Localising enzymes to biomolecular condensates increase their accumulation and benefits engineered metabolic pathway performance in Nicotiana benthamiana.

The establishment of Nicotiana benthamiana as a robust biofactory is complicated by issues such as product toxicity and proteolytic degradation of target proteins/introduced enzymes. Here we investigate whether biomolecular condensates can be used to address these problems. We engineered biomolecular condensates in N.

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.

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

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 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.

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.

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 β-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.

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 C-radiolabeled metabolically inert amino acid analogue α-aminoisobutyric acid (C-AIB).

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.

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

Hydrogen peroxide (H O ) is ubiquitous in cells and at the centre of developmental programmes and environmental responses. Its chemistry in cells makes H O 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 H O sensing in living plants.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

The 'mitoflash' probe cpYFP does not respond to superoxide.

Ageing and lifespan of organisms are determined by complicated interactions between their genetics and the environment, but the cellular mechanisms remain controversial. There have been a number of studies suggesting that cellular energy metabolism and free radical dynamics affect lifespan, implicating mitochondrial function. Recently, Shen provided apparent mechanistic insight by reporting that mitochondrial oscillations of ‘free radical production’, called ‘mitoflashes’, in the pharynx of 3-day old correlated inversely with lifespan.

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.

CDC-42 and RAC-1 regulate opposite chemotropisms in Neurospora crassa.

Cell polarization and fusion are crucial developmental processes that occur in response to intracellular and extracellular signals. Asexual spores (conidia) of the mold Neurospora crassa differentiate two types of polarized cell protrusions, germ tubes and conidial anastomosis tubes (CATs), which exhibit negative and positive chemotropism, respectively. We provide the first evidence that shared and separate functions of the Rho-type GTPases CDC-42 and RAC-1 regulate these opposite chemotropisms.

Robust anti-oxidant defences in the rice blast fungus Magnaporthe oryzae confer tolerance to the host oxidative burst.

Plants respond to pathogen attack via a rapid burst of reactive oxygen species (ROS). However, ROS are also produced by fungal metabolism and are required for the development of infection structures in Magnaporthe oryzae. To obtain a better understanding of redox regulation in M.

Studies of Physcomitrella patens reveal that ethylene-mediated submergence responses arose relatively early in land-plant evolution.

Colonization of the land by multicellular green plants was a fundamental step in the evolution of life on earth. Land plants evolved from fresh-water aquatic algae, and the transition to a terrestrial environment required the acquisition of developmental plasticity appropriate to the conditions of water availability, ranging from drought to flood. Here we show that extant bryophytes exhibit submergence-induced developmental plasticity, suggesting that submergence responses evolved relatively early in the evolution of land plants.

Taxonomies of networks from community structure.

The study of networks has become a substantial interdisciplinary endeavor that encompasses myriad disciplines in the natural, social, and information sciences. Here we introduce a framework for constructing taxonomies of networks based on their structural similarities. These networks can arise from any of numerous sources: They can be empirical or synthetic, they can arise from multiple realizations of a single process (either empirical or synthetic), they can represent entirely different systems in different disciplines, etc.