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.     

Differential antifungal and calcium channel-blocking activity among structurally related plant defensins

Plant defensins are a family of small Cys-rich antifungal proteins that play important roles in plant defense against invading fungi. Structures of several plant defensins share a Cys-stabilized alpha/beta-motif. Structural determinants in plant defensins that govern their antifungal activity and the mechanisms by which they inhibit fungal growth remain unclear. Alfalfa (Medicago sativa) seed defensin, MsDef1, strongly inhibits the growth of Fusarium graminearum in vitro, and its antifungal activity is markedly reduced in the presence of Ca(2+). By contrast, MtDef2 from Medicago truncatula, which shares 65% amino acid sequence identity with MsDef1, lacks antifungal activity against F. graminearum. Characterization of the in vitro antifungal activity of the chimeras containing portions of the MsDef1 and MtDef2 proteins shows that the major determinants of antifungal activity reside in the carboxy-terminal region (amino acids 31-45) of MsDef1. We further define the active site by demonstrating that the Arg at position 38 of MsDef1 is critical for its antifungal activity. Furthermore, we have found for the first time, to our knowledge, that MsDef1 blocks the mammalian L-type Ca(2+) channel in a manner akin to a virally encoded and structurally unrelated antifungal toxin KP4 from Ustilago maydis, whereas structurally similar MtDef2 and the radish (Raphanus sativus) seed defensin Rs-AFP2 fail to block the L-type Ca(2+) channel. From these results, we speculate that the two unrelated antifungal proteins, KP4 and MsDef1, have evolutionarily converged upon the same molecular target, whereas the two structurally related antifungal plant defensins, MtDef2 and Rs-AFP2, have diverged to attack different targets in fungi.     

Structure-activity determinants in antifungal plant defensins MsDef1 and MtDef4 with different modes of action against Fusarium graminearum

Plant defensins are small cysteine-rich antimicrobial proteins. Their three-dimensional structures are similar in that they consist of an α-helix and three anti-parallel β-strands stabilized by four disulfide bonds. Plant defensins MsDef1 and MtDef4 are potent inhibitors of the growth of several filamentous fungi including Fusarium graminearum. However, they differ markedly in their antifungal properties as well as modes of antifungal action. MsDef1 induces prolific hyperbranching of fungal hyphae, whereas MtDef4 does not. Both defensins contain a highly conserved γ-core motif (GXCX(3-9)C), a hallmark signature present in the disulfide-stabilized antimicrobial peptides, composed of β2 and β3 strands and the interposed loop. The γ-core motifs of these two defensins differ significantly in their primary amino acid sequences and in their net charge. In this study, we have found that the major determinants of the antifungal activity and morphogenicity of these defensins reside in their γ-core motifs. The MsDef1-γ4 variant in which the γ-core motif of MsDef1 was replaced by that of MtDef4 was almost as potent as MtDef4 and also failed to induce hyperbranching of fungal hyphae. Importantly, the γ-core motif of MtDef4 alone was capable of inhibiting fungal growth, but that of MsDef1 was not. The analysis of synthetic γ-core variants of MtDef4 indicated that the cationic and hydrophobic amino acids were important for antifungal activity. Both MsDef1 and MtDef4 induced plasma membrane permeabilization; however, kinetic studies revealed that MtDef4 was more efficient in permeabilizing fungal plasma membrane than MsDef1. Furthermore, the in vitro antifungal activity of MsDef1, MsDef1-γ4, MtDef4 and peptides derived from the γ-core motif of each defensin was not solely dependent on their ability to permeabilize the fungal plasma membrane. The data reported here indicate that the γ-core motif defines the unique antifungal properties of each defensin and may facilitate de novo design of more potent antifungal peptides.     

Specific domains of plant defensins differentially disrupt colony initiation,cell fusion and calcium homeostasis in Neurospora crassa

MsDef1 and MtDef4 from Medicago spp. are small cysteine‐rich defensins with potent antifungal activity against a broad range of filamentous fungi. Each defensin has a hallmark γ‐core motif (GXCX_3–9C), which contains major determinants of its antifungal activity. In this study, the antifungal activities of MsDef1, MtDef4, and peptides derived from their γ‐core motifs, were characterized during colony initiation in the fungal model, Neurospora crassa. These defensins and their cognate peptides inhibited conidial germination and accompanying cell fusion with different potencies. The inhibitory effects of MsDef1 were strongly mediated by the plasma membrane localized sphingolipid glucosylceramide. Cell fusion was selectively inhibited by the hexapeptide RGFRRR derived from the γ‐core motif of MtDef4. Fluorescent labelling of this hexapeptide showed that it strongly bound to the germ tube plasma membrane/cell wall. Using N. crassa expressing the Ca²⁺ reporter aequorin, MsDef1, MtDef4 and their cognate peptides were each shown to perturb Ca²⁺ homeostasis in specific and distinct ways, and the disruptive effects of MsDef1 on Ca²⁺ were mediated by glucosylceramide. Together, our results demonstrate that MsDef1 and MtDef4 differ markedly in their antifungal properties and specific domains within their γ‐core motifs play important roles in their different modes of antifungal action.     

Plant defensins and virally encoded fungal toxin KP4 inhibit plant root growth

Plant defensins are small, highly stable, cysteine-rich antimicrobial proteins that are thought to constitute an important component of plant defense against fungal pathogens. There are a number of such defensins expressed in various plant tissues with differing antifungal activity and spectrum. Relatively little is known about the modes of action and biological roles of these proteins. Our previous work on a virally encoded fungal toxin, KP4, from Ustilago maydis and subsequently with the plant defensin, MsDef1, from Medicago sativa demonstrated that some of these proteins specifically blocked calcium channels in both fungi and animals. The results presented here demonstrate that KP4 and three plant defensins, MsDef1, MtDef2, and RsAFP2, all inhibit root growth in germinating Arabidopsis seeds at low micromolar concentrations. We have previously demonstrated that a fusion protein composed of Rab GTPase (RabA4b) and enhanced yellow fluorescent protein (EYFP) is dependent upon calcium gradients for localization to the tips of the growing root hairs in Arabidopsis thaliana. Using this tip-localized fusion protein, we demonstrate that all four proteins rapidly depolarize the growing root hair and block growth in a reversible manner. This inhibitory activity on root and root hair is not directly correlated with the antifungal activity of these proteins and suggests that plants apparently express targets for these antifungal proteins. The data presented here suggest that plant defensins may have roles in regulating plant growth and development. A. Allen and A.K. Snyder contributed equally.     

Cell wall integrity and high osmolarity glycerol pathways are required for adaptation of Alternaria brassicicola to cell wall stress caused by brassicaceous indolic phytoalexins

Camalexin, the characteristic phytoalexin of Arabidopsis thaliana, inhibits growth of the fungal necrotroph Alternaria brassicicola. This plant metabolite probably exerts its antifungal toxicity by causing cell membrane damage. Here we observed that activation of a cellular response to this damage requires cell wall integrity (CWI) and the high osmolarity glycerol (HOG) pathways. Camalexin was found to activate both AbHog1 and AbSlt2 MAP kinases, and activation of the latter was abrogated in a AbHog1 deficient strain. Mutant strains lacking functional MAP kinases showed hypersensitivity to camalexin and brassinin, a structurally related phytoalexin produced by several cultivated Brassica species. Enhanced susceptibility to the membrane permeabilization activity of camalexin was observed for MAP kinase deficient mutants. These results suggest that the two signalling pathways have a pivotal role in regulating a cellular compensatory response to preserve cell integrity during exposure to camalexin. AbHog1 and AbSlt2 deficient mutants had reduced virulence on host plants that may, at least for the latter mutants, partially result from their inability to cope with defence metabolites such as indolic phytoalexins. This constitutes the first evidence that a phytoalexin activates fungal MAP kinases and that outputs of activated cascades contribute to protecting the fungus against antimicrobial plant metabolites.     

Arabidopsis MAP kinase phosphatase 1 and its target MAP kinases 3 and 6 antagonistically determine UV-B stress tolerance, independent of the UVR8 photoreceptor pathway

Plants perceive UV-B radiation as an informational signal by a pathway involving UVR8 as UV-B photoreceptor, activating photomorphogenic and acclimation responses. In contrast, the response to UV-B as an environmental stress involves mitogen-activated protein kinase (MAPK) signalling cascades. Whereas the perception pathway is plant specific, the UV-B stress pathway is more broadly conserved. Knowledge of the UV-B stress-activated MAPK signalling pathway in plants is limited, and its potential interplay with the UVR8-mediated pathway has not been defined. Here, we show that loss of MAP kinase phosphatase 1 in the mutant mkp1 results in hypersensitivity to acute UV-B stress, but without impairing UV-B acclimation. The MKP1-interacting proteins MPK3 and MPK6 are activated by UV-B stress and are hyperactivated in mkp1. Moreover, mutants mpk3 and mpk6 exhibit elevated UV-B tolerance and partially suppress the UV-B hypersensitivity of mkp1. We show further that the MKP1-regulated stress-response MAPK pathway is independent of the UVR8 photoreceptor, but that MKP1 also contributes to survival under simulated sunlight. We conclude that, whereas UVR8-mediated acclimation in plants promotes UV-B-induced defence measures, MKP1-regulated stress signalling results when UV-B protection and repair are insufficient and damage occurs. The combined activity of these two mechanisms is crucial to UV-B tolerance in plants.     

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.     

Structural and Functional Studies of a Phosphatidic Acid-Binding Antifungal Plant Defensin MtDef4: Identification of an RGFRRR Motif Governing Fungal Cell Entry

MtDef4 is a 47-amino acid cysteine-rich evolutionary conserved defensin from a model legume Medicago truncatula. It is an apoplast-localized plant defense protein that inhibits the growth of the ascomycetous fungal pathogen Fusarium graminearum in vitro at micromolar concentrations. Little is known about the mechanisms by which MtDef4 mediates its antifungal activity. In this study, we show that MtDef4 rapidly permeabilizes fungal plasma membrane and is internalized by the fungal cells where it accumulates in the cytoplasm. Furthermore, analysis of the structure of MtDef4 reveals the presence of a positively charged γ-core motif composed of β₂ and β₃ strands connected by a positively charged RGFRRR loop. Replacement of the RGFRRR sequence with AAAARR or RGFRAA abolishes the ability of MtDef4 to enter fungal cells, suggesting that the RGFRRR loop is a translocation signal required for the internalization of the protein. MtDef4 binds to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Amino acid substitutions in the RGFRRR sequence which abolish the ability of MtDef4 to enter fungal cells also impair its ability to bind PA. These findings suggest that MtDef4 is a novel antifungal plant defensin capable of entering into fungal cells and affecting intracellular targets and that these processes are mediated by the highly conserved cationic RGFRRR loop via its interaction with PA.     

Antifungal mechanisms of a plant defensin MtDef4 are not conserved between the ascomycete fungi Neurospora crassa and Fusarium graminearum

Defensins play an important role in plant defense against fungal pathogens. The plant defensin, MtDef4, inhibits growth of the ascomycete fungi, Neurospora crassa and Fusarium graminearum, at micromolar concentrations. We have reported that MtDef4 is transported into the cytoplasm of these fungi and exerts its antifungal activity on intracellular targets. Here, we have investigated whether the antifungal mechanisms of MtDef4 are conserved in these fungi. We show that N. crassa and F. graminearum respond differently to MtDef4 challenge. Membrane permeabilization is required for the antifungal activity of MtDef4 against F. graminearum but not against N. crassa. We find that MtDef4 is targeted to different subcellular compartments in each fungus. Internalization of MtDef4 in N. crassa is energy‐dependent and involves endocytosis. By contrast, MtDef4 appears to translocate into F. graminearum autonomously using a partially energy‐dependent pathway. MtDef4 has been shown to bind to the phospholipid phosphatidic acid (PA). We provide evidence that the plasma membrane localized phospholipase D, involved in the biosynthesis of PA, is needed for entry of this defensin in N. crassa, but not in F. graminearum. To our knowledge, this is the first example of a defensin which inhibits the growth of two ascomycete fungi via different mechanisms.     

Functional analysis of the kinome of the wheat scab fungus Fusarium graminearum

As in other eukaryotes, protein kinases play major regulatory roles in filamentous fungi. Although the genomes of many plant pathogenic fungi have been sequenced, systematic characterization of their kinomes has not been reported. The wheat scab fungus Fusarium graminearum has 116 protein kinases (PK) genes. Although twenty of them appeared to be essential, we generated deletion mutants for the other 96 PK genes, including 12 orthologs of essential genes in yeast. All of the PK mutants were assayed for changes in 17 phenotypes, including growth, conidiation, pathogenesis, stress responses, and sexual reproduction. Overall, deletion of 64 PK genes resulted in at least one of the phenotypes examined, including three mutants blocked in conidiation and five mutants with increased tolerance to hyperosmotic stress. In total, 42 PK mutants were significantly reduced in virulence or non-pathogenic, including mutants deleted of key components of the cAMP signaling and three MAPK pathways. A number of these PK genes, including Fg03146 and Fg04770 that are unique to filamentous fungi, are dispensable for hyphal growth and likely encode novel fungal virulence factors. Ascospores play a critical role in the initiation of wheat scab. Twenty-six PK mutants were blocked in perithecia formation or aborted in ascosporogenesis. Additional 19 mutants were defective in ascospore release or morphology. Interestingly, F. graminearum contains two aurora kinase genes with distinct functions, which has not been reported in fungi. In addition, we used the interlog approach to predict the PK-PK and PK-protein interaction networks of F. graminearum. Several predicted interactions were verified with yeast two-hybrid or co-immunoprecipitation assays. To our knowledge, this is the first functional characterization of the kinome in plant pathogenic fungi. Protein kinase genes important for various aspects of growth, developmental, and infection processes in F. graminearum were identified in this study.     

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.     

Identification and characterization of an ABA‐activated MAP kinase cascade in Arabidopsis thaliana

Abscisic acid (ABA) is a major phytohormone involved in important stress‐related and developmental plant processes. Recent phosphoproteomic analyses revealed a large set of ABA‐triggered phosphoproteins as putative mitogen‐activated protein kinase (MAPK) targets, although the evidence for MAPKs involved in ABA signalling is still scarce. Here, we identified and reconstituted in vivo a complete ABA‐activated MAPK cascade, composed of the MAP3Ks MAP3K17/18, the MAP2K MKK3 and the four C group MAPKs MPK1/2/7/14. In planta, we show that ABA activation of MPK7 is blocked in mkk3‐1 and map3k17mapk3k18 plants. Coherently, both mutants exhibit hypersensitivity to ABA and altered expression of a set of ABA‐dependent genes. A genetic analysis further reveals that this MAPK cascade is activated by the PYR/PYL/RCAR‐SnRK2‐PP2C ABA core signalling module through protein synthesis of the MAP3Ks, unveiling an atypical mechanism for MAPK activation in eukaryotes. Our work provides evidence for a role of an ABA‐induced MAPK pathway in plant stress signalling.     

MEKK1, MKK1/MKK2 and MPK4 function together in a mitogen-activated protein kinase cascade to regulate innate immunity in plants

Mitogen-activated protein kinase (MAPK) cascades play important roles in regulating plant innate immune responses. In a genetic screen to search for mutants with constitutive defense responses, we identified multiple alleles of mpk4 and mekk1 that exhibit cell death and constitutive defense responses. Bimolecular fluorescence complementation (BiFC) analysis showed that both MPK4 and MEKK1 interact with MKK1 and MKK2, two closely related MAPK kinases. mkk1 and mkk2 single mutant plants do not have obvious mutant phenotypes. To test whether MKK1 and MKK2 function redundantly, mkk1 mkk2 double mutants were generated. The mkk1 mkk2 double mutant plants die at seedling stage and the seedling-lethality phenotype is temperature-dependent. Similar to the mpk4 and mekk1 mutants, the mkk1 mkk2 double mutant seedlings accumulate high levels of H2O2, display spontaneous cell death, constitutively express Pathogenesis Related (PR) genes and exhibit pathogen resistance. In addition, activation of MPK4 by flg22 is impaired in the mkk1 mkk2 double mutants, suggesting that MKK1 and MKK2 function together with MPK4 and MEKK1 in a MAP kinase cascade to negatively regulate innate immune responses in plants.     

Fusarium graminearum gene deletion mutants map1 and tri5 reveal similarities and differences in the pathogenicity requirements to cause disease on Arabidopsis and wheat floral tissue

The Ascomycete pathogen Fusarium graminearum can infect all cereal species and lower grain yield, quality and safety. The fungus can also cause disease on Arabidopsis thaliana. In this study, the disease-causing ability of two F. graminearum mutants was analysed to further explore the parallels between the wheat (Triticum aestivum) and Arabidopsis floral pathosystems. Wild-type F. graminearum (strain PH-1) and two isogenic transformants lacking either the mitogen-activated protein kinase MAP1 gene or the trichodiene synthase TRI5 gene were individually spray- or point-inoculated onto Arabidopsis and wheat floral tissue. Disease development was quantitatively assessed both macroscopically and microscopically and deoxynivalenol (DON) mycotoxin concentrations determined by enzyme-linked immunosorbent assay (ELISA). Wild-type strain inoculations caused high levels of disease in both plant species and significant DON production. The map1 mutant caused minimal disease and DON accumulation in both hosts. The tri5 mutant, which is unable to produce DON, exhibited reduced pathogenicity on wheat ears, causing only discrete eye-shaped lesions on spikelets which failed to infect the rachis. By contrast, the tri5 mutant retained full pathogenicity on Arabidopsis floral tissue. This study reveals that DON mycotoxin production is not required for F. graminearum to colonize Arabidopsis floral tissue.     

Receptors and signalling components of plant hormones

Recent advances in understanding plant hormonal signalling has resulted in the identification of a variety of signalling components including receptor kinases with homology to the bacterial two component system as well as serine/threonine kinases and protein phosphatases. In addition, the existence of MAP kinase pathways in plants indicates a similar role of these signalling cascades in the relay of exogenous signals into the nucleus as has been disclosed in animal cells. The emerging signalling pathways of the plant hormone abscisic acid and ethylene are presented.     

Fungal-plant signalling in the Ustilago maydis-maize pathosystem

The smut fungus Ustilago maydis needs the host plant maize for completion of its sexual life cycle. Recent experiments highlight the importance of cAMP and mitogen-activated protein (MAP) kinase signalling for cell fusion as well as for subsequent stages of plant colonisation and induction of disease symptoms. During these distinct developmental stages the same signalling cascades must be differentially regulated and accommodate multiple inputs and outputs.