Biology subjects

Radutoiu, S.

Publications and source records attributed to Radutoiu, S..

15 recordsLinked to original sources

bioRxiv2026

Identification of Potential Regulatory Non-Coding RNAs in Lotus Japonicus Symbiosis

O_LISymbiosis between legumes and rhizobia is beneficial on nutrient-poor soils, as it enables the fixation of atmospheric N2. To establish this symbiosis, gene expression in both the host plant and the symbiont has to be regulated. To understand the underlying RNA-mediated regulation of host gene expression, we designed experiments to identify competing endogenous networks involving circular RNA, microRNA, and linear transcripts during symbiosis, using wt and symbiosis-deficient Lotus japonicus mutants with the rhizobium Mesorhizobium loti (M. loti). C_LIO_LICircRNA, miRNA, and linear transcripts were identified from Lotus japonicus wildtype and CCamK mutant (ccamk-13; snf-1) seedlings without inoculation or with M. loti inoculation using deep short-read sequencing with rRNA-depletion and random primers. C_LIO_LIDifferentially expressed miRNAs showed negative correlations to predicted target genes and may regulate symbiotic processes. The symbiosis essential iron-sensor LjnsRING/BRUTUS expresses a circRNA which was upregulated in symbiotic treatments. This circRNA may act as a target mimic and contribute to nodule longevity. CircRNAs are predicted to act predominantly as trans-regulatory molecules with similar frequencies in Arabidopsis thaliania, Oryza sativa, and Lotus japonicus. C_LIO_LIWe identified novel miRNAs, long noncoding RNAs, and circRNAs, and nominated several as potential new regulatory non-coding RNAs that may act as target mimics to stabilize genes and support symbiosis. C_LI SummarySymbiosis between Lotus japonicus and Mesorhizobium loti involves treatment-specific regulation of competing endogenous RNA networks involving circular RNA, miRNA, and linear transcripts.

plant biology↗

bioRxiv2026

The common symbiosis pathway controls plant root microbiomes in a host-specific manner

Crop nutrition depends on plant-microbe interactions, yet it remains unclear whether conserved genetic pathways impose universal rules on root microbiome assembly across plant hosts. Here, we show that the Common Symbiosis Signalling Pathway (CSSP), a conserved genetic module controlling endosymbiosis with arbuscular mycorrhizal fungi and nitrogen-fixing bacteria, regulates root microbiome assembly in a host-specific manner across contrasting fertilisation regimes. Using Lotus japonicus and Hordeum vulgare, we demonstrate that mutations in orthologous CSSP genes remodel root bacterial communities in both species, but with distinct taxonomic outcomes. In Lotus, CSSP disruption reduces rhizobial colonisation and promotes niche replacement by commensal taxa, whereas in Hordeum, the same mutations broadly restructure bacterial lineages without converging on Lotus-like responses. Root exudate profiling reveals host-specific metabolic differences, particularly in phenylpropanoid (flavonoids and coumarins) and gibberellin pathways, linking CSSP activity to chemically distinct rhizosphere environments that correlate with divergent microbiome assembly patterns across hosts. Moreover, root bacterial community composition accurately predicts plant nutritional status, highlighting tight coupling between host physiology and microbiome composition. Together, our results show that conserved symbiosis signalling regulates root microbiome assembly, while host-specific metabolic environments determine taxonomic outcomes. This extends CSSP function beyond canonical endosymbioses and positions symbiosis signalling as a general determinant of plant-microbiome interactions with implications for crop nutrition. Significance StatementRoot microbiomes influence plant nutrition, yet how conserved host genetic pathways controlling interactions with intracellular symbionts shape root-associated microbiome assembly across divergent plant species remains unresolved. The Common Symbiosis Signalling Pathway (CSSP), conserved across most land plants, regulates root microbiome composition in both a legume and a cereal, but with distinct taxonomic outcomes. These effects correlate with CSSP-dependent differences in root exudate chemistry and host metabolic profiles. Together, our results show that conserved symbiosis signalling operates within host-specific metabolic contexts, providing a framework for understanding why disruption of the same genetic pathway can lead to divergent microbiome configurations across plant species.

plant biology↗

bioRxiv2025

Circular RNAs in Lotus japonicus Responses to Nutrient Supply and Symbiotic Interactions

Symbiotic relationships, such as those formed between legumes and rhizobia or arbuscular mycorrhizal (AM) fungi, function by enhancing the nutrient uptake into the plant. The establishment and coordination of the symbiotic interactions requires changes in gene expression in the host and microbe. Circular RNAs (circRNAs) can function through sponging of microRNAs (miRNAs), resulting in changes in transcript abundances. We identified 15,252 unique nuclear circRNAs in Lotus japonicus under different nutrient conditions and symbiotic interactions with rhizobia or AMF. Our results revealed treatment-specific circRNAs and circRNAs originating from key genes in the Common Symbiosis Pathway, suggesting their potential role in the establishment of these symbioses. We validated select circRNAs potentially involved in the regulation of symbiosis and predicted miRNA recognition elements (MREs) that were only created by the backsplice junction of circRNAs. Backsplice-generated MREs represent an additional mechanism through which circRNAs may modulate abundances and translation of mRNAs. Our sequencing approach using random hexamer primers also enabled us to simultaneously characterize the transcriptome of the symbionts and host.

plant biology↗

bioRxiv2025

SyFi: generating and using sequence fingerprints to distinguish SynCom isolates

The plant root microbiome is a complex community shaped by interactions among bacteria, the plant host, and the environment. Synthetic community (SynCom) experiments help disentangle these interactions by inoculating host plants with a representative set of culturable microbial isolates from the natural root microbiome. Studying these simplified communities provides valuable insights into microbiome assembly and function. However, as SynComs become increasingly complex to better represent natural communities, bioinformatics challenges arise. Specifically, accurately identifying, and quantifying SynCom members based on, for example, 16S rRNA amplicon sequencing becomes more difficult due to the high similarity of the target amplicon, limiting downstream interpretations. Here, we present SynCom Fingerprinting (SyFi), a bioinformatics workflow designed to improve the resolution and accuracy of SynCom member identification. SyFi consists of three modules: the first module constructs a genomic fingerprint for each SynCom member based on its genome sequence, accounting for both copy number and sequence variation in the target gene. The second module then extracts a specific region from this genomic fingerprint to create a secondary fingerprint focused on the target amplicon. The third module uses these fingerprints as a reference to perform pseudoalignment-based quantification of SynCom member abundance from amplicon sequencing reads. We demonstrate that SyFi outperforms standard amplicon analysis by leveraging natural intragenomic variation, enabling more precise differentiation of closely related SynCom members. As a result, SyFi enhances the reliability of microbiome experiments using complex SynComs, which more accurately reflect natural communities. This improved resolution is essential for advancing our understanding of the root microbiome and its impact on plant health and productivity in agricultural and ecological settings. SyFi is available at https://github.com/adriangeerre/SyFi. Impact statementSyFi represents a significant advancement in microbiome research by enhancing the accuracy and resolution of synthetic community (SynCom) member identification. By leveraging natural intragenomic variation, SyFi improves the differentiation of closely related microbial strains, addressing a key challenge in amplicon-based sequencing analysis. This increased precision allows researchers to more reliably track microbial dynamics in complex SynCom experiments, leading to deeper insights into microbiome assembly, function, and host-microbe interactions. As a result, SyFi strengthens the interpretability of microbiome studies, ultimately contributing to a better understanding of plant health and productivity in both agricultural and ecological contexts. Data SummaryThe data reported in this article have been deposited in the National Center for Biotechnology Information Short Read Archive BioProject database. SyFi fingerprint generation was run on a collection of 737 human gut-derived bacterial genomes from Forster et al. (2019) (Genomic read data deposited in the ENA under project numbers ERP105624 and ERP012217) and 447 Arabidopsis-derived bacterial genomes (Selten et al., 2024b) (NCBI Project numbers PRJNA1138681, PRJNA1139421 (Genomes), and PRJNA1131834 (Genomic reads)). A list of the closed genomes used for SyFi validation can be found at https://github.com/adriangeerre/SyFi. Subsequently, SyFi was validated on a complex SynCom dataset by pseudoaligning 16S rRNA V3-V4 and V5-V7 amplicon reads (PRJNA1191388) to SyFi-generated fingerprints and comparing this to shotgun metagenomics-sequenced dataset of the same samples in Selten et al. (2024a) (PRJNA1131994). This complex SynCom dataset included the inoculation of the 447 bacterial isolates on Arabidopsis, Barley, and Lotus roots.

genomics↗

bioRxiv2025

Complete genomes of 152 root commensal bacteria from the model legume Lotus japonicus

Bacterial culture collections represent a valuable tool for mechanistic understanding of microbiome assemblies and are increasingly used to assemble tailored synthetic communities to characterize their microbe-microbe interactions and those with the environment. Given the size of these collections, short-read sequencing is primarily used to capture the encoded genetic information. Whilst sufficient for many microbiome studies, this approach is not amenable for understanding bacterial genome evolution or detailed genetic analyses at the entire genome level. Here we report the assembly of 152 full bacterial genomes from the Lj-SPHERE, the Lotus japonicus collection of root commensals. We performed long-read sequencing using Oxford Nanopore technology and used this together with pre-existing Illumina sequences to de novo assemble these into high quality genomes with improved contiguity and quality. These genomes now provide a solid platform for detailed, mechanistic understanding of microbiome assembly, dynamics and evolution in plants.

genomics↗

bioRxiv2024

The Medicago truncatula LYR4 intracellular domain serves as a scaffold in immunity signaling independent of its phosphorylation activity

Plants perceive and respond to chitin derived from fungal cell walls through lysine motif (LysM) receptor kinases. In the model legume Medicago truncatula, CERK1 and LYR4 represent the LysM receptor pair important for chitin-triggered immunity signaling. Here, we show that both the active kinase receptor CERK1 and the pseudokinase receptor LYR4 contribute to immunity signaling, leading to the production of reactive oxygen species (ROS). We determine the crystal structure of the LYR4 core intracellular domain with a bound nucleotide analog in the active site. Biochemical characterization shows that LYR4 binds ATP and has both autophosphorylation as well as transphosphorylation activity towards CERK1. However, in planta experiments demonstrate that the phosphorylation ability is not necessary for the function of LYR4 in chitin-triggered ROS production, but that the presence of its intracellular domain is indispensable. Together, we show that in chitin-triggered immunity the intracellular domain of LYR4 serves as a signaling scaffold independent of its catalytic activity.

plant biology↗

bioRxiv2024

Two residues reprogram immunity receptor kinases to signal in nitrogen-fixing symbiosis

Receptor signalling determines cellular responses and is crucial for defining specific biological outcomes. In legume root cells, highly similar and structurally conserved chitin and Nod factor receptor kinases activate immune or symbiotic pathways, respectively, upon perception of chitinous ligands. Here, we show that specific amino acid residues in the intracellular part of the Nod factor receptor NFR1 determine signalling specificity and enable the distinction between immune and symbiotic responses. Functional investigation of CERK6, NFR1 and receptor variants hereof revealed a conserved motif that we term Symbiosis Determinant 1 in the juxtamembrane region of the kinase domain that is key for symbiotic signalling. We demonstrate that two residues in Symbiosis Determinant 1 are indispensable hallmarks for NFR1-type receptors and are sufficient to convert Lotus CERK6 and barley RLK4 kinase outputs to enable symbiotic signalling in Lotus japonicus.

plant biology↗

bioRxiv2024

Functional capacities drive recruitment of bacteria into plant root microbiota

Host-associated microbiota follow predictable assembly patterns but show significant variation at the bacterial isolate level depending on the host and environmental context. This variability poses challenges for studying, predicting, and engineering microbiomes. Here we examined how Arabidopsis, Barley, and Lotus plants recruit specific bacteria from highly complex synthetic communities (SynComs) composed of hundreds of bacterial isolates originating from these plants when grown in natural soil. We discovered that, despite their taxonomic diversity, bacteria enriched by these three plant species encode largely overlapping functions. A set of 266 functions common among all host-associated communities was identified at the foundation of the microbiotas functional potential. Analysis of the differences observed between root-associated communities revealed that functions recruited by Arabidopsis and Barley were primarily driven by the SynCom composition, while Lotus selected fewer isolates but with more diverse functionalities, akin to a Swiss army knife strategy. We analysed the variation at the functional level and found this can be explained by the combined functions of bacteria at the family level. Additionally, across major taxa, the isolates covering a broader range of their familys functional diversity achieved higher relative abundance in the root communities. Our work sheds light on key functions and principles guiding the recruitment of bacterial isolates into root microbiota, offering valuable insights for microbiome engineering and inoculant discovery at a previously inaccessible taxonomic level.

plant biology↗

bioRxiv2023

Benchmarking long-read sequencing strategies for obtaining ASV-resolved rRNA operons from environmental microeukaryotes

The use of short-read metabarcoding for classifying microeukaryotes is challenged by the lack of comprehensive 18S rRNA reference databases. While recent advances in high-throughput long-read sequencing provide the potential to greatly increase the phylogenetic coverage of these databases, the performance of different sequencing technologies and subsequent bioinformatics processing remain to be evaluated, primarily because of the absence of well-defined eukaryotic mock communities. To address this challenge, we created a eukaryotic rRNA operons clone-library and turned it into a precisely defined synthetic eukaryotic mock community. This mock community was then used to evaluate the performance of three long-read sequencing techniques (PacBio HiFi, and Nanopore UMI with/without clonal pre-amplification) and three tools for resolving amplicons sequence variants (ASVs) (Uchime3, Unoise3, and DADA2). We investigated the sensitivity of the sequencing techniques based on the number of detected mock taxa, and the accuracy of the different ASV-calling tools with a specific focus on the presence of chimera among the final rRNA operon ASVs. Based on our findings, we provide recommendations and best practice protocols for how to cost-effectively obtain essential error-free rRNA operons in high-throughput. An agricultural soil sample was used to demonstrate that the sequencing and bioinformatic results from the mock community also translates to highly diverse natural samples.

microbiology↗

bioRxiv2023

Nitrogen source and Nod factor signaling map out the assemblies of Lotus japonicus root bacterial communities

Symbiosis with soil-dwelling bacteria that fix atmospheric nitrogen allows legume plants to grow in nitrogen-depleted soil. Symbiosis impacts the assembly of root microbiota, but it is not known how this process takes place and whether it is independent of nitrogen nutrition. We use plant and bacterial mutants to address the role of Nod factor signaling on Lotus japonicus root microbiota assembly. We found that Nod factors are produced by symbionts to activate Nod factor signaling in the host, and this modulates the assembly of a symbiotic root microbiota. Lotus plants grown in symbiosis-permissive or suppressive soils delineated three nitrogen-dependent nutritional states: starved, symbiotic, or inorganic. We found that root and rhizosphere microbiomes associated with these states differ in composition and connectivity, demonstrating that symbiosis and inorganic nitrogen impact the legume root microbiota differently. Finally, we demonstrated that selected bacterial genera delineating state-dependent microbiomes have a high level of accurate prediction.

plant biology↗

bioRxiv2023

Compositionally aware estimation of cross-correlations for microbiome data

In the field of microbiome studies, it is of interest to infer correlations between abundances of different microbes (here referred to as operational taxonomic units, OTUs). Several methods taking the compositional nature of the sequencing data into account exist. However, these methods cannot infer correlations between OTU abundances and other variables. In this paper we introduce the methods SparCEV (Sparse Correlations with External Variables) and SparXCC (Sparse Cross-Correlations between Compositional data) for quantifying correlations between OTU abundances and either continuous phenotypic variables or components of other compositional datasets, such as transcriptomic data. We compare these new methods to empirical Pearson cross-correlations after applying naive transformations of the data (log and log-TSS). Additionally, we test the centered log ratio transformation (CLR) and the variance stabilising transformation (VST). We find that CLR and VST outperform naive transformations, except when the correlation matrix is dense. For large numbers of OTUs, SparCEV and SparXCC perform similarly to CLR and VST. SparCEV outperforms all other tested methods when the number of OTUs is small (less than 100). SparXCC outperforms all tested methods when at least one of the compositional datasets has few variables (less than 50), and more so when both datasets have few variables. Author summarySequencing data of the microbiome posses a unique and challenging structure that renders many standard statistical tools invalid. Features such as compositionality and sparsity complicates statistical analysis, and as a result, specialized tools are needed. Practitioners have long been interested in the construction of correlation networks within the microbiome, and several methods for accomplishing this exist. However, less attention has been paid to the estimation of cross-correlations between microbial abundances and other variables (such as gene expression data or environmental and phenotypic variables). Here, we introduce novel approaches, SparCEV and SparXCC, for inferring such cross-correlations, and compare these to transformation-based approaches, namely log, log-TSS, CLR and VST. In some cases, SparCEV and SparXCC yield superior results, while in other cases, a simpler transformation-based approach suffices. The methods are used to study cross-correlations between bacterial abundances in the skin microbiome and the severity of atopic dermatitis, as well as cross-correlations between fungal and bacterial OTUs in the root microbiome of the legume Lotus japonicus.

bioinformatics↗

bioRxiv2021

Host preference and invasiveness of commensals in the Lotus and Arabidopsis root microbiota

Healthy plants are colonized by microorganisms from the surrounding environment, which form stable communities and provide beneficial services to the host. Culture-independent profiling of the bacterial root microbiota shows that different plant species are colonized by distinct bacterial communities, even if they share the same habitat. It is, however, not known to what extent the host actively selects these communities and whether commensal bacteria are adapted to a specific plant species. Here, we report a sequence-indexed culture collection from roots and nodules of the model legume Lotus japonicus that contains representatives from the majority of species found in culture-independent profiles. Using taxonomically paired synthetic communities from L. japonicus and the crucifer Arabidopsis thaliana in a multi-species gnotobiotic system, we detect clear signatures of host preference among commensal bacteria in a community context, but not in mono-associations. Sequential inoculation of either host reveals strong priority effects during the assembly of the root microbiota, where established communities are resilient to invasion by late-comers. However, we found that host preference by commensal bacteria confers a competitive advantage in their native host. We reveal that host preference is prevalent in commensal bacteria from diverse taxonomic groups and that this trait is directly linked to their invasiveness into standing root-associated communities.

plant biology↗

bioRxiv2020

Identity and provenance of neighbors, genotype-specific traits and abiotic stress affect intraspecific interactions in the annual legume Medicago truncatula

O_LIAccounting for intraspecific variation may improve our understanding of species coexistence. However, our knowledge of what factors maintain intraspecific variation is limited. We predicted that 1) a plant grows larger when with non-kin (i.e. different genotypes) than kin (i.e. same genotype) neighbors, 2) abiotic stress alters the outcome of kin vs. non-kin interactions, 3) genetic identity of plants affects composition of soil microbiome. C_LIO_LIWe set up mini-communities of Medicago truncatula, where focal genotypes were grown together with two kin or two non-kin neighbors from different origins. We analyzed how origin, identity of interacting genotypes and abiotic stress affected growth and fruit production. We also analyzed the composition of soil microbial communities. C_LIO_LIFocal plants grew larger in non-kin than in kin mini-communities. This pattern was stronger in low level of abiotic stress and when interacting genotypes were from similar origins. However, genotypic variation in growth and response to competition had a stronger effect on growth than mini-community type. Plant genotype identity did not affect soil microbiome. C_LIO_LIWe find that intraspecific variation is affected by genotype-specific traits and abiotic stress. Geographic, rather than genetic, distance among interacting genotypes affects the outcome of intraspecific interactions. C_LI

plant biology↗

bioRxiv2019

Lotus japonicus symbiosis signaling genes and their role in the establishment of root-associated bacterial and fungal communities

The wild legume Lotus japonicus engages in mutualistic symbiotic relationships with arbuscular mycorrhiza (AM) fungi and nitrogen-fixing rhizobia. Using plants grown in natural soil and community profiling of bacterial 16S rRNA genes and fungal internal transcribed spacers (ITS), we examined here the role of the Lotus symbiosis genes RAM1, NFR5, SYMRK, and CCaMK in structuring bacterial and fungal root-associated communities. We found host genotype-dependent community shifts in the root and rhizosphere compartments that were mainly confined to bacteria in nfr5 or fungi in ram1 mutants, whilst symRK and ccamk plants displayed major changes across both microbial kingdoms. We observed in all AM mutant roots an almost complete depletion of a large number of Glomeromycota taxa that was accompanied by a concomitant enrichment of Helotiales and Nectriaceae fungi, suggesting compensatory niche replacement within the fungal community. A subset of Glomeromycota whose colonization is strictly dependent on the common symbiosis pathway was retained in ram1 mutants, indicating that RAM1 is dispensable for intraradical colonization by some Glomeromycoyta fungi. However, intraradical colonization by certain Burkholderiaceae taxa is dependent on AM root infection, thereby revealing a microbial interkingdom interaction. Our findings imply a broad role for Lotus symbiosis genes in structuring the root microbiota.

plant biology↗

bioRxiv2018

A Lotus japonicus E3 ligase interacts with the Nod factor receptor 5 and positively regulates nodulation

Post-translational modification of receptor proteins is involved in activation and de-activation of signaling systems in plants. Both ubiquitination and deubiquitination have been implicated in plant interactions with pathogens and symbionts. Here we present LjPUB13, a PUB-ARMADILLO repeat E3 ligase that specifically ubiquitinates the kinase domain of the Nod Factor receptor NFR5 and has a direct role in nodule organogenesis events in Lotus japonicus. Phenotypic analyses of three LORE1 retroelement insertion plant lines revealed that pub13 plants display delayed and reduced nodulation capacity and retarded growth. LjPUB13 expression is spatially regulated during symbiosis with Mesorhizobium loti, with increased levels in young developing nodules. Thus, LjPUB13 is an E3 ligase with a positive regulatory role during the initial stages of nodulation in L. japonicus.

plant biology↗