Functional analysis of the Blumeria Haustoria - Barley interactome
Lead Research Organisation:
University of Reading
Department Name: Chemistry
Abstract
The safe, reliable, affordable and sufficient source of food and feedstock - food security- is essential for our well-being as individuals and society and for development world-wide. Agriculture has been hugely successful in delivering this but the challenge ahead is to sustain it for an ever increasing population and expectation of food security. Success so far has in great part been thanks to the ability to reduce loss to pathogens, especially fungi, by the combined us of pesticides and disease resistance bred into commercial crop varieties. The problem is that pathogens evolve to overcome these barriers: disease resistance is broken by new pathogen strains, pesticides become ineffective or obsolete as concerns emerge about their impact on health and the environment. It is therefore imperative to further our understanding of the diseases to maintain and improve our ability to feed ourselves. Some of the most successful and devastating pathogens of the main crops are biotrophic fungi which establish highly intimate and sophisticated interactions with their hosts. Examples of these are the cereal mildews. These fungi are able to penetrate the cell wall barrier and develop a highly complex intracellular feeding structure - the 'haustorium'. It has recently become apparent that in addition to nutrient uptake, pathogens are capable of taking control of the host's metabolism and immunity. This enables them to overcome resistance, suppress defence and survive unharmed inside the plant tissues for as long as it takes to reproduce and disseminate. In this proposal we aim at discovering details of the molecular mechanisms by which barley powdery mildew establishes control of the host cell through the action of the haustorium and so-called effectors: proteins that are delivered to the host and are the agents that actually effect this control. In order to do this we combine the expertise of three research groups in the UK and the USA to bring together world-class abilities in the powdery mildew genomics, protein analysis and disease resistance in barley. We plan to 1) survey candidate mildew effectors, 2) test and verify their function in modulating disease and resistance in the plant and 3) identify the molecules that interact physically with the effectors in the plant, i.e. the effector targets in the host. 1) The survey will include making use of and systematically extending the description of the proteins actually made by mildew in the haustorium. Of these a selection will be made of those proteins that bear the hall-marks of candidate effectors, for example include molecular fingerprints that target them for secretion. Additionally, we will test proteins encoded by genes that are linked to functions known to affect disease resistance and virulence. 2) The candidate effectors will be tested by delivering them to barley cells and observing how this alters the susceptibility to mildew. Delivery will be achieved either by modifying bacteria to inject specific proteins into the plant cell or by bombarding microscopic particles coated with effector-encoding DNA. The treated leaves will then be inoculated with mildew and the rates of infection scored. True effector proteins will be those that alter disease. 3) Validated effectors will then be used to 'fish' host proteins they bind to and therefore interact with - thus defining candidate effector targets. The interactions will be sought both in the plant itself and in extracts from infected plants. The molecular interactions and their biochemistry will then be characterised. The outcome of this research will lay the foundation of a detailed understanding of the molecular mechanisms underlying disease in the cereal mildews. This has the potential of aiding future developments in disease resistance, management, to promote food security and production for generations to come.
Technical Summary
Pathogens which enter into close association with their hosts must overcome host immunity. In some interactions they do this through the action of effector proteins. Many biotrophic plant pathogenic fungi develop haustoria: intracellular organs, understood to be feeding structures. In addition to the feeding function, there is mounting evidence that haustoria also deliver effectors to the host cells. The cereal powdery mildew Blumeria graminis is one of the most prominent diseases of the principal food and feedstock crops in UK agriculture. B. graminis f. sp. hordei infects barley and is the best studied powdery mildew. Here we propose to combine the expertise of three collaborating laboratories to achieve the following : 1) Define a panel of candidate effectors proteins by combining data currently available in the databases, publications and unpublished work carried with deep proteome profiling of haustoria by applying a shotgun proteomic and state-of-the-art mass spectrometry. The focus is proteins specifically associated with haustoria, proteins that are small (<10kD) and are secreted and genes that are present at loci genetically linked to avirulence genes. 2) Validate the function of effectors by delivery of the candidates into barley cells e.g. via a new Xanthomonas type III secretion vector. We will then monitor the susceptibility to Blumeria infection and disease development: bone-fide effectors are those that alter the degree of infection. 3) Identify the effector targets in the barley host by detecting proteins which interact with the validated effectors. We will achieve this by surface plasmon resonance, affinity chromatography, in vivo tandem affinity purification tagging and mass spectrometry. 4) Define targets of Blufensin (Bln-1). The techniques described in (3) will be used to determine plant and /or fungal proteins that interact with the recently described susceptibility factor Bln-1 and validate their function in vivo.
Publications
Agrawal G
(2013)
A decade of plant proteomics and mass spectrometry: Translation of technical advancements to food security and safety issues
in Mass Spectrometry Reviews
Agrawal GK
(2012)
Boosting the globalization of plant proteomics through INPPO: current developments and future prospects.
in Proteomics
Agrawal GK
(2012)
Translational plant proteomics: a perspective.
in Journal of proteomics
Bindschedler L
(2011)
Sample Preparation in Biological Mass Spectrometry
Bindschedler LV
(2009)
In planta proteomics and proteogenomics of the biotrophic barley fungal pathogen Blumeria graminis f. sp. hordei.
in Molecular & cellular proteomics : MCP
Bindschedler LV
(2011)
Quantitative plant proteomics.
in Proteomics
Bindschedler LV
(2011)
Proteogenomics and in silico structural and functional annotation of the barley powdery mildew Blumeria graminis f. sp. hordei.
in Methods (San Diego, Calif.)
Bindschedler LV
(2011)
Fully automated software solution for protein quantitation by global metabolic labeling with stable isotopes.
in Rapid communications in mass spectrometry : RCM
Bindschedler LV
(2012)
Hydroponic isotope labeling of entire plants and high-performance mass spectrometry for quantitative plant proteomics.
in Methods in molecular biology (Clifton, N.J.)
Bryant L
(2015)
Proteomic analysis of Artemisia annua--towards elucidating the biosynthetic pathways of the antimalarial pro-drug artemisinin.
in BMC plant biology
Description | The safe, reliable, affordable and sufficient source of food and feedstock - food security - is essential for our well-being as individuals and society and for development world-wide. Agriculture has been hugely successful in delivering this but the challenge ahead is to sustain it for an ever increasing population and expectation of food security. Success so far has in great part been thanks to the ability to reduce loss to pathogens, especially fungi, by the combined us of pesticides and disease resistance bred into commercial crop varieties. The problem is that pathogens evolve to overcome these barriers: disease resistance is broken by new pathogen strains, pesticides become ineffective or obsolete as concerns emerge about their impact on health and the environment. It is therefore imperative to further our understanding of the diseases to maintain and improve our ability to feed ourselves. Some of the most successful and devastating pathogens of the main crops are biotrophic fungi which establish highly intimate and sophisticated interactions with their hosts. Examples of these are the cereal mildews, of which the barley powdery mildew has been the subject of this project. These fungi are able to penetrate the cell wall barrier and develop a highly complex intracellular feeding structure - the "haustorium". It has become apparent that in addition to nutrient uptake, such pathogens are capable of taking control of the host's metabolism and immunity. This enables them to overcome resistance, suppress defence and survive unharmed inside the plant tissues for as long as it takes to reproduce and disseminate. In this project we have discovered details of the molecular machinery that enables barley powdery mildew to establish control of the host cell through the action of the haustorium and so-called effectors: proteins that are delivered to the host barley and are the agents that actually affect this control. We combined the expertise of three research groups in the UK and the USA to bring together world-class abilities in powdery mildew genomics, protein analysis and disease resistance in barley. Thus, we were able to survey candidate effectors, test and verify their function in modulating disease and resistance in the plant, and identify some candidate barley proteins that interact physically with the effector proteins, i.e. the effector targets in the host barley. Specifically, we have achieved the following: 1) A large-scale discovery study that led to the identification of hundreds of proteins in the haustorium and some tens of haustorium-specific proteins that have not been found in other parts of the pathogen, 2) A further successful survey verifying several of these haustorium-specific proteins as candidate effectors, 3) An initial interaction study of these candidate effectors, discovering some potentially important leads to effector targets and their interaction in barley. The outcome of this research now lays the foundation for understanding the molecular mechanisms in cereal diseases that are induced by mildew pathogens. This has the potential of aiding future developments in disease resistance, management, to promote food security and production for generations to come. |
Exploitation Route | Our findings have been taken forward by the groups of Prof Spanu at Imperial College and Dr Bindschedler at RHUL. |
Sectors | Agriculture Food and Drink |
Title | Cloning and purification of a recombinant Blumeria effector with similarity to a metallo-protease |
Description | A secreted Haustoria specific protein (BEC1019) was identified by proteomics and was validated for its effector activity. In lay terms this protein is involved in the host (barley) colonisation process by its powdery mildew (Blumeria graminis f.sp. hordei). The protein was expressed as a recombinant protein in E. coli to raise polyclonal antibodies. |
Type Of Material | Antibody |
Provided To Others? | No |
Title | Cloning and purification of two recombinant effector proteins to raise polyclonal antibodies |
Description | Two secreted Haustoria-specific proteins (BEC1011 and BEC54) were identified by proteomics and validated for their effector activity. In lay terms these proteins are involved in the host (barley) colonisation process by its powdery mildew (Blumeria graminis f.sp. hordei). The proteins were expressed as recombinant proteins in E. coli to raise polyclonal antibodies. |
Type Of Material | Antibody |
Provided To Others? | No |
Title | Blumeria open reading frame annotation and database |
Description | The proteogenomic approach to identify Blumeria proteins helped to cure gene models and open reading frames for the creation of an open reading frame database. |
Type Of Material | Database/Collection of data |
Year Produced | 2010 |
Provided To Others? | No |
Impact | No actual impacts realised to date |
URL | http://www.blugen.org/ |
Title | Mass spectral data of Blumeria-barley proteome |
Description | Mass spectra of the Blumeria-barley proteome were deposited into the public database PRIDE (EBI). |
Type Of Material | Database/Collection of data |
Year Produced | 2011 |
Provided To Others? | No |
Impact | No actual impacts realised to date |
URL | http://www.ebi.ac.uk/pride/ |
Title | Mass spectral data of Blumeria-barley proteome (CSEPs re-searched dataset) |
Description | The earlier submitted dataset has been re-searched using an up-to-date blumeria database |
Type Of Material | Database/Collection of data |
Year Produced | 2013 |
Provided To Others? | No |
Impact | No actual impacts realised to date |
URL | http://www.ebi.ac.uk/pride/ |
Title | Structural annotation data for CSEPs |
Description | This dataset consists of structural prediction data (e.g. 3D models) of Candidates for Secreted Effector Proteins (CSEPs) of B. graminis f.sp. hordei. |
Type Of Material | Database/Collection of data |
Year Produced | 2013 |
Provided To Others? | No |
Impact | No actual impacts realised to date |
URL | http://www.reading.ac.uk/bioinf/CSEPs |
Description | COMPARATIVE PHOSPHOPROTEOMICS ANALYSIS OF ARABIDOPSIS THALIANA UNDER ABA, SALINITY AND DROUGHT STRESS |
Organisation | University of Reading |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Co-supervision of PhD studentship in above subject area |
Start Year | 2010 |
Description | Matrix Science collaboration |
Organisation | Matrix Science |
Country | United States |
Sector | Private |
PI Contribution | In kind collaboration and input from Matrix Science Ltd who develop software for proteomics analysis |
Start Year | 2005 |
Description | Pietro Spanu Group (Imperial College, BBSRC funded) |
Organisation | Imperial College London |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Close collaboration with group through BBSRC co-funded projects. |
Start Year | 2008 |
Description | Protein 3D structure modelling |
Organisation | University of Reading |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Collaboration with a bioinformation specialised in protein 3D structure model prediction. His input has been valuable in understanding the structure features that are specific to the Blumeria proteins involved in the infection process of barley. We provided the proteomic data. This collaboration is reflected in joint published work. |
Collaborator Contribution | Structure prediction modelling |
Impact | See joint publications. |
Start Year | 2010 |
Description | Strawberry Powdery mildew |
Organisation | University of Reading |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Discussion about possible research in the field of Strawberry Powdery mildew, sharing expertise in plant pathology, proteomics and mass spectrometry, benefiting a PhD student's project. |
Start Year | 2010 |
Description | Blumeria proteogenomics and quantitative plant proteomics RHUL |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | local |
Primary Audience | Participants in your research or patient groups |
Results and Impact | A talk at a seminar at RHUL. This talk was in two parts. The first part described the approach used for the proteogenomics analysis of the barley powdery mildew, i.e. the use of large scale proteomics to annotate and validated putative genes. The second part described the methodology for the metabolic labelling of plants with 15-N nitrogen salts and its use for quantitative plant proteomic analysis. Seminar no actual impacts realised to date |
Year(s) Of Engagement Activity | 2010 |