Deliverable D10.4.1

This deliverable describes the final status of Task 10.4 of Workpackage 10 of the euHeart project. The aim of this task is to develop a prototype of an endovascular simulator of cardiac radiofrequency ablation. More precisely, its purpose is to simulate the patient-specific catheter navigation and radiofre- quency ablation of ventricular tachycardia. Since deliverable 10.4.1, work on the simulator prototype has focused on the development of a user interface and the integration of two software compo- nents : endovascular simulation and electrophysiology simulation. The first component aims at modeling the deformation of catheters and guidewires inside vessels and to generate a realistic visualization of the vis- ible X-ray images. The second component is focused on the simulation of electrophysiology. We have chosen the Mitchell-Schaeffer phenomenological model to represent the evolution of action potential on the myocardium. The integration of those 2 software components is difficult because they should both run simultaneously in real-time. To this end, we have developed a multi-thread framework allowing to parallelize the computation of the catheter deformation and the cardiac electrophysiology while sharing a minimum num- ber of information. We have also developed a user interface that can display X-ray images, 3D view of the heart and simulated electro-physiology signals measured at the tip of the catheter. An example of simulation is provided starting from the endovascular navi- gation from the veina cava and finishing with the radiofrequency ablation of endocardial tissue inside the right ventricle

Deliverable D10.4.2

This deliverable describes the final status of Task 10.4 of Workpackage 10 of the euHeart project. The aim of this task is to develop a prototype of an endovascular simulator of cardiac radiofrequency ablation. More precisely, its purpose is to simulate the patient-specific catheter navigation and radiofre- quency ablation of ventricular tachycardia. Since deliverable 10.4.1, work on the simulator prototype has focused on the development of a user interface and the integration of two software compo- nents : endovascular simulation and electrophysiology simulation. The first component aims at modeling the deformation of catheters and guidewires inside vessels and to generate a realistic visualization of the vis- ible X-ray images. The second component is focused on the simulation of electrophysiology. We have chosen the Mitchell-Schaeffer phenomenological model to represent the evolution of action potential on the myocardium. The integration of those 2 software components is difficult because they should both run simultaneously in real-time. To this end, we have developed a multi-thread framework allowing to parallelize the computation of the catheter deformation and the cardiac electrophysiology while sharing a minimum num- ber of information. We have also developed a user interface that can display X-ray images, 3D view of the heart and simulated electro-physiology signals measured at the tip of the catheter. An example of simulation is provided starting from the endovascular navi- gation from the veina cava and finishing with the radiofrequency ablation of endocardial tissue inside the right ventricle

Cell cycle-mediated regulation of plant infection by the rice blast fungus.

Final published article deposited in accordance with SHERPA RoMEO guidelinesTo gain entry to plants, many pathogenic fungi develop specialized infection structures called appressoria. Here, we demonstrate that appressorium morphogenesis in the rice blast fungus Magnaporthe oryzae is tightly regulated by the cell cycle. Shortly after a fungus spore lands on the rice (Oryza sativa) leaf surface, a single round of mitosis always occurs in the germ tube. We found that initiation of infection structure development is regulated by a DNA replication-dependent checkpoint. Genetic intervention in DNA synthesis, by conditional mutation of the Never-in-Mitosis 1 gene, prevented germ tubes from developing nascent infection structures. Cellular differentiation of appressoria, however, required entry into mitosis because nimA temperature-sensitive mutants, blocked at mitotic entry, were unable to develop functional appressoria. Arresting the cell cycle after mitotic entry, by conditional inactivation of the Blocked-in-Mitosis 1 gene or expression of stabilized cyclinB-encoding alleles, did not impair appressorium differentiation, but instead prevented these cells from invading plant tissue. When considered together, these data suggest that appressorium-mediated plant infection is coordinated by three distinct cell cycle checkpoints that are necessary for establishment of plant disease

CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus

The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific genome editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to M. oryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species

RNA interference of endochitinases in the sugarcane endophyte Trichoderma virens 223 reduces its fitness as a biocontrol agent of pineapple disease

publication-status: PublishedThe sugarcane root endophyte Trichoderma virens 223 holds enormous potential as a sustainable alternative to chemical pesticides in the control of sugarcane diseases. Its efficacy as a biocontrol agent is thought to be associated with its production of chitinase enzymes, including N-acetyl-β-D-glucosaminidases, chitobiosidases and endochitinases. We used targeted gene deletion and RNA-dependent gene silencing strategies to disrupt N-acetyl-β-D-glucosaminidase and endochitinase activities of the fungus, and to determine their roles in the biocontrol of soil-borne plant pathogens. The loss of N-acetyl-β-D-glucosaminidase activities was dispensable for biocontrol of the plurivorous damping-off pathogens Rhizoctonia solani and Sclerotinia sclerotiorum, and of the sugarcane pathogen Ceratocystis paradoxa, the causal agent of pineapple disease. Similarly, suppression of endochitinase activities had no effect on R. solani and S. sclerotiorum disease control, but had a pronounced effect on the ability of T. virens 223 to control pineapple disease. Our work demonstrates a critical requirement for T. virens 223 endochitinase activity in the biocontrol of C. paradoxa sugarcane disease, but not for general antagonism of other soil pathogens. This may reflect its lifestyle as a sugarcane root endophyte

The glycogen synthase kinase MoGsk1, regulated by Mps1 MAP kinase, is required for fungal development and pathogenicity in Magnaporthe oryzae

Magnaporthe oryzae, the causal agent of blast disease, is one of the most destructive plant pathogens, causing significant yield losses on staple crops such as rice and wheat. The fungus infects plants with a specialized cell called an appressorium, whose development is tightly regulated by MAPK signaling pathways following the activation of upstream sensors in response to environmental stimuli. Here, we show the expression of the Glycogen synthase kinase 3 (GSK3) MoGSK1 in M. oryzae is regulated by Mps1 MAP kinase, particularly under the stressed conditions. Thus, MoGSK1 is functionally characterized in this study. MoGsk1 is functionally homologues to the Saccharomyces cerevisiae GSK3 homolog MCK1. Gene replacement of MoGSK1 caused significant delay in mycelial growth, complete loss of conidiation and inability to penetrate the host surface by mycelia-formed appressorium-like structures, consequently resulting in loss of pathogenicity. However, the developmental and pathogenic defects of Delta mogsk1 are recovered via the heterologous expression of Fusarium graminearum GSK3 homolog gene FGK3, whose coding products also shows the similar cytoplasmic localization as MoGsk1 does in M. oryzae. By contrast, overexpression of MoGSK1 produced deformed appressoria in M. oryzae. In summary, our results suggest that MoGsk1, as a highly conservative signal modulator, dictates growth, conidiation and pathogenicity of M. oryzae

Two independent S-phase checkpoints regulate appressorium-mediated plant infection by the rice blast fungus Magnaporthe oryzae

To cause rice blast disease, the fungal pathogen Magnaporthe oryzae develops a specialized infection structure called an appressorium. This dome-shaped, melanin-pigmented cell generates enormous turgor and applies physical force to rupture the rice leaf cuticle using a rigid penetration peg. Appressorium-mediated infection requires septin-dependent reorientation of the F-actin cytoskeleton at the base of the infection cell, which organizes polarity determinants necessary for plant cell invasion. Here, we show that plant infection by M. oryzae requires two independent S-phase cell-cycle checkpoints. Initial formation of appressoria on the rice leaf surface requires an S-phase checkpoint that acts through the DNA damage response (DDR) pathway, involving the Cds1 kinase. By contrast, appressorium repolarization involves a novel, DDR-independent S-phase checkpoint, triggered by appressorium turgor generation and melanization. This second checkpoint specifically regulates septin- dependent, NADPH oxidase-regulated F-actin dynamics to organize the appressorium pore and facilitate entry of the fungus into host tissue

e-Fungi: a data resource for comparative analysis of fungal genomes.

BACKGROUND: The number of sequenced fungal genomes is ever increasing, with about 200 genomes already fully sequenced or in progress. Only a small percentage of those genomes have been comprehensively studied, for example using techniques from functional genomics. Comparative analysis has proven to be a useful strategy for enhancing our understanding of evolutionary biology and of the less well understood genomes. However, the data required for these analyses tends to be distributed in various heterogeneous data sources, making systematic comparative studies a cumbersome task. Furthermore, comparative analyses benefit from close integration of derived data sets that cluster genes or organisms in a way that eases the expression of requests that clarify points of similarity or difference between species. DESCRIPTION: To support systematic comparative analyses of fungal genomes we have developed the e-Fungi database, which integrates a variety of data for more than 30 fungal genomes. Publicly available genome data, functional annotations, and pathway information has been integrated into a single data repository and complemented with results of comparative analyses, such as MCL and OrthoMCL cluster analysis, and predictions of signaling proteins and the sub-cellular localisation of proteins. To access the data, a library of analysis tasks is available through a web interface. The analysis tasks are motivated by recent comparative genomics studies, and aim to support the study of evolutionary biology as well as community efforts for improving the annotation of genomes. Web services for each query are also available, enabling the tasks to be incorporated into workflows. CONCLUSION: The e-Fungi database provides fungal biologists with a resource for comparative studies of a large range of fungal genomes. Its analysis library supports the comparative study of genome data, functional annotation, and results of large scale analyses over all the genomes stored in the database. The database is accessible at http://www.e-fungi.org.uk, as is the WSDL for the web services.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

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

notes: PMCID: PMC3581893types: Journal Article; Research Support, Non-U.S. Gov'tThe 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.Biotechnology and Biological Sciences Research Council (BBSRC)National Natural Science Foundation of ChinaHalpin ScholarshipEuropean Research Council Advanced Investigator Awar