Phytophthora infestans causes the devastating late blight disease on potato and tomato. Its high adaptability allows P. infestans to easily build up pesticide resistance or avoid recognition by resistant cultivars. The resulting arms race requires continuous investments in the development of novel control strategies. To facilitate this development, we propose to set up a novel microscopic assay for quantitative screening of the P. infestans infection efficiency. By quantitatively assessing the infection process of P. infestans, we will categorize phenotypic traits such as cyst germination, hyphal growth and appressorium formation, and use these as indicators for the infection efficiency of the pathogen. In collaboration with experts specialized in developing microlayers for advanced microscopy, we will create plant cell wall mimics with a variation of physical properties to investigate hitherto unstudied physical aspects of the infection process, including adherence to the plant cell wall, penetration force of appressoria, and how physical properties of the plant cell wall affect the infection process. Through automation of microscopy and data processing, we will show the potential of this assay as a quantitative, fast and high-throughput method. Finally, we will assess the assay’s predictive value by comparing results obtained from this assay with that in vivo assays. Such a quantitative microscopic assay for infection efficiency (qMIE-assay) of P. infestans is unprecedented. It can be used to quantitatively predict the efficacy of crop protection agents, and the effects of cell wall properties on pathogen penetration, thereby serving both the agrochemical industry and plant breeding companies.

De Najaarsvergadering van de Sectie AMM is het jaarlijkse ontmoetingsmoment van de moleculaire microbiologen (Universiteiten, Instituten en Bedrijven) in Nederland. Het programma omvat (buitenlandse) keynote sprekers en voordrachten van jonge groepsleiders en biedt een ideaal platform voor het onderhouden en uitbreiden en versterken van het onderzoeksnetwerk in het veld van de algemene en moleculaire microbiologie in Nederland.

Receptors localizing to the cell surface play an essential role in immunity of plants to microbial pathogens. Receptor-like proteins (RLPs) are ubiquitous and activate defence upon recognition of an appropriate pathogen-associated ligand. RLPs frequently contain extracellular leucine-rich repeats (LRRs) involved in pathogen perception. However, in contrast to receptor-like kinases (RLKs), they lack a cytoplasmic kinase domain for cellular signalling. The Cf proteins and Ve1 of tomato (Solanum lycopersicum; Sl) are examples of LRR-RLPs mediating resistance to the fungal pathogens Cladosporium fulvum and Verticillium dahliae, respectively. The mechanism by which these proteins trigger immunity has remained enigmatic for a long time. Recently, we discovered that the LRR-RLK SOBIR1 interacts with Cfs and Ve1 in planta and that this LRR-RLK is essential for their function (Liebrand, et al. (2013) PNAS 110:10010-10015). SOBIR1, which is highly conserved in plants, interacts with additional LRR-RLPs involved in immunity or development. We propose that SOBIR1 plays a key role by acting as a regulatory RLK for LRR-RLPs. Here we will study the role of SOBIR1 in LRR-RLP-mediated plant immunity, by (I) identifying its downstream interactors, (II) determining the phosphorylation status of its kinase domain in the presence or absence of the ligand matching the interacting LRR-RLP and (III) determining where SOBIR1 and LRR-RLPs interact at the subcellular level. Understanding SOBIR1 function in relation to LRR-RLP-mediated immunity provides insight into this novel and yet unexplored segment of the defence arsenal of plants and allows exploiting this regulator for generating durable and broad-spectrum resistance of plants to pathogens.

Fire blight caused by the necrotrophic fungus Botrytis elliptica is the most destructive fungal disease on lily. Fungal secreted effector proteins play a key role in conferring pathogenicity, upon recognition by plant receptor proteins. In plant-necrotroph interactions, sensitivity to effector proteins often correlates with host susceptibility, whereas insensitivity to effectors renders the plant more resistant. Our previous studies have shown that programmed cell death-inducing activity of B. elliptica effector proteins is required for pathogenicity on lily. These effector proteins act as “host-selective toxins”, since they induce cell death exclusively in lily. However, both B. elliptica effector-proteins and the corresponding lily receptor-proteins remain to be identified. This project aims to identify and characterize the effector-protein(s) of B. elliptica, required for cell death-induction in lily. We will investigate virulence patterns in different fungal isolate-lily cultivar combinations, and identify effector genes by integrating proteomics, comparative genomics and gene expression data. Candidate effector-proteins will be expressed in yeast. Purified effector-proteins will be infiltrated into diverse lily cultivars to screen for differences in sensitivity. The prediction is that lily cultivars displaying insensitivity to effectors will show increased resistance to B. elliptica. The importance of effector-genes for virulence of B. elliptica will be tested by gene replacement. Finally, using a biochemical approach, we will attempt to identify lily receptor proteins, involved in effector recognition. This project will provide the biological tools to develop breeding programmes to eliminate effector sensitive host genes and to obtain lily cultivars with increased resistance to fire blight.