Identification and functional characterization of secreted effector proteins of the hemibiotrophic fungus Colletotrichum higginsianum

The hemibiotrophic ascomycete fungus Colletotrichum higginsianum causes anthracnose on cruciferous crops and the model plant Arabidopsis thaliana. Successful infection of wild-type plants requires sequential development of specialized infection structures, including melanized appressoria for initial penetration, and bulbous biotrophic hyphae formed inside living epidermal cells. It was hypothesized that appressoria and biotrophic hyphae secrete effector proteins that permit the fungus to evade or disarm host defence responses and to reprogram host cells. This study aimed to define the repertoire of fungal effectors expressed during plant infection and to characterise their biological activity. Discovery of Colletotrichum higginsianum Effector Candidates (ChECs) was accomplished by computational mining collections of infection stage-specific expressed sequence tags (ESTs) for genes encoding solubly secreted proteins with either no homology to known proteins or resembling presumed effectors from other pathogens. Fungal cell types and infection stages sampled for cDNA generation and pyrosequencing included developing and mature in vitro appressoria, early invasive growth in planta, biotrophy and late necrotrophy. After assembling contiguous sequences, analysis of their EST composition allowed the identification of putative plant-induced genes and the definition of a set of 69 ChEC genes that are preferentially expressed at biotrophy-relevant stages. In relation to other infection stages, the early host invasion transcriptome was enriched for genes encoding higginsianum-specific proteins and plant-induced secreted proteins, including ChECs. This suggests that the initial establishment of biotrophy requires the highest proportion of stage-specific effectors and diversified genes. One further ChEC was identified using a complementary proteomic analysis of secreted proteins produced by conidial germlings developing in vitro. Expression analysis showed that transcription of most of the ChECs chosen for further study were highly stage-specific, with ChEC3, ChEC3a, ChEC4 and ChEC6 all being plant-induced. Targeted gene replacement showed that neither ChEC1 nor ChEC2 contribute measurably to fungal virulence. Upon transient expression in tobacco, ChEC3, ChEC3a or ChEC5 all suppressed plant cell death evoked by a C. higginsianum homologue of Necrosis and Ethylene-inducing Peptide1-like proteins, but not by the Phytophthora infestans elicitin INF1, suggesting that there is functional redundancy between C. higginsianum effectors. ChEC4 was found to contain a functional nuclear localization signal and signal peptide, and was shown to be secreted by the fungus during plant infection using fluorescent protein-tagging. This raises the possibility that ChEC4 is translocated into the host nucleus for transcriptional re-programming

Politikszenarien für den Klimaschutz Band 3: Methodik-Leitfaden für die Wirkungsabschätzung von Maßnahmen zur Emissionsminderung

Die Bundesregierung hat sich im Jahr 1990 das Ziel gesetzt, den Ausstoß von Kohlendioxid in Deutschland bis zum Jahr 2005 gegenüber 1990 um 25 % zu reduzieren, und dieses Ziel mehrfach bekräftig. In den vergangenen Jahren sind bereits zahlreiche politische Maßnahmen zum Klimaschutz ergriffen worden und weitere Maßnahmen sind in der Planung oder werden derzeit noch diskutiert. Zur Bewertung der nationalen klimaschutzpolitischen Anstrengungen im Hinblick auf die Zielerreichung ist es erforderlich, daß die Wirkungen von bisherigen und weiteren Maßnahmen möglichst verläßlich quantifiziert werden können. Wirkungsanalysen von politischen Maßnahmen zum Klimaschutz sind auch Bestandteil der nationalen Berichtspflicht im Rahmen der Klimarahmenkonvention. Die entsprechenden Guidelines der FCCC "Policies and Measures" (Geneva, 17 July 1996) enthalten allgemeine Vorgaben zur Beschreibung der Maßnalunen und deren Wirkungen, die sektoral differenziert darzustellen sind. Die zugrunde liegenden Begriffe zur Kategorisierung von Maßnahmen und Wirkungen sind allerdings zum Teil nicht eindeutig definiert. Offen bleibt hierbei auch die Frage, mit welchen methodischen Verfahren die Wirkungen der Politik hinreichend genau geschätzt und einzelnen Maßnahmen zugerechnet werden können. Vor diesem Hintergrund hat das Umweltbundesamt (UBA) im Rahmen seines Umweltforschungsplanes das Forschungszentrum Jülich mit der Durchfuhrung eines Vorhabens "Politikszenarien für den Klimaschutz" beauftragt. Dabei sollten auch die Erkenntnisse und Instrumente aus dem vom BMBF geförderten IKARUS-Projekt$^{1}$ für die Berichterstattung der Bundesregierung und für Strategieüberlegungen im Rahmen der Klimarahmenkonvention nutzbar gemacht werden. Unter administrativer Koordination durch die Programmgruppe "Technikfolgenforschung" des Forschungszentrums Jülich (TFF) ist das Gesamtvorhaben vom Deutschen Institut für Wirtschaftsforschung (DIW), vom Fraunhofer-Institut für Systemtechnik und Innovationsforschung (ISI), von der Programmgruppe "Systemforschung und [...

Percutaneous transhepatic or endoscopic ultrasound-guided biliary drainage in malignant distal bile duct obstruction using a self-expanding metal stent: Study protocol for a prospective European multicenter trial (PUMa trial)

Background Endoscopic ultrasound-guided biliary drainage (EUS-BD) was associated with better clinical success and a lower rate of adverse events (AEs) than fluoroscopy-guided percutaneous transhepatic biliary drainage (PTBD) in recent single center studies with mainly retrospective design and small case numbers (< 50). The aim of this prospective European multicenter study is to compare both drainage procedures using ultrasound-guidance and primary metal stent implantation in patients with malignant distal bile duct obstruction (PUMa Trial). Methods The study is designed as a non-randomized, controlled, parallel group, non-inferiority trial. Each of the 16 study centers performs the procedure with the best local expertise (PTBD or EUS-BD). In PTBD, bile duct access is performed by ultrasound guidance. EUS-BD is performed as an endoscopic ultrasound (EUS)-guided hepaticogastrostomy (EUS-HGS), EUS-guided choledochoduodenostomy (EUS-CDS) or EUS-guided antegrade stenting (EUS-AGS). Insertion of a metal stent is intended in both procedures in the first session. Primary end point is technical success. Secondary end points are clinical success, duration pf procedure, AEs graded by severity, length of hospital stay, re-intervention rate and survival within 6 months. The target case number is 212 patients (12 calculated dropouts included). Discussion This study might help to clarify whether PTBD is non-inferior to EUS-BD concerning technical success, and whether one of both interventions is superior in terms of efficacy and safety in one or more secondary endpoints. Randomization is not provided as both procedures are rarely used after failed endoscopic biliary drainage and study centers usually prefer one of both procedures that they can perform best

Sequential Delivery of Host-Induced Virulence Effectors by Appressoria and Intracellular Hyphae of the Phytopathogen Colletotrichum higginsianum

Phytopathogens secrete effector proteins to manipulate their hosts for effective colonization. Hemibiotrophic fungi must maintain host viability during initial biotrophic growth and elicit host death for subsequent necrotrophic growth. To identify effectors mediating these opposing processes, we deeply sequenced the transcriptome of Colletotrichum higginsianum infecting Arabidopsis. Most effector genes are host-induced and expressed in consecutive waves associated with pathogenic transitions, indicating distinct effector suites are deployed at each stage. Using fluorescent protein tagging and transmission electron microscopy-immunogold labelling, we found effectors localised to stage-specific compartments at the host-pathogen interface. In particular, we show effectors are focally secreted from appressorial penetration pores before host invasion, revealing new levels of functional complexity for this fungal organ. Furthermore, we demonstrate that antagonistic effectors either induce or suppress plant cell death. Based on these results we conclude that hemibiotrophy in Colletotrichum is orchestrated through the coordinated expression of antagonistic effectors supporting either cell viability or cell death

Nachreife und Leimungsschwund – Neue interessante Effekte bei geleimten Papieren

Auch in: Wochenblatt für Papierfabrikation 139, Nr. 7, S. 532 - 539(2011) ; Vortrag beim 20. Internationalen Münchner Papier Symposium 30.3 – 1.4.201

Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules

The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors

Policy Scenarios for Climate Protection: Study on Behalf of the Federal Environmental Agency Volume 4: Methodological Guideline for Assessing the Impact of Measures for Emission Mitigation

In 1990 the German Federal Government set itself the goal of a 25% reduction of the emission of carbon dioxide in the year 2005 based on the 1990 level and has reaffirmed this goal many times. In past years, numerous policy measures for environmental protection have already been taken and additional measures are being planned or discussed at the moment. In order to evaluate national policy efforts for climate protection with respect to reaching this goal, the effects of previous and future measures must be quantified as reliably as possible. Impact analyses of policy measures for climate protection are also part of the national obligation to make an annual report under the Framework Convention on Climate Change (FCCC). The corresponding guidelines of the FCCC "Policies and Measures" (Geneva, 17 July 1996) contain general criteria for the description of the measures and their effects, which are to be displayed in a sectorally differentiated manner. However, the concepts on which the classification of the measures and effects is based are in part not clearly defined. The question also remains of the methodological procedures with which the effects of the policy can be estimated with sufficient precision and to which of the individual measures they can be allocated. Against this background, the Federal Environmental Agency (UBA) has, within the framework of its environmental research plan, charged the Research Centre Julich with the implementation of a project "Policy Scenarios for Climate Protection". In this context, the findings and instruments from the IKARUS project', which is supported by the Federal Ministry of Education, Science, Research and Technology (BMBF), should be made available for reports by the Federal Government as well as strategy assessments as part of the FCCC. The comprehensive project was implemented by the German Institute for Economic Research (DIW), the Fraunhofer Institute for Systems and Innovation Research (FhG-ISI), the Programme Group Systems Analysis and Technology Evaluation of the Research Centre Julich (STE) and the Institute of Applied Ecology [...