The filamentous fungus Sordaria macrospora develops complex fruiting bodies (perithecia) to propagate its sexual spores. Here, we present an analysis of the sterile mutant pro41 that is unable to ...produce mature fruiting bodies. The mutant carries a deletion of 4 kb and is complemented by the pro41 open reading frame that is contained within the region deleted in the mutant. In silico analyses predict PRO41 to be an endoplasmic reticulum (ER) membrane protein, and a PRO41-EGFP fusion protein colocalizes with ER-targeted DsRED. Furthermore, Western blot analysis shows that the PRO41-EGFP fusion protein is present in the membrane fraction. A fusion of the predicted N-terminal signal sequence of PRO41 with EGFP is secreted out of the cell, indicating that the signal sequence is functional. pro41 transcript levels are upregulated during sexual development. This increase in transcript levels was not observed in the sterile mutant pro1 that lacks a transcription factor gene. Moreover, microarray analysis of gene expression in the mutants pro1, pro41 and the pro1/41 double mutant showed that pro41 is partly epistatic to pro1. Taken together, these data show that PRO41 is a novel ER membrane protein essential for fruiting body formation in filamentous fungi.
Summary
Stomatal closure is known to be associated with early defence responses of plant cells triggered by microbe‐associated molecular patterns (MAMPs). However, the molecular mechanisms underlying ...these guard‐cell responses have not yet been elucidated. We therefore studied pathogen‐induced changes in ion channel activity in Hordeum vulgare guard cells. Barley mildew (Blumeria graminis) hyphae growing on leaves inhibited light‐induced stomatal opening, starting at 9 h after inoculation, when appressoria had developed. Alternatively, stomatal closure was induced by nano‐infusion of chitosan via open stomata into the sub‐stomatal cavity. Experiments using intracellular double‐barreled micro‐electrodes revealed that mildew stimulated S‐type (slow) anion channels in guard cells. These channels enable the efflux of anions from guard cells and also promote K+ extrusion by altering the plasma membrane potential. Stimulation of S‐type anion channels was also provoked by nano‐infusion of chitosan. These data suggest that MAMPs of mildew hyphae penetrating the cuticle provoke activation of S‐type anion channels in guard cells. In response, guard cells extrude K+ salts, resulting in stomatal closure. Plasma membrane anion channels probably represent general targets of MAMP signaling in plants, as these elicitors depolarize the plasma membrane of various cell types.
In ihrer Evolution mussten Pflanzen Strategien entwickeln um sich sowohl gegen Pathogene aus der Luft als auch solche im Boden zu verteidigen. Diese Resistenzmechanismen der Pflanzen zu verstehen ist ...von höchster Wichtigkeit für die moderne Gesellschaft. Die Weltbevölkerung wächst schnell, was zu der Notwendigkeit führt, die landwirtschaftlichen Flächen möglichst optimal zu nutzen. Ohne die Weiterentwicklung der landwirtschaftlichen Methoden wird eine ausreichende Versorgung mit Grundnahrungsmitteln nicht möglich sein. Obwohl nicht viele Daten zu diesem Thema vorliegen, ist es sehr wahrscheinlich, dass ein hoher Prozentsatz der jährlichen Ernteverluste auf Pflanzenkrankheiten zurückzuführen ist (Orke et al. 1994, Pinstrup-Andersen; 2001). Der Ernteverlust ist nicht ausschließlich auf den Tod der infizierten Pflanze zurückzuführen, sondern vielmehr auf die sogenannten Resistenzkosten Walters und Heil; 2007). Um sich gegen das Pathogen zu schützen müssen Ressourcen genutzt werden, die sonst für die korrekte Entwicklung der Pflanze, sowie der Samen und Früchte verwendet würden. Die pflanzliche Cuticula, welche die Blattoberfläche bedeckt, ist die erste Verteidigungslinie gegen pathogene Microorganismen, die durch die Luft verbreitet werden. Um diese Barriere zu umgehen nutzen Bakterien und einige Pilze die Stomata als Eingang in den Apoplasten der Blätter. Dies kann durch die Pflanze allerdings verhindert werden, indem diese Poren geschlossen werden. Diese Schließzellantwort wurde zunächst als Teil der Immunantwort auf Bakterien angesehen (Melotto et al. 2006). Nichtsdestotrotz konnte beobachtet werden, dass die Stomata auch während der Infektion des Mehltaupilzes schließen, obwohl dieser nicht durch die Stomata in das Blatt eindringen. Daher haben wir Einzelzellstudien an intakten Gerstenpflanzen vorgenommen um zu klären, wie die Signale erkannt und weitergeleitet werden, die schließlich zum pathogen-induzierten Stomaschluss führen (Koers et al. 2011). Zusammengefasst kann gesagt werden, dass der Stomaschluss ein wichtiger Bestandteil der pflanzlichen Immunantwort ist. Innerhalb dieser Antwort der Stomata auf durch Wind übertragene Pathogene, spielt die Aktivierung der S-Typ Anionenkanäle eine entscheidende Rolle. Es konnte dabei gezeigt werden, dass die Immunantwort die Licht-induzierte Inhibierung dieser Anionenkanäle außer Kraft setzt. S-Typ Anionenkanäle sind aber nicht allein in der Pathogenabwehr von Bedeutung, sondern auch in der Reaktion der Pflanzen auf Trockenstress. Es ist jedoch nicht bekannt, in wie weit sich die beiden Signalwege überschneiden. Zusammen mit den neuen mutierten Gerstenlinien, werden die in dieser Arbeit beschriebenen Techniken zur Messung von Einzelzellen tiefere Einsichten in das Zusammenspiel zwischen Trockenstress und Pathogenabwehr in Pflanzen ermöglichen. Die daraus resultierenden Ergebnisse können zur Optimierung von Getreide für die moderne Landwirtschaft genutzt werden. Dies wird einer der wichtigsten Ansätze sein, um die Menschheit auch in Zukunft mit ausreichend Nahrung versorgen zu können.
During evolution, plants had to evolve potent strategies to defend themselves against airborne pathogens, as well as against those encountered in the soil. Understanding the mechanisms that provide plant immunity is crucial for modern society. The world population is growing at rapid pace, leading to the necessity of using agricultural areas as productive as possible. Without improvement of agricultural practice, a sufficient supply with staple foods will not be possible. It is very likely that an important percentage of crop loss is due to plant diseases, even though precise data on this issue are lacking, (Orke et al. 1994, Pinstrup-Andersen; 2001). Crop loss is not exclusively caused by the death of infected plants, but more often by so called costs of resistance (Walters and Heil; 2007). To gain protection against an attacking pathogen, resources have to be consumed, which otherwise would be used for proper plant, crop and fruit development. Plant cuticles, that cover the leaf surface, are the first line of defence to airborne pathogenic microorganisms. To bypass this barrier, bacteria and some fungi use stomata as an entry site into the apoplastic space of leaves. The entry of pathogens through stomata can be prevented by plants upon closure of these pores. This guard cell response was proposed to contribute to plant innate immunity against bacteria (Melotto et al. 2006). However, stomata were found to close during the infection of powdery mildew fungi, which do not use open stomata to enter the leaf. We therefore pursued single cell studies within intact barley plants to elucidate the signal perception and transduction mechanisms that evoke stomatal closure during a pathogen attack (Koers et al. 2011). All results taken together, stomatal closure is an integral part of plant innate immunity. Within the stomatal response to airborne pathogens, the activation of S-type anion channels is essential. It is shown, that the immunity responses of guard cells bypass light induced inhibition of anion channels. S-type anion channels are not only crucial for responses to pathogens, but they are also involved in the response of guard cells towards drought. However, it is unknown to which extent both signals share mutual components. Together with the, now available, mutant lines of barley, the single cell techniques described in this thesis can provide a further insight into the interplay of drought and pathogen responses in plants. The results are likely to be used for optimizing crops for the future agriculture, which is a pivotal step in providing enough food for mankind in the near future.
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•P. digitatum MAPK crosstalk and have distinct roles in growth, stress and virulence.•The Hog1 and Slt2 MAPKs respond in a coordinated way to different stresses.•AfpB is an antifungal ...protein identified in P. digitatum with self-activity.•The AfpB and the fungicide fludioxonil induce the phosphorylation of Hog1 and Slt2.•The involvement of MAPKs in the response to AfpB is not defensive.
Fungi have three mitogen-activated protein kinases (MAPKs): Kss1/Fus3 involved in the invasive growth and virulence of pathogens, Hog1 in response to osmotic stress, and Slt2/Mpk1 in response to cell wall (CW) stress. We conducted comparative analyses of these MAPKs in the phytopathogen Penicillium digitatum and studied their role in the mode of action of the novel self-antifungal protein AfpB. The sensitivity to different stresses of Δhog1 and the reduced growth of Δkss1 coincided with previous reports. However, Δslt2 showed a strong reduction of growth and conidiation, abnormal morphology, and sensitivity to CW stress and temperature. The complementation of Δslt2 validated this mutant. Immunodetection of P-Hog1 and P-Slt2 confirmed the loss and gain of MAPKs in the mutant and complemented strains. Mutants Δslt2 and Δkss1 showed a strong reduction in virulence, whereas Δhog1 was the least affected, and none sporulated during infection. We studied the MAPK signalling induction in response to different treatments. Our data revealed a complex crosstalk involving the three MAPKs, the differential responses of Hog1 and Slt2 to various stresses and their induction by AfpB or the fungicide fludioxonil (FD). Δhog1 resistance to FD confirmed that Hog1 mediates the activity of FD, whereas Δkss1 sensitivity is probably due to the basal activation of Hog1 in Δkss1. None of the three MAPK mutants showed increased sensitivity to AfpB, contrary to previous reports of other antifungal proteins, which indicates that the observed AfpB-mediated activation of Hog1 and Slt2 would not have a defensive role.
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•GoldenBraid (GB) is a cloning system for plant synthetic biology (SynBio).•GB uses biodesign principles (standard pieces, designs and construction rules).•FungalBraid (FB) adapts GB ...for the genetic transformation and SynBio of fungi.•FB is a BioBrick standard, fully compatible with GB and PhytoBricks.•FB is open for the collaborative use by the fungal research community.
Current challenges in the study and biotechnological exploitation of filamentous fungi are the optimization of DNA cloning and fungal genetic transformation beyond model fungi, the open exchange of ready-to-use and standardized genetic elements among the research community, and the availability of universal synthetic biology tools and rules. The GoldenBraid (GB) cloning framework is a Golden Gate-based DNA cloning system developed for plant synthetic biology through Agrobacterium tumefaciens-mediated genetic transformation (ATMT). In this study, we develop reagents for the adaptation of GB version 3.0 from plants to filamentous fungi through: (i) the expansion of the GB toolbox with the domestication of fungal-specific genetic elements; (ii) the design of fungal-specific GB structures; and (iii) the ATMT and gene disruption of the plant pathogen Penicillium digitatum as a proof of concept. Genetic elements domesticated into the GB entry vector pUPD2 include promoters, positive and negative selection markers and terminators. Interestingly, some GB elements can be directly exchanged between plants and fungi, as demonstrated with the marker hph for HygR or the fluorescent protein reporter YFP. The iterative modular assembly of elements generates an endless number of diverse transcriptional units and other higher order combinations in the pDGB3α/pDGB3Ω destination vectors. Furthermore, the original plant GB syntax was adapted here to incorporate specific GB structures for gene disruption through homologous recombination and dual selection. We therefore have successfully adapted the GB technology for the ATMT of fungi. We propose the name of FungalBraid (FB) for this new branch of the GB technology that provides open, exchangeable and collaborative resources to the fungal research community.
Abstract Purpose To develop a new schematic scheme for efficiently recording the key parameters of gas permeable contact lens (GP) fits based on current consensus. Methods Over 100 established GP ...fitters and educators met to discuss the parameters proposed in educational material for evaluating GP fit and concluded on the key parameters that should be recorded. The accuracy and variability of evaluating the fluorescein pattern of GP fit was determined by having 35 experienced contact lens practitioners from across the world, grading 5 images of a range of fits and the topographer simulation of the same fits, in random, order using the proposed scheme. The accuracy of the grading was compared to objective image analysis of the fluorescein intensity of the same images. Results The key information to record to adequately describe the fit of an GP was agreed as: the manufacturer, brand and lens parameters; settling time; comfort on a 5 point scale; centration; movement on blink on a ±2 scale; and the Primary Fluorescein Pattern in the central, mid-peripheral and edge regions of the lens averaged along the horizontal and vertical lens axes, on a ±2 scale. On average 50–60% of practitioners selected the median grade when subjectively rating fluorescein intensity and this was correlated to objective quantification ( r = 0.602, p < 0.001). Objective grading suggesting horizontal median fluorescein intensity was generally symmetrical, as was the vertical meridian, but this was not the case for subjective grading. Simulated fluorescein patterns were subjectively and objectively graded as being less intense than real photographs ( p < 0.01). Conclusion GP fit recording can be standardised and simplified to enhance GP practice.
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