Genetic analysis of endosomal compartments using high throughput imaging
Final Report Abstract
During the DFG funded project new components important for the intracellular and spatial control of membrane trafficking events during plant immunity were identified by using a quantitative imaging approach. The plant endomembrane system regulates a plethora of cellular processes whereby vesicle distribution and trafficking pathways play essential roles during cell development and response to environmental signals. Especially during defense reactions against pathogens, it has become increasingly evident that vesicle secretion and endocytic pathways are important players in the plant's innate immune system. Plants employ a multi-layered innate immune system to fight disease. The first layer acts as an early warning system and involves the perception of conserved microbial molecules that are characteristic for microbes so called pathogen- associated molecular patterns (PAMPs). This recognition occurs at the plasma membrane by surface-localized pattern recognition receptors (PRRs) and enables plants to sense non-self molecules displayed by microbes to mount proper defense responses. The plasma membrane receptor FLAGELLIN SENSING2 (FLS2) confers immunity against bacterial infection through perception of its ligand flagellin (flg22), a conserved bacterial peptide. Following ligand elicitation, FLS2 is activated and internalized into vesicles but the role of this receptor internalization process in immune signalling is not yet clear. It is assumed that the spatio-temporal dynamics of receptor mediated endocytosis are critical for their function and to identify components controlling these membrane trafficking pathways, will help to understand how plant cells fend pathogens in such a highly localised and efficient manner. As an approach to address the role of ligand induced endocytosis of FLS2 trafficking and to understand how it does intersect and contribute to immune signalling, the endocytic route of FLS2 was indepth analysed. For this, quantitative imaging for co-localization studies in combination with chemical and functional studies of endomembrane pathways was performed. In summary, this study reported the first time of two independent endocytic pathways of FLS2 dependent on its activation status. This dynamic pattern of subcellular trafficking for FLS2 revealed a defined framework for ligand-dependent endocytosis of this receptor. During this project a quantitative imaging platform was established to study subcellular compartments in leaf cells. This method contributed also to the identification of new regulator of FLS2 endosomal sorting and revealed a new role of membrane trafficking components for plant immunity to a bacterial pathogen as well as contributed to a large scale analysis of plasmodesmata (cell to cell connections between plant cells) during plant development and stress responses. The optimized or new custom made algorithms suitable for the detection of different endomembrane compartments and for the segmentation of pant cells are freely accessible for the scientific community (http://www.plant-image-analysis.org/software. This project optimized a highly innovative imaging strategy for subcellular characterization of plant leaves and provides a framework for further cell biological studies in plant science.
Publications
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(2012) Receptor Trafficking in Plants, in Receptor-like Kinases, in: Plants, Signaling and Communication in Plants, Volume 13, 253-274, Springer
Beck M and Robatzek S
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(2012) Spatio-temporal cellular dynamics of the Arabidopsis flagellin receptor reveal activation status-dependent endosomal sorting Plant Cell.;24(10) 4205-19
Beck M, Zhou J, MacLean D and Robatzek S
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(2012) The INs and OUTs of pattern recognition receptors at the cell surface Curr Opin Plant Biol.;15(4):367-74
Beck M, Heard W, Mbengue M and Robatzek S.
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(2013) A Developmental Framework for Complex Plasmodesmata Formation Revealed by Large-Scale Imaging of the Arabidopsis Leaf Epidermis. Plant Cell 25(1):57-70
Fitzgibbon J, Beck M, Zhou J, Faulkner C, Robatzek S, and Oparka K
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(2013) ESCRT-I Mediates FLS2 Endosomal Sorting and Plant Immunity. PLoS Genet 9(12): e1004035
Spallek T, Beck M, Ben Khaled S, Salomon S, Bourdais G, Schellman S., Robatzek S.
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(2013) Salicylic acid interferes with clathrin-mediated endocytic protein trafficking. PNAS 7;110(19):7946-51
Du Y, Tejos R, Beck M, Himschoot E, Li H, Robatzek S, Vanneste S, Friml J
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(2014) High-throughput imaging of plant immune responses, in: Birch P., Jones J., Bos J. (eds) Plant-Pathogen Interactions. Methods in Molecular Biology (Methods and Protocols), vol 1127. Humana Press, Totowa, NJ , pp 67-80
Beck M, Zhou J, Faulkner C, and Robatzek S
