The Sch9 Kinase Regulates Conidium Size,Stress Responses,and Pathogenesis in Fusarium graminearum

Fusarium head blight caused by Fusarium graminearum is an important disease of wheat and barley worldwide. In a previous study on functional characterization of the F. graminearum kinome, one protein kinase gene important for virulence is orthologous to SCH9 that is functionally related to the cAMP-PKA and TOR pathways in the budding yeast. In this study, we further characterized the functions of FgSCH9 in F. graminearum and its ortholog in Magnaporthe oryzae. The ΔFgsch9 mutant was slightly reduced in growth rate but significantly reduced in conidiation, DON production, and virulence on wheat heads and corn silks. It had increased tolerance to elevated temperatures but became hypersensitive to oxidative, hyperosmotic, cell wall, and membrane stresses. The ΔFgsch9 deletion also had conidium morphology defects and produced smaller conidia. These results suggest that FgSCH9 is important for stress responses, DON production, conidiogenesis, and pathogenesis in F. graminearum. In the rice blast fungus Magnaporthe oryzae, the ΔMosch9 mutant also was defective in conidiogenesis and pathogenesis. Interestingly, it also produced smaller conidia and appressoria. Taken together, our data indicate that the SCH9 kinase gene may have a conserved role in regulating conidium size and plant infection in phytopathogenic ascomycetes.     

The FgNot3 Subunit of the Ccr4-Not Complex Regulates Vegetative Growth,Sporulation, and Virulence in Fusarium graminearum

The Ccr4-Not complex is evolutionarily conserved and important for multiple cellular functions in eukaryotic cells. In this study, the biological roles of the FgNot3 subunit of this complex were investigated in the plant pathogenic fungus Fusarium graminearum. Deletion of FgNOT3 resulted in retarded vegetative growth, retarded spore germination, swollen hyphae, and hyper-branching. The ΔFgnot3 mutants also showed impaired sexual and asexual sporulation, decreased virulence, and reduced expression of genes related to conidiogenesis. Fgnot3 deletion mutants were sensitive to thermal stress, whereas NOT3 orthologs in other model eukaryotes are known to be required for cell wall integrity. We found that FgNot3 functions as a negative regulator of the production of secondary metabolites, including trichothecenes and zearalenone. Further functional characterization of other components of the Not module of the Ccr4-Not complex demonstrated that the module is conserved. Each subunit primarily functions within the context of a complex and might have distinct roles outside of the complex in F. graminearum. This is the first study to functionally characterize the Not module in filamentous fungi and provides novel insights into signal transduction pathways in fungal development.     

Molecular characterization of HymA, an evolutionarily highly conserved and highly expressed protein of Aspergillus nidulans

Aspergillus nidulans reproduces asexually via uninucleate, haploid spores, which are produced on morphologically differentiated aerial structures, called conidiophores. These consist of four distinct cell types, a foot with a terminally swollen stalk, metulae, phialides and conidiospores. The molecular mechanisms underlying the morphological changes that occur during conidiophore development have been studied by mutant analysis. We have isolated the hymA mutant, in which conidiophore development is affected at the metula stage. In the mutant metulae do not differentiate properly but come to resemble hyphae (hym?=?hypha-like metulae). In this paper we have analyzed the corresponding gene. It encodes a highly expressed 44?kDa protein which resides in the cytoplasm and has homologues in yeast, plants, fly, worm, fish, mice and man. We constructed hym deletion strains of Saccharomyces cerevisiae and of A. nidulans and found that the gene is essential in S. cerevisiae but is dispensable in the filamentous fungus. A cellular function for the Hym protein has not yet been defined in any organism. To demonstrate functional conservation we constructed a chimeric protein comprised of the N-terminal half of the A.?nidulans and the C-terminal half of the mouse homologue MO25. This hybrid protein could fully substitute for HymA function in A. nidulans. In addition, the mouse protein itself partially rescued the hymA mutation in the fungus. HymA is thus highly conserved in evolution and probably serves similar functions. The fact that hymA is required for conidiophore development in A. nidulans suggests that homologous genes in other organisms might also be involved in morphogenesis.     

Lichen-like association of Chlamydomonas reinhardtii and Aspergillus nidulans protects algal cells from bacteria

Organismal interactions within microbial consortia and their responses to harmful intruders remain largely understudied. An important step toward the goal of understanding functional ecological interactions and their evolutionary selection is the study of increasingly complex microbial interaction systems. Here, we discovered a tripartite biosystem consisting of the fungus Aspergillus nidulans, the unicellular green alga Chlamydomonas reinhardtii, and the algicidal bacterium Streptomyces iranensis. Genetic analyses and MALDI-IMS demonstrate that the bacterium secretes the algicidal compound azalomycin F upon contact with C. reinhardtii. In co-culture, A. nidulans attracts the motile alga C. reinhardtii, which becomes embedded and surrounded by fungal mycelium and is shielded from the algicide. The filamentous fungus Sordaria macrospora was susceptible to azalomycin F and failed to protect C. reinhardtii despite chemotactically attracting the alga. Because S. macrospora was susceptible to azalomycin F, this data imply that for protection the fungus needs to be resistant. Formation of the lichen-like association between C. reinhardtii and A. nidulans increased algal growth. The protection depends on the increased amounts of membrane lipids provided by resistant fungi, thereby generating a protective shelter against the bacterial toxin. Our findings reveal a strategy whereby algae survive lethal environmental algicides through cooperation with fungi.Subject terms: Microbial ecology, Microbiome, Microbial ecology, Antibiotics, Fungal ecology     

A Network Approach to Predict Pathogenic Genes for Fusarium graminearum

Fusarium graminearum is the pathogenic agent of Fusarium head blight (FHB), which is a destructive disease on wheat and barley, thereby causing huge economic loss and health problems to human by contaminating foods. Identifying pathogenic genes can shed light on pathogenesis underlying the interaction between F. graminearum and its plant host. However, it is difficult to detect pathogenic genes for this destructive pathogen by time-consuming and expensive molecular biological experiments in lab. On the other hand, computational methods provide an alternative way to solve this problem. Since pathogenesis is a complicated procedure that involves complex regulations and interactions, the molecular interaction network of F. graminearum can give clues to potential pathogenic genes. Furthermore, the gene expression data of F. graminearum before and after its invasion into plant host can also provide useful information. In this paper, a novel systems biology approach is presented to predict pathogenic genes of F. graminearum based on molecular interaction network and gene expression data. With a small number of known pathogenic genes as seed genes, a subnetwork that consists of potential pathogenic genes is identified from the protein-protein interaction network (PPIN) of F. graminearum, where the genes in the subnetwork are further required to be differentially expressed before and after the invasion of the pathogenic fungus. Therefore, the candidate genes in the subnetwork are expected to be involved in the same biological processes as seed genes, which imply that they are potential pathogenic genes. The prediction results show that most of the pathogenic genes of F. graminearum are enriched in two important signal transduction pathways, including G protein coupled receptor pathway and MAPK signaling pathway, which are known related to pathogenesis in other fungi. In addition, several pathogenic genes predicted by our method are verified in other pathogenic fungi, which demonstrate the effectiveness of the proposed method. The results presented in this paper not only can provide guidelines for future experimental verification, but also shed light on the pathogenesis of the destructive fungus F. graminearum.     

The Role,Interaction and Regulation of the Velvet Regulator VelB in Aspergillus nidulans

The multifunctional regulator VelB physically interacts with other velvet regulators and the resulting complexes govern development and secondary metabolism in the filamentous fungus Aspergillus nidulans. Here, we further characterize VelB’s role in governing asexual development and conidiogenesis in A. nidulans. In asexual spore formation, velB deletion strains show reduced number of conidia, and decreased and delayed mRNA accumulation of the key asexual regulatory genes brlA, abaA, and vosA. Overexpression of velB induces a two-fold increase of asexual spore production compared to wild type. Furthermore, the velB deletion mutant exhibits increased conidial germination rates in the presence of glucose, and rapid germination of conidia in the absence of external carbon sources. In vivo immuno-pull-down analyses reveal that VelB primarily interacts with VosA in both asexual and sexual spores, and VelB and VosA play an inter-dependent role in spore viability, focal trehalose biogenesis and control of conidial germination. Genetic and in vitro studies reveal that AbaA positively regulates velB and vosA mRNA expression during sporogenesis, and directly binds to the promoters of velB and vosA. In summary, VelB acts as a positive regulator of asexual development and regulates spore maturation, focal trehalose biogenesis and germination by interacting with VosA in A. nidulans.     

The White Collar Complex Is Involved in Sexual Development of Fusarium graminearum

Sexual spores (ascospores) of Fusarium graminearum, a homothallic ascomycetous fungus, are believed to be the primary inocula for epidemics of the diseases caused by this species in cereal crops. Based on the light requirement for the formation of fruiting bodies (perithecia) of F. graminearum under laboratory conditions, we explored whether photoreceptors play an important role in sexual development. Here, we evaluated the roles of three genes encoding putative photoreceptors [a phytochrome gene (FgFph) and two white collar genes (FgWc-1 and FgWc-2)] during sexual development in F. graminearum. For functional analyses, we generated transgenic strains lacking one or two genes from the self-fertile Z3643 strain. Unlike the wild-type (WT) and add-back strains, the single deletion strains (ΔFgWc-1 and ΔFgWc-2) produced fertile perithecia under constant light on complete medium (CM, an unfavorable medium for sexual development) as well as on carrot agar (a perithecial induction condition). The expression of mating-type (MAT) genes increased significantly in the gene deletion strains compared to the WT under both conditions. Deletion of FgFph had no significant effect on sexual development or MAT gene expression. In contrast, all of the deletion strains examined did not show significant changes in other traits such as hyphal growth, mycotoxin production, and virulence. A split luciferase assay confirmed the in vivo protein-protein interactions among three photoreceptors along with FgLaeA, a global regulator of secondary metabolism and fungal development. Introduction of an intact copy of the A. nidulans LreA and LreB genes, which are homologs of FgWc-1 and FgWc-2, into the ΔFgWc-1 and ΔFgWc-2 strains, respectively, failed to repress perithecia formation on CM in the gene deletion strains. Taken together, these results demonstrate that FgWc-1 and FgWc-2, two central components of the blue-light sensing system, negatively regulate sexual development in F. graminearum, which differs from the regulation pattern in A. nidulans.     

Breaking the Silence: Protein Stabilization Uncovers Silenced Biosynthetic Gene Clusters in the Fungus Aspergillus nidulans

The genomes of filamentous fungi comprise numerous putative gene clusters coding for the biosynthesis of chemically and structurally diverse secondary metabolites (SMs), which are rarely expressed under laboratory conditions. Previous approaches to activate these genes were based primarily on artificially targeting the cellular protein synthesis apparatus. Here, we applied an alternative approach of genetically impairing the protein degradation apparatus of the model fungus Aspergillus nidulans by deleting the conserved eukaryotic csnE/CSN5 deneddylase subunit of the COP9 signalosome. This defect in protein degradation results in the activation of a previously silenced gene cluster comprising a polyketide synthase gene producing the antibiotic 2,4-dihydroxy-3-methyl-6-(2-oxopropyl)benzaldehyde (DHMBA). The csnE/CSN5 gene is highly conserved in fungi, and therefore, the deletion is a feasible approach for the identification of new SMs.     

Functional evaluation of a homologue of plant rapid alkalinisation factor (RALF) peptides in Fusarium graminearum

The cereal infecting fungus Fusarium graminearum is predicted to possess a single homologue of plant RALF (rapid alkalinisation factor) peptides. Fusarium mutant strains lacking FgRALF were generated and found to exhibit wildtype virulence on wheat and Arabidopsis floral tissue. Arabidopsis lines constitutively overexpressing FgRALF exhibited no obvious change in susceptibility to F. graminearum leaf infection. In contrast transient virus-mediated over-expression (VOX) of FgRALF in wheat prior to F. graminearum infection, slightly increased the rate of fungal colonisation of floral tissue. Ten putative Feronia (FER) receptors of RALF peptide were identified bioinformatically in hexaploid wheat (Triticum aestivum). Transient silencing of two wheat FER homoeologous genes prior to F. graminearum inoculation did not alter the subsequent interaction outcome. Collectively, our VOX results show that the fungal RALF peptide may be a minor contributor in F. graminearum virulence but results from fungal gene deletion experiments indicate potential functional redundancy within the F. graminearum genome. We demonstrate that virus-mediated over-expression is a useful tool to provide novel information about gene/protein function when results from gene deletion/disruption experimentation were uninformative.     

The Wor1-like Protein Fgp1 Regulates Pathogenicity,Toxin Synthesis and Reproduction in the Phytopathogenic Fungus Fusarium graminearum

WOR1 is a gene for a conserved fungal regulatory protein controlling the dimorphic switch and pathogenicity determents in Candida albicans and its ortholog in the plant pathogen Fusarium oxysporum, called SGE1, is required for pathogenicity and expression of key plant effector proteins. F. graminearum, an important pathogen of cereals, is not known to employ switching and no effector proteins from F. graminearum have been found to date that are required for infection. In this study, the potential role of the WOR1-like gene in pathogenesis was tested in this toxigenic fungus. Deletion of the WOR1 ortholog (called FGP1) in F. graminearum results in greatly reduced pathogenicity and loss of trichothecene toxin accumulation in infected wheat plants and in vitro. The loss of toxin accumulation alone may be sufficient to explain the loss of pathogenicity to wheat. Under toxin-inducing conditions, expression of genes for trichothecene biosynthesis and many other genes are not detected or detected at lower levels in Δfgp1 strains. FGP1 is also involved in the developmental processes of conidium formation and sexual reproduction and modulates a morphological change that accompanies mycotoxin production in vitro. The Wor1-like proteins in Fusarium species have highly conserved N-terminal regions and remarkably divergent C-termini. Interchanging the N- and C- terminal portions of proteins from F. oxysporum and F. graminearum resulted in partial to complete loss of function. Wor1-like proteins are conserved but have evolved to regulate pathogenicity in a range of fungi, likely by adaptations to the C-terminal portion of the protein.     

An Isoprenylation and Palmitoylation Motif Promotes Intraluminal Vesicle Delivery of Proteins in Cells from Distant Species

The C-terminal ends of small GTPases contain hypervariable sequences which may be posttranslationally modified by defined lipid moieties. The diverse structural motifs generated direct proteins towards specific cellular membranes or organelles. However, knowledge on the factors that determine these selective associations is limited. Here we show, using advanced microscopy, that the isoprenylation and palmitoylation motif of human RhoB (–CINCCKVL) targets chimeric proteins to intraluminal vesicles of endolysosomes in human cells, displaying preferential co-localization with components of the late endocytic pathway. Moreover, this distribution is conserved in distant species, including cells from amphibians, insects and fungi. Blocking lipidic modifications results in accumulation of CINCCKVL chimeras in the cytosol, from where they can reach endolysosomes upon release of this block. Remarkably, CINCCKVL constructs are sorted to intraluminal vesicles in a cholesterol-dependent process. In the lower species, neither the C-terminal sequence of RhoB, nor the endosomal distribution of its homologs are conserved; in spite of this, CINCCKVL constructs also reach endolysosomes in Xenopus laevis and insect cells. Strikingly, this behavior is prominent in the filamentous ascomycete fungus Aspergillus nidulans, in which GFP-CINCCKVL is sorted into endosomes and vacuoles in a lipidation-dependent manner and allows monitoring endosomal movement in live fungi. In summary, the isoprenylated and palmitoylated CINCCKVL sequence constitutes a specific structure which delineates an endolysosomal sorting strategy operative in phylogenetically diverse organisms.     

An aureobasidin A resistance gene isolated from Aspergillus is a homolog of yeast AUR1, a gene responsible for inositol phosphorylceramide (IPC) synthase activity

The AUR1 gene of Saccharomyces cerevisiae, mutations in which confer resistance to the antibiotic aureobasidin A, is necessary for inositol phosphorylceramide (IPC) synthase activity. We report the molecular cloning and characterization of the Aspergillus nidulans aurA gene, which is homologous to AUR1. A single point mutation in the aurA gene of A. nidulans confers a high level of resistance to aureobasidin A. The A. nidulans aurA gene was used to identify its homologs in other Aspergillus species, including A. fumigatus, A. niger, and A. oryzae. The deduced amino acid sequence of an aurA homolog from the pathogenic fungus A. fumigatus showed 87% identity to that of A. nidulans. The AurA proteins of A. nidulans and A. fumigatus shared common characteristics in primary structure, including sequence, hydropathy profile, and N-glycosylation sites, with their S. cerevisiae, Schizosaccharomyces pombe, and Candida albicans counterparts. These results suggest that the aureobasidin resistance gene is conserved evolutionarily in various fungi. Received: 27 August 1998 / Accepted: 19 November 1998     

Protein Kinase C Overexpression Suppresses Calcineurin-Associated Defects in Aspergillus nidulans and Is Involved in Mitochondrial Function

In filamentous fungi, intracellular signaling pathways which are mediated by changing calcium levels and/or by activated protein kinase C (Pkc), control fungal adaptation to external stimuli. A rise in intracellular Ca²⁺ levels activates calcineurin subunit A (CnaA), which regulates cellular calcium homeostasis among other processes. Pkc is primarily involved in maintaining cell wall integrity (CWI) in response to different environmental stresses. Cross-talk between the Ca²⁺ and Pkc-mediated pathways has mainly been described in Saccharomyces cerevisiae and in a few other filamentous fungi. The presented study describes a genetic interaction between CnaA and PkcA in the filamentous fungus Aspergillus nidulans. Overexpression of pkcA partially rescues the phenotypes caused by a cnaA deletion. Furthermore, CnaA appears to affect the regulation of a mitogen-activated kinase, MpkA, involved in the CWI pathway. Reversely, PkcA is involved in controlling intracellular calcium homeostasis, as was confirmed by microarray analysis. Furthermore, overexpression of pkcA in a cnaA deletion background restores mitochondrial number and function. In conclusion, PkcA and CnaA-mediated signaling appear to share common targets, one of which appears to be MpkA of the CWI pathway. Both pathways also regulate components involved in mitochondrial biogenesis and function. This study describes targets for PkcA and CnaA-signaling pathways in an A. nidulans and identifies a novel interaction of both pathways in the regulation of cellular respiration.