Antiactivators prevent self-sensing in Pseudomonas aeruginosa quorum sensing.

Quorum sensing is described as a widespread cell density-dependent signaling mechanism in bacteria. Groups of cells coordinate gene expression by secreting and responding to diffusible signal molecules. Theory, however, predicts that individual cells may short-circuit this mechanism by directly responding to the signals they produce irrespective of cell density. In this study, we characterize this self-sensing effect in the acyl-homoserine lactone quorum sensing system of Pseudomonas aeruginosa. We show that antiactivators, a set of proteins known to affect signal sensitivity, function to prevent self-sensing. Measuring quorum-sensing gene expression in individual cells at very low densities, we find that successive deletion of antiactivator genes qteE and qslA produces a bimodal response pattern, in which increasing proportions of constitutively induced cells coexist with uninduced cells. Comparing responses of signal-proficient and -deficient cells in cocultures, we find that signal-proficient cells show a much higher response in the antiactivator mutant background but not in the wild-type background. Our results experimentally demonstrate the antiactivator-dependent transition from group- to self-sensing in the quorum-sensing circuitry of P. aeruginosa. Taken together, these findings extend our understanding of the functional capacity of quorum sensing. They highlight the functional significance of antiactivators in the maintenance of group-level signaling and experimentally prove long-standing theoretical predictions.

The fitness benefit of pyoverdine cross-feeding by Pseudomonas protegens Pf-5.

Under iron-limiting conditions, fluorescent pseudomonads acquire iron from the environment by secreting strain-specific, iron-chelating siderophores termed pyoverdines (PVD). The rhizosphere bacterium Pseudomonas protegens Pf-5 produces its own PVD but also can cross-feed on PVDs produced by other species. Previous work has found that Pf-5 continues to produce its own PVD when allowed to cross-feed, raising questions about the benefit of heterologous PVD utilisation. Here, we investigate this question using a defined, unidirectional P. protegens Pf-5/Pseudomonas aeruginosa PAO1 cross-feeding model. Quantifying the production of PVD in the presence of heterologous PVD produced by PAO1, we show that cross-feeding Pf-5 strains reduce the production of their own PVD, while non-cross-feeding Pf-5 strains increase the production of PVD. Measuring the fitness of cross-feeding and non-cross-feeding Pf-5 strains in triple coculture with PAO1, we find that cross-feeding provides a fitness benefit to Pf-5 when the availability of heterologous PVD is high. We conclude that cross-feeding can reduce the costs of self-PVD production and may thus aid in the colonisation of iron-limited environments that contain compatible siderophores produced by other resident microbes. Taken together, these results expand our understanding of the mechanisms of interspecific competition for iron in microbial communities.

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.

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.

German Aerospace Center's advanced robotic technology for future lunar scientific missions.

The Earth's moon is currently an object of interest of many space agencies for unmanned robotic missions within this decade. Besides future prospects for building lunar gateways as support to human space flight, the Moon is an attractive location for scientific purposes. Not only will its study give insight on the foundations of the Solar System but also its location, uncontaminated by the Earth's ionosphere, represents a vantage point for the observation of the Sun and planetary bodies outside the Solar System. Lunar exploration has been traditionally conducted by means of single-agent robotic assets, which is a limiting factor for the return of scientific missions. The German Aerospace Center (DLR) is developing fundamental technologies towards increased autonomy of robotic explorers to fulfil more complex mission tasks through cooperation. This paper presents an overview of past, present and future activities of DLR towards highly autonomous systems for scientific missions targeting the Moon and other planetary bodies. The heritage from the Mobile Asteroid Scout (MASCOT), developed jointly by DLR and CNES and deployed on asteroid Ryugu on 3 October 2018 from JAXA's Hayabusa2 spacecraft, inspired the development of novel core technologies towards higher efficiency in planetary exploration. Together with the lessons learnt from the ROBEX project (2012-2017), where a mobile robot autonomously deployed seismic sensors at a Moon analogue site, this experience is shaping the future steps towards more complex space missions. They include the development of a mobile rover for JAXA's Martian Moons eXploration (MMX) in 2024 as well as demonstrations of novel multi-robot technologies at a Moon analogue site on the volcano Mt Etna in the ARCHES project. Within ARCHES, a demonstration mission is planned from the 14 June to 10 July 2021,1 during which heterogeneous teams of robots will autonomously conduct geological and mineralogical analysis experiments and deploy an array of low-frequency antennas to measure Jovian and solar bursts. This article is part of a discussion meeting issue 'Astronomy from the Moon: the next decades'.

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.

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.

Acyl-homoserine lactone quorum sensing: from evolution to application.

Quorum sensing (QS) is a widespread process in bacteria that employs autoinducing chemical signals to coordinate diverse, often cooperative activities such as bioluminescence, biofilm formation, and exoenzyme secretion. Signaling via acyl-homoserine lactones is the paradigm for QS in Proteobacteria and is particularly well understood in the opportunistic pathogen Pseudomonas aeruginosa. Despite thirty years of mechanistic research, empirical studies have only recently addressed the benefits of QS and provided support for the traditional assumptions regarding its social nature and its role in optimizing cell-density-dependent group behaviors. QS-controlled public-goods production has served to investigate principles that explain the evolution and stability of cooperation, including kin selection, pleiotropic constraints, and metabolic prudence. With respect to medical application, appreciating social dynamics is pertinent to understanding the efficacy of QS-inhibiting drugs and the evolution of resistance. Future work will provide additional insight into the foundational assumptions of QS and relate laboratory discoveries to natural ecosystems.

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.

Pseudomonas protegens Pf-5 favours self-produced siderophore over free-loading in interspecies competition for iron.

Many microorganisms compete for extracellular iron using strain-specific chelators known as siderophores. The ferric-siderophore complex limits local access to iron because import requires a suitable cognate receptor. Interestingly, many species carry receptors that enable 'cross-feeding' on heterologous siderophores made by neighboring organisms, although little is known about how this ubiquitous behaviour is regulated. Here, we investigated the soil bacterium Pseudomonas protegens Pf-5, a strain remarkable for its ability to use dozens of heterologous siderophores. We characterized the expression of six pyoverdine-type (PVD) siderophore receptors in response to their cognate PVD. In general, we found expression is tightly regulated to reflect availability of their cognate PVD. In contrast, Pf-5 continues to secrete its own primary siderophore, PVDPf-5 , despite the capability and opportunity to cross-feed. We demonstrate that this strategy is beneficial in co-culture with a competing PVDPAO1 -producer, P. aeruginosa PAO1. Although Pf-5 can cross-feed on PVDPAO1 , production of PVDPf-5 is required to maintain a competitive advantage. We attribute this to an antagonistic effect of PVDPf-5 on the growth of PAO1, presumably through limiting access to iron. Our results demonstrate the benefits of excluding competitors out-weigh the incentives associated with a free-loader lifestyle for Pf-5.

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.

NADPH oxidases regulate septin-mediated cytoskeletal remodeling during plant infection by the rice blast fungus.

The rice blast fungus Magnaporthe oryzae infects plants with a specialized cell called an appressorium, which uses turgor to drive a rigid penetration peg through the rice leaf cuticle. Here, we show that NADPH oxidases (Nox) are necessary for septin-mediated reorientation of the F-actin cytoskeleton to facilitate cuticle rupture and plant cell invasion. We report that the Nox2-NoxR complex spatially organizes a heteroligomeric septin ring at the appressorium pore, required for assembly of a toroidal F-actin network at the point of penetration peg emergence. Maintenance of the cortical F-actin network during plant infection independently requires Nox1, a second NADPH oxidase, which is necessary for penetration hypha elongation. Organization of F-actin in appressoria is disrupted by application of antioxidants, whereas latrunculin-mediated depolymerization of appressorial F-actin is competitively inhibited by reactive oxygen species, providing evidence that regulated synthesis of reactive oxygen species by fungal NADPH oxidases directly controls septin and F-actin dynamics.

Uncovering effects of antibiotics on the host and microbiota using transkingdom gene networks.

OBJECTIVE: Despite widespread use of antibiotics for the treatment of life-threatening infections and for research on the role of commensal microbiota, our understanding of their effects on the host is still very limited. DESIGN: Using a popular mouse model of microbiota depletion by a cocktail of antibiotics, we analysed the effects of antibiotics by combining intestinal transcriptome together with metagenomic analysis of the gut microbiota. In order to identify specific microbes and microbial genes that influence the host phenotype in antibiotic-treated mice, we developed and applied analysis of the transkingdom network. RESULTS: We found that most antibiotic-induced alterations in the gut can be explained by three factors: depletion of the microbiota; direct effects of antibiotics on host tissues and the effects of remaining antibiotic-resistant microbes. Normal microbiota depletion mostly led to downregulation of different aspects of immunity. The two other factors (antibiotic direct effects on host tissues and antibiotic-resistant microbes) primarily inhibited mitochondrial gene expression and amounts of active mitochondria, increasing epithelial cell death. By reconstructing and analysing the transkingdom network, we discovered that these toxic effects were mediated by virulence/quorum sensing in antibiotic-resistant bacteria, a finding further validated using in vitro experiments. CONCLUSIONS: In addition to revealing mechanisms of antibiotic-induced alterations, this study also describes a new bioinformatics approach that predicts microbial components that regulate host functions and establishes a comprehensive resource on what, why and how antibiotics affect the gut in a widely used mouse model of microbiota depletion by antibiotics.

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.