Analysis of Magnaporthe grisea pathogenicity by insertional mutagenesis and hierarchical metabolomics

Lead Research Organisation: University of Exeter
Department Name: Biosciences

Abstract

The project will utilize high throughput analytical biochemistry techniques to provide new information concerning the infection of cereals by an economically significant plant pathogenic fungus. Fungi cause the world's most serious and devastating crop diseases and this project will provide new information about the process of plant infection by these microbial agents of disease. We will investigate a fungus called Magnaporthe grisea which causes rice blast disease, a disease that destroys enough rice each year to feed 60 million people. We have produced mutants of the fungus that are impaired in plant infection, and these will be subject to metabolome analysis. Metabolomics techniques will allow us to resolve many of the low molecular weight compounds in the cells of the fungus (organic acids, amino acids, sugars, and polyols) and produce metabolite fingerprints for each mutant strain investigated. We will investigate a set of mutants affected in glycogen, lipid and trehalose metabolism that are required for development of turgor pressure in the specialised infection structures produced by the fungus. To break into a rice leaf, the fungus generates enormous pressure that allows the cuticle of the cereal leaf to be broken. We will also investigate a novel set of mutants that are affected in their ability to colonize living plant tissue, resulting in small, or mis-timed disease lesions. The techniques we propose to use allow the simultaneous global analysis of all the small molecules (several hundreds of metabolites) contained within a cell. This comprehensive analysis will ensure that all major areas of metabolism are studied which will allow us to develop new ideas of the precise differences exist between the normal, wild type fungus and each mutant. These complex data require specialised methods for data analysis to determine metabolic differences between strains. In the future, we expect that novel fungicides and disease-control strategies may be developed that disrupt the virulence-associated metabolic processes identified in this study.

Technical Summary

The project aims to utilise metabolomics approaches to identify metabolic processes associated with pathogenicity in the fungus Magnaporthe grisea, a major disease of a range of cereals and grasses. The genome sequence of the fungus has been determined and tools are available for generating targeted gene replacement mutants, studying gene expression using genome microarrays, and carrying out detailed cell biological studies of plant infection (for review see Talbot, 2003). M. grisea is being subjected to intensive functional genomics analysis, including large-scale insertion mutagenesis projects. To date, mutants affecting pathogenicity are almost without exception impaired in ability to form infection structures (appressoria) and penetrate host epidermal cells. However, in new mutant screens carried out at Exeter and elsewhere, several new classes of mutant are emerging where the timing of lesion formation and subsequent lesion expansion is impaired. We hypothesize that the corresponding genes may make important contributions to plant tissue colonization and disease symptom formation by M. grisea. Molecular genetic analysis of early-phase infection mutants in M. grisea have largely been carried out ex planta by germination of spores on inert plastic surfaces, providing large synchronous populations of infection structures for biochemical analysis. In parallel, we have developed an accurate sampling system for in planta infection sites based on microscopy and GFP-tagging of the pathogen, and by using such sampling approaches have shown that metabolomic fingerprinting and supervised data analysis can detect reproducible major changes in metabolome during lesion development. We will carry out detailed metabolome phenotyping of M. grisea in order to understand the precise roles of genes involved in the development of fungal infection structures using already available mutants. We will also refine and carry out a screen for mutants affected in the timing, rate of growth and sporulation of disease lesions. Mutants representative of different phenotypic classes will be inoculated onto hosts in controlled environments and lesion material collected at several time points. Metabolome analysis will follow a hierarchical procedure initiated with high-throughput, low-resolution ESI-MS fingerprinting (LTQ linear ion trap) and GC-tof-MS fingerprinting (LECO Pegasus II). Discrimination of appropriate sample combinations will be determined by supervised data analysis. If there is evidence for metabolome differences (e.g. comparing mutants with an isogenic wild type strain at the same stage of infection) then a further subset of the same samples will be subjected to ESI-FTMS fingerprinting to generate high resolution peak tables. Corresponding GC-tof-MS chromatograms will also be processed to deconvolute and annotate peaks for data mining. Explanatory metabolite signals between different sample classes will be determined using machine learning procedures. In ESI-MS data, high ranked m/z signals will be examined in chromatograms of further LC-FTMS analyses to predict the mass of possible parent ions which will be further fragmented to obtain MS(n) spectral data. Metabolite mass tables and spectral libraries will be searched for matches with spectra representing discriminatory peaks, and predicted metabolites will be quantified against standards using targeted GC-tof-MS or LC-MS as appropriate. Metabolome differences centred on specific metabolites in the various mutants will be used to determine areas of metabolism that may impact on fungal pathogenicity in future experiments. Parallel genetic analysis of insertional mutant collections of M. grisea will focus on those in which metabolome differences are apparent during plant tissue invasion. Gene isolation by inverse PCR, complementation and validation by targeted gene replacement experiments will be used to define genes associated with disease lesion formation by M. grisea.

Publications


10 25 50
Caracuel-Rios Z (2007) Cellular differentiation and host invasion by the rice blast fungus Magnaporthe grisea. in Current opinion in microbiology
 
Description The project carried out the first metabolomic analysis of a plant pathogenic fungus during invasion of its host plant. Infection by Magnaporthe grisea on three cereal species,identified a conserved metabolite signature associated with infection of all three hosts. (Plant Journal 2009 59:723-37). Procedures from the project were published in Nature Protocols and associated software resources generated. The project also characterised the metabolite profile of infection structure development by the fungus on hydrophobic surfaces. Metabolite profiles of a MAP kinase mutant and associated transcription factor mutant, glyoxylate cycle, trehalose synthesis and glycogen metabolism mutants were compared, revealing key changes in metabolite profile during infection-related development and its impairment by morphogenetic mutants. A library of 10,000 T-DNA insertion mutants of M. grisea was generated and corresponding genes are being identified by TAIL-PCR, complementation analysis and subsequent metabolite profiling.



Two major outputs from the team in Exeter were the development of procedures for the large-scale production of appressoria on hydrophobic surfaces in a reproducible way that yielded uniform metabolite profiles in repetitions carried out by different scientists (Caracuel-Rios/Egan) at different times, and the generation of metabolite profiles revealing key differences associated with morphogenetic mutants of M. grisea impaired in appressorium formation or function. For example, a profound effect of the pmk1 MAPK mutant on secondary metabolite spectra was observed. This large-scale comparative study, integrating results from a full timecourse of appressorium development, morphogenetic and metabolic mutants, will be developed into a significant publication. We aim to integrate proteomic data from identical samples and newly generated transcriptional profile analysis, using Illumina sequencing of superSAGE samples from extracted mRNA.



A library of 10,000 insertion mutants of M. grisea was also generated and screened for pathogenicity and virulence mutants, identifying 92 putative mutants so far that will be further analysed in crosses, by identification of corresponding loci and complementation analysis. This will be a valuable resource for the fungal research community which we will make publicly available. With our collaborators in Aberystwyth, we published a series of procedures in Nature Protocols (Parker et al., 2008, providing a detailed guide on sample preparation and subsequent MS fingerprinting analysis for infected Brachypodium leaves. A number of software resources were generated in Aberystwyth utilizing data generated from biological material generated at Exeter, including ARMeC, a LC-MS signal annotation tool, and MZedDB, an accurate mass matrix analysis reference database.
Exploitation Route My research group has a strong and longstanding commitment to the public understanding of science. Rice blast-related research at Exeter has featured widely in the media during the period of this project, including interviews on BBC TV Spotlight, Radio Devon, The Independent, The Guardian, Western Morning News, BBC News Web (2006-08). Research from the principal applicant's laboratory has also been highlighted in Science magazine (Editor's Choice August 2009). Metabolite accurate mass information will be available at (http://maltese.dbs.aber.ac.uk:8888/hrmet/ & http://users.aber.ac.uk/jhd).

ARMeC (http://www.armec.org/MetaboliteLibrary/index.html).
Sectors Agriculture, Food and Drink,Education
URL http://www.exeter.ac.uk/nicktalbot/
 
Description The PI and research assistant have been involved in developing activities at two Science in Education & Teaching (SET) weeks. Of particular relevance to the present grant is a demonstration for school children to explain the "Chemistry of Colour" in the natural world which, apart from poster display, has hands-on demonstrations of the principle of chromatography by analysing highly pigmented vegetables using paper chromatography. Further activities have centred on Institute Open days in which six form students are invited to learn about the basic principles of analytical chemistry.
First Year Of Impact 2006
Sector Agriculture, Food and Drink
Impact Types Economic
 
Description School Engagement 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Primary Audience Schools
Results and Impact The PI has interacted with a local Primary School for scientific engagement and organised several school open days in the School of Biosciences at the University of Exeter, involving both of the postdoctoral fellows employed on this grant. The School of Biosciences produces a newsletter for sixth-form pupils and their teachers, Excite, which has featured research from the principal applicant. This is a project in the School that has received funding from the Sutton Trust to enhance the take-up of science training by school children from families with no previous experience of higher education. An event 'Britain needs Bioscientists' took place in September 2009 involving school children from Devon attending a series of lectures, demonstrations and practical classes. The principal applicant's laboratory has also worked with an artist-in-residence, and two bio-ethics PhD students on secondment as part of its interaction with E-genis, the ESRC Centre for the Ethics of Genomics at the University of Exeter.

no actual impacts realised to date
Year(s) Of Engagement Activity 2009
URL http://www.ox.ac.uk/media/news_stories/2011/110825.html