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. benthamiana leaves using transient expression of synthetic modular scaffolds. The in vivo properties of the condensates that resulted were consistent with them being liquid-like bodies with thermodynamic features typical of multicomponent phase-separating systems. We show that recruitment of enzymes to condensates in vivo led to several-fold yield increases in one- and three-step metabolic pathways (citramalate biosynthesis and poly-3-hydroxybutyrate (PHB) biosynthesis, respectively). This enhanced yield could be for several reasons including improved enzyme kinetics, metabolite channelling or avoidance of cytotoxicity by retention of the pathway product within the condensate, which was demonstrated for PHB. However, we also observed a several-fold increase in the amount of the enzymes that accumulated when they were targeted to the condensates. This suggests that the enzymes were more stable when localised to the condensate than when freely diffusing in the cytosol. We hypothesise that this stability is likely the main driver for increased pathway product production. Our findings provide a foundation for leveraging biomolecular condensates in plant metabolic engineering and advance N. benthamiana as a versatile biofactory for industrial applications.

Phenotypic plasticity (i.e. the ability of a genotype to alter its phenotype in response to environmental changes) can play a crucial role in maximizing fitness in fluctuating environments. However, plasticity comes at a cost, as the energy required to transform a phenotype imposes limits on its extent. While limits to plasticity are well-documented in many organisms, they are not well understood in modular organisms like filamentous fungi. Fungi have highly flexible morphologies, allowing individual organisms to adjust their phenotype in response to local environmental conditions. This flexibility has led to speculation that fungi exhibit "extreme phenotypic plasticity," making them an interesting system for studying plasticity mechanisms. To test this idea, we analyzed the grazer-induced morphological plasticity of four cord-forming fungal species exposed to different soil microfauna with varying feeding behaviors, using a phenotypic trajectory analysis framework. We hypothesized that grazing would drive convergence towards a common "grazing-resistant phenotype" across species, due to the extreme flexibility of fungal mycelia. Alternatively, we proposed that species would follow unique developmental trajectories in response to grazers, reflecting species-specific plasticity limits. Our results showed that fungal species accounted for most of the morphological variation, with grazers having only a small but significant effect. Moreover, we did not observe convergence towards a common grazing-resistant phenotype; instead, each species followed distinct developmental pathways within species-specific limits, which varied widely. Some species altered up to 30% of their phenotype in response to grazers, while others showed as little as 1% variation. These findings suggest a more nuanced view of fungal plasticity, where morphological plasticity might not be the most effective response to grazing pressure. We speculate that the observed differences in plasticity limits may correlate with the complexity of cord formation in different species. These constraints on fungal morphological plasticity offer new insights into how fungi adapt to dynamic ecological conditions and underscore the need to explore alternative phenotypic responses to environmental pressures. Our study highlights the need to refine plasticity theories, particularly for organisms with complex, modular structures like fungi. ### Competing Interest Statement The authors have declared no competing interest.

Modular organisms such as fungi are assumed to exhibit extreme morphological plasticity, yet this assumption has rarely been tested experimentally. Their morphology emerges from local, independent responses of constituent modules, suggesting strong plastic responses to environmental conditions. While such levels of plasticity decouple morphology from ecological function, they make these organisms an ideal system for studying the evolution of plasticity. Here we quantified the plasticity of modular fungi to grazers with known strong effects on their fitness and tested two competing hypotheses: (1) fungal morphology converges on a common 'grazing-resistant' phenotype across species (i.e., extreme plasticity) or (2) grazer-induced plasticity remains limited and species-specific. We found support for the latter, suggesting a more nuanced plasticity for fungi than would be expected based on their modularity. Our study calls for refining assumptions about plasticity in modular organisms and informs the use of morphological traits as predictors of ecological function.

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.

Simultaneous stoichiometric expression of multiple genes plays a major part in modern research and biotechnology. Traditional methods for incorporating multiple transgenes (or “gene stacking”) have drawbacks such as long time frames, uneven gene expression, gene silencing, and segregation derived from the use of multiple promoters. 2A self-cleaving peptides have emerged over the last two decades as a functional gene stacking method and have been used in plants for the co-expression of multiple genes under a single promoter. Here we describe design features of multicistronic polyproteins using 2A peptides for co-expression in plant cells and targeting to the endoplasmic reticulum (ER). We designed up to quad-cistronic vectors that could target proteins in tandem to the ER. We also exemplify the incorporation of self-excising intein domains within 2A polypeptides, to remove residue additions. These features could aid in the design of stoichiometric protein co-expression strategies in plants in combination with targeting to different subcellular compartments.

The dataset contains leaf venation architecture and functional traits for a phylogenetically diverse set of 122 plant species (including ferns, basal angiosperms, monocots, basal eudicots, asterids, and rosids) collected from the living collections of the University of California Botanical Garden at Berkeley (37.87° N, 122.23° W; CA, USA) from February to September 2021. The sampled species originated from all continents, except Antarctica, and are distributed in different growth forms (aquatic, herb, climbing, tree, shrub). The functional dataset comprises 31 traits (mechanical, hydraulic, anatomical, physiological, economical, and chemical) and describes six main leaf functional axes (hydraulic conductance, resistance and resilience to damages caused by drought and herbivory, mechanical support, and construction cost). It also describes how architecture features vary across venation networks. Our trait dataset is suitable for (1) functional and architectural characterization of plant species; (2) identification of venation architecture-function trade-offs; (3) investigation of evolutionary trends in leaf venation networks; and (4) mechanistic modeling of leaf function. Data are made available under the Open Data Commons Attribution License.

Variation in leaf venation network architecture may reflect trade-offs among multiple functions including efficiency, resilience, support, cost, and resistance to drought and herbivory. However, our knowledge about architecture-function trade-offs is mostly based on studies examining a small number of functional axes, so we still lack a more integrative picture of multidimensional trade-offs. Here, we measured architecture and functional traits on 122 ferns and angiosperms species to describe how trade-offs vary across phylogenetic groups and vein spatial scales (small, medium, and large vein width) and determine whether architecture traits at each scale have independent or integrated effects on each function. We found that generalized architecture-function trade-offs are weak. Architecture strongly predicts leaf support and damage resistance axes but weakly predicts efficiency and resilience axes. Architecture traits at different spatial scales contribute to different functional axes, allowing plants to independently modulate different functions by varying network properties at each scale. This independence of vein architecture traits within and across spatial scales may enable evolution of multiple alternative leaf network designs with similar functioning.

The plant endoplasmic reticulum forms a network of tubules connected by three-way junctions or sheet-like cisternae. Although the network is three-dimensional, in many plant cells, it is constrained to thin volume sandwiched between the vacuole and plasma membrane, effectively restricting it to a 2-D planar network. The structure of the network, and the morphology of the tubules and cisternae can be automatically extracted following intensity-independent edge-enhancement and various segmentation techniques to give an initial pixel-based skeleton, which is then converted to a graph representation. ER dynamics can be determined using optical flow techniques from computer vision or persistency analysis. Collectively, this approach yields a wealth of quantitative metrics for ER structure and can be used to describe the effects of pharmacological treatments or genetic manipulation. The software is publicly available.

ABSTRACT Calcium (Ca2+) is an important second messenger for activating stress response signaling and cell adaptation in eukaryotic cells yet intracellular Ca2+-dynamics in fungi are poorly understood due to lack of effective real-time Ca2+ reporters. We engineered the GCaMP6f construct for use in the fungal pathogen, Candida albicans, and used live-cell imaging to observe both dynamic Ca2+ spiking and slower changes in non-spiking Ca2+-GCaMP signals elicited by stress or gene deletion. Short-term exposure to membrane, osmotic or oxidative stress generated immediate stress-specific responses and repeated exposure revealed differential recovery signatures. Osmotic stress caused yeast cell shrinkage and no adaptation response, where Ca2+-GCaMP spiking was inhibited by 1 M NaCl but not by 0.666 M CaCl2. Treatment with sodium dodecylsulfate (SDS) caused a spike-burst, raised the non-spiking Ca2+-GCaMP signals, and caused significant cell death, but surviving cells adapted over subsequent exposures. Treatment with 5 mM H2O2 abolished spiking and caused transient non-GCaMP-related autofluorescence, but cells adapted such that spiking returned and autofluorescence diminished on repeated exposure. Adaptation to H2O2 was dependent on Cap1, extracellular Ca2+, and calcineurin but not on its downstream target, Crz1. Ca2+-dynamics were not affected by H2O2 in the hog1Δ or yvc1Δ mutants, suggesting a pre-adapted, resistant state, possibly due to changes in membrane permeability. Live-cell imaging of Ca2+-GCaMP responses in individual cells has, therefore, revealed the dynamics of Ca2+-influx, signaling and homeostasis, and their role in the temporal stress response signatures of C. albicans. IMPORTANCE Intracellular calcium signaling plays an important role in the resistance and adaptation to stresses encountered by fungal pathogens within the host. This study reports the optimization of the GCaMP fluorescent calcium reporter for live-cell imaging of dynamic calcium responses in single cells of the pathogen, Candida albicans, for the first time. Exposure to membrane, osmotic or oxidative stress generated both specific changes in single cell intracellular calcium spiking and longer calcium transients across the population. Repeated treatments showed that calcium dynamics become unaffected by some stresses but not others, consistent with known cell adaptation mechanisms. By expressing GCaMP in mutant strains and tracking the viability of individual cells over time, the relative contributions of key signaling pathways to calcium flux, stress adaptation, and cell death were demonstrated. This reporter, therefore, permits the study of calcium dynamics, homeostasis, and signaling in C. albicans at a previously unattainable level of detail.

Methane is a potent greenhouse gas, which has contributed to approximately a fifth of global warming since pre-industrial times. The agricultural sector produces significant methane emissions, especially from livestock, waste management and rice cultivation. Rice fields alone generate around 9% of total anthropogenic emissions. Methane is produced in waterlogged paddy fields by methanogenic archaea, and transported to the atmosphere through the aerenchyma tissue of rice plants. Thus, bioengineering rice with catalysts to detoxify methane en route could contribute to an efficient emission mitigation strategy. Particulate methane monooxygenase (pMMO) is the predominant methane catalyst found in nature, and is an enzyme complex expressed by methanotrophic bacteria. Recombinant expression of pMMO has been challenging, potentially due to its membrane localization, multimeric structure, and polycistronic operon. Here we show the first steps towards the engineering of plants for methane detoxification with the three pMMO subunits expressed in the model systems tobacco and Arabidopsis. Membrane topology and protein–protein interactions were consistent with correct folding and assembly of the pMMO subunits on the plant ER. Moreover, a synthetic self-cleaving polypeptide resulted in simultaneous expression of all three subunits, although low expression levels precluded more detailed structural investigation. The work presents plant cells as a novel heterologous system for pMMO allowing for protein expression and modification.

Engineering of subcellular compartmentalisation is one of synthetic biology’s key challenges. Among different approaches, de novo construction of a synthetic compartment is the most coveted but also most difficult option. Restructuring the endoplasmic reticulum (ER), via the introduction of recombinant oligomerising ER-membrane resident proteins, is an alternative starting point for building a new compartment. The presence of such proteins leads to a massive expansion of the ER and the formation of organised smooth endoplasmic reticulum (OSER), a large membranous compartment. However, OSER is poorly characterised and our understanding of its effect on the underlying biology of the plant is limited. Here we characterise a range of OSER compartments and show how the structure of the inducing polyprotein constructs affect the final compartment morphology, with the cytosolic-facing antiparallel oligomerisation domain demonstrated to be an essential component to trigger OSER formation. We show that while OSER retains a connection to the ER, a diffusional barrier exists to both the ER and the cytosol. Using high-resolution quantitative image analysis, we also show that the presence of this large compartment does not disrupt the rest of the ER network. Moreover, transgenic Arabidopsis constitutively expressing the compartment-forming polyproteins grew and developed normally. These properties collectively suggest that OSER could be developed as a plant synthetic biology tool for compartmentalisation, combining the benefits of several existing strategies. Only a single protein construct is necessary to induce its formation, and the compartment retains a delimiting membrane and a diffusional barrier to the rest of the cell.

Hydrogen peroxide (H2O2) has key signaling roles at physiological levels, while causing molecular damage at elevated concentrations. H2O2 production by mitochondria is implicated in regulating processes inside and outside these organelles. However, it remains unclear whether and how mitochondria in intact cells release H2O2. Here, we employed a genetically encoded high-affinity H2O2 sensor, HyPer7, in mammalian tissue culture cells to investigate different modes of mitochondrial H2O2 release. We found substantial heterogeneity of HyPer7 dynamics between individual cells. We further observed mitochondria-released H2O2 directly at the surface of the organelle and in the bulk cytosol, but not in the nucleus or at the plasma membrane, pointing to steep gradients emanating from mitochondria. Gradient formation is controlled by cytosolic peroxiredoxins, which act redundantly and with a substantial reserve capacity. Dynamic adaptation of cytosolic thioredoxin reductase levels during metabolic changes results in improved H2O2 handling and explains previously observed differences between cell types. Our data suggest that H2O2-mediated signaling is initiated only in close proximity to mitochondria and under specific metabolic conditions.

Emergence of cities and road networks have characterised human activity and movement over millennia. However, this anthropogenic infrastructure does not develop in isolation, but is deeply embedded in the natural landscape, which strongly influences the resultant spatial patterns. Nevertheless, the precise impact that landscape has on the location, size and connectivity of cities is a long-standing, unresolved problem. To address this issue, we incorporate high-resolution topographic maps into a Turing-like pattern forming system, in which local reinforcement rules result in co-evolving centres of population and transport networks. Using Italy as a case study, we show that the model constrained solely by topography results in an emergent spatial pattern that is consistent with Zipf’s Law and comparable to the census data. Thus, we infer the natural landscape may play a dominant role in establishing the baseline macro-scale population pattern, that is then modified by higher-level historical, socio-economic or cultural factors.

Colonization of terrestrial environments by filamentous fungi relies on their ability to form networks that can forage for and connect resource patches. Despite the importance of these networks, ecologists rarely consider network features as functional traits because their measurement and interpretation are conceptually and methodologically difficult. To address these challenges, we have developed a pipeline to translate images of fungal mycelia, from both micro- and macro-scales, to weighted network graphs that capture ecologically relevant fungal behaviour. We focus on four properties that we hypothesize determine how fungi forage for resources, specifically: connectivity; relative construction cost; transport efficiency; and robustness against attack by fungivores. Constrained ordination and Pareto front analysis of these traits revealed that foraging strategies can be distinguished predominantly along a gradient of connectivity for micro- and macro-scale mycelial networks that is reminiscent of the qualitative 'phalanx' and 'guerilla' descriptors previously proposed in the literature. At one extreme are species with many inter-connections that increase the paths for multidirectional transport and robustness to damage, but with a high construction cost; at the other extreme are species with an opposite phenotype. Thus, we propose this approach represents a significant advance in quantifying ecological strategies for fungi using network information.

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.

Summary 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. 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.

Fungal mycelia consist of an interconnected network of filamentous hyphae and represent the dominant phase of the lifecycle in all major fungal phyla, from basal to more recent clades. Indeed, the ecological success of fungi on land is partly due to such filamentous network growth. Nevertheless, fungal ecologists rarely use network features as functional traits. Given the widespread occurrence of this body type, we hypothesized that interspecific variation in network features may reflect both phylogenetic affiliation and distinct ecological strategies among species. We show first that there is high interspecific variation in network parameters of fungi, which partly correlates with taxonomy; and second that network parameters, related to predicted-mycelial transport mechanisms during the exploration phase, reveal the trait space in mycelium architecture across species. This space predicts a continuum of ecological strategies along two extremes: from highly connected mycelia with high resilience to damage but limited transport efficiency, to poorly connected mycelia with low resilience but high transport efficiency. We argue that mycelial networks are potentially a rich source of information to inform functional trait analysis in fungi, but we also note the challenges in establishing common principles and processing pipelines that are required to facilitate widespread use of network properties as functional traits in fungal ecology.

SUMMARYH2O2performs central roles in signaling at physiological levels, while at elevated levels it causes molecular damage. Mitochondria are major producers of H2O2, which has been implied in regulating diverse processes inside and outside the organelle. However, it still remains unclear whether and how mitochondria in intact cells release H2O2. Here we employed the genetically encoded high-affinity H2O2sensor HyPer7 in mammalian tissue culture cells to investigate different modes of mitochondrial H2O2release. We found substantial heterogeneity of HyPer7 dynamics between individual cells, and observed H2O2released from mitochondria directly at the surface of the organelle and in the bulk cytosol, but not in the nucleus nor on the plasma membrane, pointing to steep gradients emanating from mitochondria. These gradients are controlled by cytosolic peroxiredoxins that act redundantly and are present with a substantial reserve capacity. Furthermore, dynamic adaptation of cytosolic thioredoxin reductase levels during metabolic changes results in improved H2O2handling and explains previously observed cell-to-cell differences. Thus, our data indicate that H2O2-mediated signaling likely occurs close to mitochondria during specific metabolic conditions.HIGHLIGHTSMitochondrial H2O2can be detected in the cytosol in intact human cellsMitochondrial H2O2gradients are steep and controlled by peroxiredoxins 1 and 2Peroxiredoxins 1 and 2 complement for each otherPeroxiredoxins 1 and 2 are present with a substantial reserve capacityMetabolism-induced changes of reducing processes control peroxiredoxin activity

Images of fungal mycelia from basidiomycetes, ascomycetes and zygomycetes with associated parameter files for processing using the Fungal Network Analysis software available from DOI: 10.5281/zenodo.5187932. Please cite: Aguilar-Trigueros, C.A., Boddy, L., Rillig, M.C. and Fricker, M.D. (2022) Network traits predict ecological strategies in fungi. ISME Communications. 2, 2 https://doi.org/10.1038/s43705-021-00085-1