Multi-site fungicides suppress banana Panama disease, caused by Fusarium oxysporum f. sp. cubense Tropical Race 4.

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.

Asynchronous development of Zymoseptoria tritici infection in wheat.

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.

Conditional promoters to investigate gene function during wheat infection by Zymoseptoria tritici.

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.

Modelling the motion of organelles in an elongated cell via the coordination of heterogeneous drift-diffusion and long-range transport.

 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.

Alloxan Disintegrates the Plant Cytoskeleton and Suppresses mlo-Mediated Powdery Mildew Resistance.

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.

The fungicide dodine primarily inhibits mitochondrial respiration in Ustilago maydis, but also affects plasma membrane integrity and endocytosis, which is not found in Zymoseptoria tritici.

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.

Fungi, fungicide discovery and global food security.

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.

Optimal timing for Agrobacterium-mediated DNA transformation of Trichoderma reesei conidia revealed by live cell imaging.

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.

Class V chitin synthase and ß(1,3)-glucan synthase co-travel in the same vesicle in Zymoseptoria tritici.

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.

Optimised red- and green-fluorescent proteins for live cell imaging in the industrial enzyme-producing fungus Trichoderma reesei.

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.

ATP prevents Woronin bodies from sealing septal pores in unwounded cells of the fungus Zymoseptoria tritici.

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.

Spatial organization of organelles in fungi: Insights from mathematical modelling.

Mathematical modelling in cellular systems aims to describe biological processes in a quantitative manner. Most accurate modelling is based on robust experimental data. Here we review recent progress in the theoretical description of motor behaviour, early endosome motility, ribosome distribution and peroxisome transport in the fungal model system Ustilago maydis and illustrate the power of modelling in our quest to understand molecular details and cellular roles of membrane trafficking in filamentous fungi.

Woronin body-based sealing of septal pores.

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.

New insights into the peroxisomal protein inventory: Acyl-CoA oxidases and -dehydrogenases are an ancient feature of peroxisomes.

Peroxisomes are ubiquitous organelles which participate in a variety of essential biochemical pathways. An intimate interrelationship between peroxisomes and mitochondria is emerging in mammals, where both organelles cooperate in fatty acid ß-oxidation and cellular lipid homeostasis. As mitochondrial fatty acid ß-oxidation is lacking in yeast and plants, suitable genetically accessible model systems to study this interrelationship are scarce. Here, we propose the filamentous fungus Ustilago maydis as a suitable model for those studies. We combined molecular cell biology, bioinformatics and phylogenetic analyses and provide the first comprehensive inventory of U. maydis peroxisomal proteins and pathways. Studies with a peroxisome-deficient Δpex3 mutant revealed the existence of parallel and complex, cooperative ß-oxidation pathways in peroxisomes and mitochondria, mimicking the situation in mammals. Furthermore, we provide evidence that acyl-CoA dehydrogenases (ACADs) are bona fide peroxisomal proteins in fungi and mammals and together with acyl-CoA oxidases (ACOX) belong to the basic enzymatic repertoire of peroxisomes. A genome comparison with baker's yeast and human gained new insights into the basic peroxisomal protein inventory shared by humans and fungi and revealed novel peroxisomal proteins and functions in U. maydis. The importance of our findings for the evolution and function of the complex interrelationship between peroxisomes and mitochondria in fatty acid ß-oxidation is discussed.

Myosin-5, kinesin-1 and myosin-17 cooperate in secretion of fungal chitin synthase.

Plant infection by pathogenic fungi requires polarized secretion of enzymes, but little is known about the delivery pathways. Here, we investigate the secretion of cell wall-forming chitin synthases (CHSs) in the corn pathogen Ustilago maydis. We show that peripheral filamentous actin (F-actin) and central microtubules (MTs) form independent tracks for CHSs delivery and both cooperate in cell morphogenesis. The enzyme Mcs1, a CHS that contains a myosin-17 motor domain, is travelling along both MTs and F-actin. This transport is independent of kinesin-3, but mediated by kinesin-1 and myosin-5. Arriving vesicles pause beneath the plasma membrane, but only ~15% of them get exocytosed and the majority is returned to the cell centre by the motor dynein. Successful exocytosis at the cell tip and, to a lesser extent at the lateral parts of the cell requires the motor domain of Mcs1, which captures and tethers the vesicles prior to secretion. Consistently, Mcs1-bound vesicles transiently bind F-actin but show no motility in vitro. Thus, kinesin-1, myosin-5 and dynein mediate bi-directional motility, whereas myosin-17 introduces a symmetry break that allows polarized secretion.

The mechanism of peroxisome motility in filamentous fungi.

Peroxisomes (POs) are an essential part of the fungal cell's inventory. They are involved in cellular lipid homeostasis, reactive oxygen metabolism and in the synthesis of secondary metabolites. These functions are thought to require frequent organelle-organelle interactions and the even-distribution of POs in fungal hypha. Recent work in the basidiomycete Ustilago maydis and the ascomycete Aspergillus nidulans reveals a crucial role of early endosomes (EEs) in the dynamic behavior of POs required for their cellular distribution and interaction. This article summarizes the main findings, which provided unexpected insight into the mechanism by which fungal cells organize themselves.

Controlled and stochastic retention concentrates dynein at microtubule ends to keep endosomes on track.

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.

Kinesin-3 in the basidiomycete Ustilago maydis transports organelles along the entire microtubule array.

The molecular motor kinesin-3 transports early endosomes along microtubules in filamentous fungi. It was reported that kinesin-3 from the ascomycete fungi Aspergillus nidulans and Neurospora crassa use a subset of post-translationally modified and more stable microtubules. Here, I show that kinesin-3 from the basidiomycete Ustilago maydis moves along all hyphal microtubules. This difference is likely due to variation in cell cycle control and associated organization of the microtubule array.

Cell biology of Zymoseptoria tritici: Pathogen cell organization and wheat infection.

Cell biological research in the wheat pathogen Zymoseptoria tritici (formerly Mycosphaerella graminicola) has led to a good understanding of the histology of the infection process. Expression profiling and bioinformatic approaches, combined with molecular studies on signaling pathways, effectors and potential necrosis factors provides first insight into the complex interplay between the host and the pathogen. Cell biological studies will help to further our understanding of the infection strategy of the fungus. The cellular organization and intracellular dynamics of the fungus itself is largely unexplored. Insight into essential cellular processes within the pathogen will expand our knowledge of the basic biology of Z. tritici, thereby providing putative new anti-fungal targets.

Measurement of virulence in Zymoseptoria tritici through low inoculum-density assays.

Hitherto, pathogenicity assays with mutants or wildtype variants of Zymoseptoria tritici have been based on pycnidial counts, following inoculation of host leaves with high density inoculum. Here, we present data which suggest that high inoculum densities may mask deficiencies in virulence due to symptom saturation. We describe a low inoculum-density method which obviates this problem. This method can also be used to (i) interrogate the process of lesion formation in Z. tritici (ii) determine whether individuals of the same or different genotypes co-operate or compete during the establishment of apoplastic infections (iii) dissect the determinants of virulence, by assessing a given strain's stomatal penetration efficiency (SPE), its ability to spread within the apoplast and its pycnidiation efficiency. Such methodology can thus be used to investigate the reasons underpinning attenuated virulence in mutant or avirulent wildtype strains.