Characterization of intracellular membrane structures derived from a massive expansion of endoplasmic reticulum (ER) membrane due to synthetic ER-membrane-resident polyproteins.

The endoplasmic reticulum (ER) is a dynamic organelle that is amenable to major restructuring. Introduction of recombinant ER-membrane-resident proteins that form homo oligomers is a known method of inducing ER proliferation: interaction of the proteins with each other alters the local structure of the ER network, leading to the formation large aggregations of expanded ER, sometimes leading to the formation of organized smooth endoplasmic reticulum (OSER). However, these membrane structures formed by ER proliferation are poorly characterized and this hampers their potential development for plant synthetic biology. Here, we characterize a range of ER-derived membranous compartments in tobacco and show how the nature of the polyproteins introduced into the ER membrane affect the morphology of the final compartment. We show that a cytosol-facing oligomerization domain is an essential component for compartment formation. Using fluorescence recovery after photobleaching, we demonstrate that although the compartment retains a connection to the ER, a diffusional barrier exists to both the ER and the cytosol associated with the compartment. Using quantitative image analysis, we also show that the presence of the compartment does not disrupt the rest of the ER network. Moreover, we demonstrate that it is possible to recruit a heterologous, bacterial enzyme to the compartment, and for the enzyme to accumulate to high levels. Finally, transgenic Arabidopsis constitutively expressing the compartment-forming polyproteins grew and developed normally under standard conditions.

Stress-Activated Protein Kinase Signalling Regulates Mycoparasitic Hyphal-Hyphal Interactions in Trichoderma atroviride.

Trichoderma atroviride is a mycoparasitic fungus used as biological control agent against fungal plant pathogens. The recognition and appropriate morphogenetic responses to prey-derived signals are essential for successful mycoparasitism. We established microcolony confrontation assays using T. atroviride strains expressing cell division cycle 42 (Cdc42) and Ras-related C3 botulinum toxin substrate 1 (Rac1) interactive binding (CRIB) reporters to analyse morphogenetic changes and the dynamic displacement of localized GTPase activity during polarized tip growth. Microscopic analyses showed that Trichoderma experiences significant polarity stress when approaching its fungal preys. The perception of prey-derived signals is integrated via the guanosine triphosphatase (GTPase) and mitogen-activated protein kinase (MAPK) signalling network, and deletion of the MAP kinases Trichoderma MAPK 1 (Tmk1) and Tmk3 affected T. atroviride tip polarization, chemotropic growth, and contact-induced morphogenesis so severely that the establishment of mycoparasitism was highly inefficient to impossible. The responses varied depending on the prey species and the interaction stage, reflecting the high selectivity of the signalling process. Our data suggest that Tmk3 affects the polarity-stress adaptation process especially during the pre-contact phase, whereas Tmk1 regulates contact-induced morphogenesis at the early-contact phase. Neither Tmk1 nor Tmk3 loss-of-function could be fully compensated within the GTPase/MAPK signalling network underscoring the crucial importance of a sensitive polarized tip growth apparatus for successful mycoparasitism.

IntEResting structures: formation and applications of organized smooth endoplasmic reticulum in plant cells.

The endoplasmic reticulum (ER) is an organelle with remarkable plasticity, capable of rapidly changing its structure to accommodate different functions based on intra- and extracellular cues. One of the ER structures observed in plants is known as "organized smooth endoplasmic reticulum" (OSER), consisting of symmetrically stacked ER membrane arrays. In plants, these structures were first described in certain specialized tissues, e.g. the sieve elements of the phloem, and more recently in transgenic plants overexpressing ER membrane resident proteins. To date, much of the investigation of OSER focused on yeast and animal cells but research into plant OSER has started to grow. In this update, we give a succinct overview of research into the OSER phenomenon in plant cells with case studies highlighting both native and synthetic occurrences of OSER. We also assess the primary driving forces that trigger the formation of OSER, collating evidence from the literature to compare two competing theories for the origin of OSER: that OSER formation is initiated by oligomerizing protein accumulation in the ER membrane or that OSER is the result of ER membrane proliferation. This has long been a source of controversy in the field and here we suggest a way to integrate arguments from both sides into a single unifying theory. Finally, we discuss the potential biotechnological uses of OSER as a tool for the nascent plant synthetic biology field with possible applications as a synthetic microdomain for metabolic engineering and as an extensive membrane surface for synthetic chemistry or protein accumulation.

Chitosan inhibits septin-mediated plant infection by the rice blast fungus Magnaporthe oryzae in a protein kinase C and Nox1 NADPH oxidase-dependent manner.

Chitosan is a partially deacetylated linear polysaccharide composed of ß-1,4-linked units of d-glucosamine and N-acetyl glucosamine. As well as a structural component of fungal cell walls, chitosan is a potent antifungal agent. However, the mode of action of chitosan is poorly understood. Here, we report that chitosan is effective for control of rice blast disease. Chitosan application impairs growth of the blast fungus Magnaporthe oryzae and has a pronounced effect on appressorium-mediated plant infection. Chitosan inhibits septin-mediated F-actin remodelling at the appressorium pore, thereby preventing repolarization of the infection cell. Chitosan causes plasma membrane permeabilization of M. oryzae and affects NADPH oxidase-dependent synthesis of reactive oxygen species, essential for septin ring formation and fungal pathogenicity. We further show that toxicity of chitosan to M. oryzae requires the protein kinase C-dependent cell wall integrity pathway, the Mps1 mitogen-activated protein kinase and the Nox1 NADPH oxidase. A conditionally lethal, analogue (PP1)-sensitive mutant of Pkc1 is partially remediated for growth in the presence of chitosan, while ∆nox1 mutants increase their glucan : chitin cell wall ratio, rendering them resistant to chitosan. Taken together, our data show that chitosan is a potent fungicide which requires the cell integrity pathway, disrupts plasma membrane function and inhibits septin-mediated plant infection.

Growth induced translocation effectively directs an amino acid analogue to developing zones in Agaricus bisporus.

The vegetative mycelium of Agaricus bisporus supplies developing white button mushrooms with water and nutrients. However, it is not yet known which part of the mycelium contributes to the feeding of the mushrooms and how this depends on growth conditions. Here we used photon counting scintillation imaging to track translocation of the 14C-radiolabeled metabolically inert amino acid analogue α-aminoisobutyric acid (14C-AIB). Translocation to the periphery of the mycelium was observed in actively growing vegetative mycelium with a velocity of up to 6.6 mm h-1, which was 30-fold higher than the growth rate. Furthermore, 14C-AIB translocated to neighboring colonies after fusion by anastomosis depending on the relative growth rate in these colonies. When mushrooms started to develop, translocation of 14C-AIB was redirected to the fruiting bodies via mycelium and hyphal cords. More abundant mycelial cord formation and a 5-fold higher rate of translocation was observed for cultures growing directionally from inoculum located at one side of the substrate, when compared to non-directional growth (inoculum mixed throughout the substrate). The maximum translocation distance was also greater (≥50 and 22 cm, respectively). In conclusion, 14C-AIB translocation switches between vegetative growth and towards developing mushrooms, especially via cords and when source-sink relationships change.

A mechanistic explanation of the transition to simple multicellularity in fungi.

Development of multicellularity was one of the major transitions in evolution and occurred independently multiple times in algae, plants, animals, and fungi. However recent comparative genome analyses suggest that fungi followed a different route to other eukaryotic lineages. To understand the driving forces behind the transition from unicellular fungi to hyphal forms of growth, we develop a comparative model of osmotrophic resource acquisition. This predicts that whenever the local resource is immobile, hard-to-digest, and nutrient poor, hyphal osmotrophs outcompete motile or autolytic unicellular osmotrophs. This hyphal advantage arises because transporting nutrients via a contiguous cytoplasm enables continued exploitation of remaining resources after local depletion of essential nutrients, and more efficient use of costly exoenzymes. The model provides a mechanistic explanation for the origins of multicellular hyphal organisms, and explains why fungi, rather than unicellular bacteria, evolved to dominate decay of recalcitrant, nutrient poor substrates such as leaf litter or wood.

The fluorescent protein sensor roGFP2-Orp1 monitors in vivo H2 O2 and thiol redox integration and elucidates intracellular H2 O2 dynamics during elicitor-induced oxidative burst in Arabidopsis.

Hydrogen peroxide (H2 O2 ) is ubiquitous in cells and at the centre of developmental programmes and environmental responses. Its chemistry in cells makes H2 O2 notoriously hard to detect dynamically, specifically and at high resolution. Genetically encoded sensors overcome persistent shortcomings, but pH sensitivity, silencing of expression and a limited concept of sensor behaviour in vivo have hampered any meaningful H2 O2 sensing in living plants. We established H2 O2 monitoring in the cytosol and the mitochondria of Arabidopsis with the fusion protein roGFP2-Orp1 using confocal microscopy and multiwell fluorimetry. We confirmed sensor oxidation by H2 O2 , show insensitivity to physiological pH changes, and demonstrated that glutathione dominates sensor reduction in vivo. We showed the responsiveness of the sensor to exogenous H2 O2 , pharmacologically-induced H2 O2 release, and genetic interference with the antioxidant machinery in living Arabidopsis tissues. Monitoring intracellular H2 O2 dynamics in response to elicitor exposure reveals the late and prolonged impact of the oxidative burst in the cytosol that is modified in redox mutants. We provided a well defined toolkit for H2 O2 monitoring in planta and showed that intracellular H2 O2 measurements only carry meaning in the context of the endogenous thiol redox systems. This opens new possibilities to dissect plant H2 O2 dynamics and redox regulation, including intracellular NADPH oxidase-mediated ROS signalling.

ATP sensing in living plant cells reveals tissue gradients and stress dynamics of energy physiology.

Growth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here, we establish MgATP2- measurement in living plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensor in vitro under conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP2- changes in planta. A MgATP2- map of the Arabidopsis seedling highlights different MgATP2- concentrations between tissues and within individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant.

The Mycelium as a Network.

The characteristic growth pattern of fungal mycelia as an interconnected network has a major impact on how cellular events operating on a micron scale affect colony behavior at an ecological scale. Network structure is intimately linked to flows of resources across the network that in turn modify the network architecture itself. This complex interplay shapes the incredibly plastic behavior of fungi and allows them to cope with patchy, ephemeral resources, competition, damage, and predation in a manner completely different from multicellular plants or animals. Here, we try to link network structure with impact on resource movement at different scales of organization to understand the benefits and challenges of organisms that grow as connected networks. This inevitably involves an interdisciplinary approach whereby mathematical modeling helps to provide a bridge between information gleaned by traditional cell and molecular techniques or biophysical approaches at a hyphal level, with observations of colony dynamics and behavior at an ecological level.

Microcompartmentation of cytosolic aldolase by interaction with the actin cytoskeleton in Arabidopsis.

Evidence is accumulating for molecular microcompartments formed when proteins interact in localized domains with the cytoskeleton, organelle surfaces, and intracellular membranes. To understand the potential functional significance of protein microcompartmentation in plants, we studied the interaction of the glycolytic enzyme fructose bisphosphate aldolase with actin in Arabidopsis thaliana. Homology modelling of a major cytosolic isozyme of aldolase, FBA8, suggested that the tetrameric holoenzyme has two actin binding sites and could therefore act as an actin-bundling protein, as was reported for animal aldolases. This was confirmed by in vitro measurements of an increase in viscosity of F-actin polymerized in the presence of recombinant FBA8. Simultaneously, interaction with F-actin caused non-competitive inhibition of aldolase activity. We did not detect co-localization of an FBA8-RFP fusion protein, expressed in an fba8-knockout background, with the actin cytoskeleton using confocal laser-scanning microscopy. However, we did find evidence for a low level of interaction using FRET-FLIM analysis of FBA8-RFP co-expressed with the actin-binding protein GFP-Lifeact. Furthermore, knockout of FBA8 caused minor alterations of guard cell actin cytoskeleton morphology and resulted in a reduced rate of stomatal closure in response to decreased humidity. We conclude that cytosolic aldolase can be microcompartmented in vivo by interaction with the actin cytoskeleton and may subtly modulate guard cell behaviour as a result.

Effective delivery of volatile biocides employing mesoporous silicates for treating biofilms.

Nanoparticulate delivery of biocides has the potential to decrease levels of exposure to non-target organisms, and miminize long-term exposure that can promote the development of resistance. Silica nanoparticles are an ideal vehicle since they are inert, biocompatible, biodegradable, and thermally and chemically stable. Encapsulation of biocides within nanoparticulates can improve their stability and longevity and maximize the biocidal potential of hydrophobic volatile compounds. Herein, we have shown that the plant secondary metabolites allyl isothiocyanate and cinnamaldehyde demonstrated increased antimicrobial activity against Escherichia coli in planktonic form, when packaged into mesoporous silica nanoparticles. Furthermore, the biocide-loaded nanoparticles showed activity against Pseudomonas aeruginosa biofilms that have inherent resistance to antimicrobial agents. The delivery platform can also be expanded to traditional biocides and other non-conventional antimicrobial agents.

A C-terminal amphipathic helix is necessary for the in vivo tubule-shaping function of a plant reticulon.

Reticulons (RTNs) are a class of endoplasmic reticulum (ER) membrane proteins that are capable of maintaining high membrane curvature, thus helping shape the ER membrane into tubules. The mechanism of action of RTNs is hypothesized to be a combination of wedging, resulting from the transmembrane topology of their conserved reticulon homology domain, and scaffolding, arising from the ability of RTNs to form low-mobility homo-oligomers within the membrane. We studied the plant RTN isoform RTN13, which has previously been shown to locate to ER tubules and the edges of ER cisternae and to induce constrictions in ER tubules when overexpressed, and identified a region in the C terminus containing a putative amphipathic helix (APH). Here we show that deletion of this region or disruption of the hydrophobic face of the predicted helix abolishes the ability of RTN13 to induce constrictions of ER tubules in vivo. These mutants, however, still retain their ability to interact and form low-mobility oligomers in the ER membrane. Hence, our evidence indicates that the conserved APH is a key structural feature for RTN13 function in vivo, and we propose that RTN, like other membrane morphogens, rely on APHs for their function.

Making microscopy count: quantitative light microscopy of dynamic processes in living plants.

Cell theory has officially reached 350 years of age as the first use of the word 'cell' in a biological context can be traced to a description of plant material by Robert Hooke in his historic publication 'Micrographia: or some physiological definitions of minute bodies'. The 2015 Royal Microscopical Society Botanical Microscopy meeting was a celebration of the streams of investigation initiated by Hooke to understand at the subcellular scale how plant cell function and form arises. Much of the work presented, and Honorary Fellowships awarded, reflected the advanced application of bioimaging informatics to extract quantitative data from micrographs that reveal dynamic molecular processes driving cell growth and physiology. The field has progressed from collecting many pixels in multiple modes to associating these measurements with objects or features that are meaningful biologically. The additional complexity involves object identification that draws on a different type of expertise from computer science and statistics that is often impenetrable to biologists. There are many useful tools and approaches being developed, but we now need more interdisciplinary exchange to use them effectively. In this review we show how this quiet revolution has provided tools available to any personal computer user. We also discuss the oft-neglected issue of quantifying algorithm robustness and the exciting possibilities offered through the integration of physiological information generated by biosensors with object detection and tracking.

Immobilized Subpopulations of Leaf Epidermal Mitochondria Mediate PENETRATION2-Dependent Pathogen Entry Control in Arabidopsis.

The atypical myrosinase PENETRATION2 (PEN2) is required for broad-spectrum invasion resistance to filamentous plant pathogens. Previous localization studies suggested PEN2-GFP association with peroxisomes. Here, we show that PEN2 is a tail-anchored protein with dual-membrane targeting to peroxisomes and mitochondria and that PEN2 has the capacity to form homo-oligomer complexes. We demonstrate pathogen-induced recruitment and immobilization of mitochondrial subpopulations at sites of attempted fungal invasion and show that mitochondrial arrest is accompanied by peripheral accumulation of GFP-tagged PEN2. PEN2 substrate production by the cytochrome P450 monooxygenase CYP81F2 is localized to the surface of the endoplasmic reticulum, which focally reorganizes close to the immobilized mitochondria. Exclusive targeting of PEN2 to the outer membrane of mitochondria complements the pen2 mutant phenotype, corroborating the functional importance of the mitochondrial PEN2 protein subpool for controlled local production of PEN2 hydrolysis products at subcellular plant-microbe interaction domains. Moreover, live-cell imaging shows that mitochondria arrested at these domains exhibit a pathogen-induced redox imbalance, which may lead to the production of intracellular signals.

Energetic Constraints on Fungal Growth.

Saprotrophic fungi are obliged to spend energy on growth, reproduction, and substrate digestion. To understand the trade-offs involved, we developed a model that, for any given growth rate, identifies the strategy that maximizes the fraction of energy that could possibly be spent on reproduction. Our model's predictions of growth rates and bioconversion efficiencies are consistent with empirical findings, and it predicts the optimal investment in reproduction, resource acquisition, and biomass recycling for a given environment and timescale of reproduction. Thus, if the timescale of reproduction is long compared to the time required for the fungus to double in size, the model suggests that the total energy available for reproduction is maximal when a very small fraction of the energy budget is spent on reproduction. The model also suggests that fungi growing on substrates with a high concentration of low-molecular-weight compounds will not benefit from recycling: they should be able to grow more rapidly and allocate more energy to reproduction without recycling. In contrast, recycling offers considerable benefits to fungi growing on recalcitrant substrates, where the individual hyphae are not crowded and the time taken to consume resource is significantly longer than the fungus doubling time.

Quantitative Redox Imaging Software.

SIGNIFICANCE: A wealth of fluorescent reporters and imaging systems are now available to characterize dynamic physiological processes in living cells with high spatiotemporal resolution. The most reliable probes for quantitative measurements show shifts in their excitation or emission spectrum, rather than just a change in intensity, as spectral shifts are independent of optical path length, illumination intensity, probe concentration, and photobleaching, and they can be easily determined by ratiometric measurements at two wavelengths. RECENT ADVANCES: A number of ratiometric fluorescent reporters, such as reduction-oxidation-sensitive green fluorescent protein (roGFP), have been developed that respond to the glutathione redox potential and allow redox imaging in vivo. roGFP and its derivatives can be expressed in the cytoplasm or targeted to different organelles, giving fine control of measurements from sub-cellular compartments. Furthermore, roGFP can be imaged with probes for other physiological parameters, such as reactive oxygen species or mitochondrial membrane potential, to give multi-channel, multi-dimensional 4D (x,y,z,t) images. CRITICAL ISSUES: Live cell imaging approaches are needed to capture transient or highly spatially localized physiological behavior from intact, living specimens, which are often not accessible by other biochemical or genetic means. FUTURE DIRECTIONS: The next challenge is to be able to extract useful data rapidly from such large (GByte) images with due care given to the assumptions used during image processing. This article describes a suite of software programs, available for download, that provide intuitive user interfaces to conduct multi-channel ratio imaging, or alternative analysis methods such as pixel-population statistics or image segmentation and object-based ratio analysis. Antioxid. Redox Signal. 24, 752-762.

The EF-Hand Ca2+ Binding Protein MICU Choreographs Mitochondrial Ca2+ Dynamics in Arabidopsis.

Plant organelle function must constantly adjust to environmental conditions, which requires dynamic coordination. Ca(2+) signaling may play a central role in this process. Free Ca(2+) dynamics are tightly regulated and differ markedly between the cytosol, plastid stroma, and mitochondrial matrix. The mechanistic basis of compartment-specific Ca(2+) dynamics is poorly understood. Here, we studied the function of At-MICU, an EF-hand protein of Arabidopsis thaliana with homology to constituents of the mitochondrial Ca(2+) uniporter machinery in mammals. MICU binds Ca(2+) and localizes to the mitochondria in Arabidopsis. In vivo imaging of roots expressing a genetically encoded Ca(2+) sensor in the mitochondrial matrix revealed that lack of MICU increased resting concentrations of free Ca(2+) in the matrix. Furthermore, Ca(2+) elevations triggered by auxin and extracellular ATP occurred more rapidly and reached higher maximal concentrations in the mitochondria of micu mutants, whereas cytosolic Ca(2+) signatures remained unchanged. These findings support the idea that a conserved uniporter system, with composition and regulation distinct from the mammalian machinery, mediates mitochondrial Ca(2+) uptake in plants under in vivo conditions. They further suggest that MICU acts as a throttle that controls Ca(2+) uptake by moderating influx, thereby shaping Ca(2+) signatures in the matrix and preserving mitochondrial homeostasis. Our results open the door to genetic dissection of mitochondrial Ca(2+) signaling in plants.

Analysis of plant mitochondrial function using fluorescent protein sensors.

Mitochondrial physiology sets the basis for function of the organelle and vice versa. While a limited range of in vivo parameters, such as oxygen consumption, has been classically accessible for measurement, a growing collection of fluorescent protein sensors can now give insights into the physiology of plant mitochondria. Nevertheless, the meaningful application of these sensors in mitochondria is technically challenging and requires rigorous experimental standards. Here we exemplify the application of three genetically encoded sensors to monitor glutathione redox potential, pH, and calcium in the matrix of mitochondria in intact plants. We describe current methods for quantitative imaging and analysis in living root tips by confocal microscopy and discuss methodological limitations.

An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology.

Plant leaves are optically complex, which makes them difficult to image by light microscopy. Careful sample preparation is therefore required to enable researchers to maximize the information gained from advances in fluorescent protein labeling, cell dyes and innovations in microscope technologies and techniques. We have previously shown that mounting leaves in the non-toxic, non-fluorescent perfluorocarbon (PFC), perfluorodecalin (PFD) enhances the optical properties of the leaf with minimal impact on physiology. Here, we assess the use of the PFCs, PFD, and perfluoroperhydrophenanthrene (PP11) for in vivo plant leaf imaging using four advanced modes of microscopy: laser scanning confocal microscopy (LSCM), two-photon fluorescence microscopy, second harmonic generation microscopy, and stimulated Raman scattering (SRS) microscopy. For every mode of imaging tested, we observed an improved signal when leaves were mounted in PFD or in PP11, compared to mounting the samples in water. Using an image analysis technique based on autocorrelation to quantitatively assess LSCM image deterioration with depth, we show that PP11 outperformed PFD as a mounting medium by enabling the acquisition of clearer images deeper into the tissue. In addition, we show that SRS microscopy can be used to image PFCs directly in the mesophyll and thereby easily delimit the "negative space" within a leaf, which may have important implications for studies of leaf development. Direct comparison of on and off resonance SRS micrographs show that PFCs do not to form intracellular aggregates in live plants. We conclude that the application of PFCs as mounting media substantially increases advanced microscopy image quality of living mesophyll and leaf vascular bundle cells.