High-throughput confocal imaging of intact live tissue enables quantification of membrane trafficking in Arabidopsis.
Membrane compartmentalization and trafficking within and between cells is considered an essential cellular property of higher eukaryotes. We established a high-throughput imaging method suitable for the quantitative detection of membrane compartments at subcellular resolution in intact epidermal tissue. Whole Arabidopsis (Arabidopsis thaliana) cotyledon leaves were subjected to quantitative confocal laser microscopy using automated image acquisition, computational pattern recognition, and quantification of membrane compartments. This revealed that our method is sensitive and reliable to detect distinct endomembrane compartments. We applied quantitative confocal laser microscopy to a transgenic line expressing GFP-2xFYVE as a marker for endosomal compartments during biotic or abiotic stresses, and detected markedly quantitative adaptations in response to changing environments. Using a transgenic line expressing the plasma membrane-resident syntaxin GFP-PEN1, we quantified the pathogen-inducible extracellular accumulation of this fusion protein at fungal entry sites. Our protocol provides a platform to study the quantitative and dynamic changes of endomembrane trafficking, and potential adaptations of this machinery to physiological stress.
Salomon
Susanne
S
Department of Plant Microbe Interactions, Max-Planck Institut für Pflanzenzüchtungsforschung, Cologne, Germany.
Grunewald
Dorit
D
Stüber
Kurt
K
Schaaf
Sebastian
S
MacLean
Dan
D
Schulze-Lefert
Paul
P
Robatzek
Silke
S
D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D010363:Pattern Recognition, Automated; D019441:Plant Epidermis / Q000166:cytology; D018515:Plant Leaves / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000378:metabolism; D021381:Protein Transport; D050765:Qa-SNARE Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013312:Stress, Physiological
D029681:Arabidopsis Proteins; C479425:PEN1 protein, Arabidopsis; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins