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International audience; 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.

International audience; 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 4,5bisphosphate for the regulation of the peripherical cytoskeleton during cellular processes such as cytokinesis, polar growth, biotic and abiotic responses.

International audience; 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 analyzed 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

Plant cytokinesis, which fundamentally differs from that in animals, involves de novo assembly of a plasma membrane precursor named the cell plate. How the transition from the cell plate to a plasma membrane occurs at the end of the plant cytokinesis remains poorly understood. Here, we describe with unprecedented spatiotemporal precision, the acquisition of plasma membrane identity upon cytokinesis through the lateral patterning of phosphatidylinositol 4,5-bisphosphate PI(4,5)P 2 at the newly formed cell plate membrane. We show that during late cytokinesis, opposing polarity domains are formed along the cell plate. Exclusion of PI(4,5)P 2 from the leading edge of the cell plate is controlled by SAC9, a putative phosphoinositide phosphatase. SAC9 colocalizes with MAP65-3, a key regulator of the cytokinesis, at the cell plate leading zone and regulates its function. In the sac9-3 mutant, the polar distribution of PI(4,5)P 2 at the cell plate is altered, leading to de-novo recruitment of the cytokinesis apparatus and to formation of an additional, ectopic cell plate insertion site. We proposed that PI(4,5)P 2 acts as a polar cue to spatially separate the expansion and maturation domains of the forming cell plate during the final steps of cytokinesis. One Sentence Summary The phosphoinositide PI(4,5)P 2 acts as an hallmark to guide the final step of 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.

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., This work was supported by two Seed Fund ENS LYON-2016 and LYON-2019 (to MCC), a Junior Investigator grant ANR-16-CE13-0021 (to MCC), ERC no. 3363360-APPL under FP/2007–2013 (to YJ). This work was supported by an Emmy Noether Fellowship GZ: UE188/2-1 from the Deutsche Forschungsgemeinschaft (DFG; to SÜ). MD and AL were funded by Ph.D. fellowships from the French Ministry of Research and Higher Education., Funding: Agence Nationale de la Recherche (ANR-16-CE13-0021); European Research Council (3363360-APPL); Deutsche Forschungsgemeinschaft (UE188/2-1)

International audience; Phosphoinositides (PIs) play 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, activity), membrane physicochemical properties (e.g., curvature, surface charges, packing), and the generation of second messengers. PIs act at very precise locations within the cell in a dosedependent manner, and their local concentration can vary drastically during signaling and trafficking. Techniques able to manipulate PI amounts acutely and with subcellular accuracy are thus paramount to understand the role of these lipids in vivo. Here, we review these methods and emphasize the approaches recently developed to perturb PI levels in multicellular organisms. Phosphoinositides (PIs) constitute a dynamic and interlinked network of landmark lipids in cellular membranes PIs are phosphorylated derivatives from the glycerophospholipid phosphatidylinositol (PtdIns) [1]. The inositol head group can be phosphorylated in position three, four or five, forming up to seven distinct species of phosphorylated PtdIns, collectively known as phosphoinositides

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.

During the life cycle of any multicellular organism, cell division contributes to the proliferation of the cell in the tissues as well as the generation of specialized cells, both necessary to form a functional organism. Therefore, the mechanisms of cell division need to be tightly regulated, as malfunctions in their control can lead to tumor formation or developmental defects. This is particularly true in land plants, where cells cannot relocate and therefore cytokinesis is key for morphogenesis. In the green lineage, cell division is executed in radically different manners than animals, with the appearance of new structures (the preprophase band (PPB), cytokinetic the cell plate and phragmoplast), and the disappearance of ancestral mechanisms (cleavage, centrosomes). While F-actin and microtubules closely co-exist to allow the orientation and the progression of the plant cell division, recent studies mainly focused on the microtubule’s involvement in this key process. Here, we used our recently developed root tracking system to follow actin dynamics in dividing Arabidopsis meristematic root cells. In this study, we imaged in time and space the fluorescent-tagged F-actin reporter Lifeact together with cell division markers in dividing cells embedded in their tissues. In addition to the F-actin accumulation’s in the phragmoplasts, we observed and quantified a dynamic apical-basal enrichment of the F-actin during cytokinesis. The role and the possible actors responsible for F-actin dynamics during cytokinesis are discussed.

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 consequence of PI(4,5)P2 misspatterning in plants is to impact the endocytic trafficking.One-sentence summarySAC9 prevents the accumulation of PI(4,5)P2 in intracellular compartments, thereby contributing to clathrin-mediated endocytosis at the plasma membrane and regulating the cortical localization of its protein partner SH3P2.

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)P

International audience; PI(4,5)P-2 is importantly involved in a broad array of cellular processes, including polar auxin transport, vesicle trafficking and anisotropic cell growth. An inducible system is developed in Arabidopsis to conduct tunable depletion of PI(4,5)P-2 and reveal new roles of this membrane lipid.Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2) is a low-abundance membrane lipid essential for plasma membrane function(1,2). In plants, mutations in phosphatidylinositol 4-phosphate (PI4P) 5-kinases (PIP5K) suggest that PI(4,5)P-2 production is involved in development, immunity and reproduction(3-5). However, phospholipid synthesis is highly intricate(6). It is thus likely that steady-state depletion of PI(4,5)P-2 triggers confounding indirect effects. Furthermore, inducible tools available in plants allow PI(4,5)P-2 to increase(7-9) but not decrease, and no PIP5K inhibitors are available. Here, we introduce iDePP (inducible depletion of PI(4,5)P-2 in plants), a system for the inducible and tunable depletion of PI(4,5)P-2 in plants in less than three hours. Using this strategy, we confirm that PI(4,5)P-2 is critical for various aspects of plant development, including root growth, root-hair elongation and organ initiation. We show that PI(4,5)P-2 is required to recruit various endocytic proteins, including AP2-mu, to the plasma membrane, and thus to regulate clathrin-mediated endocytosis. Finally, we find that inducible PI(4,5)P-2 perturbation impacts the dynamics of the actin cytoskeleton as well as microtubule anisotropy. Together, we propose that iDePP is a simple and efficient genetic tool to test the importance of PI(4,5)P-2 in given cellular or developmental responses, and also to evaluate the importance of this lipid in protein localization.

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., Specific examples are used to illustrate some of the challenges of live cell imaging, from designing genetically encoded probes to choosing a pipeline for image analysis and quantification.

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., PI4Kα1 is targeted to plasma membrane nanodomains by a lipid-anchored heterotetrameric complex essential for plant cell survival, including gametophytic, embryonic, and post-embryonic development.

Phosphoinositides are low-abundant lipids that participate in the acquisition of membrane identity through their spatiotemporal enrichment in specific compartments. 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 PI4Kα1 to the plasma membrane via a nanodomain-anchored scaffolding complex. We identified that PI4Kα1 is part of a complex composed of proteins from the NO-POLLEN-GERMINATION, EFR3-OF-PLANTS, and HYCCIN-CONTAINING families. Comprehensive knock-out and knock-down 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 new perspectives on the mechanisms and function of plasma membrane nanopatterning by lipid kinases.

Activation of intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) results in immunity and a localized cell death response of infected cells. Cell death activity of many NLRs requires oligomerization and in some cases plasma membrane (PM) localization. However, the exact mechanisms underlying PM localization of NLRs lacking recognizable N- or C-terminal lipidation motifs or predicted transmembrane domains remains elusive. Here we show that the PM localization and stability of members of the RPW8-like coiled-coil (CCR) domain NLRs (RNLs) and a CC-type NLR (CNL) depend on the interaction with PM phospholipids. Depletion of phosphatidylinositol-4-phosphate (PI4P) from the PM led to a mislocalization of the analyzed NLRs and consequently inhibited their cell death activity. We further demonstrate activation-dependent self-association of cell death inducing RNLs. Our results provide new insights into the molecular mechanism of NLR PM localization and defines an important role of phospholipids for CNL and RNL activity during immunity.

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] is a low abundant lipid present at the plasma membrane of eukaryotic cells. Extensive studies in animal cells revealed the pleiotropic functions of PI(4,5)P 2 . In plant cells, PI(4,5)P 2 is involved in various cellular processes including the regulation of cell polarity and tip growth, clathrin-mediated endocytosis, polar auxin transport, actin dynamics or membrane-contact sites. To date, most studies investigating the role of PI(4,5)P 2 in plants have relied on mutants lacking enzymes responsible for PI(4,5)P 2 synthesis and degradation. However, such genetic perturbations only allow steady-state analysis of plants undergoing their life cycle in PI(4,5)P 2 deficient conditions and the corresponding mutants are likely to induce a range of non-causal (untargeted) effects driven by compensatory mechanisms. In addition, there are no small molecule inhibitors that are available in plants to specifically block the production of this lipid. Thus, there is currently no system to fine tune PI(4,5)P 2 content in plant cells. Here we report a genetically encoded and inducible synthetic system, iDePP (Inducible De pletion of P I(4,5)P 2 in P lants), that efficiently removes PI(4,5)P 2 from the plasma membrane in different organs of Arabidopsis thaliana , including root meristem, root hair and shoot apical meristem. We show that iDePP allows the inducible depletion of PI(4,5)P 2 in less than three hours. Using this strategy, we reveal that PI(4,5)P 2 is critical for cortical microtubule organization. Together, we propose that iDePP is a simple and efficient genetic tool to test the importance of PI(4,5)P 2 in given cellular or developmental responses but also to evaluate the importance of this lipid in protein localization.; Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a low abundant lipid present at the plasma membrane of eukaryotic cells. Extensive studies in animal cells revealed the pleiotropic functions of PI(4,5)P2. In plant cells, PI(4,5)P2 is involved in various cellular processes including the regulation of cell polarity and tip growth, clathrin-mediated endocytosis, polar auxin transport, actin dynamics or membrane-contact sites. To date, most studies investigating the role of PI(4,5)P2 in plants have relied on mutants lacking enzymes responsible for PI(4,5)P2 synthesis and degradation. However, such genetic perturbations only allow steady-state analysis of plants undergoing their life cycle in PI(4,5)P2 deficient conditions and the corresponding mutants are likely to induce a range of non-causal (untargeted) effects driven by compensatory mechanisms. In addition, there are no small molecule inhibitors that are available in plants to specifically block the production of this lipid. Thus, there is currently no system to fine tune PI(4,5)P2 content in plant cells. Here we report a genetically encoded and inducible synthetic system, iDePP (Inducible Depletion of PI(4,5)P2 in Plants), that efficiently removes PI(4,5)P2 from the plasma membrane in different organs of Arabidopsis thaliana, including root meristem, root hair and shoot apical meristem. We show that iDePP allows the inducible depletion of PI(4,5)P2 in less than three hours. Using this strategy, we reveal that PI(4,5)P2 is critical for cortical microtubule organization. Together, we propose that iDePP is a simple and efficient genetic tool to test the importance of PI(4,5)P2 in given cellular or developmental responses but also to evaluate the importance of this lipid in protein localization.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P

International audience; 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.

Membrane surface charge is critical for the transient, yet specific recruitment of proteins with polybasic regions to certain organelles. In all eukaryotes, the plasma membrane (PM) is the most electronegative compartment of the cell, which specifies its identity. As such, membrane electrostatics is a central parameter in signaling, intracellular trafficking and polarity. Here, we explore which are the lipids that control membrane electrostatics using plants as a model. We show that phosphatidic acidic (PA), phosphatidylserine (PS) and phosphatidylinositol-4-phosphate (PI4P) are separately required to generate the electrostatic signature of the plant PM. In addition, we reveal the existence of an electrostatic territory that is organized as a gradient along the endocytic pathway and is controlled by PS/PI4P combination. Altogether, we propose that combinatorial lipid composition of the cytosolic leaflet of cellular organelles not only defines the plant electrostatic territory but also distinguishes different compartments within this territory by specifying their varying surface charges.

Here we describe a protocol that enables to automatically perform time-lapse imaging of growing root tips for several hours. Plants roots expressing fluorescent proteins or stained with dyes are imaged while they grow using automatic movement of the microscope stage that compensates for root growth and allows to follow a given region of the root over time. The protocol makes possible the image acquisition of multiple growing root tips, therefore increasing the number of recorded mitotic events in a given experiment. The protocol also allows the visualization of more than one fluorescent protein or dye simultaneously, using multiple channel acquisition. We particularly focus on imaging of cytokinesis in Arabidopsis root tip meristem, but this protocol is also suitable to follow root hair growth, pollen tube growth, and other regions of root over time, in various plant species. It may as well be amendable to automatically track non-plant structures with an apical growth.

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.

International audience; Mitosis which is a major step during plant development can also be observed in physiopathological conditions. During the compatible interaction between the root-knot nematode Meloidogyne incognita and its host Arabidopsis, the pathogen induce through repeated divisions without complete cytokinesis the formation of hypertrophied and multinucleate feeding cells, named giant cells. Due to the presence of hypertrophied plant cell material surrounding the giant cells, classical live cell imaging gave therefore very poor resolution. Here, we describe a protocol which allows the in vivo observation of the mitotic apparatus in developing giant cells using confocal imaging of vibrosliced tissues. This approach can also be used to visualize in vivo other cellular processes occurring in different steps of giant cells.

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 additional to their more characterized role of suppressing plant defense. Recent studies suggest that effectors may manipulate host transcription or other nuclear regulatory components for the benefit of pathogen development. However, the specific mechanisms by which these effectors promote susceptibility remain unclear. Of two recent screenings, we identified 15 nuclear-localized Hpa effectors (HaRxLs) that interact directly or indirectly with host nuclear components. When stably expressed in planta, nuclear HaRxLs cause diverse developmental phenotypes highlighting that nuclear effectors might interfere with fundamental plant regulatory mechanisms. Here, we report recent advances in understanding how a pathogen can manipulate nuclear processes in order to cause disease.

The infection of plants by obligate parasitic nematodes constitutes an interesting model for investigating plant cytoskeleton functions. Root knot nematodes have evolved the ability to manipulate host functions to their own advantage by redifferentiating root cells into multinucleate and hypertrophied feeding cells. These giant cells result from repeated rounds of karyokinesis without cell division. Detailed functional analyses demonstrated that Arabidopsis thaliana Microtubule-Associated Protein65-3 (MAP65-3) was essential for giant cell ontogenesis and that cytokinesis was initiated but not completed in giant cells. In developing giant cells, MAP65-3 was associated with a novel kind of cell plate—the giant cell mini cell plate—that separates daughter nuclei. In the absence of functional MAP65-3, giant cells developed but failed to fully differentiate and were eventually destroyed. These defects in giant cells impaired the maturation of nematode larvae. Thus, MAP65-3 is essential for giant cell development during root knot nematode infection. Subcellular localization of MAP65-3 and analysis of microtubule organization in the dyc283 T-DNA map65-3 mutant demonstrated that MAP65-3 played a critical role in organizing the mitotic microtubule array during both early and late mitosis in all plant organs. Here, we propose a model for the role of MAP65-3 in giant cell ontogenesis.

International audience; Sedentary endoparasitic nematodes are root parasites that interact with their hosts in a remarkable way. These obligate biotrophic pathogens establish an intimate relationship with their host plants, inducing the redifferentiation of root cells into specialized feeding cells. The successful establishment of feeding cells is essential for nematode development. Root-knot nematodes, of the genus Meloidogyne, have evolved strategies enabling them to induce feeding cell formation in thousands of plant species, probably by manipulating fundamental elements of plant cell development. Feeding cells enlarge and are converted into multinucleate giant cells through synchronous nuclear divisions without cell division. Fully differentiated giant cells may contain more than a hundred polyploid nuclei that may have undergone extensive endoreduplication. Hyperplasia and hypertrophy of the surrounding cells lead to the formation of the typical root gall. Giant cell formation requires extensive changes to gene expression. The induction of feeding cells remains poorly understood, but it is thought that effectors secreted by the nematode play a key role in parasitism, with potential direct effects on recipient host cells. In this review, we focus on the most recent investigations of the molecular basis of the plant–root-knot nematode interaction. Recently, microarray technology has been used to study the plant response to Meloidogyne spp. infection. Such a genome-wide expression profiling provides a global view of transcriptional changes, especially for genes involved in cell wall, transport processes and plant defense responses during giant cell formation. The identification of nematode-responsive plant genes constitutes a major step toward understanding how root-knot nematodes dramatically alter root development to induce and maintain giant cells. The characterization of nematode secretions as parasitism effectors and the development of RNAi technology should improve our understanding of the molecular events and regulatory mechanisms involved in plant parasitism. Finally, Meloidogyne genome sequences should provide further insight into plant–root-knot nematode interactions.

Plant-parasitic nematodes are major agricultural pests worldwide and novel approaches to control them are sorely needed. We report the draft genome sequence of the root-knot nematode Meloidogyne incognita, a biotrophic parasite of many crops, including tomato, cotton and coffee. Most of the assembled sequence of this asexually reproducing nematode, totaling 86 Mb, exists in pairs of homologous but divergent segments. This suggests that ancient allelic regions in M. incognita are evolving toward effective haploidy, permitting new mechanisms of adaptation. The number and diversity of plant cell wall-degrading enzymes in M. incognita is unprecedented in any animal for which a genome sequence is available, and may derive from multiple horizontal gene transfers from bacterial sources. Our results provide insights into the adaptations required by metazoans to successfully parasitize immunocompetent plants, and open the way for discovering new antiparasitic strategies

Mitosis which is a major step during plant development can also be observed in physiopathological conditions. During the compatible interaction between the root-knot nematode Meloidogyne incognita and its host Arabidopsis, the pathogen induce through repeated divisions without complete cytokinesis the formation of hypertrophied and multinucleate feeding cells, named giant cells. Due to the presence of hypertrophied plant cell material surrounding the giant cells, classical live cell imaging gave therefore very poor resolution. Here, we describe a protocol which allows the in vivo observation of the mitotic apparatus in developing giant cells using confocal imaging of vibrosliced tissues. This approach can also be used to visualize in vivo other cellular processes occurring in different steps of giant cells.

International audience; Many signaling proteins permanently or transiently localize to specific organelles for function. It is well established that certain lipids act as biochemical landmarks to specify compartment identity. However, they also influence membrane biophysical properties, which emerge as important features in specifying cellular territories. Such parameters include the membrane inner surface potential, which varies according to the lipid composition of each organelle. Here, we found that the plant plasma membrane (PM) and the cell plate of dividing cells have a unique electrostatic signature controlled by phosphatidylinositol-4-phosphate (PI4P). Our results further reveal that, contrarily to other eukaryotes, PI4P massively accumulates at the PM, establishing it as a critical hallmark of this membrane in plants. Membrane surface charges control the PM localization and function of the polar auxin transport regulator PINOID, as well as proteins from the BRI1 KINASE INHIBITOR1 (BKI1)/MEMBRANE ASSOCIATED KINASE REGULATORs (MAKRs) family, which are involved in brassinosteroid and receptor-like kinase signaling. We anticipate that this PI4P-driven physical membrane property will control the localization and function of many proteins involved in development, reproduction, immunity and nutrition. Each membrane compartment has a unique identity and thereby recruits a specific set of proteins 1-3. It has been established for decades that these identities are acquired by the combined presence of specific lipid and protein molecules that act as biochemical landmark on each membrane. For example, small GTPases from the Rab and ADP ribosylation factor (ARF) family as well as Soluble N-ethylmaleimide-sensitive-factor Attachment protein Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#termsReprints and permissions information is available at www.nature.com/reprints.

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. Fil: Wirthmueller, Lennart. John Innes Institute; Reino Unido. The Sainsbury Laboratory; Reino Unido Fil: Roth, Charlotte. Georg‐August‐University; Alemania Fil: Fabro, Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Caillaud, Marie-Cécile. The Sainsbury Laboratory; Reino Unido Fil: Rallapalli, Ghanasyam. The Sainsbury Laboratory; Reino Unido Fil: Asai, Shuta. The Sainsbury Laboratory; Reino Unido Fil: Sklenar, Jan. John Innes Institute; Reino Unido Fil: Jones, Alexandra M. E.. John Innes Institute; Reino Unido Fil: Wiermer, Marcel. Georg‐August‐University; Alemania Fil: Jones, Jonathan D. G.. The Sainsbury Laboratory; Reino Unido Fil: Banfield, Mark J.. Georg‐August‐University; Alemania

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., Author Summary Haustoria are specialised invasive structures that project from fungal or oomycete hyphae into host plant cells during infection, acting as sites for molecular exchange between host and pathogen. Haustoria are targets of plant defence responses, including the deposition of membranes and polysaccharides in an encasement structure that surrounds the haustorium. It is assumed that the encasement physically seals the haustorium off from the host cell. Here we have used cell biological and genetic approaches to reveal that the plasmodesmata-associated receptor-like protein PDLP1 plays a role in infection success of the Arabidopsis downy mildew pathogen, specifically in the development of the encasement. Using live cell imaging, we observed that PDLP1 relocates to the extra-haustorial membrane, and this is required for deposition of the polysaccharide callose in the encasement. This directly correlates pathogen success with the structure of the encasement, verifying the significance of the encasement in host defence. Further, our data pose the possibility that callose deposition at plasmodesmata and the haustorial encasement exploit similar mechanisms. Our findings shed light on plant defences at haustoria and how they inhibit pathogen success.

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., Author Summary A comprehensive understanding of host-pathogen interactions requires knowledge of the dynamics of gene expression changes in both the host and the pathogen during a time course of infection. However, expression profiling has often focused on either the host or the pathogen due to limitations of methods that involve microarrays. We report here gene expression changes in both Arabidopsis and its parasite Hyaloperonospora arabidopsidis (Hpa) simultaneously during infection using a high-throughput RNA sequencing method. By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of cognate recognized effector ATR1. We also found that Hpa suppresses responsiveness to salicylic acid (SA) in haustoriated cells into which host-translocated effectors are delivered. An Hpa effector HaRxL62, previously shown to enhance host susceptibility, was highly expressed in this assay, and we found it suppresses responsiveness to SA. Expression profiling of both pathogen effector genes and host genes involved in immunity allows us to suggest distinct mechanisms of effector-mediated susceptibility and reveals interesting Hpa effectors for detailed mechanistic investigation in future experiments.

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.

HaRxL44, a secreted effector from the Arabidopsis downy mildew pathogen Hyaloperonospora arabidopsidis, enhances disease susceptibility by interacting with and degrading Mediator subunit MED19a, thereby perturbing plant defense gene transcription., 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., Author Summary The highly conserved Mediator complex plays an essential role in transcriptional regulation by providing a molecular bridge between transcription factors and RNA polymerase II. Recent studies in Arabidopsis have revealed that it also performs an essential role in plant defence. However, it remains unknown how pathogens manipulate Mediator function in order to increase a plant's susceptibility to infection. In this article, we show that a secreted effector, HaRxL44, from the Arabidopsis downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa), interacts with and degrades the Mediator subunit MED19a, resulting in the alteration of plant defence gene transcription. This effector-mediated interference with host transcriptional regulation perturbs the balance between jasmonic acid/ethylene (JA/ET) and salicylic acid (SA)–dependent defence. HaRxL44 interaction with MED19a results in reduced SA-regulated gene expression, indicating that this pathogen effector modulates host transcription to promote virulence. The resulting alteration in defence transcription patterns compromises the plant's ability to defend itself against pathogens, such as Hpa, that establish long-term parasitic interactions with living host cells via haustoria (a pathogen structure that creates an expanded host/parasite interface to extract nutrients) but not against necrotrophic pathogens that kill host cells. HaRxL44 is unlikely to be the sole effector that accomplishes this shift in hormonal balance, and other nuclear HaRxL proteins were reported by other researchers to interact with Mediator components, as well as with other regulators of the JA/ET signalling pathway. Functional analyses of these effectors should facilitate the discovery of new components of the plant immune system. These data show that pathogens can target fundamental mechanisms of host regulation in order to tip the balance of signalling pathways to suppress defence and favour parasitism.

We examined changes to subcellular architecture during the compatible interaction between the biotroph pathogen Hyaloperonospora arabidopsidis (Hpa) and its host Arabidopsis. Live-cell imaging highlighted rearrangements in plant cell membranes upon infection. In particular, the tonoplast appeared close to the extrahaustorial membrane surrounding the haustorium. We investigated the subcellular localization patterns of Hpa RxLR effector candidates (HaRxLs) in planta. This subcellular localization screening led to the identification of an extrahaustorial membrane-localized effector, HaRxL17 that when stably expressed in Arabidopsis increased plant susceptibility to Hpa during compatible and incompatible interactions. Here, we report that the N-terminal part of HaRxL17 is sufficient to target the plant cell membranes. We showed that both C- or N-terminal fluorescent-tagged HaRxL17 localizes around Hpa haustoria, in early and in late stages of infection. As with Hpa infection, GFP-HaRxL17 also localizes around haustoria during infection with Albugo laibachii. Thus, HaRxL17 that increases plant susceptibility to Hpa during both compatible and incompatible interactions, localizes around oomycete haustoria when stably expressed in Arabidopsis.

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., Author Summary Hyaloperonospora arabidopsidis (Hpa) is an obligate biotroph whose population coevolves with its host, Arabidopsis thaliana. The Hpa isolate Emoy2 genome has been sequenced, allowing the discovery of dozens of secreted candidate effectors. We set out to assign functions to these candidate effectors, investigating if they suppress host defenses. We analyzed a sub-set of Hpa candidate effectors (HaRxLs) that carry the RxLR motif, using a bacterial system for in planta delivery. To our surprise, we found that most of the HaRxLs enhanced plant susceptibility on at least some accessions, while few decreased it. These phenotypes were mostly confirmed on Arabidopsis transgenic lines stably expressing HaRxLs that became more susceptible to compatible Hpa isolates. Furthermore, effectors that conferred enhanced virulence generally suppressed callose deposition, a hallmark of plant defense. This indicates that the “effectorome” of Hpa comprises multiple distinct effectors that can attenuate Arabidopsis immunity. We found that many HaRxLs did not confer enhanced virulence on all host accessions, and also that only ∼50% of the effectors that conferred enhanced Pst growth on Arabidopsis, were able to do so on turnip, a non-host for Hpa. Our data reveal interesting HaRxLs for detailed mechanistic investigation in future experiments.

Summary 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. Fil: Caillaud, Marie-Cécile. John Innes Institute; Reino Unido Fil: Piquerez, Sophie J. M.. John Innes Institute; Reino Unido Fil: Fabro, Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Steinbrenner, Jens. University of Warwick; Reino Unido Fil: Ishaque, Naveed. John Innes Institute; Reino Unido Fil: Beynon, Jim. University of Warwick; Reino Unido Fil: Jones, Jonathan D. G.. John Innes Institute; Reino Unido

International audience; Background: In eukaryotes, the spindle assembly checkpoint (SAC) ensures that chromosomes undergoing mitosis do not segregate until they are properly attached to the microtubules of the spindle. Methodology/Principal Findings: We investigated the mechanism underlying this surveillance mechanism in plants, by characterising the orthogolous SAC proteins BUBR1, BUB3 and MAD2 from Arabidopsis. We showed that the cell cycle-regulated BUBR1, BUB3.1 and MAD2 proteins interacted physically with each other. Furthermore, BUBR1 and MAD2 interacted specifically at chromocenters. Following SAC activation by global defects in spindle assembly, these three interacting partners localised to unattached kinetochores. In addition, in cases of 'wait anaphase', plant SAC proteins were associated with both kinetochores and kinetochore microtubules. Unexpectedly, BUB3.1 was also found in the phragmoplast midline during the final step of cell division in plants. Conclusions/Significance: We conclude that plant BUBR1, BUB3.1 and MAD2 proteins may have the SAC protein functions conserved from yeast to humans. The association of BUB3.1 with both unattached kinetochore and phragmoplast suggests that in plant, BUB3.1 may have other roles beyond the spindle assembly checkpoint itself. Finally, this study of the SAC dynamics pinpoints uncharacterised roles of this surveillance mechanism in plant cell division.

International audience; Root-knot nematodes are plant parasitic worms that establish and maintain an intimate relationship with their host plants. RKN induce the redifferentiation of root cells into multinucleate and hypertrophied giant cells essential for nematode growth and reproduction. Major rearrangements of the cytoskeleton occur during giant cell formation. We characterized the first plant candidate genes implicated in giant cell actin and microtubule cytoskeleton reorganization. We showed previously that formins may regulate giant cell isotropic growth by controlling the assembly of actin cables. Recently we demonstrated that a Microtubule-Associated Protein, MAP65-3, is essential for giant cell development. In the absence of functional MAP65-3, giant cells started to develop but failed to fully differentiate and were eventually destroyed. In developing giant cells, MAP65-3 was associated with a novel kind of cell plate — the giant cell mini cell plate — that separates daughter nuclei. Despite karyokinesis occurs without cell division in giant cell, we demonstrated that cytokinesis is initiated and required for successful pathogen growth and development.