Leaf microbiome dysbiosis triggered by T2SS-dependent enzyme secretion from opportunistic Xanthomonas pathogens.

In healthy plants, the innate immune system contributes to maintenance of microbiota homoeostasis, while disease can be associated with microbiome perturbation or dysbiosis, and enrichment of opportunistic plant pathogens like Xanthomonas. It is currently unclear whether the microbiota change occurs independently of the opportunistic pathogens or is caused by the latter. Here we tested if protein export through the type-2 secretion system (T2SS) by Xanthomonas causes microbiome dysbiosis in Arabidopsis thaliana in immunocompromised plants. We found that Xanthomonas strains secrete a cocktail of plant cell wall-degrading enzymes that promote Xanthomonas growth during infection. Disease severity and leaf tissue degradation were increased in A. thaliana mutants lacking the NADPH oxidase RBOHD. Experiments with gnotobiotic plants, synthetic bacterial communities and wild-type or T2SS-mutant Xanthomonas revealed that virulence and leaf microbiome composition are controlled by the T2SS. Overall, a compromised immune system in plants can enrich opportunistic pathogens, which damage leaf tissues and ultimately cause microbiome dysbiosis by facilitating growth of specific commensal bacteria.

Mechanical activation of mitochondria in germ cell differentiation.

Mitochondria are activated during stem cell differentiation. Recently, Wang et al. found that mechanical stimulation from tissue surrounding differentiating germ cells in the female fly ovary is necessary to sustain intracellular calcium levels, promoting mitochondrial activity. This suggests a molecular link between cell mechanics and developmental metabolic transitions in eukaryotes.

Engineering Endosymbiotic Growth of E. coli in Mammalian Cells.

Endosymbioses are cellular mergers in which one cell lives within another cell and have led to major evolutionary transitions, most prominently to eukaryogenesis. Generation of synthetic endosymbioses aims to provide a defined starting point for studying fundamental processes in emerging endosymbiotic systems and enable the engineering of cells with novel properties. Here, we tested the potential of different bacteria for artificial endosymbiosis in mammalian cells. To this end, we adopted the fluidic force microscopy technology to inject diverse bacteria directly into the cytosol of HeLa cells and examined the endosymbiont-host interactions by real-time fluorescence microscopy. Among them, Escherichia coli grew exponentially within the cytoplasm, however, at a faster pace than its host cell. To slow down the intracellular growth of E. coli, we introduced auxotrophies in E. coli and demonstrated that the intracellular growth rate can be reduced by limiting the uptake of aromatic amino acids. In consequence, the survival of the endosymbiont-host pair was prolonged. The presented experimental framework enables studying endosymbiotic candidate systems at high temporal resolution and at the single cell level. Our work represents a starting point for engineering a stable, vertically inherited endosymbiosis.

Live-seq enables temporal transcriptomic recording of single cells.

Single-cell transcriptomics (scRNA-seq) has greatly advanced our ability to characterize cellular heterogeneity1. However, scRNA-seq requires lysing cells, which impedes further molecular or functional analyses on the same cells. Here, we established Live-seq, a single-cell transcriptome profiling approach that preserves cell viability during RNA extraction using fluidic force microscopy2,3, thus allowing to couple a cell's ground-state transcriptome to its downstream molecular or phenotypic behaviour. To benchmark Live-seq, we used cell growth, functional responses and whole-cell transcriptome read-outs to demonstrate that Live-seq can accurately stratify diverse cell types and states without inducing major cellular perturbations. As a proof of concept, we show that Live-seq can be used to directly map a cell's trajectory by sequentially profiling the transcriptomes of individual macrophages before and after lipopolysaccharide (LPS) stimulation, and of adipose stromal cells pre- and post-differentiation. In addition, we demonstrate that Live-seq can function as a transcriptomic recorder by preregistering the transcriptomes of individual macrophages that were subsequently monitored by time-lapse imaging after LPS exposure. This enabled the unsupervised, genome-wide ranking of genes on the basis of their ability to affect macrophage LPS response heterogeneity, revealing basal Nfkbia expression level and cell cycle state as important phenotypic determinants, which we experimentally validated. Thus, Live-seq can address a broad range of biological questions by transforming scRNA-seq from an end-point to a temporal analysis approach.

Dynamic character displacement among a pair of bacterial phyllosphere commensals in situ.

Differences between species promote stable coexistence in a resource-limited environment. These differences can result from interspecies competition leading to character shifts, a process referred to as character displacement. While character displacement is often interpreted as a consequence of genetically fixed trait differences between species, it can also be mediated by phenotypic plasticity in response to the presence of another species. Here, we test whether phenotypic plasticity leads to a shift in proteome allocation during co-occurrence of two bacterial species from the abundant, leaf-colonizing families Sphingomonadaceae and Rhizobiaceae in their natural habitat. Upon mono-colonizing of the phyllosphere, both species exhibit specific and shared protein functions indicating a niche overlap. During co-colonization, quantitative differences in the protein repertoire of both bacterial populations occur as a result of bacterial coexistence in planta. Specifically, the Sphingomonas strain produces enzymes for the metabolization of xylan, while the Rhizobium strain reprograms its metabolism to beta-oxidation of fatty acids fueled via the glyoxylate cycle and adapts its biotin acquisition. We demonstrate the conditional relevance of cross-species facilitation by mutagenesis leading to loss of fitness in competition in planta. Our results show that dynamic character displacement and niche facilitation mediated by phenotypic plasticity can contribute to species coexistence.

Mitochondria transplantation between living cells.

Mitochondria and the complex endomembrane system are hallmarks of eukaryotic cells. To date, it has been difficult to manipulate organelle structures within single live cells. We developed a FluidFM-based approach to extract, inject, and transplant organelles from and into living cells with subcellular spatial resolution. The technology combines atomic force microscopy, optical microscopy, and nanofluidics to achieve force and volume control with real-time inspection. We developed dedicated probes that allow minimally invasive entry into cells and optimized fluid flow to extract specific organelles. When extracting single or a defined number of mitochondria, their morphology transforms into a pearls-on-a-string phenotype due to locally applied fluidic forces. We show that the induced transition is calcium independent and results in isolated, intact mitochondria. Upon cell-to-cell transplantation, the transferred mitochondria fuse to the host cells mitochondrial network. Transplantation of healthy and drug-impaired mitochondria into primary keratinocytes allowed monitoring of mitochondrial subpopulation rescue. Fusion with the mitochondrial network of recipient cells occurred 20 minutes after transplantation and continued for over 16 hours. After transfer of mitochondria and cell propagation over generations, donor mitochondrial DNA (mtDNA) was replicated in recipient cells without the need for selection pressure. The approach opens new prospects for the study of organelle physiology and homeostasis, but also for therapy, mechanobiology, and synthetic biology.

Injection into and extraction from single fungal cells.

The direct delivery of molecules and the sampling of endogenous compounds into and from living cells provide powerful means to modulate and study cellular functions. Intracellular injection and extraction remain challenging for fungal cells that possess a cell wall. The most common methods for intracellular delivery into fungi rely on the initial degradation of the cell wall to generate protoplasts, a step that represents a major bottleneck in terms of time, efficiency, standardization, and cell viability. Here, we show that fluidic force microscopy enables the injection of solutions and cytoplasmic fluid extraction into and out of individual fungal cells, including unicellular model yeasts and multicellular filamentous fungi. The approach is strain- and cargo-independent and opens new opportunities for manipulating and analyzing fungi. We also perturb individual hyphal compartments within intact mycelial networks to study the cellular response at the single cell level.

Modularized CRISPR/dCas9 effector toolkit for target-specific gene regulation.

The ability to control mammalian genes in a synergistic mode using synthetic transcription factors is highly desirable in fields of tissue engineering, stem cell reprogramming and fundamental research. In this study, we developed a standardized toolkit utilizing an engineered CRISPR/Cas9 system that enables customizable gene regulation in mammalian cells. The RNA-guided dCas9 protein was implemented as a programmable transcriptional activator or repressor device, including targeting of endogenous loci. For facile assembly of single or multiple CRISPR RNAs, our toolkit comprises a modular RNAimer plasmid, which encodes the required noncoding RNA components.