sequences encoding proteins involved in protein synthesis and metabolism occurred more frequently among ESTs up-regulated in the mutant, while sequences involved in cell signaling and communication were more frequent in the down-regulated subset of ESTs. The lack of fertility in fungi with no known sexual stage and the limited fertility of local populations of sexually reproducing fungi are probably due to changes in one or more of numerous genes that cause female sterility. A number of gene disruption mutants of Fusarium proliferatum were assessed for their mating capabilities. Fphch (a Het-C homologue), Fpmtr (an amino acid transporter gene), and Fpnitr1 (a putative nitrilase encoding gene) encode proteins in seemingly unrelated pathways, but mutations at any of these loci can reduce female fertility. Thus, a number of genes, with functions not related directly to mating, can influence the frequency of sexual reproduction indicating that this process requires the concerted operation of many factors not obviously connected to female fertility/sterility.

Mating type in filamentous ascomycetes is controlled by idiomorphic alleles, named MAT1-1 and MAT1-2, which contain 1-3 genes. Of these genes MAT1-1-1 and MAT1-2-1 encode putative transcription factors and are thus considered to be the major regulators of sexual communication and mating. Fungi with no known sexual stage may also have fully functional mating type genes and therefore it was plausible to hypothesize that the MAT products may also regulate other types of genes not involved directly in the mating process. To identify putative target genes of these transcription factors in Fusarium verticillioides, DeltaMAT1-2-1 knock out mutants were produced and transcript profiles of mutant and wild type were compared by means of differential cDNA hybridization. Clones, either up- or down-regulated in the DeltaMAT1-2-1 mutant were sequenced and a total of 248 sequences were blasted against the NCBI database as well as the Gibberella zeae and Gibberella moniliformis genomes. Fifty-five percent of the clones were down-regulated in the mutant, indicating that the MAT1-2-1 product positively affected these tagged sequences. On the other hand, 45% were found to be up-regulated in the mutant, suggesting that the MAT1-2-1 product also exerted a negative regulatory function on this set of genes. Sequences involved in protein synthesis and metabolism occurred more frequently among the clones up-regulated in the mutant, whereas genes belonging to cell signalling and communication were especially frequently tagged among the sequences down-regulated in the mutant.

The mimp1 element previously identified in the ascomycete fungus Fusarium oxysporum has hallmarks of miniature inverted-repeat transposable elements (MITEs): short size, terminal inverted repeats (TIRs), structural homogeneity, and a stable secondary structure. Since mimp1 has no coding capacity, its mobilization requires a transposase-encoding element. On the basis of the similarity of TIRs and target-site preference with the autonomous Tcl-like element impala, together with a correlated distribution of both elements among the Fusarium genus, we investigated the ability of mimp1 to jump upon expression of the impala transposase provided in trans. Under these conditions, we present evidence that mimp1 transposes by a cut-and-paste mechanism into TA dinucleoticles, which are duplicated upon insertion. Our results also show that mimp1 reinserts very frequently in genic regions for at least one-third of the cases. We also show that the mimp1/impala, double-component system is fully functional in the heterologous species F. graminearum, allowing the development of a highly efficient tool for gene tagging in filamentous fungi.

Aspects of manganese metabolism during normal and acidogenic growth of Aspergillus niger were explored. Arginase from this fungus was a Mn[II]-enzyme. The contribution of the arginase protein towards A. niger manganese metabolism was investigated using arginase knockout (D-42) and arginase over-expressing (Delta XCA-29) strains of A. niger NCIM 565. The Mn[II] contents of various mycelial fractions were found in the order: D-42 strain < parent strain < Delta XCA-29 strain. While the soluble fraction forms 60 % of the total mycelial Mn[II] content, arginase accounted for a significant fraction of this soluble Mn[II] pool. Changes in the arginase levels affected the absolute mycelial Mn[II] content but not its distribution in the various mycelial fractions. The A. niger mycelia harvested from acidogenic growth media contain substantially less Mn[II] as compared to those from normal growth media. Nevertheless, acidogenic mycelia harbor considerable Mn[II] levels and a functional arginase. Altered levels of mycelial arginase protein did not significantly influence citric acid production. The relevance of arginase to cellular Mn[II] pool and homeostasis was evaluated and the results suggest that arginase regulation could occur via manganese availability.

Background The understanding of enzymatic polysaccharide degradation has progressed intensely in the past few years with the identification of a new class of fungal-secreted enzymes, the lytic polysaccharide monooxygenases (LPMOs) that enhance cellulose conversion. In the fungal kingdom, saprotrophic fungi display a high number of genes encoding LPMOs from family AA9 but the functional relevance of this redundancy is not fully understood. Results In this study, we investigated a set of AA9 LPMOs identified in the secretomes of the coprophilous ascomycete Podospora anserina, a biomass degrader of recalcitrant substrates. Their activity was assayed on cellulose in synergy with the cellobiose dehydrogenase from the same organism. We showed that the total release of oxidized oligosaccharides from cellulose was higher for PaLPMO9A, PaLPMO9E, and PaLPMO9H that harbored a carbohydrate-binding module from the family CBM1. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and β-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan). Conclusions This study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0274-3) contains supplementary material, which is available to authorized users.

International audience; The ascomycete fungus Ophiostoma novo-ulmi is responsible for the pandemic of Dutch elm disease that has been ravaging Europe and North America for 50 years. We proceeded to annotate the genome of the O. novo-ulmi strain H327 that was sequenced in 2012. The 31.784-Mb nuclear genome (50.1% GC) is organized into 8 chromosomes containing a total of 8,640 protein-coding genes that we validated with RNA sequencing analysis. Approximately 53% of these genes have their closest match to Grosmannia clavigera kw1407, followed by 36% in other close Sordariomycetes, 5% in other Pezizomycotina, and surprisingly few (5%) orphans. A relatively small portion (~3.4%) of the genome is occupied by repeat sequences; however, the mechanism of repeat-induced point mutation appears active in this genome. Approximately 76% of the proteins could be assigned functions using Gene Ontology analysis; we identified 311 carbohydrate-active enzymes, 48 cytochrome P450s, and 1,731 proteins potentially involved in pathogen– host interaction, along with 7 clusters of fungal secondary metabolites. Complementary mating-type locus sequencing, mating tests, and culturing in the presence of elm terpenes were conducted. Our analysis identified a specific genetic arsenal impacting the sexual and vegetative growth, phytopathogenicity, and signaling/plant–defense–degradation relationship between O. novo-ulmi and its elm host and insect vectors. Introduction During the last centuries, increased movements of people and goods across countries and continents have favored the emergence and global spread of plant pathogens, insect pests, and invasive weeds which have substantially altered the landscape of several parts of the world. One well-documented example is Dutch elm disease (DED), the most destructive disease of elms. It has been estimated that over 1 billion mature elms were killed by two successive pandemics since the early 1900s (Paoletti et al. 2005). The first pandemic, which prompted initial investigations by Dutch scientists shortly after the First World War (Holmes and Heybroek 1990), was caused by the ascomycete fungus Ophiostoma ulmi (Buisman) Nannf. As it spread relentlessly over Western Europe and, a few decades

Allorecognition, the ability to distinguish “self” from “nonself” based on allelic differences at allorecognition loci, is common in all domains of life. Allorecognition restricts the opportunities for social parasitism, and is therefore crucial for the evolution of cooperation. However, the maintenance of allorecognition diversity provides a paradox. If allorecognition is costly relative to cooperation, common alleles will be favored. Thus, the cost of allorecognition may reduce the genetic variation upon which allorecognition crucially relies, a prediction now known as “Crozier's paradox.” We establish the relative costs of allorecognition, and their consequences for the short-term evolution of recognition labels theoretically predicted by Crozier. We use fusion among colonies of the fungus Neurospora crassa, regulated by highly variable allorecognition genes, as an experimental model system. We demonstrate that fusion among colonies is mutually beneficial, relative to absence of fusion upon allorecognition. This benefit is due not only to absence of mutual antagonism, which occurs upon allorecognition, but also to an increase in colony size per se. We then experimentally demonstrate that the benefit of fusion selects against allorecognition diversity, as predicted by Crozier. We discuss what maintains allorecognition diversity

The daily recurrence of activity and rest are so common as to seem trivial. However, they reflect a ubiquitous temporal programme called the circadian clock. In the absence of either anatomical clock structures or clock genes, the timing of sleep and wakefulness is disrupted. The complex nature of circadian behaviour is evident in the fact that phasing of the cycle during the day varies widely for individuals, resulting in extremes colloquially called 'larks' and 'owls'. These behavioural oscillations are mirrored in the levels of physiology and gene expression. Deciphering the underlying mechanisms will provide important insights into how the circadian clock affects health and disease.

The daily recurrence of activity and rest are so common as to seem trivial. However, they reflect a ubiquitous temporal programme called the circadian clock. In the absence of either anatomical clock structures or clock genes, the timing of sleep and wakefulness is disrupted. The complex nature of circadian behaviour is evident in the fact that phasing of the cycle during the day varies widely for individuals, resulting in extremes colloquially called 'larks' and 'owls'. These behavioural oscillations are mirrored in the levels of physiology and gene expression. Deciphering the underlying mechanisms will provide important insights into how the circadian clock affects health and disease.

Aim: To understand the conditions promoting activation and germination of spores, and to contribute to the control of tempe starters. Methods and Results: Using microscopic counts of fluorescent labelled spores, the following results were obtained: (1) L-alanine plays an important role (of the same order as that of peptone) in stimulation of germination of dormant spores. Alanine can satisfy the requirements of carbon as well as nitrogen for spore germination; (2) L-proline, on the other hand, inhibits alanine uptake presumably by blocking/congesting transporters of spore cells, resulting in apparent low viability on agar media; (3) L-leucine and L-isoleucine slightly favour spore germination while L-arginine and L-lysine do not have any stimulating effect; (4) The stimulatory role of glucose was only evident in the presence of phosphate (in minimal medium); when glucose is used in the absence of phosphate, either alone or in combination with single amino acids its role is hardly distinguishable; (5) Phosphate plays a facilitating role in spore germination. Conclusions: Glucose and amino acids play important roles in activation and germination of sporangiospores of Rhizopus oligosporus in tempe starter (stored for 12 months). The ability and rate of germination of dormant/old sporangiospores of R. oligosporus, depend on their ability for uptake of individual amino acids and/or glucose. Significance and Impact of Study: New light was shed on the counteractive role of proline and the stimulating effect of phosphate. Soybeans subjected to traditional preparation for tempe making are heavily leached; germination of starter spores on such beans is sub-optimal, and bean processing could be optimized.

The gene encoding an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum was cloned, functionally expressed and characterized in Saccharomyces cerevisiae M4276. The permease, designated PcMtr, is structurally and functionally homologous to Mtr of Neurospora crassa, and unrelated to the Amino Acid Permease (AAP) family which includes most amino acid permeases in fungi. Database searches of completed fungal genome sequences reveal that Mtr type permeases are not widely distributed among fungi, suggesting a specialized function. (C) 2004 Elsevier B.V. All rights reserved.

The overall structure of V-ATPase complexes resembles that of F-type ATPases, but the stalk region is different and more complex. Database searches followed by sequence analysis of the five water-soluble stalk region subunits C-G revealed that (i) to date V-ATPases are found in 16 bacterial species, (ii) bacterial V-ATPases are closer to archaeal A-ATPases than to eukaryotic V-ATPases, and (iii) different groups of bacterial V-ATPases exist. Inconsistencies in the nomenclature of types and subunits are addressed. Attempts to assign subunit positions in V-ATPases based on biochemical experiments, chemical cross-linking, and electron microscopy are discussed. A structural model for prokaryotic and eukaryotic V-ATPases is proposed. The prokaryotic V-ATPase is considered to have a central stalk between headpiece and membrane flanked by two peripheral stalks. The eukaryotic V-ATPases have one additional peripheral stalk.

This review describes the different approaches that have been used to manipulate and improve carotenoid production in Xanthophyllomyces dendrorhous. The red yeast X dendrorhous (formerly known as Phaffia rhodozyma) is one of the microbiological production systems for natural astaxanthin. Astaxanthin is applied in food and feed industry and can be used as a nutraceutical because of its strong antioxidant properties. However, the production levels of astaxanthin in wild-type isolates are rather low. To increase the astaxanthin content in X. dendrorhous, cultivation protocols have been optimized and astaxanthin-hyperproducing mutants have been obtained by screening of classically mutagenized X. dendrorhous strains. The knowledge about the regulation of carotenogenesis in X dendrorhous is still limited in comparison to that in other carotenogenic fungi. The X dendrorhous carotenogenic genes have been cloned and a X dendrorhous transformation system has been developed. These tools allowed the directed genetic modification of the astaxanthin pathway in X. dendrorhous. The crt YB gene, encoding the bifunctional enzyme phytoene synthase/lycopene cyclase, was inactivated by insertion of a vector by single and double cross-over events, indicating that it is possible to generate specific carotenoid-biosynthetic mutants. Additionally, overexpression of crt YB resulted in the accumulation of P-carotene and echinone, which indicates that the oxygenation reactions are rate-limiting in these recombinant strains. Furthermore, overexpression of the phytoene desaturase-encoding gene (crt1) showed an increase in monocyclic carotenoids such as torulene and HDCO (3-hydroxy-3',4'-didehydro-beta,-psi-carotene-4-one) and a decrease in bicyclic carotenoids such as echinone, P-carotene and astaxanthin. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

The promoter of avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum contains 12 sequences within a region of 0.6 kb that are rermniscent of the binding sequences of the GATA-type regulator involved in nitrogen utilisation of the filamentous fungi Aspergillus nidulans and Neurospora crassa. Mutational analysis of this 0.6-kb promoter region, fused to the beta-glueuronidase reporter gene, revealed that two regions, each containing two TAGATA boxes in inverted orientation and overlapping by two base pairs, are important for induction of Avr9 promoter activity in A. nidulans. Each overlapping TAGATA box differentially affected Avr9 promoter activity when shifted apart by nucleotide insertions. The other regions, which do not contain two overlapping TAGATA boxes have no, or only a limited, contribution to the inducibility of promoter activity.

Background: RNA interference (RNAi) is involved in genome defense as well as diverse cellular, developmental, and physiological processes. Key components of RNAi are Argonaute, Dicer, and RNA-dependent RNA polymerase (RdRP), which have been functionally characterized mainly in model organisms. The key components are believed to exist throughout eukaryotes; however, there is no systematic platform for archiving and dissecting these important gene families. In addition, few fungi have been studied to date, limiting our understanding of RNAi in fungi. Here we present funRNA http://funrna.riceblast.snu.ac.kr/, a fungal kingdom-wide comparative genomics platform for putative genes encoding Argonaute, Dicer, and RdRP. Description: To identify and archive genes encoding the abovementioned key components, protein domain profiles were determined from reference sequences obtained from UniProtKB/SwissProt. The domain profiles were searched using fungal, metazoan, and plant genomes, as well as bacterial and archaeal genomes. 1,163, 442, and 678 genes encoding Argonaute, Dicer, and RdRP, respectively, were predicted. Based on the identification results, active site variation of Argonaute, diversification of Dicer, and sequence analysis of RdRP were discussed in a fungus-oriented manner. funRNA provides results from diverse bioinformatics programs and job submission forms for BLAST, BLASTMatrix, and ClustalW. Furthermore, sequence collections created in funRNA are synced with several gene family analysis portals and databases, offering further analysis opportunities. Conclusions: funRNA provides identification results from a broad taxonomic range and diverse analysis functions, and could be used in diverse comparative and evolutionary studies. It could serve as a versatile genomics workbench for key components of RNAi.

International audience; A few yeasts, including Hansenula polymorpha are able to assimilate nitrate and use it as nitrogen source. The genes necessary for nitrate assimilation are organised in this organism as a cluster comprising those encoding nitrate reductase (YNR1), nitrite reductase (YNI1), a high affinity transporter (YNT1), as well as the two pathway specific Zn(II)2Cys2 transcriptional activators (YNA1, YNA2). Yna1p and Yna2p mediate induction of the system and here we show that their functions are interdependent. Yna1p activates YNA2 as well as its own (YNA1) transcription thus forming a nitrate-dependent autoactivation loop. Using a split-YFP approach we demonstrate here that Yna1p and Yna2p form a heterodimer independently of the inducer and despite both Yna1p and Yna2p can occupy the target promoter as mono- or homodimer individually, these proteins are transcriptionally incompetent. Subsequently, the transcription factors target genes containing a conserved DNA motif (termed nitrate-UAS) determined in this work by in vitro and in vivo protein-DNA interaction studies. These events lead to a rearrangement of the chromatin landscape on the target promoters and are associated with the onset of transcription of these target genes. In contrast to other fungi and plants, in which nuclear accumulation of the pathway-specific transcription factors only occur in the presence of nitrate, Yna1p and Yna2p are constitutively nuclear in H. polymorpha. Yna2p is needed for this nuclear accumulation and Yna1p is incapable of strictly positioning in the nucleus without Yna2p. In vivo DNA footprinting and ChIP analyses revealed that the permanently nuclear Yna1p/Yna2p heterodimer only binds to the nitrate-UAS when the inducer is present. The nitrate-dependent up-regulation of one partner protein in the heterodimeric complex is functionally similar to the nitrate-dependent activation of nuclear accumulation in other systems.

In filamentous fungi, allorecognition takes the form of heterokaryon incompatibility, a cell death reaction triggered when genetically distinct hyphae fuse. Heterokaryon incompatibility is controlled by specific loci termed het-loci. In this article, we analyzed the natural variation in one such fungal allorecognition determinant, the het-c heterokaryon incompatibility locus of the filamentous ascomycete Podospora anserina. The het-c locus determines an allogenic incompatibility reaction together with two unlinked loci termed het-d and het-e. Each het-c allele is incompatible with a specific subset of the het-d and het-e alleles. We analyzed variability at the het-c locus in a population of 110 individuals, and in additional isolates from various localities. We identified a total of 11 het-c alleles, which define 7 distinct incompatibility specificity classes in combination with the known het-d and het-e alleles. We found that the het-c allorecognition gene of P. anserina is under diversifying selection. We find a highly unequal allele distribution of het-c in the population, which contrasts with the more balanced distribution of functional groups of het-c based on their allorecognition function. One explanation for the observed het-c diversity in the population is its function in allorecognition. However, alleles that are most efficient in allorecognition are rare. An alternative and not exclusive explanation for the observed diversity is that het-c is involved in pathogen recognition. In Arabidopsis thaliana, a homolog of het-c is a pathogen effector target, supporting this hypothesis. We hypothesize that the het-c diversity in P. anserina results from both its functions in pathogen-defense, and allorecognition.

The actin cytoskeleton is a dynamic but well organized intracellular framework that is essential for proper functioning of eukaryotic cells. Here, we use the actin binding peptide Lifeact to investigate the in vivo actin cytoskeleton dynamics in the oomycete plant pathogen Phytophthora infestans. Lifeact-eGFP labelled thick and thin actin bundles and actin filament plaques allowing visualization of actin dynamics. All actin structures in the hyphae were cortically localized. In growing hyphae actin filament cables were axially oriented in the sub-apical region whereas in the extreme apex in growing hyphae, waves of fine F-actin polymerization were observed. Upon growth termination, actin filament plaques appeared in the hyphal tip. The distance between a hyphal tip and the first actin filament plaque correlated strongly with hyphal growth velocity. The actin filament plaques were nearly immobile with average lifetimes exceeding one hour, relatively long when compared to the lifetime of actin patches known in other eukaryotes. Plaque assembly required ~30 seconds while disassembly was accomplished in ~10 sec. Remarkably, plaque disassembly was not accompanied with internalization and the formation of endocytic vesicles. These findings suggest that the functions of actin plaques in oomycetes differ from those of actin patches present in other organisms.

Although fungi have contributed tremendously to understanding biological phenomena common to all eukaryotic organisms, some of their properties testify as to their uniqueness. Among these are growth by apical extension of hyphae, the manufacture of hydrophobins for emergence into the air, and the possession of an extended somatic heterokaryon in basidiomycetes. This justifies studies on the molecular basis of development aimed particularly at this group of organisms, which are of great importance to life on earth and human society. (C) 1999 Academic Press.

In industrial fermentations, Penicillium chrysogenum uses sulfate as the source of sulfur for the biosynthesis of penicillin. By a PCR-based approach, two genes, sutA and sutB, whose encoded products belong to the SulP superfamily of sulfate permeases were isolated. Transformation of a sulfate uptake-negative sB3 mutant of Aspergillus nidulans with the sutB gene completely restored sulfate uptake activity. The sutA gene did not complement the A. nidulans sB3 mutation, even when expressed under control of the sutB promoter. Expression of both sutA and sutB in P. chrysogenum is induced by growth under sulfur starvation conditions. However, sutA is expressed to a much lower level than is sutB. Disruption of sutB resulted in a loss of sulfate uptake ability. Overall, the results show that SutB is the major sulfate permease involved in sulfate uptake by P. chrysogenum.

After introduction of extra copies of the SC3 hydrophobin gene into a wild-type strain of Schizophyllum commune, gene silencing was observed acting on both endogenous and introduced SC3 genes in primary vegetative transformants. Nuclear run-on experiments indicated that silencing acted at the transcriptional level. Southern analysis revealed that cytosine methylation of genomic DNA occurred. Moreover, SC3 silencing was suppressed by exposure to 5-azacytidine during growth. After growth of SC3-suppressed colonies from homogenized mycelium or from colonies stored at 4 degrees, SC3 transcription was restored. However, after prolonged growth SC3 silencing was again observed. Introduction of a promoterless SC3 fragment into wild type gave less SC3 silencing.

Information on the genetics and metabolism of 4-aminobutyrate (GABA) in yeasts and fungi is reviewed. In spite of ubiquitous occurrence, there is limited information on its function and biological role. Most fungi utilize GABA both as a carbon and a nitrogen source. Fungal endogenous GABA largely originates from the decarboxylation of L-glutamate and is associated with sporulation/spore metabolism. Whatever its source, GABA is catabolized to succinate via succinicsemialdehyde. Taken together these steps define a potential bypass outside the classical tricarboxylic acid cycle. Evidence for the existence of such a functional bypass in fungi is reviewed. The role of GABA and its metabolism in various facets of fungal biology is gradually emerging.

This work was supported by the Irish Department of Agriculture, Fisheries & Food Research Stimulus Fund (RSF 07 513). peer-reviewed Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km(glucose) was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing. This work was supported by the Irish Department of Agriculture, Fisheries & Food Research Stimulus Fund (RSF 07 513).

Repetitive DNA elements are ubiquitous constituents of eukaryotic genomes. The biological roles of these repetitive elements, supposed to impact genome organization and evolution, are not completely elucidated yet. The availability of whole genome sequence offers the opportunity to draw a picture of the genome-wide distribution of these elements and provide insights into potential mechanisms of genome plasticity. The present study uses in silico approaches to describe tandem repeats and transposable elements distribution in the genome of the button mushroom, Agaricus bisporus . Transposable elements comprised 12.43% of the assembled genome, and 66% of them were found clustered in the centromeric or telomeric regions. Methylation of retrotransposon has been demonstrated. A total of 1996 mini-, 4062 micro-, and 37 satellites motifs were identified. The microsatellites appeared widely and evenly spread over the whole genome sequence, whereas the minisatellites were not randomly distributed. Indeed, minisatellites were found to be associated with transposable elements clusters. Telomeres exhibited a specific sequence with a T n AG n signature. A comparison between the two available genome sequences of A. bisporus was also performed and sheds light on the genetic divergence between the two varieties. Beyond their role in genome structure, repeats provide a virtually endless source of molecular markers useful for genetic studies in this cultivated species.

Transport studies with Penicillium chrysogenum plasma membranes fused with cytochrome c oxidase liposomes demonstrate that sulfate uptake is driven by the transmembrane pH gradient and not by the transmembrane electrical potential. Ca2+ and other divalent cations are not required. It is concluded that the sulfate transport system catalyzes the symport of two protons with one sulfate anion.

Hyphae of higher fungi are compartmentalized by septa. These septa contain a central pore that allows for inter-compartmental and inter-hyphal cytoplasmic streaming. The cytoplasm within the mycelium is therefore considered to be a continuous system. In this study, however, we demonstrate by laser dissection that 40% of the apical septa of exploring hyphae of Aspergillus oryzae are closed. Closure of septa correlated with the presence of a peroxisome-derived organelle, known as Woronin body, near the septal pore. The location of Woronin bodies in the hyphae was dynamic and, as a result, plugging of the septal pore was reversible. Septal plugging was abolished in a ?Aohex1 strain that cannot form Woronin bodies. Notably, hyphal heterogeneity was also affected in the ?Aohex1 strain. Wild-type strains of A.?oryzae showed heterogeneous distribution of GFP between neighbouring hyphae at the outer part of the colony when the reporter was expressed from the promoter of the glucoamylase gene glaA or the a-glucuronidase gene aguA. In contrast, GFP fluorescence showed a normal distribution in the case of the ?Aohex1 strain. Taken together, it is concluded that Woronin bodies maintain hyphal heterogeneity in a fungal mycelium by impeding cytoplasmic continuity.