Arabinosylation of cell wall extensin is required for the directional response to salinity in roots.

Soil salinity is a major contributor to crop yield losses. To improve our understanding of root responses to salinity, we developed and exploited a real-time salt-induced tilting assay. This assay follows root growth upon both gravitropic and salt challenges, revealing that root bending upon tilting is modulated by Na+ ions, but not by osmotic stress. Next, we measured this salt-specific response in 345 natural Arabidopsis (Arabidopsis thaliana) accessions and discovered a genetic locus, encoding the cell wall-modifying enzyme EXTENSIN ARABINOSE DEFICIENT TRANSFERASE (ExAD) that is associated with root bending in the presence of NaCl (hereafter salt). Extensins are a class of structural cell wall glycoproteins known as hydroxyproline (Hyp)-rich glycoproteins, which are posttranslationally modified by O-glycosylation, mostly involving Hyp-arabinosylation. We show that salt-induced ExAD-dependent Hyp-arabinosylation influences root bending responses and cell wall thickness. Roots of exad1 mutant seedlings, which lack Hyp-arabinosylation of extensin, displayed increased thickness of root epidermal cell walls and greater cell wall porosity. They also showed altered gravitropic root bending in salt conditions and a reduced salt-avoidance response. Our results suggest that extensin modification via Hyp-arabinosylation is a unique salt-specific cellular process required for the directional response of roots exposed to salinity.

Kinesin-4 optimizes microtubule orientations for responsive tip growth guidance in moss.

Tip-growing cells of, amongst others, plants and fungi secrete wall materials in a highly polarized fashion for fast and efficient colonization of the environment. A polarized microtubule cytoskeleton, in which most microtubule ends are directed toward the growing apex, has been implicated in directing growth. Its organizing principles, in particular regarding maintenance of network unipolarity, have remained elusive. We show that a kinesin-4 protein, hitherto best known for a role in cytokinesis, suppresses encounters between antiparallel microtubules. Without this activity, microtubules hyper-aligned along the growth axis and increasingly grew away from the apex. Cells themselves displayed an overly straight growth path and a delayed gravitropic response. This result revealed conflicting systemic needs for a stable growth direction and an ability to change course in response to extracellular cues. Thus, the use of selective inhibition of microtubule growth at antiparallel overlaps constitutes a new organizing principle within a unipolar microtubule array.

Fatal attraction: How Phytophthora zoospores find their host.

Oomycete plant pathogens, such as Phytophthora and Pythium species produce motile dispersal agents called zoospores that actively target host plants. Zoospores are exceptional in their ability to display taxis to chemical, electrical and physical cues to navigate the phyllosphere and reach stomata, wound sites and roots. Many components of root exudates have been shown attractive or repulsive to zoospores. Although some components possess very strong attractiveness, it seems that especially the mix of components exuded by the primary host is most attractive to zoospores. Zoospores actively approach attractants with swimming behaviour reminiscent of other microswimmers. To achieve a unified description of zoospore behaviour when sensing an attractant, we propose the following terms for the successive stages of the homing response: reorientation, approaching, retention and settling. How zoospores sense and process attractants is poorly understood but likely involves signal perception via cell surface receptors. Since zoospores are important for infection, undermining their activity by luring attractants or blocking receptors seem promising strategies for disease control.

An actin mechanostat ensures hyphal tip sharpness in Phytophthora infestans to achieve host penetration.

Filamentous plant pathogens apply mechanical forces to pierce their hosts surface and penetrate its tissues. Devastating Phytophthora pathogens harness a specialized form of invasive tip growth to slice through the plant surface, wielding their hypha as a microscopic knife. Slicing requires a sharp hyphal tip that is not blunted at the site of the mechanical interaction. How tip shape is controlled, however, is unknown. We uncover an actin-based mechanostat in Phytophthora infestans that controls tip sharpness during penetration. Mechanical stimulation of the hypha leads to the emergence of an aster-like actin configuration, which shows fast, local, and quantitative feedback to the local stress. We evidence that this functions as an adaptive mechanical scaffold that sharpens the invasive weapon and prevents it from blunting. The hyphal tip mechanostat enables the efficient conversion of turgor into localized invasive pressures that are required to achieve host penetration.

Molecular sensors reveal the mechano-chemical response of Phytophthora infestans walls and membranes to mechanical and chemical stress.

Phytophthora infestans, causal agent of late blight in potato and tomato, remains challenging to control. Unravelling its biomechanics of host invasion, and its response to mechanical and chemical stress, could provide new handles to combat this devastating pathogen. Here we introduce two fluorescent molecular sensors, CWP-BDP and NR12S, that reveal the micromechanical response of the cell wall-plasma membrane continuum in P. infestans during invasive growth and upon chemical treatment. When visualized by live-cell imaging, CWP-BDP reports changes in cell wall (CW) porosity while NR12S reports variations in chemical polarity and lipid order in the plasma membrane (PM). During invasive growth, mechanical interactions between the pathogen and a surface reveal clear and localized changes in the structure of the CW. Moreover, the molecular sensors can reveal the effect of chemical treatment to CW and/or PM, thereby revealing the site-of-action of crop protection agents. This mechano-chemical imaging strategy resolves, non-invasively and with high spatio-temporal resolution, how the CW-PM continuum adapts and responds to abiotic stress, and provides information on the dynamics and location of cellular stress responses for which, to date, no other methods are available.

A slicing mechanism facilitates host entry by plant-pathogenic Phytophthora.

Phytophthora species, classified as oomycetes, are among the most destructive plant pathogens worldwide and pose a substantial threat to food security. Plant pathogens have developed various methods to breach the cuticle and walls of plant cells. For example, plant-pathogenic fungi use a 'brute-force' approach by producing a specialized and fortified invasion organ to generate invasive pressures. Unlike in fungi, the biomechanics of host invasion in oomycetes remains poorly understood. Here, using a combination of surface-deformation imaging, molecular-fracture sensors and modelling, we find that Phytophthora infestans, Phytophthora palmivora and Phytophthora capsici slice through the plant surface to gain entry into host tissues. To distinguish this mode of entry from the brute-force approach of fungi that use appressoria, we name this oomycete entry without appressorium formation 'naifu' invasion. Naifu invasion relies on polarized, non-concentric, force generation onto the surface at an oblique angle, which concentrates stresses at the site of invasion to enable surface breaching. Measurements of surface deformations during invasion of artificial substrates reveal a polarized mechanical geometry that we describe using a mathematical model. We confirm that the same mode of entry is used on real hosts. Naifu invasion uses actin-mediated polarity, surface adherence and turgor generation to enable Phytophthora to invade hosts without requiring specialized organs or vast turgor generation.

Phytophthora infestans RXLR effector AVR1 disturbs the growth of Physcomitrium patens without affecting Sec5 localization.

Plant pathogens often exploit a whole range of effectors to facilitate infection. The RXLR effector AVR1 produced by the oomycete plant pathogen Phytophthora infestans suppresses host defense by targeting Sec5. Sec5 is a subunit of the exocyst, a protein complex that is important for mediating polarized exocytosis during plant development and defense against pathogens. The mechanism by which AVR1 manipulates Sec5 functioning is unknown. In this study, we analyzed the effect of AVR1 on Sec5 localization and functioning in the moss Physcomitrium patens. P. patens has four Sec5 homologs. Two (PpSec5b and PpSec5d) were found to interact with AVR1 in yeast-two-hybrid assays while none of the four showed a positive interaction with AVR1ΔT, a truncated version of AVR1. In P. patens lines carrying ß-estradiol inducible AVR1 or AVR1ΔT transgenes, expression of AVR1 or AVR1ΔT caused defects in the development of caulonemal protonema cells and abnormal morphology of chloronema cells. Similar phenotypes were observed in Sec5- or Sec6-silenced P. patens lines, suggesting that both AVR1 and AVR1ΔT affect exocyst functioning in P. patens. With respect to Sec5 localization we found no differences between ß-estradiol-treated and untreated transgenic AVR1 lines. Sec5 localizes at the plasma membrane in growing caulonema cells, also during pathogen attack, and its subcellular localization is the same, with or without AVR1 in the vicinity.

Long-term single-cell imaging and simulations of microtubules reveal principles behind wall patterning during proto-xylem development.

Plants are the tallest organisms on Earth; a feature sustained by solute-transporting xylem vessels in the plant vasculature. The xylem vessels are supported by strong cell walls that are assembled in intricate patterns. Cortical microtubules direct wall deposition and need to rapidly re-organize during xylem cell development. Here, we establish long-term live-cell imaging of single Arabidopsis cells undergoing proto-xylem trans-differentiation, resulting in spiral wall patterns, to understand microtubule re-organization. We find that the re-organization requires local microtubule de-stabilization in band-interspersing gaps. Using microtubule simulations, we recapitulate the process in silico and predict that spatio-temporal control of microtubule nucleation is critical for pattern formation, which we confirm in vivo. By combining simulations and live-cell imaging we further explain how the xylem wall-deficient and microtubule-severing KATANIN contributes to microtubule and wall patterning. Hence, by combining quantitative microscopy and modelling we devise a framework to understand how microtubule re-organization supports wall patterning.

Time-gated confocal microscopy reveals accumulation of exocyst subunits at the plant-pathogen interface.

Polarized exocytosis is essential for plant development and defence. The exocyst, an octameric protein complex that tethers exocytotic vesicles to the plasma membrane, targets exocytosis. Upon pathogen attack, secreted materials form papillae to halt pathogen penetration. To determine if the exocyst is directly involved in targeting exocytosis to infection sites, information about its localization is instrumental. Here, we investigated exocyst subunit localization in the moss Physcomitrella patens upon pathogen attack and infection by Phytophthora capsici. Time-gated confocal microscopy was used to eliminate autofluorescence of deposited material around infection sites, allowing the visualization of the subcellular localization of exocyst subunits and of v-SNARE Vamp72A1-labelled exocytotic vesicles during infection. This showed that exocyst subunits Sec3a, Sec5b, Sec5d, and Sec6 accumulated at sites of attempted pathogen penetration. Upon pathogen invasion, the exocyst subunits accumulated on the membrane surrounding papilla-like structures and hyphal encasements. Vamp72A1-labelled vesicles were found to localize in the cytoplasm around infection sites. The re-localization of exocyst subunits to infection sites suggests that the exocyst is directly involved in facilitating polarized exocytosis during pathogenesis.

The Flower Petal Training System in Microsurgery: Validation of a Training Model Using a Randomized Controlled Trial.

INTRODUCTION: Despite hundreds of training models for microsurgery being available in the literature, very few of them are scientifically validated. We chose to validate our low-fidelity training model on flower petals by comparing it head-to-head with a moderate fidelity training model, the anastomosis on chicken leg femoral artery. MATERIALS AND METHODS: A total of 16 participants of different levels of expertise were randomized into 2 groups, 1 training on flower petals and 1 on chicken leg femoral arteries. The groups were evaluated on performing a rat femoral artery anastomosis using the validated Stanford Microsurgical Assessment (SMaRT) Scale. The Mann-Whitney U test was used to check for statistically significant differences between the groups. The flower petal sutures were also evaluated and Pearson correlation was used to check for associations between better petal anastomosis scores and better final SMaRT results. RESULTS: After 6 weeks of flower petal training, microsurgical trainees had significantly better overall SMaRT scores than trainees using chicken leg training, better fine tissue feeling, and better scores in knot tying. The anastomosis times for the rat femoral arteries did not differ between the 2 groups. Good scores for flower petals strongly correlated with a better SMaRT score for the anastomosis. The number of rats used in training reduced after the implementation of this model in continuous training. CONCLUSIONS: The flower petal technique, despite being a low-fidelity model, shows superiority in developing fine tissue feeling and improved knot tying in microsurgery beginners and intermediate level practitioners adding this training model to their program. Further research needs to establish if the improvements also apply to already seasoned microsurgeons and whether the petal score has predictive value for future clinical application.

Optical Trapping in Plant Cells.

Optical tweezers allow for noninvasive manipulation of subcellular compartments to study their physical interactions and attachments. By measuring (delay of) displacements, (semi)quantitative force measurements within a living cell can be performed. In this chapter, we provide practical tips for setting up such experiments paying special attention to the technical considerations for integrating optical tweezers into a confocal microscope. Next, we describe experimental approaches we have taken to trap intracellular structures in plant cells.

Live Cell Imaging of Arabidopsis Root Hairs.

Root hairs are tubular extensions from the root surface that expand by tip growth. This highly focused type of cell expansion, combined with position of root hairs on the surface of the root, makes them ideal cells for microscopic observation. This chapter describes the method that is routinely used in our laboratory for live cell imaging of Arabidopsis root hairs.

Exocyst subunit Sec6 is positioned by microtubule overlaps in the moss phragmoplast prior to cell plate membrane arrival.

During plant cytokinesis a radially expanding membrane-enclosed cell plate is formed from fusing vesicles that compartmentalizes the cell in two. How fusion is spatially restricted to the site of cell plate formation is unknown. Aggregation of cell-plate membrane starts near regions of microtubule overlap within the bipolar phragmoplast apparatus of the moss Physcomitrella patens Since vesicle fusion generally requires coordination of vesicle tethering and subsequent fusion activity, we analyzed the subcellular localization of several subunits of the exocyst, a tethering complex active during plant cytokinesis. We found that the exocyst complex subunit Sec6 but not the Sec3 or Sec5 subunits localized to microtubule overlap regions in advance of cell plate construction in moss. Moreover, Sec6 exhibited a conserved physical interaction with an ortholog of the Sec1/Munc18 protein KEULE, an important regulator for cell-plate membrane vesicle fusion in Arabidopsis Recruitment of the P. patens protein KEULE and vesicles to the early cell plate was delayed upon Sec6 gene silencing. Our findings, thus, suggest that vesicle-vesicle fusion is, in part, enabled by a pool of exocyst subunits at microtubule overlaps, which is recruited independently of vesicle delivery.

CLASP stabilization of plus ends created by severing promotes microtubule creation and reorientation.

Central to the building and reorganizing cytoskeletal arrays is creation of new polymers. Although nucleation has been the major focus of study for microtubule generation, severing has been proposed as an alternative mechanism to create new polymers, a mechanism recently shown to drive the reorientation of cortical arrays of higher plants in response to blue light perception. Severing produces new plus ends behind the stabilizing GTP-cap. An important and unanswered question is how these ends are stabilized in vivo to promote net microtubule generation. Here we identify the conserved protein CLASP as a potent stabilizer of new plus ends created by katanin severing in plant cells. Clasp mutants are defective in cortical array reorientation. In these mutants, both rescue of shrinking plus ends and the stabilization of plus ends immediately after severing are reduced. Computational modeling reveals that it is the specific stabilization of severed ends that best explains CLASP's function in promoting microtubule amplification by severing and array reorientation.

Stacks off tracks: a role for the golgin AtCASP in plant endoplasmic reticulum-Golgi apparatus tethering.

The plant Golgi apparatus modifies and sorts incoming proteins from the endoplasmic reticulum (ER) and synthesizes cell wall matrix material. Plant cells possess numerous motile Golgi bodies, which are connected to the ER by yet to be identified tethering factors. Previous studies indicated a role for cis-Golgi plant golgins, which are long coiled-coil domain proteins anchored to Golgi membranes, in Golgi biogenesis. Here we show a tethering role for the golgin AtCASP at the ER-Golgi interface. Using live-cell imaging, Golgi body dynamics were compared in Arabidopsis thaliana leaf epidermal cells expressing fluorescently tagged AtCASP, a truncated AtCASP-ΔCC lacking the coiled-coil domains, and the Golgi marker STtmd. Golgi body speed and displacement were significantly reduced in AtCASP-ΔCC lines. Using a dual-colour optical trapping system and a TIRF-tweezer system, individual Golgi bodies were captured in planta. Golgi bodies in AtCASP-ΔCC lines were easier to trap and the ER-Golgi connection was more easily disrupted. Occasionally, the ER tubule followed a trapped Golgi body with a gap, indicating the presence of other tethering factors. Our work confirms that the intimate ER-Golgi association can be disrupted or weakened by expression of truncated AtCASP-ΔCC and suggests that this connection is most likely maintained by a golgin-mediated tethering complex.

Shortening of Microtubule Overlap Regions Defines Membrane Delivery Sites during Plant Cytokinesis.

Different from animal cells that divide by constriction of the cortex inward, cells of land plants divide by initiating a new cell-wall segment from their center. For this, a disk-shaped, membrane-enclosed precursor termed the cell plate is formed that radially expands toward the parental cell wall [1-3]. The synthesis of the plate starts with the fusion of vesicles into a tubulo-vesicular network [4-6]. Vesicles are putatively delivered to the division plane by transport along microtubules of the bipolar phragmoplast network that guides plate assembly [7-9]. How vesicle immobilization and fusion are then locally triggered is unclear. In general, a framework for how the cytoskeleton spatially defines cell-plate formation is lacking. Here we show that membranous material for cell-plate formation initially accumulates along regions of microtubule overlap in the phragmoplast of the moss Physcomitrella patens. Kinesin-4-mediated shortening of these overlaps at the onset of cytokinesis proved to be required to spatially confine membrane accumulation. Without shortening, the wider cell-plate membrane depositions evolved into cell walls that were thick and irregularly shaped. Phragmoplast assembly thus provides a regular lattice of short overlaps on which a new cell-wall segment can be scaffolded. Since similar patterns of overlaps form in central spindles of animal cells, involving the activity of orthologous proteins [10, 11], we anticipate that our results will help uncover universal features underlying membrane-cytoskeleton coordination during cytokinesis.

Filamentous actin accumulates during plant cell penetration and cell wall plug formation in Phytophthora infestans.

The oomycete Phytophthora infestans is the cause of late blight in potato and tomato. It is a devastating pathogen and there is an urgent need to design alternative strategies to control the disease. To find novel potential drug targets, we used Lifeact-eGFP expressing P. infestans for high resolution live cell imaging of the actin cytoskeleton in various developmental stages. Previously, we identified actin plaques as structures that are unique for oomycetes. Here we describe two additional novel actin configurations; one associated with plug deposition in germ tubes and the other with appressoria, infection structures formed prior to host cell penetration. Plugs are composed of cell wall material that is deposited in hyphae emerging from cysts to seal off the cytoplasm-depleted base after cytoplasm retraction towards the growing tip. Preceding plug formation there was a typical local actin accumulation and during plug deposition actin remained associated with the leading edge. In appressoria, formed either on an artificial surface or upon contact with plant cells, we observed a novel aster-like actin configuration that was localized at the contact point with the surface. Our findings strongly suggest a role for the actin cytoskeleton in plug formation and plant cell penetration.

Low-Phosphate Induction of Plastidal Stromules Is Dependent on Strigolactones But Not on the Canonical Strigolactone Signaling Component MAX2.

Stromules are highly dynamic protrusions of the plastids in plants. Several factors, such as drought and light conditions, influence the stromule frequency (SF) in a positive or negative way. A relatively recently discovered class of plant hormones are the strigolactones; strigolactones inhibit branching of the shoots and promote beneficial interactions between roots and arbuscular mycorrhizal fungi. Here, we investigate the link between the formation of stromules and strigolactones. This research shows a strong link between strigolactones and the formation of stromules: SF correlates with strigolactone levels in the wild type and strigolactone mutants (max2-1 max3-9), and SF is stimulated by strigolactone GR24 and reduced by strigolactone inhibitor D2.

Interaction between the moss Physcomitrella patens and Phytophthora: a novel pathosystem for live-cell imaging of subcellular defence.

Live-cell imaging of plant-pathogen interactions is often hampered by the tissue complexity and multicell layered nature of the host. Here, we established a novel pathosystem with the moss Physcomitrella patens as host for Phytophthora. The tip-growing protonema cells of this moss are ideal for visualizing interactions with the pathogen over time using high-resolution microscopy. We tested four Phytophthora species for their ability to infect P. patens and showed that P. sojae and P. palmivora were only rarely capable to infect P. patens. In contrast, P. infestans and P. capsici frequently and successfully penetrated moss protonemal cells, showed intracellular hyphal growth and formed sporangia. Next to these successful invasions, many penetration attempts failed. Here the pathogen was blocked by a barrier of cell wall material deposited in papilla-like structures, a defence response that is common in higher plants. Another common response is the upregulation of defence-related genes upon infection and also in moss we observed this upregulation in tissues infected with Phytophthora. For more advanced analyses of the novel pathosystem we developed a special set-up that allowed live-cell imaging of subcellular defence processes by high-resolution microscopy. With this set-up, we revealed that Phytophthora infection of moss induces repositioning of the nucleus, accumulation of cytoplasm and rearrangement of the actin cytoskeleton, but not of microtubules.

Effect of Flumorph on F-Actin Dynamics in the Potato Late Blight Pathogen Phytophthora infestans.

Oomycetes are fungal-like pathogens that cause notorious diseases. Protecting crops against oomycetes requires regular spraying with chemicals, many with an unknown mode of action. In the 1990s, flumorph was identified as a novel crop protection agent. It was shown to inhibit the growth of oomycete pathogens including Phytophthora spp., presumably by targeting actin. We recently generated transgenic Phytophthora infestans strains that express Lifeact-enhanced green fluorescent protein (eGFP), which enabled us to monitor the actin cytoskeleton during hyphal growth. For analyzing effects of oomicides on the actin cytoskeleton in vivo, the P. infestans Lifeact-eGFP strain is an excellent tool. Here, we confirm that flumorph is an oomicide with growth inhibitory activity. Microscopic analyses showed that low flumorph concentrations provoked hyphal tip swellings accompanied by accumulation of actin plaques in the apex, a feature reminiscent of tips of nongrowing hyphae. At higher concentrations, swelling was more pronounced and accompanied by an increase in hyphal bursting events. However, in hyphae that remained intact, actin filaments were indistinguishable from those in nontreated, nongrowing hyphae. In contrast, in hyphae treated with the actin depolymerizing drug latrunculin B, no hyphal bursting was observed but the actin filaments were completely disrupted. This difference demonstrates that actin is not the primary target of flumorph.