Protein Detection and Localization in Plant Cells Using Spot-Tagging

ABSTRACTFluorescent labelling of proteins without compromising their activity is crucial for determining their spatiotemporal localization while retaining their functionality. Spot-tag is a 12-amino acid peptide recognized by a single-domain nanobody. Here we introduce the spot-tag as a labelling strategy for proteins in fixed and living plant cells, using as an example the microtubule motor centromeric protein E-related Kinesin 7.3. Spot-tagging of ectopically introduced Kinesin 7.3 does not interfere with microtubules and spot staining results in a close-grained fluorophore labelling revealing a localization pattern that resembles “beads-on-a-string”. We anticipate that our protocol will apply to many more demanding protein cellular targets, offsetting activity perturbations and low photon quantum yields imposed by other protein-tagging approaches.

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Confocal microscopy of the cytoskeleton in living plant cells following microinjection of fluorescent probes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146497 ◽

1993 ◽

Vol 51 ◽

pp. 130-131

Author(s):

Ann Cleary

Keyword(s):

Cell Division ◽

Fluorescent Probes ◽

Actin Filaments ◽

Intracellular Transport ◽

Cell Structure ◽

Plant Cells ◽

Cell Plate ◽

Cell Cortex ◽

Living Plant ◽

Rhodamine Phalloidin

Microinjection of fluorescent probes into living plant cells reveals new aspects of cell structure and function. Microtubules and actin filaments are dynamic components of the cytoskeleton and are involved in cell growth, division and intracellular transport. To date, cytoskeletal probes used in microinjection studies have included rhodamine-phalloidin for labelling actin filaments and fluorescently labelled animal tubulin for incorporation into microtubules. From a recent study of Tradescantia stamen hair cells it appears that actin may have a role in defining the plane of cell division. Unlike microtubules, actin is present in the cell cortex and delimits the division site throughout mitosis. Herein, I shall describe actin, its arrangement and putative role in cell plate placement, in another material, living cells of Tradescantia leaf epidermis.The epidermis is peeled from the abaxial surface of young leaves usually without disruption to cytoplasmic streaming or cell division. The peel is stuck to the base of a well slide using 0.1% polyethylenimine and bathed in a solution of 1% mannitol +/− 1 mM probenecid.

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Faculty Opinions recommendation of Smaller, faster, brighter: advances in optical imaging of living plant cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718084605.793482669 ◽

2013 ◽

Author(s):

Staffan Persson ◽

Clara Sanchez-Rodriguez

Keyword(s):

Optical Imaging ◽

Plant Cells ◽

Living Plant

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Faculty Opinions recommendation of Complete microviscosity maps of living plant cells and tissues with a toolbox of targeting mechanoprobes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.738332148.793578370 ◽

2020 ◽

Author(s):

Ram Dixit

Keyword(s):

Plant Cells ◽

Living Plant

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Behavior of Microtubules in Living Plant Cells

PLANT PHYSIOLOGY ◽

10.1104/pp.112.2.455 ◽

1996 ◽

Vol 112 (2) ◽

pp. 455-461 ◽

Cited By ~ 53

Author(s):

P. K. Hepler ◽

J. M. Hush

Keyword(s):

Plant Cells ◽

Living Plant

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Effectors of biotrophic fungal plant pathogens

Functional Plant Biology ◽

10.1071/fp10072 ◽

2010 ◽

Vol 37 (10) ◽

pp. 913 ◽

Cited By ~ 12

Author(s):

Pamela H. P. Gan ◽

Maryam Rafiqi ◽

Adrienne R. Hardham ◽

Peter N. Dodds

Keyword(s):

Plant Tissue ◽

Fungal Pathogen ◽

Plant Pathogens ◽

Plant Cells ◽

Host Cells ◽

Plant Proteins ◽

Living Plant ◽

Host Cytoplasm ◽

Fungal Plant Pathogens ◽

Biotrophic Fungi

Plant pathogenic biotrophic fungi are able to grow within living plant tissue due to the action of secreted pathogen proteins known as effectors that alter the response of plant cells to pathogens. The discovery and identification of these proteins has greatly expanded with the sequencing and annotation of fungal pathogen genomes. Studies to characterise effector function have revealed that a subset of these secreted pathogen proteins interact with plant proteins within the host cytoplasm. This review focuses on the effectors of intracellular biotrophic and hemibiotrophic fungal plant pathogens and summarises advances in understanding the roles of these proteins in disease and in elucidating the mechanism of fungal effector uptake into host cells.

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