Molecular Tracer Systems for Visualizing Plant-Pathogen Interactions Compatible with Fluorescence Imaging and Cryo-Electron and X-Ray Tomography
Authors:
Dara Dowlatshahi1,2* ([email protected]), Jacob Summers2, David Nana Agyeman-Budu1,2, Deepak Bhandari3, Jung-Gun Kim2, Rose Knight1,2, Adam Bowman2,4, James Safranek1, Johanna Nelson Weker1, Christopher Takacs1,
Mary Beth Mudgett2, Federica Brandizzi3, Mark Kasevich2, Soichi Wakatsuki1,2 (PI)
Institutions:
1SLAC National Accelerator Laboratory; 2Stanford University; 3Michigan State University; 4Salk Institute for Biological Studies
Abstract
Plant–pathogen interactions are complex and dynamic phenomena, relevant to fundamental, environmental and bioenergy biology. Imaging plays important roles in understanding the interactions at the atomic, molecular, subcellular, cellular, to tissue and whole plant scales. Each bioimaging method has its intrinsic limitations in spatial and/or temporal resolution, field of view and depth, and sensitivity. Fluorescence-based optical microscopes have huge advantages in monitoring dynamics of cellular and subcellular events using wide spectral ranges with super-resolution, light-sheet 3D, and wide-field imaging, but cannot go beyond 10 or 20 nanometer spatial resolution. X-ray imaging and tomography can penetrate deeper than light, and with high brilliance synchrotron sources one can reach 10 nm spatial resolution, but with a high risk of sample damage from radiation dose. Cryogenic electron tomography (cryo-ET) can offer tremendous insight into the subcellular organization of organelles and macromolecules down to several nm resolution, but information gained is largely static.
Experimental validation of the spatial position and size of molecules observed in these methods typically requires some sort of reference probe or fiducial marker in the case of tomography. These markers are typically exogenously added for correlation, and this project arm aims to improve upon this by developing molecular tracers with protein nanocages containing metal nanocrystals that also serve as intrinsic fiducials. The group presents advances towards fluorescent protein- and nanocrystal-containing cages as molecular tracers for X-ray imaging/microscopy and EO-FLIM and fiducial markers for cryo-ET, including proof-of-concept synchrotron TXM images of leaf samples bombarded with 400 nm nanogold particles. The group shares ongoing development and discuss further plans for the initial application examining fungal-plant pathogen interactions via chitin binding domains at the plant cell surface using split fluorescence complementation probes.
References
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Bowman, A. J., et al. 2019. “Electro-Optic Imaging Enables Efficient Wide-Field Fluorescence Lifetime Microscopy,” Nature Communications 10(1) DOI:10.1038/s41467-019-12535-5.
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Zhang, K., et al. 2022. “Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics Against Acute Myeloid Leukemia,” ACS Central Science 8(2), 214–22. DOI:10.1021/acscentsci.1c01090.
Funding Information
This research is supported by the U. S. DOE, Office of Science, through the Biomolecular Characterization and Imaging Sciences program, BER program, under BCIS FWP 100878.