Chloroplasts alter their morphology and accumulate at the pathogen interface during infection by Phytophthora infestans.

Upon immune activation, chloroplasts switch off photosynthesis, produce antimicrobial compounds and associate with the nucleus through tubular extensions called stromules. Although it is well established that chloroplasts alter their position in response to light, little is known about the dynamics of chloroplast movement in response to pathogen attack. Here, we report that during infection with the Irish potato famine pathogen Phytophthora infestans, chloroplasts accumulate at the pathogen interface, associating with the specialized membrane that engulfs the pathogen haustorium. The chemical inhibition of actin polymerization reduces the accumulation of chloroplasts at pathogen haustoria, suggesting that this process is partially dependent on the actin cytoskeleton. However, chloroplast accumulation at haustoria does not necessarily rely on movement of the nucleus to this interface and is not affected by light conditions. Stromules are typically induced during infection, embracing haustoria and facilitating chloroplast interactions, to form dynamic organelle clusters. We found that infection-triggered stromule formation relies on BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1)-mediated surface immune signaling, whereas chloroplast repositioning towards haustoria does not. Consistent with the defense-related induction of stromules, effector-mediated suppression of BAK1-mediated immune signaling reduced stromule formation during infection. On the other hand, immune recognition of the same effector stimulated stromules, presumably via a different pathway. These findings implicate chloroplasts in a polarized response upon pathogen attack and point to more complex functions of these organelles in plant-pathogen interactions.

Applying Optical Tweezers with TIRF Microscopy to Quantify Physical Interactions Between Organelles in the Plant Endomembrane System.

Plant organelles are associated with each other through tethering proteins at membrane contact sites (MCS). Methods such as total internal reflection fluorescence (TIRF) optical tweezers allow us to probe organelle interactions in live plant cells. Optical tweezers (focused infrared laser beams) can trap organelles that have a different refractive index to their surrounding medium (cytosol), whilst TIRF allows us to simultaneously image behaviors of organelles in the thin region of cortical cytoplasm. However, few MCS tethering proteins have so far been identified and tested in a quantitative manner. Automated routines (such as setting trapping laser power and controlling the stage speed and distance) mean we can quantify organelle interactions in a repeatable and reproducible manner. Here we outline a series of protocols which describe laser calibrations required to collect robust data sets, generation of fluorescent plant material (Nicotiana tabacum, tobacco), how to set up an automated organelle trapping routine, and how to quantify organelle interactions (particularly organelle interactions with the endoplasmic reticulum). TIRF-optical tweezers enable quantitative testing of putative tethering proteins to reveal their role in plant organelle associations at MCS. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Microscope system set-up and stability Basic Protocol 2: Generation of transiently expressed fluorescent tobacco tissue by Agrobacterium-mediated infiltration Basic Protocol 3: Setting up an automated organelle trapping routine Basic Protocol 4: Quantifying organelle interactions.

The smfBox is an open-source platform for single-molecule FRET.

Single-molecule Förster Resonance Energy Transfer (smFRET) is a powerful technique capable of resolving both relative and absolute distances within and between structurally dynamic biomolecules. High instrument costs, and a lack of open-source hardware and acquisition software have limited smFRET's broad application by non-specialists. Here, we present the smfBox, a cost-effective confocal smFRET platform, providing detailed build instructions, open-source acquisition software, and full validation, thereby democratising smFRET for the wider scientific community.

Miro2 tethers the ER to mitochondria to promote mitochondrial fusion in tobacco leaf epidermal cells.

Mitochondria are highly pleomorphic, undergoing rounds of fission and fusion. Mitochondria are essential for energy conversion, with fusion favouring higher energy demand. Unlike fission, the molecular components involved in mitochondrial fusion in plants are unknown. Here, we show a role for the GTPase Miro2 in mitochondria interaction with the ER and its impacts on mitochondria fusion and motility. Mutations in AtMiro2's GTPase domain indicate that the active variant results in larger, fewer mitochondria which are attached more readily to the ER when compared with the inactive variant. These results are contrary to those in metazoans where Miro predominantly controls mitochondrial motility, with additional GTPases affecting fusion. Synthetically controlling mitochondrial fusion rates could fundamentally change plant physiology by altering the energy status of the cell. Furthermore, altering tethering to the ER could have profound effects on subcellular communication through altering the exchange required for pathogen defence.

Super-resolution Microscopy at Cryogenic Temperatures Using Solid Immersion Lenses.

Our mechanistic understanding of cell function depends on imaging biological processes in cells with molecular resolution. Super-resolution fluorescence microscopy plays a crucial role by reporting cellular ultrastructure with 20-30 nm resolution. However, this resolution is insufficient to image macro-molecular machinery at work. A path to improve resolution is to image under cryogenic conditions, which substantially increases the brightness of most fluorophores and preserves native ultrastructure much better than chemical fixatives. Cryogenic conditions are, however, underutilized because of the lack of compatible high numerical aperture (NA) objectives. Here we describe a protocol for the use of super-hemispherical solid immersion lenses (superSILs) to achieve super-resolution imaging at cryogenic temperatures with an effective NA of 2.17 and resolution of ~10 nm.