Functional analysis of the Blumeria Haustoria - Barley interactome

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.

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.