Imaging the living plant cell: From probes to quantification.

At the center of cell biology is our ability to image the cell and its various components, either in isolation or within an organism. Given its importance, biological imaging has emerged as a field of its own, which is inherently highly interdisciplinary. Indeed, biologists rely on physicists and engineers to build new microscopes and imaging techniques, chemists to develop better imaging probes, and mathematicians and computer scientists for image analysis and quantification. Live imaging collectively involves all the techniques aimed at imaging live samples. It is a rapidly evolving field, with countless new techniques, probes, and dyes being continuously developed. Some of these new methods or reagents are readily amenable to image plant samples, while others are not and require specific modifications for the plant field. Here, we review some recent advances in live imaging of plant cells. In particular, we discuss the solutions that plant biologists use to live image membrane-bound organelles, cytoskeleton components, hormones, and the mechanical properties of cells or tissues. We not only consider the imaging techniques per se, but also how the construction of new fluorescent probes and analysis pipelines are driving the field of plant cell biology.

A nanodomain-anchored scaffolding complex is required for the function and localization of phosphatidylinositol 4-kinase alpha in plants.

Phosphoinositides are low-abundant lipids that participate in the acquisition of membrane identity through their spatiotemporal enrichment in specific compartments. Phosphatidylinositol 4-phosphate (PI4P) accumulates at the plant plasma membrane driving its high electrostatic potential, and thereby facilitating interactions with polybasic regions of proteins. PI4Kα1 has been suggested to produce PI4P at the plasma membrane, but how it is recruited to this compartment is unknown. Here, we pin-point the mechanism that tethers Arabidopsis thaliana phosphatidylinositol 4-kinase alpha1 (PI4Kα1) to the plasma membrane via a nanodomain-anchored scaffolding complex. We established that PI4Kα1 is part of a complex composed of proteins from the NO-POLLEN-GERMINATION, EFR3-OF-PLANTS, and HYCCIN-CONTAINING families. Comprehensive knockout and knockdown strategies revealed that subunits of the PI4Kα1 complex are essential for pollen, embryonic, and post-embryonic development. We further found that the PI4Kα1 complex is immobilized in plasma membrane nanodomains. Using synthetic mis-targeting strategies, we demonstrate that a combination of lipid anchoring and scaffolding localizes PI4Kα1 to the plasma membrane, which is essential for its function. Together, this work opens perspectives on the mechanisms and function of plasma membrane nanopatterning by lipid kinases.

Arabidopsis ADR1 helper NLR immune receptors localize and function at the plasma membrane in a phospholipid dependent manner.

Activation of nucleotide-binding leucine-rich repeat receptors (NLRs) results in immunity and a localized cell death. NLR cell death activity requires oligomerization and in some cases plasma membrane (PM) localization. The exact mechanisms underlying PM localization of NLRs lacking predicted transmembrane domains or recognizable lipidation motifs remain elusive. We used confocal microscopy, genetically encoded molecular tools and protein-lipid overlay assays to determine whether PM localization of members of the Arabidopsis HeLo-/RPW8-like domain 'helper' NLR (RNL) family is mediated by the interaction with negatively charged phospholipids of the PM. Our results show that PM localization and stability of some RNLs and one CC-type NLR (CNL) depend on the direct interaction with PM phospholipids. Depletion of phosphatidylinositol-4-phosphate from the PM led to a mis-localization of the analysed NLRs and consequently inhibited their cell death activity. We further demonstrate homo- and hetero-association of members of the RNL family. Our results provide new insights into the molecular mechanism of NLR localization and defines an important role of phospholipids for CNL and RNL PM localization and consequently, for their function. We propose that RNLs interact with anionic PM phospholipids and that RNL-mediated cell death and immune responses happen at the PM.

Probing formation of cargo/importin-α transport complexes in plant cells using a pathogen effector.

Importin-αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin-α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin-α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin-α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co-opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin-α paralogs from Arabidopsis thaliana. A crystal structure of the importin-α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin-αs expressed in rosette leaves have an almost identical NLS-binding site. Comparison of the importin-α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin-α, sequence variation at the importin-α NLS-binding sites and tissue-specific expression levels of importin-αs determine formation of cargo/importin-α transport complexes in plant cells.

The plasmodesmal protein PDLP1 localises to haustoria-associated membranes during downy mildew infection and regulates callose deposition.

The downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa) is a filamentous oomycete that invades plant cells via sophisticated but poorly understood structures called haustoria. Haustoria are separated from the host cell cytoplasm and surrounded by an extrahaustorial membrane (EHM) of unknown origin. In some interactions, including Hpa-Arabidopsis, haustoria are progressively encased by host-derived, callose-rich materials but the molecular mechanisms by which callose accumulates around haustoria remain unclear. Here, we report that PLASMODESMATA-LOCATED PROTEIN 1 (PDLP1) is expressed at high levels in Hpa infected cells. Unlike other plasma membrane proteins, which are often excluded from the EHM, PDLP1 is located at the EHM in Hpa-infected cells prior to encasement. The transmembrane domain and cytoplasmic tail of PDLP1 are sufficient to convey this localization. PDLP1 also associates with the developing encasement but this association is lost when encasements are fully mature. We found that the pdlp1,2,3 triple mutant is more susceptible to Hpa while overexpression of PDLP1 enhances plant resistance, suggesting that PDLPs enhance basal immunity against Hpa. Haustorial encasements are depleted in callose in pdlp1,2,3 mutant plants whereas PDLP1 over-expression elevates callose deposition around haustoria and across the cell surface. These data indicate that PDLPs contribute to callose encasement of Hpa haustoria and suggests that the deposition of callose at haustoria may involve similar mechanisms to callose deposition at plasmodesmata.

Expression profiling during arabidopsis/downy mildew interaction reveals a highly-expressed effector that attenuates responses to salicylic acid.

Plants have evolved strong innate immunity mechanisms, but successful pathogens evade or suppress plant immunity via effectors delivered into the plant cell. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on Arabidopsis thaliana, and a genome sequence is available for isolate Emoy2. Here, we exploit the availability of genome sequences for Hpa and Arabidopsis to measure gene-expression changes in both Hpa and Arabidopsis simultaneously during infection. Using a high-throughput cDNA tag sequencing method, we reveal expression patterns of Hpa predicted effectors and Arabidopsis genes in compatible and incompatible interactions, and promoter elements associated with Hpa genes expressed during infection. By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of the cognate recognized effector ATR1. Arabidopsis salicylic acid (SA)-responsive genes including PR1 were activated not only at early time points in the incompatible interaction but also at late time points in the compatible interaction. By histochemical analysis, we found that Hpa suppresses SA-inducible PR1 expression, specifically in the haustoriated cells into which host-translocated effectors are delivered, but not in non-haustoriated adjacent cells. Finally, we found a highly-expressed Hpa effector candidate that suppresses responsiveness to SA. As this approach can be easily applied to host-pathogen interactions for which both host and pathogen genome sequences are available, this work opens the door towards transcriptome studies in infection biology that should help unravel pathogen infection strategies and the mechanisms by which host defense responses are overcome.

The Arabidopsis microtubule-associated protein MAP65-3 supports infection by filamentous biotrophic pathogens by down-regulating salicylic acid-dependent defenses.

The oomycete Hyaloperonospora arabidopsidis and the ascomycete Erysiphe cruciferarum are obligate biotrophic pathogens causing downy mildew and powdery mildew, respectively, on Arabidopsis. Upon infection, the filamentous pathogens induce the formation of intracellular bulbous structures called haustoria, which are required for the biotrophic lifestyle. We previously showed that the microtubule-associated protein AtMAP65-3 plays a critical role in organizing cytoskeleton microtubule arrays during mitosis and cytokinesis. This renders the protein essential for the development of giant cells, which are the feeding sites induced by root knot nematodes. Here, we show that AtMAP65-3 expression is also induced in leaves upon infection by the downy mildew oomycete and the powdery mildew fungus. Loss of AtMAP65-3 function in the map65-3 mutant dramatically reduced infection by both pathogens, predominantly at the stages of leaf penetration. Whole-transcriptome analysis showed an over-represented, constitutive activation of genes involved in salicylic acid (SA) biosynthesis, signaling, and defense execution in map65-3, whereas jasmonic acid (JA)-mediated signaling was down-regulated. Preventing SA synthesis and accumulation in map65-3 rescued plant susceptibility to pathogens, but not the developmental phenotype caused by cytoskeleton defaults. AtMAP65-3 thus has a dual role. It positively regulates cytokinesis, thus plant growth and development, and negatively interferes with plant defense against filamentous biotrophs. Our data suggest that downy mildew and powdery mildew stimulate AtMAP65-3 expression to down-regulate SA signaling for infection.

A downy mildew effector attenuates salicylic acid-triggered immunity in Arabidopsis by interacting with the host mediator complex.

Plants are continually exposed to pathogen attack but usually remain healthy because they can activate defences upon perception of microbes. However, pathogens have evolved to overcome plant immunity by delivering effectors into the plant cell to attenuate defence, resulting in disease. Recent studies suggest that some effectors may manipulate host transcription, but the specific mechanisms by which such effectors promote susceptibility remain unclear. We study the oomycete downy mildew pathogen of Arabidopsis, Hyaloperonospora arabidopsidis (Hpa), and show here that the nuclear-localized effector HaRxL44 interacts with Mediator subunit 19a (MED19a), resulting in the degradation of MED19a in a proteasome-dependent manner. The Mediator complex of ∼25 proteins is broadly conserved in eukaryotes and mediates the interaction between transcriptional regulators and RNA polymerase II. We found MED19a to be a positive regulator of immunity against Hpa. Expression profiling experiments reveal transcriptional changes resembling jasmonic acid/ethylene (JA/ET) signalling in the presence of HaRxL44, and also 3 d after infection with Hpa. Elevated JA/ET signalling is associated with a decrease in salicylic acid (SA)-triggered immunity (SATI) in Arabidopsis plants expressing HaRxL44 and in med19a loss-of-function mutants, whereas SATI is elevated in plants overexpressing MED19a. Using a PR1::GUS reporter, we discovered that Hpa suppresses PR1 expression specifically in cells containing haustoria, into which RxLR effectors are delivered, but not in nonhaustoriated adjacent cells, which show high PR1::GUS expression levels. Thus, HaRxL44 interferes with Mediator function by degrading MED19, shifting the balance of defence transcription from SA-responsive defence to JA/ET-signalling, and enhancing susceptibility to biotrophs by attenuating SA-dependent gene expression.

Three BUB1 and BUBR1/MAD3-related spindle assembly checkpoint proteins are required for accurate mitosis in Arabidopsis.

The spindle assembly checkpoint (SAC) is a refined surveillance mechanism which ensures that chromosomes undergoing mitosis do not segregate until they are properly attached to the spindle microtubules (MT). The SAC has been extensively studied in metazoans and yeast, but little is known about its role in plants. We identified proteins interacting with a MT-associated protein MAP65-3, which plays a critical role in organising mitotic MT arrays, and carried out a functional analysis of previously and newly identified SAC components. We show that Arabidopsis SAC proteins BUB3.1, MAD2, BUBR1/MAD3s and BRK1 interact with each other and with MAP65-3. We found that two BUBR1/MAD3s interacted specifically at centromeres. When stably expressed in Arabidopsis, BRK1 localised to the kinetochores during all stages of the mitotic cell cycle. Early in mitosis, BUB3.1 and BUBR1/MAD3.1 localise to the mitotic spindle, where MAP65-3 organises spindle MTs. A double-knockout mad3.1 mad3.2 mutant presented spindle MT abnormalities, chromosome misalignments on the metaphase plate and the production of lagging chromosomes and micronuclei during mitosis. We conclude that BRK1 and BUBR1/MAD3-related proteins play a key role in ensuring faithful chromosome segregation during mitosis and that their interaction with MAP65-3 may be important for the regulation of MT-chromosome attachment.

Subcellular localization of the Hpa RxLR effector repertoire identifies a tonoplast-associated protein HaRxL17 that confers enhanced plant susceptibility.

Filamentous phytopathogens form sophisticated intracellular feeding structures called haustoria in plant cells. Pathogen effectors are likely to play a role in the establishment and maintenance of haustoria in addition to their better-characterized role in suppressing plant defence. However, the specific mechanisms by which these effectors promote virulence remain unclear. To address this question, we examined changes in subcellular architecture using live-cell imaging during the compatible interaction between the oomycete Hyaloperonospora arabidopsidis (Hpa) and its host Arabidopsis. We monitored host-cell restructuring of subcellular compartments within plant mesophyll cells during haustoria ontogenesis. Live-cell imaging highlighted rearrangements in plant cell membranes upon infection, in particular to the tonoplast, which was located close to the extra-haustorial membrane surrounding the haustorium. We also investigated the subcellular localization patterns of Hpa RxLR effector candidates (HaRxLs) in planta. We identified two major classes of HaRxL effector based on localization: nuclear-localized effectors and membrane-localized effectors. Further, we identified a single effector, HaRxL17, that associated with the tonoplast in uninfected cells and with membranes around haustoria, probably the extra-haustorial membrane, in infected cells. Functional analysis of selected effector candidates in planta revealed that HaRxL17 enhances plant susceptibility. The roles of subcellular changes and effector localization, with specific reference to the potential role of HaRxL17 in plant cell membrane trafficking, are discussed with respect to Hpa virulence.

Multiple candidate effectors from the oomycete pathogen Hyaloperonospora arabidopsidis suppress host plant immunity.

Oomycete pathogens cause diverse plant diseases. To successfully colonize their hosts, they deliver a suite of effector proteins that can attenuate plant defenses. In the oomycete downy mildews, effectors carry a signal peptide and an RxLR motif. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on the model plant Arabidopsis thaliana (Arabidopsis). We investigated if candidate effectors predicted in the genome sequence of Hpa isolate Emoy2 (HaRxLs) were able to manipulate host defenses in different Arabidopsis accessions. We developed a rapid and sensitive screening method to test HaRxLs by delivering them via the bacterial type-three secretion system (TTSS) of Pseudomonas syringae pv tomato DC3000-LUX (Pst-LUX) and assessing changes in Pst-LUX growth in planta on 12 Arabidopsis accessions. The majority (~70%) of the 64 candidates tested positively contributed to Pst-LUX growth on more than one accession indicating that Hpa virulence likely involves multiple effectors with weak accession-specific effects. Further screening with a Pst mutant (ΔCEL) showed that HaRxLs that allow enhanced Pst-LUX growth usually suppress callose deposition, a hallmark of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We found that HaRxLs are rarely strong avirulence determinants. Although some decreased Pst-LUX growth in particular accessions, none activated macroscopic cell death. Fewer HaRxLs conferred enhanced Pst growth on turnip, a non-host for Hpa, while several reduced it, consistent with the idea that turnip's non-host resistance against Hpa could involve a combination of recognized HaRxLs and ineffective HaRxLs. We verified our results by constitutively expressing in Arabidopsis a sub-set of HaRxLs. Several transgenic lines showed increased susceptibility to Hpa and attenuation of Arabidopsis PTI responses, confirming the HaRxLs' role in Hpa virulence. This study shows TTSS screening system provides a useful tool to test whether candidate effectors from eukaryotic pathogens can suppress/trigger plant defense mechanisms and to rank their effectiveness prior to subsequent mechanistic investigation.

Maintaining asymmetry in cell division.

Promoting asymmetric division through microtubule dynamics establishes cell fate.

Connecting the plant cytoskeleton to the cell surface via the phosphoinositides.

Plants have developed fine-tuned cellular mechanisms to respond to a variety of intracellular and extracellular signals. These responses often necessitate the rearrangement of the plant cytoskeleton to modulate cell shape and/or to guide vesicle trafficking. At the cell periphery, both actin filaments and microtubules associate with the plasma membrane that acts as an integrator of the intrinsic and extrinsic environments. At this membrane, acidic phospholipids such as phosphatidic acid, and phosphoinositides contribute to the selection of peripheral proteins and thereby regulate the organization and dynamic of the actin and microtubules. After recognition of the importance of phosphatidic acid on cytoskeleton dynamics and rearrangement, it became apparent that the other lipids might play a specific role in shaping the cytoskeleton. This review focuses on the emerging role of the phosphatidylinositol 4,5-bisphosphate for the regulation of the peripherical cytoskeleton during cellular processes such as cytokinesis, polar growth, biotic and abiotic responses.

The phosphoinositide signature guides the final step of plant cytokinesis.

Plant cytokinesis, which fundamentally differs from that in animals, requires the outward expansion of a plasma membrane precursor named the cell plate. How the transition from a cell plate to a plasma membrane occurs remains poorly understood. Here, we report that the acquisition of plasma membrane identity occurs through lateral patterning of the phosphatidylinositol 4,5-bisphosphate PI(4,5)P2 at the newly formed cell plate membrane. There, the phosphoinositide phosphatase SAC9 emerges as a key regulator, colocalizing with and regulating the function of the microtubule-associated protein MAP65-3 at the cell plate leading zone. In sac9-3 mutant, the polar distribution of PI(4,5)P2 at the cell plate is altered, leading to ectopic recruitment of the cytokinesis apparatus and formation of an additional cell plate insertion site. We propose that at the cell plate, SAC9 drives the depletion of PI(4,5)P2, which acts as a polar cue to spatially separate cell plate expansion from the acquisition of plasma membrane identity during final step of cytokinesis.

Tools for studying the cytoskeleton during plant cell division.

The plant cytoskeleton regulates fundamental biological processes, including cell division. How to experimentally perturb the cytoskeleton is a key question if one wants to understand the role of both actin filaments (AFs) and microtubules (MTs) in a given biological process. While a myriad of mutants are available, knock-out in cytoskeleton regulators, when nonlethal, often produce little or no phenotypic perturbation because such regulators are often part of a large family, leading to functional redundancy. In this review, alternative techniques to modify the plant cytoskeleton during plant cell division are outlined. The different pharmacological and genetic approaches already developed in cell culture, transient assays, or in whole organisms are presented. Perspectives on the use of optogenetics to perturb the plant cytoskeleton are also discussed.

Methods to Visualize the Actin Cytoskeleton During Plant Cell Division.

Cell division in plants consists of separating the mother cell in two daughter cells by the centrifugal growth of a new wall. This process involves the reorganization of the structural elements of the cell, namely the microtubules and actin cytoskeleton which allow the coordination, the orientation, and the progression of mitosis. In addition to its implication in those plant-specific structures, the actin cytoskeleton, in close association with the plasma membrane, exhibits specific patterning at the cortex of the dividing cells, and might act as a signaling component. This review proposes an overview of the techniques available to visualize the actin cytoskeleton in fixed tissues or living cells during division, including electron, fluorescent, and super-resolution microscopy techniques.

Experimental manipulation of phosphoinositide lipids: from cells to organisms.

Phosphoinositides (PIs) have critical roles in various cellular, physiological, developmental, pathological, and infectious processes. They are signaling phospholipids that can affect every aspect of membrane biology, including protein function (e.g., recruitment and activity), membrane physicochemical properties (e.g., curvature, surface charges, and packing), and the generation of secondary messengers. PIs act at precise locations within the cell in a dose-dependent manner, and their local concentration can vary drastically during signaling and trafficking. Thus, techniques able to manipulate PI amounts acutely and with subcellular accuracy are paramount to understanding the role of these lipids in vivo. Here, we review these methods and emphasize approaches recently developed to perturb PI levels in multicellular organisms.

Dynamic apico-basal enrichment of the F-actin during cytokinesis in Arabidopsis cells embedded in their tissues.

Cell division is a tightly regulated mechanism, notably in tissues where malfunctions can lead to tumour formation or developmental defects. This is particularly true in land plants, where cells cannot relocate and therefore cytokinesis determines tissue topology. In plants, cell division is executed in radically different manners than in animals, with the appearance of new structures and the disappearance of ancestral mechanisms. Whilst F-actin and microtubules closely co-exist, recent studies mainly focused on the involvement of microtubules in this key process. Here, we used a root tracking system to image the spatio-temporal dynamics of both F-actin reporters and cell division markers in dividing cells embedded in their tissues. In addition to the F-actin accumulation at the phragmoplast, we observed and quantified a dynamic apico-basal enrichment of F-actin from the prophase/metaphase transition until the end of the cytokinesis.

The Arabidopsis SAC9 enzyme is enriched in a cortical population of early endosomes and restricts PI(4,5)P2 at the plasma membrane.

Membrane lipids, and especially phosphoinositides, are differentially enriched within the eukaryotic endomembrane system. This generates a landmark code by modulating the properties of each membrane. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] specifically accumulates at the plasma membrane in yeast, animal, and plant cells, where it regulates a wide range of cellular processes including endocytic trafficking. However, the functional consequences of mispatterning PI(4,5)P2 in plants are unknown. Here, we functionally characterized the putative phosphoinositide phosphatase SUPPRESSOR OF ACTIN9 (SAC9) in Arabidopsis thaliana (Arabidopsis). We found that SAC9 depletion led to the ectopic localization of PI(4,5)P2 on cortical intracellular compartments, which depends on PI4P and PI(4,5)P2 production at the plasma membrane. SAC9 localizes to a subpopulation of trans-Golgi Network/early endosomes that are enriched in a region close to the cell cortex and that are coated with clathrin. Furthermore, it interacts and colocalizes with Src Homology 3 Domain Protein 2 (SH3P2), a protein involved in endocytic trafficking. In the absence of SAC9, SH3P2 localization is altered and the clathrin-mediated endocytosis rate is reduced. Together, our results highlight the importance of restricting PI(4,5)P2 at the plasma membrane and illustrate that one of the consequences of PI(4,5)P2 misspatterning in plants is to impact the endocytic trafficking.