1,721,080 research outputs found
Comparative functional proteomics of MAP Kinase signalling in Magnaporthe oryzae
No full textRice blast disease is caused by the heterothallic ascomycete fungus Magnaporthe oryzae and is one of the most severe diseases of cultivated rice throughout the world. The PMK1 (Pathogenicity Mitogen-activated protein Kinase) gene in M. oryzae has been identified to play diverse roles during pathogenesis-related development. PMK1 regulates appressorium formation and infectious hyphal growth. PMK1 is functionally related to Saccharomyces cerevisiae FUS3 and KSS1 MAPK genes which regulate the transcription factor Ste12. The STE12 homologue in M. oryzae, MST12, has also been identified and is known to be essential for appressorium mediated penetration and infectious growth. These observations imply that Pmk1 regulates a diverse set of targets important in both the initiation of appressorium development and the subsequent stages of invasive growth. In addition, the Mst12 transcription factor may function downstream of Pmk1 to regulate genes involved in appressorial penetration and infectious hyphal growth. We have used a comparative proteomic study between Guy11 (isogenic wild type), ∆mst12 and ∆pmk1 mutants to understand which genes are induced during appressorium formation and may be regulated by Mst12 and Pmk1. We carried out two-dimensional gel electrophoresis with proteins extracted from conidia that had germinated on a hydrophobic surface after 6h, 12h, 16h and 24h of incubation. M. oryzae underwent major changes in protein abundance and expression during the first 6 hours of spore germination in Guy11 which suggested that much of protein synthesis associated with appressorium morphogenesis and virulence occurs precociously during conidium germination on rice leaf surface. More than 394 differentially expressed proteins during conidium germination and appressorium formation have been identified by mass spectrometry. We confirmed many proteins already known as determinants of pathogenicity in M. oryzae such as enzymes involved in melanin biosynthesis or fatty acid β-oxidation. However, we also identified proteins absent or lowly induced in the Δpmk1 and Δmst12 mutants involved in cell wall reorganisation, secondary metabolism, lipid metabolism, photomorphogenesis or found as proteins of unknown functions. We generated 28 targeted gene deletion mutants to test the putative function of proteins predicted by proteomics analysis to be associated with appressorium development. We obtained 7 mutants showing a significant reduction in virulence and confirmed importance of regulated proteolysis during appressorium development but also elucidated new processes involved in infection such as the phosphoinositide pathway and three proteins of unknown function
Signalling Circuitry Controlling Fungal Virulence in the Rice Blast Fungus Magnaporthe Oryzae
No full textRice blast disease is caused by the filamentous ascomycete fungus Magnaporthe oryzae and is the most destructive disease of cultivated rice. The pathogen elaborates a specialized infection structure called the appressorium. The morphological and physiological transitions that lead to appressorium formation of M. oryzae are stimulated through perception of environmental signals and are tightly regulated by cell cycle checkpoints. External stimuli are internalized by a variety of intracellular MAP kinase signaling pathways, and the major pathway regulating appressorium morphogenesis and plant infection is the Pmk1 MAP kinase signaling pathway. The central kinase, Pmk1, is required for appressorium morphogenesis and the homeobox and C2/H2 Zn-finger domain transcription factor, called Mst12, is required for appressorium formation and tissue invasion. The Mst12 null mutant is able to form melanised appressoria, but it is non-pathogenic. To understand the mechanism of appressorium morphogenesis and penetration peg formation, genome-wide comparative transcriptional profiling analysis was performed for the Δpmk1 and Δmst12 mutant using RNA-seq and HiSeq 2000 sequencing. This thesis reports the identification of gene sets regulated by the Pmk1 signalling pathway and defines the sub-set of these genes regulated by Mst12. I show that a hierarchy of transcription factors is likely to operate downstream of Pmk1 to regulate the main processes required for appressorium morphogenesis and plant infection. I also report the role of Mst12 in cytoskeletal re-organisation and show that it is necessary for septin-dependent F-actin polymerisation at the base on the appressorium prior to plant infection. This is consistent with the major transcriptional changes observed by RNA-seq. The thesis also reports experiments that strongly suggest that appressorium mediated plant penetration is regulated by an S-phase checkpoint which operates independently of the conventional DNA damage and repair response, and the Cds1 and Chk1 checkpoint kinases. Transcriptional profiling results are consistent with the S-phase checkpoint operating downstream of the Pmk1 MAP kinase signalling pathway. An integrated model for the operation of the Pmk1/Mst12 signalling pathways and the hierarchical control of appressorium morphogenesis in the rice blast fungus is presented.Marie Curie Action
Investigating The Regulation of Host Tissue Colonisation by the Rice Blast Fungus Magnaporthe oryzae
Get PDFThe filamentous fungus Magnaporthe oryzae is a devastating pathogen of cultivated rice. M. oryzae elaborates a pressurized dome-shaped infection structure, called the appressorium, which physically ruptures the cuticle and gains entry into host tissue. Intracellular invasive hyphae invade neighbouring host cells through plasmodesmata. The Pmk1 MAPK cascade is well known for its roles in regulating the formation and function of the appressorium. Interestingly, ∆pmk1 mutants cannot infect host plant tissue through wounds, suggesting a role in invasive growth. Here, I define biological functions of the Pmk1 MAPK at various stages of the life cycle, by using a controllable version of Pmk1 that is specifically inhibited by a cell-permeable compound without disturbing other wild-type kinases. The Pmk1 MAPK signalling regulates morphogenesis of narrow invasive hyphae traversing the host cell wall, and modulates production of several putative secreted effectors, providing a direct link between the signalling cascade and effector-driven host immune suppression. These results indicate that the Pmk1 pathway is a central regulator of infection-related development necessary for many stages of plant infection including appressorium development, plant penetration, and importantly tissue colonisation. I also report the role of cell cycle progression in the development of plant infection structure. By using two novel conditional mutants that arrest in S and G2 phases, I defined that S-phase progression is crucial for appressorium-mediated plant penetration.The Halpin Trus
The role of cellular morphogenesis in the pathogenicity of the rice blast fungus Magnaporthe oryzae
Get PDFAppressorium-mediated plant infection is a common strategy used by many plant pathogenic fungi. Understanding the underlying genetic network that controls cellular differentiation of appressorium is therefore pivotal to design durable resistance strategies for these devastating pathogens. This thesis describes four published studies, which investigate the role of septin GTPases in infection and the role of secretion during plant tissue invasion by the rice blast pathogen Magnaporthe oryzae.
Appressorium development involves a series of morphogenetic changes that are tightly regulated by cell cycle checkpoints. Entry into mitosis allows differentiation of an appressorium, while penetration peg emergence appears to require progression through subsequent cell cycle checkpoints and cytokinesis. The studies presented here show that symmetry-breaking events that occur during appressorium differentiation are mediated by scaffold proteins, named septins. Septin GTPases recruit actomyosin ring components during septation and define the site of cytokinesis. They also recruit a toroidal cortical F-actin network to the appressorium pore that provides cortical rigidity to facilitate plant infection. Septins act as diffusion barriers for proteins that mediate membrane curvature necessary for penetration peg formation. Repolarization of the F-actin cytoskeleton at the appressorium pore is essential for plant penetration and is controlled by cell polarity regulators, such as Cdc42 and Chm1. Septin-mediated plant infection is regulated by NADPH oxidase (Nox) dependent generation of reactive oxygen species (ROS). The Nox2/NoxR complex is essential for septin organization at the appressorium pore. Septins are therefore key determinants of appressorium repolarization. I also report an investigation of fungal secretory processes during tissue invasion and present evidence that distinct pathways are involved in effector secretion by Magnaporthe oryzae. A BrefeldinA-sensitive pathway is necessary for secretion of apoplastic effectors, such as Bas4 and Slp1, while a BrefeldinA-insensitive pathway is necessary for secretion of effectors destined for delivery to rice cells.The Halpin Scholarship for Rice Blast Researc
Investigating the Role of the Exocyst Complex in Infection-related Development of the Rice Blast Fungus Magnaporthe Oryzae
No full textHost colonization is mediated through the secretion of effector proteins in order to neutralize host immune responses. However, the mechanism of the effector delivery during biotrophic invasion is not well defined in M. oryzae. In this thesis, I define the role of the exocyst complex, an evolutionarily conserved octameric protein complex involved in vesicle docking to the plasma membrane (composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84), during infection-related development in M. oryzae. Like other filamentous fungi, M. oryzae, exocyst components localize to the vegetative hyphal tip distinct from the Spitzenkörper. However, at the initial stage of infection-related development all the exocyst components localise as a ring at the cortex of the appressorium and re-assembles around the appressorium pore in an actin-dependent manner in mature appressoria. I report that the septin network is required for the transition of exocyst ring from periphery to the appressorium pore. Deletion of Exo70 and Sec5 showed significant reduction in protein secretion and plant infection. I show that Sec6 is required for the exocyst assembly around the appressorium pore and effector secretion from the appressorium. I report that, during biotrophic invasion, effectors are secreted through a distinct pathway. Apoplastic effectors, Bas4 and Slp1 are secreted via a Golgi-dependent pathway while secretion of cytoplasmic effectors, Pwl2 and Bas1 meditates through a Golgi-independent pathway in which exocyst components Exo70 and Sec5 are involved.The Halpin Scholarship for Rice Blast Researc
Evolutionary history of clathrin-mediated endocytosis and the eisosome
Get PDFEndocytosis is both an ancient and a diverse feature of the eukaryotic cell. Studying how it evolved can provide insight into the nature of the last common eukaryotic ancestor, and the diversification of eukaryotes into the known extant lineages. In this thesis, I present two studies on the evolution of endocytosis. In the first part of the thesis I report results from a large-scale, phylogenetic and comparative genomic study of clathrin-mediated endocytosis (CME). The CME pathway has been studied to a great level of detail in yeast to mammal model organisms. Several protein families have now been identified as part of the complex set of protein-protein and protein-lipid interactions which mediate endocytosis. To investigate how such complexity evolved, first, I defined the modular nature of the CME interactome (CME-I) by literature review, and then I carried out a systematic phylogenetic and protein domain architecture analysis of the proteins involved. These data were used to construct a model of the evolution of the CME-I network, and to map the expansion of the network's complexity to the eukaryotic tree of life. In the second part of the thesis, I present results from evolutionary and functional studies of the eisosome, a protein complex which has been proposed to regulate the spatial distribution of endocytosis in S. cerevisiae. The phylogeny of eisosomes components Pil1 and Lsp1 reported here, suggests that eisosomes are likely to have originated at the base of the fungi, and then diversified significantly via multiple gene duplications. I thus studied the localisation and function of Pil1 and Lsp1 homologues in Magnaporthe oryzae to investigate the role of eisosomes in filamentous fungi. Results suggests that eisosomes are linked with septal formation and integrity in M. oryzae, and that the septal specific Pil2 paralogue was lost
in budding yeasts. Together, the data presented in this thesis describe the evolutionary history of a complex biological system, but also highlights the problem of asymmetry in the understanding of endocytic diversity in the eukaryotes.BBSR
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Insertional Mutagenesis to Identify Novel Determinants of Pathogenicity in Magnaporthe oryzae
No full textRice blast disease is caused by the filamentous fungus Magnaporthe oryzae and is the most destructive disease of cultivated rice. It was the first plant pathogenic fungus to have its genome sequence published which opened up the opportunities to discern the principal genetic components that confer pathogenicity on the fungus. The availability of the genome sequence has also presented fresh challenges in terms of converting sequence data into meaningful biological information. Functional genomics studies involve the generation of genome-wide mutant collections and comprehensive screens with potential to identify novel pathogenicity determinants. In this study I utilized Agrobacterium tumefaciens mediated random insertional mutagenesis to study the infection mechanism of M. oryzae. A collection 10,200 M. oryzae T-DNA insertion mutants were generated as part of this study and pathogenicity was assayed by high-throughput disease screening. From the primary qualitative screening I obtained 200 mutants that were reduced or lacking in pathogenicity. Quantitative re-screening allowed selection of 71 T-DNA mutants, including 9 non-pathogenic and 63 reduced virulence mutants exhibiting at least a 50% reduction in disease symptoms. Finally, we selected 8 non-pathogenic mutants for detailed phenotypic and gene functional analysis. A novel approach was used to retrieve T-DNA tagged genes from mutants of interest. Next generation DNA sequencing (NGS) was used to retrieve T-DNA flanking sequences in a high-throughput manner. The efficiency of NGS to facilitate the high-throughput large scale insertional mutagenesis was therefore demonstrated. Out of 8 selected mutants, I identified three novel genes that putatively encode a transcription factor, a PH domain containing signalling protein and a MAP kinase. I also provided evidence that, MGG_05343 is a functional C6 zinc finger transcription factor involved in conidiogenesis. The PH domain containing protein MGG_12956 is involved in vegetative growth, condiogenesis and virulence. The novel kinase MGG_15325 is a S. cerevisiae IME2 homolog that belongs to the Ime2 class of non-classical MAP kinase subfamily. Intriguingly, M. oryzae IME2 seems to have an essential role in growth in planta because the mutant was able to penetrate and colonize plant tissue but failed to cause necrotic rice blast lesions. Identification of these novel genes will allow us greater insight into the processes required for condiogenesis, vegetative and invasive growth and a more integrated understanding of the post-penetration phases of plant tissue colonization. Interestingly, I identified two mutants tagged with T-DNA insertion in the autophagy genes ATG2 and ATG3, reaffirming the importance of infection-associated autophagy in plant infection by M. oryzae and we characterized the ATG3 gene. In addition, I generated a resource of 63 unidentified T-DNA mutants which can potentially lead to identification of more novel determinants of pathogenicity in rice blast disease.The Halpin PhD Studentship Programm
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