A Palette of Minimally Tagged Sucrose Analogues for Real‐Time Raman Imaging of Intracellular Plant Metabolism

Sucrose is the main saccharide used for long-distance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real-time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small (≈50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne-tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real-time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy

Simple, but Not Branched, Plasmodesmata Allow the Nonspecific Trafficking of Proteins in Developing Tobacco Leaves

AbstractLeaves undergo a sink–source transition during which a physiological change occurs from carbon import to export. In sink leaves, biolistic bombardment of plasmids encoding GFP-fusion proteins demonstrated that proteins with an Mr up to 50 kDa could move freely through plasmodesmata. During the sink–source transition, the capacity to traffic proteins decreased substantially and was accompanied by a developmental switch from simple to branched forms of plasmodesmata. Inoculation of sink leaves with a movement protein-defective virus showed that virally expressed GFP, but not viral RNA, was capable of trafficking between sink cells during infection. Contrary to dogma that plasmodesmata have a size exclusion limit below 1 kDa, the data demonstrate that nonspecific “macromolecular trafficking” is a general feature of simple plasmodesmata in sink leaves

The TGB1 Movement Protein of Potato virus X Reorganizes Actin and Endomembranes into the X-Body, a Viral Replication Factory

Potato virus X (PVX) requires three virally encoded proteins, the triple gene block (TGB), for movement between cells. TGB1 is a multifunctional protein that suppresses host gene silencing and moves from cell to cell through plasmodesmata, while TGB2 and TGB3 are membrane-spanning proteins associated with endoplasmic reticulum-derived granular vesicles. Here, we show that TGB1 organizes the PVX “X-body,” a virally induced inclusion structure, by remodeling host actin and endomembranes (endoplasmic reticulum and Golgi). Within the X-body, TGB1 forms helically arranged aggregates surrounded by a reservoir of the recruited host endomembranes. The TGB2/3 proteins reside in granular vesicles within this reservoir, in the same region as nonencapsidated viral RNA, while encapsidated virions accumulate at the outer (cytoplasmic) face of the X-body, which comprises a highly organized virus “factory.” TGB1 is both necessary and sufficient to remodel host actin and endomembranes and to recruit TGB2/3 to the X-body, thus emerging as the central orchestrator of the X-body. Our results indicate that the actin/endomembrane-reorganizing properties of TGB1 function to compartmentalize the viral gene products of PVX infection

Architecture and permeability of post-cytokinesis plasmodesmata lacking cytoplasmic sleeves

This work was supported by the grants by the Region Aquitaine (to E.M.B) and PEPS (Initial Support for Exploratory Projects to E.M.B) and National Agency for Research (Grant ANR-14-CE19-0006-01 to E.M.B).Plasmodesmata are remarkable cellular machines responsible for the controlled exchange of proteins, small RNAs and signalling molecules between cells. They are lined by the plasma membrane (PM), contain a strand of tubular endoplasmic reticulum (ER), and the space between these two membranes is thought to control plasmodesmata permeability. Here, we have reconstructed plasmodesmata three-dimensional (3D) ultrastructure with an unprecedented level of 3D information using electron tomography. We show that within plasmodesmata, ER-PM contact sites undergo substantial remodelling events during cell differentiation. Instead of being open pores, post-cytokinesis plasmodesmata present such intimate ER-PM contact along the entire length of the pores that no intermembrane gap is visible. Later on, during cell expansion, the plasmodesmata pore widens and the two membranes separate, leaving a cytosolic sleeve spanned by tethers whose presence correlates with the appearance of the intermembrane gap. Surprisingly, the post-cytokinesis plasmodesmata allow diffusion of macromolecules despite the apparent lack of an open cytoplasmic sleeve, forcing the reassessment of the mechanisms that control plant cell-cell communication.PostprintPeer reviewe

Tracking the green invaders: advances in imaging virus infection in plants

Bioimaging contributes significantly to our understanding of plant virus infections. In the present review, we describe technical advances that enable imaging of the infection process at previously unobtainable levels. We highlight how such new advances in subcellular imaging are contributing to a detailed dissection of all stages of the viral infection process. Specifically, we focus on: (i) the increasingly detailed localizations of viral proteins enabled by a diversifying palette of cellular markers; (ii) approaches using fluorescence microscopy for the functional analysis of proteins in vivo; (iii) the imaging of viral RNAs; (iv) methods that bridge the gap between optical and electron microscopy; and (v) methods that are blurring the distinction between imaging and structural biology. We describe the advantages and disadvantages of such techniques and place them in the broader perspective of their utility in analysing plant virus infection.</p