RIKEN Center for Sustainable Resource Science

Auxin plays diverse roles in plant growth and development, including sensing environmental changes. Quantifying the interaction between auxin coreceptors provides the molecular basis for cells to sense and adapt to environmental cues. Although several assays are available, a more high-throughput method is necessary to efficiently evaluate the auxin-induced binding of coreceptors. We developed a homogeneous time-resolved fluorescence (HTRF) assay to quantitatively measure the binding between the Arabidopsis thaliana TRANSPORT INHIBITOR RESPONSE 1 (TIR1) and indole-3-acetic acid 7 (IAA7) auxin coreceptor proteins. The HTRF assay provides a rapid analysis with sensitivity similar to the enzyme-linked immunosorbent assay. We demonstrated its effectiveness by analyzing the potency of several auxin analogs to induce binding between TIR1 and IAA7. We also found that a mild increase in temperature impairs the binding activity of TIR1 to IAA7. This method provides a rapid and robust tool to evaluate the auxin-induced binding between TIR1 and Aux/IAA auxin coreceptors. A similar strategy may also be applicable to study other plant hormone heterodimer coreceptors.

Makou disease is a grave disease in the production of Angelica sinensis, seriously affecting this plant's yield and quality. Its typical symptoms include yellowing and necrotic spots on leaves of infected plants, and the belowground symptoms are longitudinal brown cracks (depth is about 1 to 2 mm) in the root epidermis. The symptoms of Makou disease are completely different from those of root rot disease, and the pathogen responsible has yet to be officially reported and is highly controversial surrounding the existing research. Symptomatic roots and infested soils were collected from 18 fields, from which 161 fungal isolates were isolated. Through morphological observation, 105 isolates were taxonomically identified by comparing the sequences of their internal transcribed spacer (ITS) region to those of known species in the NCBI database. Ceratobasidium sp. AG-K was the most abundant species (61.0%), followed by Fusarium spp. (31.4%) and Plectosphaerella cucumerina (5.7%). Phylogenetic analyses based on sequences of ITS of those isolates showed that those belonging to the same species were clearly separated in the derived dendrogram. Two representative isolates, R2 and P3, were selected for the pathogenicity test. Pathogenicity testing revealed Ceratobasidium sp. AG-K was the pathogenic fungus causing Makou disease in A. sinensis seedlings. Additionally, a strain of Bacillus sp. with inhibitory activity against Ceratobasidium sp. AG-K was identified in vitro. The RT-qPCR results showed that the abundance of Ceratobasidium sp. AG-K in the rhizosphere soil of seedlings inoculated with this pathogen reached 10 times that of the control group. Understanding the influence of Ceratobasidium sp. AG-K upon the occurrence and severity of Angelica Makou disease outbreaks will provide key information for devising prevention and control strategies. Furthermore, the detection method we developed can provide future guidance and a sound basis for studying the pathogen's dynamics in the rhizosphere of A. sinensis.

Iron (Fe) is a transition metal necessary for achieving essential physiological processes throughout the plant lifecycle. In Arabidopsis thaliana, secreting Fe-mobilizing coumarins (FMC) is a key mechanism enabling roots to acquire nonbioavailable Fe present in soils. Here, we unveil the pivotal role played by NRT1/PTR FAMILY 7.2 (NPF7.2) in coumarin secretion by conducting phenotypic analyses of NPF7.2 loss-of-function mutants, NPF7.2 expression-localization analyses and direct transport assay of NPF7.2 protein with coumarin compounds using yeast cells. NPF7.2 loss-of-function impairs coumarin secretion and Fe acquisition in Arabidopsis seedlings, and the corresponding mutants are hypersensitive to Fe deficiency. NPF7.2 protein colocalizes with the main coumarin exporter, PDR9 (PLEIOTROPIC DRUG RESISTANCE PROTEIN 9), in both cortex and epidermis cell layers in root areas where coumarin secretion occurs. A site-specific accumulation of fraxin, a storage form of fraxetin, in root peripheral tissues is disturbed in the mutants under Fe deficiency. When expressed in yeast cells, NPF7.2 protein has an uptake activity for fraxetin and scopoletin, a major FMC and its precursor, respectively. However, fraxin and scopolin, their storage form, are not transported. We propose that NPF7.2 facilitates PDR9-mediated FMC secretion from the epidermis into the rhizosphere by the cellular loading of coumarins from apoplastic spaces, leading to optimal Fe acquisition.

Constructed floating wetlands (CFWs) provide a passive pathway for the retention and accumulation of nutrients and perfluoroalkyl and polyfluoroalkyl substances (PFAS) present in treated wastewater (TWW). This two-year field-scale study investigated the accumulation of PFAS and nutrients in three native wetland species in a lagoon setting. During the plant establishment period and despite measurable nutrient and PFAS uptake in plant tissues, their removal from the water was limited due to short residence times (∼10–12 h). The highest nutrient accumulation in shoots was observed in Phragmites australis (0.14 g N/m2/day and 0.02 g P/m2/day), which showed the highest biomass (12.1 g/m2/day). Phragmites australis also exhibited the highest PFAS and precursor accumulation in tissues. PFAS removal improved after plant establishment, and the daily removal rate for PFAS (based on PFAS accumulation in plant tissue and sediment) was 240 ng/m2/d. Overall, CFWs show promise as a passive, modular solution for PFAS reduction in TWW, particularly after root system development and biofilm formation. However, long-term monitoring across multiple growth and harvest cycles and assessing the contribution of other mechanisms (such as sorption to growth media and accumulation in biofilms) are required to evaluate the CFW system sustainability under varying environmental conditions.

The significance of gut microbiota in human health hasgained increasingattention. Accordingly, metabolomics has been used to elucidate host–microbiotainteractions. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)is an ideal choice for metabolome analysis of gut microbiota due toits quantitative capabilities. However, conventional LC-MS/MS requiresmultiple columns, multiple mobile phases, and complex procedures tooptimize conditions for each target metabolite. To address these limitations,we developed a quantitative serial LC-MS/MS method, termed the KobeUniversity Serial LC-MS/MS Analysis using Multiple columns with aSingle mobile phase (KUSLAMS). This platform integrates two columns(PFPP and C18) and a derivatization method for seamless, high-throughputquantification of 215 metabolites, including amino acids, nucleotides,carboxylic acids, amines, and fatty acids. Reproducibility for repeatedanalysis was assessed using 82 intracellular gut microbiota metabolites,for which new analytical methods were developed. Among these, 64 metaboliteswere detected with coefficients of variation (CV) below 15%. The applicationof KUSLAMS to an in vitro gut microbiota culture system with and withoutinulin revealed differences in the concentrations of 21 intracellularand 14 extracellular metabolites. Notably, several metabolites exhibitedincreased intracellular and decreased extracellular concentrations,suggesting a possible link between intracellular accumulation andextracellular depletion, although this interpretation is exploratory.These results indicate that KUSLAMS allows for the simultaneous monitoringof intra- and extracellular metabolite dynamics. Together, these findingsdemonstrate that KUSLAMS is a robust and versatile platform for theexploration of microbiota-derived metabolites relevant to human health.

ABSTRACT Precise control over protein self‐assembly is central to the design of functional and adaptive biomaterials. Recombinant non‐canonical collagens offer a biosafe and sustainable alternative to animal‐derived collagens, with superior tunability and reduced batch‐to‐batch variability. However, their assembly mechanisms remain poorly understood, particularly due to the absence of hydroxyproline residues that stabilize canonical collagens. In this work, we investigate the self‐assembly of a recombinantly produced collagen‐like silk, elucidating the process from the molecular to the material level. Circular dichroism spectroscopy confirms the formation of collagen triple helices, whose thermal stability is enhanced by non‐collagenous domains. Furthermore, the terminal domains facilitate liquid–liquid phase separation (LLPS), which is essential for fiber pulling. Solution‐state NMR relaxation analysis reveals increased rigidity of the C‐terminal domain, likely arising from enhanced hydrophobic interactions that restrict conformational dynamics and thereby contribute to the molecular mechanism underlying liquid–liquid phase separation. We further demonstrate that recombinant collagen‐like silk forms diverse material architectures, including fibers and films. Remarkably, the assembly is reversible, as evidenced by water‐assisted self‐healing behavior. These findings highlight the potential of recombinant non‐canonical collagens as an adaptable biomaterial platform and provide new insights into molecular mechanisms governing collagen self‐assembly.

Abstract Soils harbour immense biosynthetic gene cluster (BGC) diversity that can mediate microbial interactions, yet this potential is still mapped unevenly across the tree of life. Ktedonobacteria —a class of actinomycete-like bacteria within phylum Chloroflexota —are widespread in terrestrial environments and repeatedly dominate pioneer communities in extremely oligotrophic volcanic bare-ground soils; however, their secondary metabolism and genome architecture remain poorly characterised. Here, we integrate targeted cultivation using volcanic soils from Mount Zao with genome-resolved metagenomics and public genomes to analyse 183 ktedonobacterial genomes. Using antiSMASH and BiG-SLiCE, we identified 1,546 BGCs comprising 1,162 non-redundant gene-cluster families (GCFs). In our dataset, nearly one quarter of genomes encode ≥10 distinct GCFs, and several family-level clades show mean GCF counts comparable to those in genus Streptomyces . Most ktedonobacterial BGCs are highly divergent from reference collections and exhibit unusually low intra-genomic redundancy, suggesting broad, underexplored chemotypes. Long-read assemblies from ten strains reveal recurrent 1.6–3.5 Mb chromid-like secondary replicons with chromosome-like composition but distinct maintenance signatures. These replicons are consistently enriched in BGCs and mobility-associated genes, with mobility loci concentrated near BGC boundaries. Collectively, our results expand the current knowledge of the phylogenetic landscape of soil biosynthetic diversity and highlight chromid-like secondary replicons as major genomic reservoirs for specialised metabolism in Ktedonobacteria .

Aminoacyl-tRNAs, charged by aminoacyl-tRNA synthetases with cognate amino acids, are essential for protein synthesis in primary metabolism. Beyond this canonical role, increasing evidence highlights their involvement in natural product biosynthesis. In this review, we first describe the biosynthesis of the aminoacyl nucleoside sulfamate ascamycin from Streptomyces sp. 80H647, highlighting the discovery of the alanyl-tRNA synthetase-like enzyme AcmF through an AI-driven "Forecasting Biosynthesis" approach. Leveraging recent advances in AlphaFold 3, we constructed complex models of a broadened repertoire of aminoacyl-tRNA-dependent enzymes to provide preliminary structure-function insights. These include the isoleucyl-tRNA synthetase-like enzyme SbzA, Gcn5-related N-acetyltransferase-fold transferases, cyclodipeptide synthase family enzymes, and lantibiotic dehydratase-like peptide aminoacyl-tRNA ligases. The catalytic mechanisms of these aminoacyl-tRNA-dependent enzymes are summarized in detail in this review.

Deciphering protein-protein interactions (PPIs) is crucial for a comprehensive understanding of biological processes, yet current methodologies often provide incomplete interactome maps. Here, we present a sensitive bimolecular NanoBiT-based protein complementation assay platform for robust PPI detection in the fission yeast Schizosaccharomyces pombe. Our platform utilizes two NanoLuc moieties, SmBiT and LgBiT, fused to interacting protein partners, generating a quantifiable luminescent signal upon interaction. To maximize the chances of detection and mitigate potential issues arising from tag position-dependent inactivation of bait proteins, our system enables simultaneous expression of two distinct bait constructs within a single cell: one with the LgBiT-tag fused at its N-terminus and another at its C-terminus. For the prey, we constructed a comprehensive ORFeome library of fission yeast proteins, each fused with SmBiT at its C-terminus, leveraging homologous recombination tools. We established efficient high-throughput methods for cloning and selection of single-copy integrants, enabling the rapid construction of the prey library and reliable identification of true yeast transformants. Validating the platform, high-throughput screening using the general transcription elongation factor Tfs1 successfully identified previously undetectable interactors. This versatile platform not only significantly expands the scope of interactome discovery but also offers a powerful tool for future protein-compound interaction studies.