Institut Jean-Pierre Bourgin

ABSTRACTSeed immune responses are an underexplored area in host–pathogen interactions, leaving seed–pathogen interactions poorly understood despite their considerable economic impact. This study examined tomato seed defences by assessing the antimicrobial activity (AA) of seed exudates during germination. Results showed genotype‐dependent and constitutive defence responses from seeds showing AA in exudates. Seed priming with a panel of elicitors such as methyl jasmonate (MeJA) enhanced AA in certain genotypes, highlighting an inducible defence response. Both constitutive and inducible (elicitor‐dependent) seed defences were genotype‐dependent and more effective against the non‐host pathogen Alternaria brassicicola (Abra43), while host pathogens seemed resistant to exudates' AA, suggesting that they developed strategies to neutralise exudates' AA. Multi‐omic analyses revealed distinct hormonal and molecular pathways involved in constitutive and inducible defences. By characterising exudates and correlating genotype‐ and elicitor‐specific AA, candidate antimicrobial compounds were identified. As proof of concept, we functionally validated the AA of a putative defensin (Solyc07g007755) whose expression was highly correlated with the observed AA of the seed exudate against Abra43, demonstrating the potential of our dataset for the development of phytosanitary strategies to protect seeds during germination.

ABSTRACT Nitrogen is essential for plant growth, yet its availability often limits agricultural productivity. Some legumes have evolved a unique ability to form symbiotic relationships with nitrogen-fixing soil bacteria called rhizobia, enabling them to thrive in nitrogen-deficient soils. In five legume clades, an exploitive strategy has evolved in which rhizobia undergo Terminal Bacteroid Differentiation (TBD), where the bacteria become larger, polyploid, and have a permeabilized membrane. Terminally differentiated bacteria are associated with higher N 2 -fixation and, thus, a higher return on investment to the plant. In several members of the IRLC (Inverted Repeat-Lacking Clade) and the Dalbergioid clades of legumes, this differentiation process is triggered by a set of apparently unrelated plant antimicrobial peptides with membrane-damaging activity, known as Nodule-specific Cysteine-Rich (NCR) peptides. However, whether NCR peptides are also implicated in symbiotic TBD in other legume clades and whether they are evolutionarily related remains unknown. Here, to address the molecular identity of NCR peptides and their evolution in different legume clades, we performed inter- and intra-clade comparisons of NCR peptides in representative species of four TBD-inducing legume clades. First, we collected genomic and proteomic data of species for which NCR peptides are known (1523 NCR peptides). We then used sequence similarity-based clustering to regroup the NCR peptides, resulting in over 400 different NCR clusters, each clade-specific. We obtained Hidden Markov Models for each cluster and used them to predict NCR peptides in 21 legume genomes (6 clades), including newly generated deep-sequenced root and nodule RNA-seq data of Indigofera argentea (Indigoferoid clade) and newly assembled high-quality transcriptomes of Lupinus luteus and Lupinus mariae-josephae (Genistoid clade), using tailored gene prediction pipeline and transcriptome matching. This resulted in 3710 NCR peptides in species that induce TBD. To date, the rapid diversification of NCR peptides that reduces the sequence similarities has masked the origin of NCR peptide evolution. We obtained high-confidence structural models for one sequence of each cluster. We performed structure-based clustering and phylogenetics, which resulted in 23 superclusters (14 inter-clade and nine clade-specific) that we represent in a structural distance-based tree. Our study revealed that the evolution of NCR peptides is a mix of divergent and convergent processes within each clade. We further chose nine independently evolved NCR peptides to test in vitro whether they are functional analogs in symbiosis. Graphical abstract Overview of the experimental and computational workflow for NCR peptide detection, characterization, and structural analysis. Nodule and root samples from Indigofera argentea (8 weeks post-inoculation) were collected and subjected to RNA extraction, library preparation, and Illumina PE150 sequencing. Raw RNA-seq reads from two Lupinus species were also included ( Lupinus luteus and Lupinus mariae-josephae) . Bacteroid differentiation of I. argentea was assessed by flow cytometry and confocal microscopy. Transcriptomes were assembled de novo and analyzed for differential gene expression between root and nodule tissues. NCR peptides were identified from them and other legume genomes and transcriptomes using the SPADA pipeline and HMM profiles from NCR clusters of the known NCR peptides. The putative NCR peptides were filtered based on conserved cysteine motifs, length, and nodule expression to build an exhaustive NCR peptide database. 3D structural predictions of NCR clusters were performed using AlphaFold2 (pLDDT >70), followed by structural clustering (Foldseek) and phylogenetic analysis (Foldtree). Functional validation involved flow cytometry and antimicrobial assays (against Eschericha coli , Sinorhizobium meliloti , and Bacillus subtilis ), enabling structural and evolutionary characterization of NCR peptides. The green box at the top represents the experimental analysis, the blue box represents the sequence-based computational pipeline, the red box represents the structure-based computational pipeline, and the grey box at the bottom left represents the functional validation and interpretation of the results.

Factors controlling plant photosynthesis and primary production include parameters dictating photosynthetic activity together with export and allocation properties. In fact, phloem loading activity as well as phloem sap redistribution and velocity are

Abstract Interindividual epigenetic variability, particularly in DNA methylation, is now recognized as a significant contributor to phenotypic diversity in humans and mammals. These epivariable regions, which make up a small fraction of the genome, are strongly influenced by genetic factors and environmental factors, especially during early development. In this context, epigenetic variability of DNA methylation has been proposed as an adaptive force involved in various environmental responses. In fish and other vertebrates, environmental factors are known to influence the health, performance and welfare, likely through the alteration of the epigenetic landscape. However, whether interindividual epigenetic variability may contribute to the phenotypic plasticity of fishes is unknown. Here we provide a first description of the rainbow trout methylome variability using a whole-genome bisulfite sequencing approach in an isogenic line to minimize genetic variation. Variable methylation regions were identified in both liver and hypothalamus tissues of 12 replicate fishes and were found enriched at gene regulatory elements, such as promoters and first introns. Gene Ontology analysis revealed functional clusters related to cellular development, neural communication, metabolic balance, and immune response. Interestingly, some variably methylated regions are found at the same genomic loci in both tissues and showed a strong intraindividual correlation in methylation levels, suggesting establishment during early embryogenesis. Overall, our work demonstrates the existence of interindividual epigenetic variability in rainbow trout and provides valuable insights into the regulatory function of DNA methylation variation that is likely involved in developmental and physiological processes.

The unfolded protein response (UPR) is a signalling pathway activated when endoplasmic reticulum (ER) proteostasis is disturbed. We investigated the contribution of the UPR in the Arabidopsis thaliana response to two necrotrophic fungi, Botrytis cinerea and Alternaria brassicicola. The IRE1-bZIP60 branch of the UPR was specifically activated upon infection with both pathogenic fungi, as evidenced by the production of active bZIP60 transcription factor forms and the increased expression of UPR-responsive genes. We also demonstrated using reverse genetics that the IRE1-bzIP60 axis was necessary to restrict foliar necrotic symptoms induced by both fungi. Furthermore, mutants deficient in two ER quality control components were more susceptible to infection by B. cinerea. By contrast, investigating the involvement of CDC48, an AAA+ ATPAse that assists the ER-associated degradation pathway, we showed that a series of mutants and transgenics are more resistant to B. cinerea. To gain molecular insights into how the ER shapes the Arabidopsis immune response to B. cinerea, we quantified defence gene and cell death marker expression in bzip60 single and ire1 double mutants. None of these genes were misregulated in mutant backgrounds, indicating that the IRE1-bZIP60 branch of the UPR modulates the Arabidopsis response to B. cinerea by a yet-to-be-identified mechanism. Interestingly, we identified NAC053 as a potential actor of this unknown mechanism.

ABSTRACTAn efficient nitrate uptake system contributes to the improvement of crop nitrogen use efficiency under low nitrogen availability. The High Affinity nitrate Transport System (HATS) in plants is active in low range of external nitrate and is mediated by a two‐component system (high affinity transporters NRT2 associated to a partner protein NRT3 (NAR2)). In Brachypodium, the model plant for C3 cereals, we investigated the role of BdNRT2A and BdNRT3.2 through various experimental approaches. Expression profile of BdNRT2.A and BdNRT3.2 genes in response to nitrate availability fits perfectly with the characteristics of the HATS components. 15Nitrate influx measurements decreased in bdnrt2a mutants (one NaN3 induced mutant with a truncated NRT2A protein and two amiRNA mutants). In addition, the N limited phenotype of the mutant with a truncated NRT2A protein confirmed that BdNRT2A is a major contributor of the HATS in Brachypodium. An effective nitrate transport in the heterologous expression system Xenopus oocytes required the coexpression of BdNRT2A and BdNRT3.2 that characterizes two‐component system of the HATS. Functional interaction between BdNRT2A‐GFP and BdNRT3.2‐RFP fusion proteins was observed at the plasma membrane in Arabidopsis protoplasts in transient expression experiments with BdNRT3.2 being necessary for the plasma membrane localization of BdNRT2A. The role of a conserved Ser residue in BdNRT2A (S461) specific to monocotyledons was evaluated in the BdNRT2A and BdNRT3.2 interaction leading to plasma membrane targeting. Assuming that S461 could be regulated by phosphorylation, a directed mutagenesis was performed to mimic a nonphosphorylated (S461A) or a constitutively phosphorylated (S461D), However, the mimicking the phosphorylation status of S461 by mutagenesis did not modify the BdNRT2A and BdNRT3.2 interaction, suggesting a more complex regulating mechanism. In conclusion, our data show that BdNRT2A and BdNRT3.2 are the main components of the nitrate HATS activity in Brachypodium (Bd21‐3) and allow an optimal growth in low N conditions.

Cadmium (Cd) is a major environmental pollutant with high toxicity. While Cd exposure reduces root growth, its specific impact on the root meristem and differentiating parts remains poorly understood. This study investigates the spatial and temporal responses of Arabidopsis thaliana roots to Cd stress by dividing roots into root tips (RT) and remaining roots (RR) and employing transcriptomic, ionomic, and metabolomic analyses. Cd exposure altered mineral profiles, with RT accumulating less Cd but showing distinct changes in other elements compared to RR. Metabolomic analysis revealed root part-specific changes in phytochelatins, flavonoids, and glucosinolates. Transcriptomic data highlighted constitutive differences between RT and RR, reflecting functional specialization. Also, they revealed Cd-induced root part-specific and time-dependent transcriptional responses, including modulation of Fe-related genes. Phenotypic validation identified ELONGATED HYPOCOTYL 5 as a key regulator limiting Cd accumulation and promoting tolerance, as hy5 mutants exhibited increased Cd sensitivity and accumulation. Additionally, mutants of genes regulated by HY5, such as xyloglucan endotransglucosylase/hydrolase genes (XTH) and MYB12, also showed altered root growth under Cd stress, implicating cell wall remodeling and flavonoid biosynthesis in Cd responses. This study provides a spatially and temporally resolved understanding of Cd's impact on root growth, and highlights HY5's role in Cd tolerance, thereby advancing our knowledge of plant responses to trace metal excess.

Antimicrobial peptides (AMPs) are often produced by eukaryotes to control bacterial populations in both pathogenic and mutualistic symbioses. Several pathogens and nitrogen-fixing legume symbionts depend on transporters called SbmA (or BacA) or BclA (BacA-like) to survive exposure to AMPs. However, how broadly these transporters are distributed amongst bacteria, and their evolutionary history, is poorly understood. We used computational approaches, including phylogenetic and sequence similarity analyses, to examine the distribution of SbmA/BacA and BclA proteins across 1,255 species spanning the domain Bacteria, leading to the identification of 71 and 177 SbmA/BacA and BclA proteins, respectively. In vitro sensitivity assays using legume AMPs and several BclA proteins confirmed that AMP transport is a common feature of BclA homologues. Our analyses indicated that SbmA/BacA homologues are encoded only by species in the phylum Pseudomonadota and are primarily found in just two orders: Hyphomicrobiales and Enterobacterales. BclA homologues are somewhat more broadly distributed and were found in clusters across four phyla. These included several orders of the phyla Pseudomonadota and Cyanobacteriota, the order Mycobacteriales (phylum Actinomycetota) and the class Negativicutes (phylum Bacillota). Many of the clades enriched for species encoding SbmA/BacA or BclA homologues are rich in species that interact with eukaryotic hosts in mutualistic or pathogenic interactions. These observations suggest that SbmA/BacA and BclA proteins have been repeatedly co-opted to facilitate associations with eukaryotic hosts by allowing bacteria to cope with host-encoded AMPs.