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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. Antifungal azoles inhibit ergosterol biosynthesis, but how that leads to fungistatic or fungicidal activities in many pathogenic fungi is poorly understood. Here, Schuster, Kilaru & Steinberg show that azole lethality in the plant pathogens Zymoseptoria tritici and Magnaporthe oryzae is due to a combination of reactive oxygen species-induced apoptosis and macroautophagy.

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 (C 18 -SMe 2 + ), also induces production of reactive oxygen species at the level of respiratory complex I, thus triggering fungal apoptosis. In addition, C 18 -SMe 2 + activates innate plant defense. This multiple activity effectively protects cereals against Septoria tritici blotch and rice blast disease. C 18 -SMe 2 + has low toxicity in Daphnia magna , and is not mutagenic or phytotoxic. Thus, MALCs hold potential as effective and non-toxic crop fungicides. New fungicides are needed due to emerging resistance shown by crop pathogens. Here, the authors show that a mono-alkyl lipophilic cation protects plants from fungal pathogens by inhibiting fungal mitochondrial respiration, inducing production of reactive oxygen species, triggering fungal apoptosis, and activating innate plant defense.

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.

Filamentous fungi colonise substrates by invasive growth of multi-cellular hyphae. It is commonly accepted that hyphae expand by tip growth that is restricted to the first apical cell, where turgor pressure, exocytosis and endocytosis cooperate to expand the apex. Here we show that, contrary to expectations, subapical cells play important roles in hyphal growth in the industrial enzyme-producing fungus Trichoderma reesei . We find that the second and third cells are crucial for hyphal extension, which correlates with tip-ward cytoplasmic streaming, and the fourth-to-sixth cells support rapid growth rates. Live cell imaging reveals exocytotic and endocytic activity in both apical and subapical cells, associated with microtubule-based bi-directional transport of secretory vesicles and early endosomes across septa. Moreover, visualisation of 1,3-β-glucan synthase in subapical cells reveals that these compartments deliver cell wall-forming enzymes to the apical growth region. Thus, subapical cells are active in exocytosis and endocytosis, and deliver growth supplies and enzymes to the expanding hyphal apex. It is commonly accepted that the hyphae of filamentous fungi expand by tip growth that is restricted to the first apical cell. Here, Schuster et al. show that, contrary to expectations, subapical cells play important roles in hyphal growth in the industrial enzyme-producing fungus Trichoderma reesei .

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. Transitioning from spores to hyphae is crucial for host invasion by the plant pathogenic fungus Zymoseptoria tritici . Here, the authors show that the spore-to-hypha transition is enhanced by wheat leaf surface compounds and is regulated by the white-collar complex, which integrates light with biotic and abiotic cues to allow host invasion through open stomata.

To cause plant disease, pathogenic fungi can secrete effector proteins into plant cells to suppress plant immunity and facilitate fungal infection. Most fungal pathogens infect plants using very long strand-like cells, called hyphae, that secrete effectors from their tips into host tissue. How fungi undergo long-distance cell signalling to regulate effector production during infection is not known. Here we show that long-distance retrograde motility of early endosomes (EEs) is necessary to trigger transcription of effector-encoding genes during plant infection by the pathogenic fungus Ustilago maydi s. We demonstrate that motor-dependent retrograde EE motility is necessary for regulation of effector production and secretion during host cell invasion. We further show that retrograde signalling involves the mitogen-activated kinase Crk1 that travels on EEs and participates in control of effector production. Fungal pathogens therefore undergo long-range signalling to orchestrate host invasion. It is unclear how the nuclei of very long fungal cells (hyphae) receive information from the hyphal tips during the invasion of plant tissues. Here, the authors show that retrograde movement of early endosomes, from the hyphal tip to the nucleus, is required for this signalling process.

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.

Semi-synthetic derivatives of the tricyclic diterpene antibiotic pleuromutilin from the basidiomycete Clitopilus passeckerianus are important in combatting bacterial infections in human and veterinary medicine. These compounds belong to the only new class of antibiotics for human applications, with novel mode of action and lack of cross-resistance, representing a class with great potential. Basidiomycete fungi, being dikaryotic, are not generally amenable to strain improvement. We report identification of the seven-gene pleuromutilin gene cluster and verify that using various targeted approaches aimed at increasing antibiotic production in C. passeckerianus , no improvement in yield was achieved. The seven-gene pleuromutilin cluster was reconstructed within Aspergillus oryzae giving production of pleuromutilin in an ascomycete, with a significant increase (2106%) in production. This is the first gene cluster from a basidiomycete to be successfully expressed in an ascomycete, and paves the way for the exploitation of a metabolically rich but traditionally overlooked group of fungi.

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. Woronin bodies are fungal‐specific organelles. They seal septal pores upon injury to prevent catastrophic leakage of cell contents in multicellular hyphae. Steinberg et al. reveal that movement of Woronin bodies into the pore occurs independently of the cytoskeleton or ATP. Surprisingly, however, ATP is required to prevent Woronin body movement into the septal pore. This result implies a novel mechanism by which fungi control communication between individual hyphal cells. This ATP‐dependent process may have physiological implications beyond wound repair.

Bidirectional transport of early endosomes (EEs) involves microtubules (MTs) and associated motors. In fungi, the dynein/dynactin motor complex concentrates in a comet‐like accumulation at MT plus‐ends to receive kinesin‐3‐delivered EEs for retrograde transport. Here, we analyse the loading of endosomes onto dynein by combining live imaging of photoactivated endosomes and fluorescent dynein with mathematical modelling. Using nuclear pores as an internal calibration standard, we show that the dynein comet consists of ∼55 dynein motors. About half of the motors are slowly turned over ( T 1/2 : ∼98 s) and they are kept at the plus‐ends by an active retention mechanism involving an interaction between dynactin and EB1. The other half is more dynamic ( T 1/2 : ∼10 s) and mathematical modelling suggests that they concentrate at MT ends because of stochastic motor behaviour. When the active retention is impaired by inhibitory peptides, dynein numbers in the comet are reduced to half and ∼10% of the EEs fall off the MT plus‐ends. Thus, a combination of stochastic accumulation and active retention forms the dynein comet to ensure capturing of arriving organelles by retrograde motors. The dynein motor is required for retrograde transport of endosomes in fungal hyphae. This work investigates how dynein is delivered to and retained at the hyphal tip, and how this in turn controls endosome trafficking.