RESUMEN
Triazoles are widely used to control pathogenic fungi. They inhibit the ergosterol biosynthetic pathway, but the precise mechanisms leading to fungicidal activities in many fungal pathogens are poorly understood. Here, we elucidate the mode of action of epoxiconazole and metconazole in the wheat pathogen Zymoseptoria tritici and the rice blast fungus Magnaporthe oryzae. We show that both azoles have fungicidal activity and reduce fluidity, but not integrity, of the plasma membrane. This impairs localisation of Cdc15-like F-BAR proteins, resulting in defective actin ring assembly and incomplete septation. However, mutant studies and pharmacological experiments in vitro and in planta show that azole lethality is due to a combination of reactive oxygen species-induced apoptosis and macroautophagy. Simultaneous inhibition of both programmed cell death pathways abolishes azole-induced cell death. Other classes of ergosterol biosynthesis inhibitors also induce apoptosis and macroautophagy, suggesting that activation of these two cell death pathways is a hallmark of ergosterol synthesis-targeting fungicides. This knowledge will inform future crop protection strategies.
Asunto(s)
Apoptosis , Ascomicetos , Fungicidas Industriales , Enfermedades de las Plantas , Especies Reactivas de Oxígeno , Apoptosis/efectos de los fármacos , Enfermedades de las Plantas/microbiología , Ascomicetos/efectos de los fármacos , Ascomicetos/metabolismo , Ascomicetos/patogenicidad , Fungicidas Industriales/farmacología , Especies Reactivas de Oxígeno/metabolismo , Triticum/microbiología , Azoles/farmacología , Ergosterol/biosíntesis , Ergosterol/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Autofagia/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacos , Oryza/microbiología , Oryza/metabolismo , Triazoles/farmacología , Productos Agrícolas/microbiologíaRESUMEN
Global banana production is currently challenged by Panama disease, caused by Fusarium oxysporum f.sp. cubense Tropical Race 4 (FocTR4). There are no effective fungicide-based strategies to control this soil-borne pathogen. This could be due to insensitivity of the pathogen to fungicides and/or soil application per se. Here, we test the effect of 12 single-site and 9 multi-site fungicides against FocTR4 and Foc Race1 (FocR1) in quantitative colony growth, and cell survival assays in purified FocTR4 macroconidia, microconidia and chlamydospores. We demonstrate that these FocTR4 morphotypes all cause Panama disease in bananas. These experiments reveal innate resistance of FocTR4 to all single-site fungicides, with neither azoles, nor succinate dehydrogenase inhibitors (SDHIs), strobilurins or benzimidazoles killing these spore forms. We show in fungicide-treated hyphae that this innate resistance occurs in a subpopulation of "persister" cells and is not genetically inherited. FocTR4 persisters respond to 3 µg ml-1 azoles or 1000 µg ml-1 strobilurins or SDHIs by strong up-regulation of genes encoding target enzymes (up to 660-fold), genes for putative efflux pumps and transporters (up to 230-fold) and xenobiotic detoxification enzymes (up to 200-fold). Comparison of gene expression in FocTR4 and Zymoseptoria tritici, grown under identical conditions, reveals that this response is only observed in FocTR4. In contrast, FocTR4 shows little innate resistance to most multi-site fungicides. However, quantitative virulence assays, in soil-grown bananas, reveals that only captan (20 µg ml-1) and all lipophilic cations (200 µg ml-1) suppress Panama disease effectively. These fungicides could help protect bananas from future yield losses by FocTR4.
Asunto(s)
Fungicidas Industriales , Fusarium , Musa , Fungicidas Industriales/farmacología , Succinato Deshidrogenasa , Estrobilurinas , Captano , Xenobióticos , Enfermedades de las Plantas/genética , Esporas Fúngicas , Suelo , Azoles , BencimidazolesRESUMEN
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.
Asunto(s)
Enfermedades de las Plantas , Caracteres Sexuales , Ascomicetos , Señales (Psicología) , Opsinas , Enfermedades de las Plantas/microbiología , Temperatura , Factores de Transcripción , Triticum/genética , Triticum/microbiologíaRESUMEN
Cellular distribution of organelles in living cells is achieved via a variety of transport mechanisms, including directed motion, mediated by molecular motors along microtubules (MTs), and diffusion which is predominantly heterogeneous in space. In this paper, we introduce a model for particle transport in elongated cells that couples poleward drift, long-range bidirectional transport and diffusion with spatial heterogeneity in a three-dimensional space. Using stochastic simulations and analysis of a related population model, we find parameter regions where the three-dimensional model can be reduced to a coupled one-dimensional model or even a one-dimensional scalar model. We explore the efficiency with which individual model components can overcome drift towards one of the cell poles to reach an approximately even distribution. In particular, we find that if lateral movement is well mixed, then increasing the binding ability of particles to MTs is an efficient way to overcome a poleward drift, whereas if lateral motion is not well mixed, then increasing the axial diffusivity away from MTs becomes an efficient way to overcome the poleward drift. Our three-dimensional model provides a new tool that will help to understand the mechanisms by which eukaryotic cells organize their organelles in an elongated cell, and in particular when the one-dimensional models are applicable.
Asunto(s)
Basidiomycota/metabolismo , Microtúbulos/metabolismo , Orgánulos/metabolismo , Basidiomycota/crecimiento & desarrollo , Simulación por Computador , Difusión , Microtúbulos/ultraestructura , Modelos Biológicos , Movimiento (Física) , Orgánulos/ultraestructuraRESUMEN
The fungus Zymoseptoria tritici causes Septoria tritici blotch of wheat. Pathogenicity begins with spore germination, followed by stomata invasion by hyphae, mesophyll colonization and fruiting body formation. It was previously found that entry into the plant via stomata occurs in a non-synchronized way over several days, while later developmental steps, such as early and late fruiting body formation, were reported to follow each other in time. This suggests synchronization of the pathogen population in planta prior to sporulation. Here, we image a fluorescent Z. tritici IPO323-derived strain during infection. We describe 6 morphologically distinct developmental stages, and determine their abundance in infected leaves, with time post inoculation. This demonstrates that 3-5 stages co-exist in infected tissues at any given time. Thus, later stages of pathogen development also occur asynchronously amongst the population of infecting cells. This merits consideration when interpreting transcriptomics or proteomics data gathered from infected plants.
Asunto(s)
Ascomicetos/crecimiento & desarrollo , Enfermedades de las Plantas/microbiología , Transcriptoma/genética , Triticum/genética , Ascomicetos/genética , Ascomicetos/patogenicidad , Interacciones Huésped-Patógeno/genética , Enfermedades de las Plantas/genética , Hojas de la Planta/microbiología , Proteómica , Triticum/crecimiento & desarrollo , Triticum/microbiologíaRESUMEN
The fungus Zymoseptoria tritici causes Septoria tritici leaf blotch, which poses a serious threat to temperate-grown wheat. Recently, we described a raft of molecular tools to study the biology of this fungus in vitro. Amongst these are 5 conditional promoters (Pnar1, Pex1A, Picl1, Pgal7, PlaraB), which allow controlled over-expression or repression of target genes in cells grown in liquid culture. However, their use in the host-pathogen interaction in planta was not tested. Here, we investigate the behaviour of these promoters by quantitative live cell imaging of green-fluorescent protein-expressing cells during 6 stages of the plant infection process. We show that Pnar1 and Picl1 are repressed in planta and demonstrate their suitability for studying essential gene expression and function in plant colonisation. The promoters Pgal7 and Pex1A are not fully-repressed in planta, but are induced during pycnidiation. This indicates the presence of inducing galactose or xylose and/or arabinose, released from the plant cell wall by the activity of fungal hydrolases. In contrast, the PlaraB promoter, which normally controls expression of an α-l-arabinofuranosidase B, is strongly induced inside the leaf. This suggests that the fungus is exposed to L-arabinose in the mesophyll apoplast. Taken together, this study establishes 2 repressible promoters (Pnar1 and Picl1) and three inducible promoters (Pgal7, Pex1A, PlaraB) for molecular studies in planta. Moreover, we provide circumstantial evidence for plant cell wall degradation during the biotrophic phase of Z. tritici infection.
Asunto(s)
Ascomicetos/genética , Interacciones Huésped-Patógeno/genética , Hojas de la Planta/genética , Triticum/genética , Ascomicetos/patogenicidad , Genes Esenciales/genética , Proteínas Fluorescentes Verdes/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Regiones Promotoras Genéticas , Triticum/microbiologíaRESUMEN
Securing sufficient food for a growing world population is of paramount importance for social stability and the well-being of mankind. Recently, it has become evident that fungal pathogens pose the greatest biotic challenge to our calorie crops. Moreover, the loss of commodity crops to fungal disease destabilises the economies of developing nations, thereby increasing the dimension of the threat. Our best weapon to control these pathogens is fungicides, but increasing resistance puts us in an arms race against them. New anti-fungal compounds need to be discovered, such as mono-alky lipophilic cations (MALCs) described herein. Collaborations between academia and industry are imperative to establish new and efficient ways to develop these new fungicides and to bring them to the market-place.
Asunto(s)
Productos Agrícolas/efectos de los fármacos , Seguridad Alimentaria , Fungicidas Industriales/química , Enfermedades de las Plantas/microbiología , Productos Agrícolas/crecimiento & desarrollo , Farmacorresistencia Fúngica/efectos de los fármacos , Farmacorresistencia Fúngica/genética , Hongos/efectos de los fármacos , Hongos/patogenicidad , Fungicidas Industriales/síntesis química , Fungicidas Industriales/farmacología , Humanos , Enfermedades de las Plantas/genéticaRESUMEN
Trichoderma reesei is the foremost fungal producer of enzymes for industrial processes. Here, we use fluorescent live cell imaging of germinating conidia to improve Agrobacterium tumefaciens-mediated transformation (ATMT) efficiency. We define the timing of (a) morphological changes and (b) nuclear reorganisation during initial conidia germination. This reveals that conidia swell for 7 h, during which nuclei undergo 2 non-synchronised mitotic divisions. Histones are recruited to the nucleus during the first 2 h, suggesting that conidia enter S-phase immediately after activation. This correlates with a significantly increased ATMT efficiency at 2 h after germination initiation. This finding promises to improve genetic manipulation efficiency in T. reesei.
Asunto(s)
Agrobacterium tumefaciens/genética , Hypocreales/genética , Esporas Fúngicas/genética , Transformación Genética/genética , ADN Bacteriano/genética , Vectores Genéticos/genética , Hypocreales/crecimiento & desarrollo , Mutagénesis Insercional , Esporas Fúngicas/crecimiento & desarrolloRESUMEN
Early reports in the fungus Ustilago maydis suggest that the amphipathic fungicide dodine disrupts the fungal plasma membrane (PM), thereby killing this corn smut pathogen. However, a recent study in the wheat pathogen Zymoseptoria tritici does not support such mode of action (MoA). Instead, dodine inhibits mitochondrial ATP-synthesis, both in Z. tritici and U. maydis. This casts doubt on an fungicidal activity of dodine at the PM. Here, we use a cell biological approach and investigate further the effect of dodine on the plasma membrane in both fungi. We show that dodine indeed breaks the integrity of the PM in U. maydis, indicated by a concentration-dependent cell depolarization. In addition, the fungicide reduces PM fluidity and arrests endocytosis by inhibiting the internalization of endocytic vesicles at the PM. This is likely due to impaired recruitment of the actin-crosslinker fimbrin to endocytic actin patches. However, quantitative data reveal that the effect on mitochondria represents the primary MoA in U. maydis. None of these plasma membrane-associated effects were found in dodine-treated Z. tritici cells. Thus, the physiological effect of an anti-fungal chemistry can differ between pathogens. This merits consideration when characterizing a given fungicide.
Asunto(s)
Basidiomycota/efectos de los fármacos , Guanidinas/farmacología , Mitocondrias/efectos de los fármacos , Respiración/efectos de los fármacos , Ascomicetos/efectos de los fármacos , Basidiomycota/genética , Basidiomycota/crecimiento & desarrollo , Membrana Celular/efectos de los fármacos , Endocitosis/efectos de los fármacos , Fungicidas Industriales/farmacologíaRESUMEN
The emerging resistance of crop pathogens to fungicides poses a challenge to food security and compels discovery of new antifungal compounds. Here, we show that mono-alkyl lipophilic cations (MALCs) inhibit oxidative phosphorylation by affecting NADH oxidation in the plant pathogens Zymoseptoria tritici, Ustilago maydis and Magnaporthe oryzae. One of these MALCs, consisting of a dimethylsulfonium moiety and a long alkyl chain (C18-SMe2+), also induces production of reactive oxygen species at the level of respiratory complex I, thus triggering fungal apoptosis. In addition, C18-SMe2+ activates innate plant defense. This multiple activity effectively protects cereals against Septoria tritici blotch and rice blast disease. C18-SMe2+ has low toxicity in Daphnia magna, and is not mutagenic or phytotoxic. Thus, MALCs hold potential as effective and non-toxic crop fungicides.
Asunto(s)
Cationes/farmacología , Productos Agrícolas/efectos de los fármacos , Fungicidas Industriales/farmacología , Enfermedades de las Plantas/prevención & control , Sustancias Protectoras/farmacología , Animales , Ascomicetos/efectos de los fármacos , Cationes/química , Daphnia/efectos de los fármacos , Descubrimiento de Drogas , Grano Comestible/microbiología , Fibroblastos/efectos de los fármacos , Fungicidas Industriales/química , Humanos , Mitocondrias/efectos de los fármacos , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Sustancias Protectoras/química , Triticum/microbiología , Ustilago/efectos de los fármacosRESUMEN
The filamentous fungus Trichoderma reesei is a major source of cellulolytic enzymes in biofuel production. Despite its economic relevance, our understanding of its secretory pathways is fragmentary. A major challenge is to visualise the dynamic behaviour of secretory vesicles in living cells. To this end, we establish a location juxtaposing the succinate dehydrogenase locus as a "soft-landing" site for controlled expression of 4 green-fluorescent and 5 red-fluorescent protein-encoding genes (GFPs, RFPs). Quantitative and comparative analysis of their fluorescent signals in living cells demonstrates that codon-optimised monomeric superfolder GFP (TrmsGFP) and codon-optimised mCherry (TrmCherry) combine highest signal intensity with significantly improved signal-to-noise ratios. Finally, we show that integration of plasmid near the sdi1 locus does not affect secretion of cellulase activity in RUT-C30. The molecular and live cell imaging tools generated in this study will help our understanding the secretory pathway in the industrial fungus T. reesei.
Asunto(s)
Proteínas Fluorescentes Verdes/genética , Hypocreales , Proteínas Luminiscentes/genética , Colorantes Fluorescentes , Proteínas Fúngicas/genética , Expresión Génica , Genes Fúngicos , Hypocreales/citología , Hypocreales/genética , Microscopía Intravital/métodos , Microscopía Fluorescente/métodos , Biología Molecular/métodos , Proteínas Recombinantes/genética , Trichoderma/citología , Trichoderma/genética , Proteína Fluorescente RojaRESUMEN
Emerging fungal and oomycete pathogens infect staple calorie crops and economically important commodity crops, thereby posing a significant risk to global food security. Our current agricultural systems - with emphasis on intensive monoculture practices - and globalized markets drive the emergence and spread of new pathogens and problematic traits, such as fungicide resistance. Climate change further promotes the emergence of pathogens on new crops and in new places. Here we review the factors affecting the introduction and spread of pathogens and current disease control strategies, illustrating these with the historic example of the Irish potato famine and contemporary examples of soybean rust, wheat blast and blotch, banana wilt and cassava root rot. Our Review looks to the future, summarizing what we see as the main challenges and knowledge gaps, and highlighting the direction that research must take to face the challenge of emerging crop pathogens.
RESUMEN
The fungal cell wall consists of proteins and polysaccharides, formed by the co-ordinated activity of enzymes, such as chitin or glucan synthases. These enzymes are delivered via secretory vesicles to the hyphal tip. In the ascomycete Neurospora crassa, chitin synthases and ß(1,3)-glucan synthase are transported in different vesicles, whereas they co-travel along microtubules in the basidiomycete Ustilago maydis. This suggests fundamental differences in wall synthesis between taxa. Here, we visualize the class V chitin synthase ZtChs5 and the ß(1,3)-glucan synthase ZtGcs1 in the ascomycete Zymoseptoria tritici. Live cell imaging demonstrate that both enzymes co-locate to the apical plasma membrane, but are not concentrated in the Spitzenkörper. Delivery involves co-transport along microtubules of the chitin and glucan synthase. Live cell imaging and electron microscopy suggest that both cell wall synthases locate in the same vesicle. Thus, microtubule-dependent co-delivery of cell wall synthases in the same vesicle is found in asco- and basidiomycetes.
Asunto(s)
Ascomicetos/enzimología , Quitina Sintasa/metabolismo , Glucosiltransferasas/metabolismo , Vesículas Secretoras/fisiología , Ascomicetos/genética , Basidiomycota/metabolismo , Quitina Sintasa/genética , Citoplasma/metabolismo , Citoplasma/ultraestructura , Glucosiltransferasas/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Electrónica , Neurospora crassa/metabolismo , Vesículas Secretoras/ultraestructuraRESUMEN
Recessively inherited mutant alleles of Mlo genes (mlo) confer broad-spectrum penetration resistance to powdery mildew pathogens in angiosperm plants. Although a few components are known to be required for mlo resistance, the detailed molecular mechanism underlying this type of immunity remains elusive. In this study, we identified alloxan (5,5-dihydroxyl pyrimidine-2,4,6-trione) and some of its structural analogs as chemical suppressors of mlo-mediated resistance in monocotyledonous barley (Hordeum vulgare) and dicotyledonous Arabidopsis thaliana. Apart from mlo resistance, alloxan impairs nonhost resistance in Arabidopsis. Histological analysis revealed that the chemical reduces callose deposition and hydrogen peroxide accumulation at attempted fungal penetration sites. Fluorescence microscopy revealed that alloxan interferes with the motility of cellular organelles (peroxisomes, endosomes and the endoplasmic reticulum) and the pathogen-triggered redistribution of the PEN1/SYP121 t-SNARE protein. These cellular defects are likely the consequence of disassembly of actin filaments and microtubules upon alloxan treatment. Similar to the situation in animal cells, alloxan elicited the temporary accumulation of reactive oxygen species (ROS) in cotyledons and rosette leaves of Arabidopsis plants. Our results suggest that alloxan may destabilize cytoskeletal architecture via induction of an early transient ROS burst, further leading to the failure of molecular and cellular processes that are critical for plant immunity.
Asunto(s)
Aloxano/metabolismo , Ascomicetos/patogenicidad , Citoesqueleto/metabolismo , Resistencia a la Enfermedad/fisiología , Microtúbulos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cotiledón/metabolismo , Resistencia a la Enfermedad/genética , Glucanos , Hordeum/genética , Hordeum/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB) disease of wheat. Z. tritici is an apoplastic fungal pathogen, which does not penetrate plant cells at any stage of infection, and has a long initial period of symptomless leaf colonisation. During this phase it is unclear to what extent the fungus can access host plant nutrients or communicate with plant cells. Several important primary and secondary metabolite pathways in fungi are regulated by the post-translational activator phosphopantetheinyl transferase (Ppt) which provides an essential co-factor for lysine biosynthesis and the activities of non-ribosomal peptide synthases (NRPS) and polyketide synthases (PKS). To investigate the relative importance of lysine biosynthesis, NRPS-based siderophore production and PKS-based DHN melanin biosynthesis, we generated deletion mutants of ZtPpt. The ∆ZtPpt strains were auxotrophic for lysine and iron, non-melanised and non-pathogenic on wheat. Deletion of the three target genes likely affected by ZtPpt loss of function (Aar- lysine; Nrps1-siderophore and Pks1- melanin), highlighted that lysine auxotrophy was the main contributing factor for loss of virulence, with no reduction caused by loss of siderophore production or melanisation. This reveals Ppt, and the lysine biosynthesis pathway, as potential targets for fungicides effective against Z. tritici.
Asunto(s)
Ascomicetos/patogenicidad , Proteínas Bacterianas/metabolismo , Lisina/biosíntesis , Melaninas/metabolismo , Enfermedades de las Plantas/microbiología , Sideróforos/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Triticum/microbiología , Virulencia , Proteínas Fúngicas/metabolismo , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Esporas Fúngicas/metabolismo , Triticum/crecimiento & desarrollo , Triticum/metabolismoRESUMEN
Optimal disease management depends on the ability to monitor the development of fungicide resistance in plant pathogen populations. Benzimidazole resistance is caused by the point mutations of the ß-tubulin gene in Botrytis cinerea, and three mutations (E198A, E198K, and E198V) at codon 198 account for more than 98% of all resistant strains. Although traditional methods remain a cornerstone in monitoring fungicide resistance, molecular methods that do not require the isolation of pathogens can detect resistance alleles present at low frequencies, and require less time and labor than traditional methods. In this study, we present an efficient, rapid, and highly specific method for detecting highly benzimidazole-resistant B. cinerea isolates based on loop-mediated isothermal amplification (LAMP). By using specific primers, we could simultaneously detect all three resistance-conferring mutations at codon 198. The LAMP reaction components and conditions were optimized, and the best reaction temperatures and times were 60 to 62°C and 45 min, respectively. When B. cinerea field isolates were assessed for benzimidazole resistance, similar results were obtained with LAMP, minimal inhibition concentration, and sequencing. The LAMP assay developed in the current study was highly suitable for detection of highly benzimidazole-resistant field isolates of B. cinerea.
Asunto(s)
Bencimidazoles/farmacología , Botrytis/efectos de los fármacos , Farmacorresistencia Fúngica , Variación Genética , Tubulina (Proteína)/genética , Antihelmínticos/farmacología , Técnicas de Amplificación de Ácido NucleicoRESUMEN
In ascomycete fungi, hyphal cells are separated by perforate septa, which allow cell-to-cell communication. To protect against extensive wound-induced damage, septal pores are sealed by peroxisome-derived Woronin bodies (WBs). The mechanism underpinning WB movement is unknown, but cytoplasmic bulk flow may "flush" WBs into the pore. However, some studies suggest a controlled and active mechanism of WB movement. Indeed, in the wheat pathogen Zymoseptoria tritici cellular ATP prevents WBs from pore sealing in unwounded cells. Thus, cells appear to exert active control over WB closure. Here, we summarize our current understanding of WB-based pore sealing in ascomycete fungi.
Asunto(s)
Ascomicetos/fisiología , Proteínas Fúngicas/fisiología , Ascomicetos/ultraestructura , Hifa/fisiologíaRESUMEN
Septa of filamentous ascomycetes are perforated by septal pores that allow communication between individual hyphal compartments. Upon injury, septal pores are plugged rapidly by Woronin bodies (WBs), thereby preventing extensive cytoplasmic bleeding. The mechanism by which WBs translocate into the pore is not known, but it has been suggested that wound-induced cytoplasmic bleeding "flushes" WBs into the septal opening. Alternatively, contraction of septum-associated tethering proteins may pull WBs into the septal pore. Here, we investigate WB dynamics in the wheat pathogen Zymoseptoria tritici. Ultrastructural studies showed that 3.4 ± 0.2 WBs reside on each side of a septum and that single WBs of 128.5 ± 3.6 nm in diameter seal the septal pore (41 ± 1.5 nm). Live cell imaging of green fluorescent ZtHex1, a major protein in WBs, and the integral plasma membrane protein ZtSso1 confirms WB translocation into the septal pore. This was associated with the occasional formation of a plasma membrane "balloon," extruding into the dead cell, suggesting that the plasma membrane rapidly seals the wounded septal pore wound. Minor amounts of fluorescent ZtHex1-enhanced green fluorescent protein (eGFP) appeared associated with the "ballooning" plasma membrane, indicating that cytoplasmic ZtHex1-eGFP is recruited to the extending plasma membrane. Surprisingly, in ~15% of all cases, WBs moved from the ruptured cell into the septal pore. This translocation against the cytoplasmic flow suggests that an active mechanism drives WB plugging. Indeed, treatment of unwounded and intact cells with the respiration inhibitor carbonyl cyanide m-chlorophenyl hydrazone induced WB translocation into the pores. Moreover, carbonyl cyanide m-chlorophenyl hydrazone treatment recruited cytoplasmic ZtHex1-eGFP to the lateral plasma membrane of the cells. Thus, keeping the WBs out of the septal pores, in Z. tritici, is an ATP-dependent process.
Asunto(s)
Ascomicetos/metabolismo , Membrana Celular/metabolismo , Proteínas Fúngicas/metabolismo , Hifa/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Proteínas Fluorescentes Verdes , Microscopía Electrónica , Enfermedades de las Plantas/microbiología , Triticum/microbiologíaRESUMEN
The polysaccharide-rich wall, which envelopes the fungal cell, is pivotal to the maintenance of cellular integrity and for the protection of the cell from external aggressors - such as environmental fluxes and during host infection. This review considers the commonalities in the composition of the wall across the fungal kingdom, addresses how little is known about the assembly of the polysaccharide matrix, and considers changes in the wall of plant-pathogenic fungi during on and in planta growth, following the elucidation of infection structures requiring cell wall alterations. It highlights what is known about the phytopathogenic fungal wall and what needs to be discovered.