Glycosylphosphatidylinositol-Anchored Proteins in Fusarium graminearum: Inventory,Variability, and Virulence

The contribution of cell surface proteins to plant pathogenicity of fungi is not well understood. As such, the objective of this study was to investigate the functions and importance of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the wheat pathogen F. graminearum. GPI-APs are surface proteins that are attached to either the membrane or cell wall. In order to simultaneously disrupt several GPI-APs, a phosphoethanolamine transferase-encoding gene gpi7 was deleted and the resultant mutant characterized in terms of growth, development, and virulence. The Δgpi7 mutants exhibited slower radial growth rates and aberrantly shaped macroconidia. Furthermore, virulence tests and microscopic analyses indicated that Gpi7 is required for ramification of the fungus throughout the rachis of wheat heads. In parallel, bioinformatics tools were utilized to predict and inventory GPI-APs within the proteome of F. graminearum. Two of the genes identified in this screen (FGSG_01588 and FGSG_08844) displayed isolate-specific length variability as observed for other fungal cell wall adhesion genes. Nevertheless, deletion of these genes failed to reveal obvious defects in growth, development, or virulence. This research demonstrates the global importance of GPI-APs to in planta proliferation in F. graminearum, and also highlights the potential of individual GPI-APs as diagnostic markers.     

The velvet gene, FgVe1, affects fungal development and positively regulates trichothecene biosynthesis and pathogenicity in Fusarium graminearum

Trichothecenes are a group of toxic secondary metabolites produced mainly by Fusarium graminearum (teleomorph: Gibberella zeae) during the infection of crop plants, including wheat, maize, barley, oats, rye and rice. Some fungal genes involved in trichothecene biosynthesis have been shown to encode regulatory proteins. However, the global regulation of toxin biosynthesis is still enigmatic. In addition to the production of secondary metabolites belonging to the trichothecene family, F. graminearum produces the red pigment aurofusarin. The gene regulation underlying the production of aurofusarin is not well understood. The velvet gene (veA) is conserved in various genera of filamentous fungi. Recently, the veA gene from Aspergillus nidulans has been shown to be the key component of the velvet complex regulating development and secondary metabolism. Using blast analyses, we identified the velvet gene from F. graminearum, FgVe1. Disruption of FgVe1 causes several phenotypic effects. However, the complementation of this mutant with the FgVe1 gene restores the wild-type phenotypes. The in vitro phenotypes include hyperbranching of the mycelium, suppression of aerial hyphae formation, reduced hydrophobicity of the mycelium and highly reduced sporulation. Our data also show that FgVe1 modulates the production of the aurofusarin pigment and is essential for the expression of Tri genes and the production of trichothecenes. Pathogenicity studies performed on flowering wheat plants indicate that FgVe1 is a positive regulator of virulence in F. graminearum.     

A mitogen-activated protein kinase gene (MGV1) in Fusarium graminearum is required for female fertility,heterokaryon formation,and plant infection

Fusarium graminearum is an important pathogen of small grains and maize in many areas of the world. Infected grains are often contaminated with mycotoxins harmful to humans and animals. During the past decade, F. graminearum has caused several severe epidemics of head scab in wheat and barley. In order to understand molecular mechanisms regulating fungal development and pathogenicity in this pathogen, we isolated and characterized a MAP kinase gene, MGV1, which is highly homologous to the MPS1 gene in Magnaporthe grisea. The MGV1 gene was dispensable for conidiation in F. graminearum but essential for female fertility during sexual reproduction. Vegetative growth of mgv1 deletion mutants was normal in liquid media but reduced on solid media. Mycelia of the mgv1 mutants had weak cell walls and were hypersensitive to cell wall degrading enzymes. Interestingly, the mgv1 mutants were self-incompatible when tested for heterokaryon formation, and their virulence was substantially reduced. The ability of the mutants to accumulate trichothecene mycotoxins on inoculated wheat was also greatly reduced. Our data suggest that MGV1 in F. graminearum is involved in multiple developmental processes related to sexual reproduction, plant infection, and cell wall integrity.     

Control of glucosylceramide production and morphogenesis by the Bar1 ceramide synthase in Fusarium graminearum

The contribution of plasma membrane proteins to the virulence of plant pathogenic fungi is poorly understood. Accordingly, the objective of this study was to characterize the acyl-CoA dependent ceramide synthase Bar1 (previously implicated in plasma membrane organization) in the wheat pathogen Fusarium graminearum. The role of Bar1 in mediating cell membrane organization was confirmed as ΔBAR1 mutants failed to display a distinct sterol-rich domain at the hyphal tip. The ΔBAR1 mutants were non-pathogenic when inoculated onto wheat heads, and their in vitro growth also was severely perturbed. ΔBAR1 mutants were incapable of producing perithecia (sexual fruiting structures) and only produced macroconidia (asexual spores) in the presence of NaCl. Sphingolipid analyses indicated that Bar1 is specifically necessary for the production of glucosylceramides in both F. graminearum and Aspergillus nidulans. Interestingly, glucosylceramides appear to mediate sensitivity to heat stable antifungal factor (HSAF), as, in addition to ΔBAR1 mutants, a glucosylceramide synthase deficient mutant of Yarrowia lipolytica is also resistant to HSAF.     

BDM1, a phosducin-like gene of Fusarium graminearum, is involved in virulence during infection of wheat and maize

Fusarium graminearum is a common pathogen of wheat and maize throughout the world. Despite recent advances in the elucidation of the genetic basis of virulence, significant gaps in the regulatory network underlying pathogenesis remain to be filled. In particular, little is known at the molecular level about the overlap among mechanisms of pathogenicity on maize and wheat. G-protein signalling has been implicated in pathogenesis in F. graminearum, although the underlying mechanisms are not fully understood. In this study, we investigated the involvement of a putative phosducin-like gene (BDM1) in growth, development and pathogenesis in F. graminearum. Targeted deletion of BDM1 revealed roles in sexual and asexual sporulation, germ tube development, hyphal branching and mycelial morphology. During pathogenesis, BDM1 is required for wild-type levels of colonization of maize silk tissue and stalks, but is dispensable for the colonization of kernels. The deletion of BDM1 also reduced the virulence of F. graminearum during the infection of wheat seedlings and heads, resulting in a significant reduction in fungal biomass and a delayed spread of visual symptom expression (i.e. bleaching in heads). Furthermore, BDM1 is required for wild-type levels of deoxynivalenol biosynthesis during the infection of wheat heads and maize silks. In summation, BDM1 is one of the few genes characterized to date in F. graminearum involved in virulence during infection of both maize and wheat. Thus, the functional characterization of BDM1 has established a new regulatory link between pathogenesis in maize and wheat, and provides a genetic resource through which the regulatory networks underlying virulence in F. graminearum can be further elucidated.     

The infection biology of Fusarium graminearum: defining the pathways of spikelet to spikelet colonisation in wheat ears

Fusarium graminearum is one of the main causal agents of Fusarium Ear Blight on wheat. How the pathogen colonises the entire ear is not known. There is controversy over whether this mycotoxin producing pathogenic fungus invades wheat floral tissue using a necrotrophic or another mode of nutrition. A detailed microscopic investigation has revealed how wild-type fungal hyphae, of the sequenced strain PH-1, colonised susceptible wheat ears and spread from spikelet to spikelet. At the advancing infection front, colonisation of the host cortex occurred ahead of any vascular colonisation and the hyphae adapted to the available intercellular space between host cells. Intercellular hyphae then became abundant and host cells lost their entire cellular contents just prior to intracellular colonisation. No host cells died ahead of the infection. However, while these deep cortex infections progressed, just below the surface the highly photosynthetic chlorenchyma cells were observed to have died prior to colonisation. Behind the infection front, hyphae were abundant in the vasculature and the cortex, often growing through the pit fields of thick walled cells. This high level of inter- and intracellular fungal colonisation resulted in the collapse of the non-lignified cell-types. In this middle zone of infection, hyphal diameters were considerably enlarged. Far behind the infection front inter- and intracellular hyphae were devoid of contents and had often collapsed. At later stages of infection, the pathogen switched from predominately vertical to lateral growth and accumulated below the surface of the rachis. Here the lignified host cell walls became heavily degraded and hyphae ruptured the epidermis and produced an aerial mycelium.     

Three LysM effectors of Zymoseptoria tritici collectively disarm chitin-triggered plant immunity

Chitin is a major structural component of fungal cell walls and acts as a microbe-associated molecular pattern (MAMP) that, on recognition by a plant host, triggers the activation of immune responses. To avoid the activation of these responses, the Septoria tritici blotch (STB) pathogen of wheat, Zymoseptoria tritici, secretes LysM effector proteins. Previously, the LysM effectors Mg1LysM and Mg3LysM were shown to protect fungal hyphae against host chitinases. Furthermore, Mg3LysM, but not Mg1LysM, was shown to suppress chitin-induced reactive oxygen species (ROS) production. Whereas initially a third LysM effector gene was disregarded as a presumed pseudogene, we now provide functional data to show that this gene also encodes a LysM effector, named Mgx1LysM, that is functional during wheat colonization. While Mg3LysM confers a major contribution to Z. tritici virulence, Mgx1LysM and Mg1LysM contribute to Z. tritici virulence with smaller effects. All three LysM effectors display partial functional redundancy. We furthermore demonstrate that Mgx1LysM binds chitin, suppresses the chitin-induced ROS burst, and is able to protect fungal hyphae against chitinase hydrolysis. Finally, we demonstrate that Mgx1LysM is able to undergo chitin-induced polymerization. Collectively, our data show that Z. tritici utilizes three LysM effectors to disarm chitin-triggered wheat immunity.     

Degradation of salicylic acid by Fusarium graminearum

Fusarium head blight (FHB) is a major cereal crop disease, caused most frequently by the fungus Fusarium graminearum. We have previously demonstrated that F. graminearum can utilize SA as sole source of carbon to grow. In this current study, we further characterized selected four fungal SA-responsive genes that are predicted to encode salicylic acid (SA)-degrading enzymes and we used a gene replacement approach to characterize them further. These included two genes predicted to encode a salicylate 1-monooxygenase, FGSG_03657 and FGSG_09063, a catechol 1, 2-dioxygenase gene, FGSG_03667, and a 2, 3-dihydroxybenzoic acid decarboxylase gene, FGSG_09061. For each gene, three independent gene replacement strains were assayed for their ability to degrade salicylic acid in liquid culture. Salicylate 1-monooxygenase FGSG_03657 and catechol 1, 2-dioxygenase FGSG_03667 were shown to be essential for SA degradation, while a loss of 2, 3-dihydroxybenzoic acid decarboxylase FGSG_09061 caused only a partial reduction of SA degradation and a loss of salicylate 1-monooxygenase FGSG_09063 had no effect when compared to wild type culture. Salicylate 1-monooxygenase FGSG_03657 and catechol 1, 2-dioxygenase FGSG_03667 were identified as the first two key enzyme steps of SA degradation via catechol in the β-ketoadipate pathway. Expression profiles for all four genes were also determined in liquid culture and in planta. Salicylate 1-monooxygenase FGSG_03657 and catechol 1, 2-dioxygenase FGSG_03667 were co-expressed and their expression was substrate dependent in liquid culture; however their expression was uncoupled in planta. Disruption of the gene for catechol 1, 2-dioxygenase FGSG_03667 was shown to have no effect on fungal virulence on wheat. Our results with 2, 3-dihydroxybenzoic acid decarboxylase FGSG_09061 raise the possibility of an alternate non-oxidative decarboxylation pathway for the conversion of SA to catechol via 2, 3-dihydrozybenzoic acid and for a connection between the oxidative and the non-oxidative decarboxylation pathways for SA conversion.     

Real-time PCR assay to quantify Fusarium graminearum wild-type and recombinant mutant DNA in plant material

Fusarium graminearum (teleomorph, Gibberella zeae) is the predominant causal agent of Fusarium head blight (FHB) of wheat resulting in yearly losses through reduction in grain yield and quality and accumulation of fungal generated toxins in grain. Numerous fungal genes potentially involved in virulence have been identified and studies with deletion mutants to ascertain their role are in progress. Although wheat field trials with wild-type and mutant strains are critical to understand the role these genes may play in the disease process, the interpretation of field trial data is complicated by FHB generated by indigenous species of F. graminearum. This report describes the development of a SYBR green-based real time PCR assay that quantifies the total F. graminearum genomic DNA in a plant sample as well as the total F. graminearum genomic DNA contributed from a strain containing a common fungal selectable marker used to create deletion mutants. We found our method more sensitive, reproducible and accurate than other similar recently described assays and comparable to the more expensive probe-based assays. This assay will allow investigators to correlate the amount of disease observed in wheat field trials to the F. graminearum mutant strains being examined.     

Involvement of a velvet protein FgVeA in the regulation of asexual development, lipid and secondary metabolisms and virulence in Fusarium graminearum

The velvet protein, VeA, is involved in the regulation of diverse cellular processes. In this study, we explored functions of FgVeA in the wheat head blight pathogen, Fusarium graminearum,using a gene replacement strategy. The FgVEA deletion mutant exhibited a reduction in aerial hyphae formation, hydrophobicity, and deoxynivalenol (DON) biosynthesis. Deletion of FgVEA gene led to an increase in conidial production, but a delay in conidial germination. Pathogencity assays showed that the mutant was impaired in virulence on flowering wheat head. Sensitivity tests to various stresses exhibited that the FgVEA deletion mutant showed increased resistance to osmotic stress and cell wall-damaging agents, but increased sensitivity to iprodione and fludioxonil fungicides. Ultrastructural and histochemical analyses revealed that conidia of FgVeA deletion mutant contained an unusually high number of large lipid droplets, which is in agreement with the observation that the mutant accumulated a higher basal level of glycerol than the wild-type progenitor. Serial analysis of gene expression (SAGE) in the FgVEA mutant confirmed that FgVeA was involved in various cellular processes. Additionally, six proteins interacting with FgVeA were identified by yeast two hybrid assays in current study. These results indicate that FgVeA plays a critical role in a variety of cellular processes in F. graminearum.     

Lipid rafts in Cryptococcus neoformans concentrate the virulence determinants phospholipase B1 and Cu/Zn superoxide dismutase

Lipid rafts have been identified in the membranes of mammalian cells, the yeast Saccharomyces cerevisiae, and the pathogenic fungus Candida albicans. Formed by a lateral association of sphingolipids and sterols, rafts concentrate proteins carrying a glycosylphosphatidylinositol (GPI) anchor. We report the isolation of membranes with the characteristics of rafts from the fungal pathogen Cryptococcus neoformans. These characteristics include insolubility in Triton X-100 (TX100) at 4 degrees C, more-buoyant density within a sucrose gradient than the remaining membranes, and threefold enrichment with sterols. The virulence determinant phospholipase B1 (PLB1), a GPI-anchored protein, was highly concentrated in raft membranes and could be displaced from them by treatment with the sterol-sequestering agent methyl-beta-cyclodextrin (MbetaCD). Phospholipase B enzyme activity was inhibited in the raft environment and increased 15-fold following disruption of rafts with TX100 at 37 degrees C. Treatment of viable cryptococcal cells in suspension with MbetaCD also released PLB1 protein and enzyme activity, consistent with localization of PLB1 in plasma membrane rafts prior to secretion. The antioxidant virulence factor Cu/Zn superoxide dismutase (SOD1) was concentrated six- to ninefold in raft membrane fractions compared with nonraft membranes, whereas the cell wall-associated virulence factor laccase was not detected in membranes. We hypothesize that raft membranes function to cluster certain virulence factors at the cell surface to allow efficient access to enzyme substrate and/or to provide rapid release to the external environment.     

Apoplastic extracts from a transgenic wheat line exhibiting lesion-mimic phenotype have multiple pathogenesis-related proteins that are antifungal

A transgenic wheat line constitutively expressing genes encoding a class IV acidic chitinase and an acidic beta-1,3-glucanase, showed significant delay in spread of Fusarium head blight (scab) disease under greenhouse conditions. In an earlier work, we observed a lesion-mimic phenotype in this transgenic line when homozygous for transgene loci. Apoplastic fluid (AF) extracted from the lesion-mimic plants had pathogenesis-related (PR) proteins belonging to families of beta-1,3-glucanases, chitinases, and thaumatin-like proteins (TLPs). AF had growth inhibitory activity against certain fungal pathogens, including Fusarium graminearum and Gaeumannomyces graminis var. tritici. Through a two-step ion-exchange chromatography protocol, we recovered many PR proteins and a few uncharacterized proteins. Three individual protein bands corresponding to a TLP (molecular mass, 16 kDa) and two beta-1,3-glucanases (molecular mass, 32 kDa each) were purified and identified by tandem mass spectrometry. We measured the in vitro antifungal activity of the three purified enzymes and a barley class II chitinase (purified earlier in our laboratory) in microtiter plate assays with macroconidia or conidiophores of F. graminearum and Pyrenophora tritici-repentis. Mixtures of proteins revealed synergistic or additive inhibitory activity against F. graminearum and P. tritici-repentis hyphae. The concentrations of PR proteins at which these effects were observed are likely to be those reached in AF of cells exhibiting a hypersensitive response. Our results suggest that apoplastic PR proteins are antifungal and their antimicrobial potency is dependent on concentrations and combinations that are effectively reached in plants following microbial attack.     

Glucosylceramide synthase is essential for alfalfa defensin-mediated growth inhibition but not for pathogenicity of Fusarium graminearum

Antifungal defensins, MsDef1 and MtDef4, from Medicago spp., inhibit the growth of a fungal pathogen, Fusarium graminearum, at micromolar concentrations. However, molecular mechanisms by which they inhibit the growth of this fungus are not known. We have characterized a functional role of the fungal sphingolipid glucosylceramide in regulating sensitivity of the fungus to MsDef1 and MtDef4. A null mutation of the FgGCS1 gene encoding glucosylceramide synthase results in a mutant lacking glucosylceramide. The DeltaFggcs1-null mutant becomes resistant to MsDef1, but not to MtDef4. It shows a significant change in the conidial morphology and displays dramatic polar growth defect, and its mycelia are resistant to cell wall degrading enzymes. Contrary to its essential role in the pathogenicity of a human fungal pathogen, Cryptococcus neoformans, GCS1 is not required for the pathogenicity of F. graminearum. The DeltaFggcs1 mutant successfully colonizes wheat heads and corn silk, but its ability to spread in these tissues is significantly reduced as compared with the wild-type PH-1 strain. In contrast, it retains full virulence on tomato fruits and Arabidopsis thaliana floral and foliar tissues. Based on our findings, we conclude that glucosylceramide is essential for MsDef1-mediated growth inhibition of F. graminearum, but its role in fungal pathogenesis is host-dependent.     

Transgenic expression of polygalacturonase-inhibiting proteins in Arabidopsis and wheat increases resistance to the flower pathogen Fusarium graminearum

Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most important diseases of wheat worldwide, resulting in yield losses and mycotoxin contamination. The molecular mechanisms regulating Fusarium penetration and infection are poorly understood. Beside mycotoxin production, cell wall degradation may play a role in the development of FHB. Many fungal pathogens secrete polygalacturonases (PGs) during the early stages of infection, and plants have evolved polygalacturonase-inhibiting proteins (PGIPs) to restrict pectin degradation during fungal infection. To investigate the role of plant PGIPs in restricting the development of FHB symptoms, we first used Arabidopsis thaliana, whose genome encodes two PGIPs (AtPGIP1 and AtPGIP2). Arabidopsis transgenic plants expressing either of these PGIPs under control of the CaMV 35S promoter accumulate inhibitory activity against F.?graminearum PG in their inflorescences, and show increased resistance to FHB. Second, transgenic wheat plants expressing the bean PvPGIP2 in their flowers also had a significant reduction of symptoms when infected with F.?graminearum. Our data suggest that PGs likely play a role in F.?graminearum infection of floral tissues, and that PGIPs incorporated into wheat may be important for increased resistance to FHB.     

Characterization of the four GH12 Endoxylanases from the plant pathogen Fusarium graminearum

Four putative GH12 genes were found in the Fusarium graminearum genome. The corresponding proteins were expressed in Escherichia coli, purified, and evaluated. FGSG_05851 and FGSG_11037 displayed high activities towards xyloglucan (V(max) of 4 and 11 micronmol/min, respectively), whereas FGSG_07892 and FGSG_16349 were much less active with this substrate (0.081 and 0.004 micronmol/min, respectively). However, all four of these enzymes had a similar binding affinity for xyloglucan. Xyloglucan was the substrate preferred by FGSG_05851, in contrast to the three other enzymes, which preferred beta-glucan or lichenan. Therefore, FGSG_05851 is a xyloglucan-specific glucanase (E.C. 3.2.1.151) rather than an endoglucanase (E.C. 3.2.1.4) with broad substrate specificity. FGSG_11037 displayed a peculiar behavior in that the xyloglucan binding was highly cooperative, with a Hill coefficient of 2.5. Finally, FGSG_05851 essentially degraded xyloglucan into hepta-, octa-, and nonasaccharides, whereas the three other enzymes yielded hepta- and octa-saccharides as well as larger molecules.     

Neuronal Orphan G-Protein Coupled Receptor Proteins Mediate Plasmalogens-Induced Activation of ERK and Akt Signaling

The special glycerophospholipids plasmalogens (Pls) are enriched in the brain and reported to prevent neuronal cell death by enhancing phosphorylation of Akt and ERK signaling in neuronal cells. Though the activation of Akt and ERK was found to be necessary for the neuronal cells survival, it was not known how Pls enhanced cellular signaling. To answer this question, we searched for neuronal specific orphan GPCR (G-protein coupled receptor) proteins, since these proteins were believed to play a role in cellular signal transduction through the lipid rafts, where both Pls and some GPCRs were found to be enriched. In the present study, pan GPCR inhibitor significantly reduced Pls-induced ERK signaling in neuronal cells, suggesting that Pls could activate GPCRs to induce signaling. We then checked mRNA expression of 19 orphan GPCRs and 10 of them were found to be highly expressed in neuronal cells. The knockdown of these 10 neuronal specific GPCRs by short hairpin (sh)-RNA lentiviral particles revealed that the Pls-mediated phosphorylation of ERK was inhibited in GPR1, GPR19, GPR21, GPR27 and GPR61 knockdown cells. We further found that the overexpression of these GPCRs enhanced Pls-mediated phosphorylation of ERK and Akt in cells. Most interestingly, the GPCRs-mediated cellular signaling was reduced significantly when the endogenous Pls were reduced. Our cumulative data, for the first time, suggest a possible mechanism for Pls-induced cellular signaling in the nervous system.     

Lipid raft polarization contributes to hyphal growth in Candida albicans

The polarization of sterol- and sphingolipid-enriched domains (lipid rafts) has been linked to morphogenesis and cell movement in diverse cell types. In the yeast Saccharomyces cerevisiae, a dramatic polarization of sterol-rich domains to the shmoo tip was observed in pheromone-induced cells (M. Bagnat and K. Simons, Proc. Natl. Acad. Sci. USA 99:14183-14188, 2002). We therefore examined whether plasma membrane lipid polarization contributes to the ability of the fungal pathogen Candida albicans to grow in a highly polarized manner to form hyphae. Interestingly, staining with filipin revealed that membrane sterols were highly polarized to the leading edge of growth during all stages of hyphal growth. Budding and pseudohyphal cells did not display polarized staining. Filipin staining was also enriched at septation sites in hyphae, where colocalization with septin proteins was observed, suggesting a role for the septins in forming a boundary domain. Actin appeared to play a role in sterol polarization and hyphal morphogenesis in that both were disrupted by low concentrations of latrunculin A that did not prevent budding. Furthermore, blocking either sphingolipid biosynthesis with myriocin or sterol biosynthesis with ketoconazole resulted in a loss of ergosterol polarization and caused abnormal hyphal morphogenesis, suggesting that lipid rafts are involved. Since hyphal growth is required for the full virulence of C. albicans, these results suggest that membrane polarization may contribute to the pathogenesis of this organism.