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Quantification of cell growth is central to any study of photoautotrophic microorganisms. However, cellular self-shading and limited CO2 control in conventional photobioreactors lead to heterogeneous conditions that obscure distinct correlations between the environment and cellular physiology. Here we present a microfluidic cultivation platform that enables precise analysis of cyanobacterial growth with spatio-temporal resolution. Since cyanobacteria are cultivated in monolayers, cellular self-shading does not occur, allowing homogeneous illumination and precise knowledge of the photon-flux density at single-cell resolution. A single chip contains multiple channels, each connected to several hundred growth chambers. In combination with an externally applied light gradient, this setup enables high-throughput multi-parameter analysis in short time. In addition, the multilayered microfluidic design allows continuous perfusion of defined gas mixtures. Transversal CO2 diffusion across the intermediate polydimethylsiloxane membrane results in homogeneous CO2 supply, with a unique exchange-surface to cultivation-volume ratio. Three cyanobacterial model strains were examined under various, static and dynamic environmental conditions. Phase-contrast and chlorophyll fluorescence images were recorded by automated time-lapse microscopy. Deep-learning trained cell segmentation was used to efficiently analyse large image stacks, thereby generating statistically reliable data. Cell division was highly synchronized, and growth was robust under continuous illumination but stopped rapidly upon initiating dark phases. CO2-Limitation, often a limiting factor in photobioreactors, was only observed when the device was operated under reduced CO2 between 50 and 0 ppm. Here we provide comprehensive and precise data on cyanobacterial growth at single-cell resolution, accessible for further growth studies and modeling.

Transcript and metabolite changes during the early phase of abscisic acid-mediated induction of crassulacean acid metabolism in Talinum triangulare

Host preference and invasiveness of commensal bacteria in the Lotus and Arabidopsis root microbiota

Fungal pathogens depend on sophisticated gene expression programs for successful infection. A crucial component is RNA regulation mediated by RNA-binding proteins (RBPs). However, little is known about the spatiotemporal RNA control mechanisms during fungal pathogenicity. Here, we discover that the RBP Khd4 defines a distinct mRNA regulon to orchestrate membrane trafficking during pathogenic development of Ustilago maydis. By establishing hyperTRIBE for fungal RBPs, we generated a comprehensive transcriptome-wide map of Khd4 interactions in vivo. We identify a defined set of target mRNAs enriched for regulatory proteins involved, e.g., in GTPase signaling. Khd4 controls the stability of target mRNAs via its cognate regulatory element AUACCC present in their 3′ untranslated regions. Studying individual examples reveals a unique link between Khd4 and vacuole maturation. Thus, we uncover a distinct role for an RNA stability factor defining a specific mRNA regulon for membrane trafficking during pathogenicity.

Microtubule-dependent endosomal transport is crucial for polar growth, ensuring the precise distribution of cellular cargos such as proteins and mRNAs. However, the molecular mechanism linking mRNAs to the endosomal surface remains poorly understood. Here, we present a structural analysis of the key RNA-binding protein Rrm4 from Ustilago maydis. Our findings reveal a different type of MademoiseLLE domain (MLLE) featuring a seven-helical bundle that provides a distinct binding interface. A comparative analysis with the canonical MademoiseLLE domain of the poly(A)-binding protein Pab1 disclosed unique characteristics of both domains. Deciphering the MLLE binding code enabled prediction and verification of previously unknown Rrm4 interactors containing short linear motifs. Importantly, we demonstrated that the human MLLE domains, such as those of PABPC1 and UBR5, employed a similar principle to distinguish among interaction partners. Thus, our study provides detailed mechanistic insights into how structural variations in the widely distributed MLLE domain facilitate mRNA attachment during endosomal transport.

Reversible Burst of Transcriptional Changes during Induction of Crassulacean Acid Metabolism in Talinum triangulare.

Cofunctioning of bacterial exometabolites drives root microbiota establishment

The iron-containing porphyrin heme is of high interest for the food industry for the production of artificial meat as well as for medical applications, e.g. for anemia treatment. Recently, the biotechnological platform strain Corynebacterium glutamicum has emerged as a promising host for animal-free heme production. Beyond engineering of complex heme biosynthetic pathways, improving heme export offers significant yet untapped potential for enhancing production strains. In this study, a growth-coupled biosensor was designed to impose a selection pressure on the increased expression of the hrtBA operon encoding an ABC-type heme exporter in C. glutamicum. For this purpose, the promoter region PhrtB was replaced with that of the growth-regulating genes pfkA (phosphofructokinase) and aceE (pyruvate dehydrogenase), creating biosensor strains with a selection pressure for hrtBA activation. Resulting sensor strains were used for plate-based selections and for a repetitive batch f(luorescent)ALE using a robotics platform. Genome sequencing of isolated clones featuring increased hrtBA expression revealed three distinct mutational hotspots: (i) chrS, (ii) chrA, and (iii) cydD. Mutations in the genes of the ChrSA two-component system, which regulates hrtBA in response to heme levels, were identified as a promising target to enhance export activity. Furthermore, causal mutations within cydD, encoding an ABC-transporter essential for cytochrome bd oxidase assembly, were confirmed by the construction of a deletion mutant, which showed strongly increased hrtBA expression as well as increased cellular heme levels. These results further support the proposed role of CydDC as a heme transporter. Mutations identified in this study therefore underline the potential of biosensor-based growth coupling and provide promising engineering targets to improve microbial heme production.

PPD-H1 Improves Stress Resistance and Energy Metabolism to Boost Spike Fertility under High Ambient Temperatures

Genome sequencing of blackcurrant (Ribes nigrum)

MAdLand Project - Erika Csicsely, Oguz Top, Wolfgang Frank et al.

This ARC accompanies the publication in Plant Journal: https://doi.org/10.1111/tpj.17236

DICER-LIKE (DCL) proteins play a central role in plant small RNA (sRNA) biogenesis. The genome of the early land plant Marchantia polymorpha encodes four DCL proteins: MpDCL1a, MpDCL1b, MpDCL3, and MpDCL4. While MpDCL1a, MpDCL3 and MpDCL4 show high similarities to their orthologs in Physcomitrium patens and Arabidopsis thaliana, MpDCL1b shares only a limited homology with PpDCL1b, but it is very similar, in terms of functional domains, to orthologs in other moss and fern species. We generated Mpdclge mutant lines for all MpDCL genes with the CRISPR/Cas9 system and conducted phenotypic analyses under control, salt stress, and phytohormone treatments to uncover specific MpDCL functions. The mutants displayed severe developmental aberrations, altered responses to salt and phytohormones, and disturbed sexual organ development. By combining mRNA and sRNA analyses, we demonstrate that MpDCLs and their associated sRNAs play pivotal roles in regulating development, abiotic stress tolerance and phytohormone response in M. polymorpha. We identified MpDCL1a in microRNA biogenesis, MpDCL4 in trans-acting small interfering RNA generation, and MpDCL3 in the regulation of pathogen-related genes. Notably, salt sensitivity in M. polymorpha is dependent on MpDCL1b and Mpdcl1bge mutants display enhanced tolerance and reduced miRNA expression in response to salt stress. We propose that M. polymorpha employs specific mechanisms for regulating MpDCL1b associated miRNAs under high salinity conditions, potentially shared with other species harboring MpDCL1b homologs.

Characterization of the FNR-type regulator GoxR

ARC repo for MARS testing

Global expression patterns of R-genes in tomato and potato