dataesr/scientific-paragraphs-categorization huggingface dataset:
Data availability All RNA-seq data reported in this paper are deposited into the NCBI Gene Expression Omnibus database. The accession number is GSE203051. The telogen melanocyte dataset, which was previously deposited, has the identifier GSE113502. The mm10/GRCm38 reference genome (mm10-2020-A) utilized in the scRNA-seq analysis is available at https://support. 10xgenomics. com/single-cell-gene-expression/ software/release-notes/build#mm10_2020A. The GOBP_DENDRITE_ DEVELOPMENT dataset used in the GSEA analysis was downloaded from the MSigDB (http://www. gsea-msigdb. org/gsea/msigdb/index. jsp) at http://www. gsea-msigdb. org/gsea/msigdb/mouse/geneset/ GOBP_DENDRITE_DEVELOPMENT. html. The GOBP_DENDRITE_MOR-PHOGENESIS dataset used in the GSEA analysis was downloaded from the MSigDB at http://www.
Code availability All the bioinformatics analyses were performed using the following previously published packages with publicly available codes: Cell Ranger Single-Cell software (https://support. 10xgenomics. com/ single-cell-gene-expression/software/pipelines/latest/what-is-cellranger); Seurat (https://satijalab. org/seurat/); slingshot (https://bioconductor. org/packages/devel/bioc/vignettes/slingshot/inst/doc/ vignette. html); and GSEA Broad Institute (https://software. broadinstitute. org/gsea/index. jsp).
1AE200 strain (Ledermann and Couzigou, unpublished). L. albus forms peculiar lupinoid nodules  30  and its genome has been recently sequenced  31. In brief, we identified 3,976/4,944 (up/ down) differentially regulated genes in mature nodules in response to Bradyrhizobium sp. 1AE200 compared to non-inoculated roots (Supplementary Tables 1, 3 and 4). These differentially expressed genes (DEGs) represent around 33% of the 26,204 L. albus expressed genes. To obtain comparable datasets, raw RNA-seq reads obtained in the presence or absence of their respective bacterial symbionts from seven other nodulating species (Supplementary Table  1 ) were remapped on their respective genomes, and differentially expressed genes were computed following the same approach as for M. pudica and L. albus. Due to sampling and sequencing depth heterogeneity among species, we used different fold-change thresholds to obtain comparable numbers of differentially expressed genes (see Methods).
Identification of an ancestral RNS transcriptomic signature The two largest groups of RNS-forming species, the Papilionoideae subfamily and the Mimosoid clade which is nested in the largely non-nodulating Caesalpinioideae subfamily  17, 22  , belong to the Fabales order. While transcriptomic data have been obtained in response to RNS in a number of Papilionoideae  [23] [24] [25] [26] [27] [28]  , the Mimosoids have been ignored. To fill this gap, we conducted a time-course experiment with Mimosa pudica inoculated with its bacterial symbiont Cupriavidus taiwanensis (Supplementary Table  1 ). As a preliminary to gene expression studies, we de novo sequenced the genome of M. pudica. This Mimosa species is tetraploid (2n = 4x = 52)  29  and its genome size was previously estimated to be around 900 Mb (ref.
16). To generate a high-quality genomic sequence, we used a combination of long-read sequencing and optical mapping (see Methods) leading to a near-chromosome-level assembly (Supplementary Table  2 ). This method produced 74 hybrid scaffolds (from 128 kbp to 25. 5 Mbp with N50 = 16. 1 Mbp) for 52 expected chromosomes and a total genome size of 797. 25 Mb. Automated structural annotation of the genome yielded 73,541 protein-coding genes and 5,134 non-coding RNAs. Finally, the high completeness of the annotated genome was evidenced by a 97% (2,255 genes) Busco recovery score on eudicots_odb10 (C:97. 0% (S:10. 2%, D:86. 8%), F:1. 1%, M:1. 9%, n: 2,326). As expected for a tetraploid genome, most of the genes are duplicated. The expression of 51,214 Mimosa genes was detected in our complete transcriptomic dataset, 43% of which were differentially expressed (9,034 genes up/16,470 genes downregulated) during the symbiotic interaction with C.
The ancestral transcriptome supported multiple symbiotic traits Next, we sought to trace the evolutionary history of transcriptomic recruitment or innovation associated with each of the following symbiotic traits: response to NF, nodule organogenesis and infection, symbiosome release, symbiosome persistence and nitrogen fixation. To do so, we exploited the gradual improvement in symbiotic abilities of Ralstonia strains and compared the responses of M. pudica to couples of strains that are able or unable to realize the different symbiotic traits (Fig. 3a  and Supplementary Table  3 ). Transcriptomic responses to direct NF treatments were also available for two other Fabales, M. truncatula and L. japonicus  55, 56. Another dataset available for M. truncatula was obtained from laser-capture microdissection associated with nodule tissue differentiation, corresponding to symbiosome release (FIId) and symbiosome persistence (FIIp), plant and bacteroid cell differentiation (FIIp, IZ) and nitrogen fixation (ZIII) (Supplementary Tables  3  and 4 )  57.
truncatula (Fig. 4f  and Supplementary Table  9 ). Most of these genes encode enzymes that have not been characterized yet (Supplementary Table  4 ). Although symbiosome release, inferred from the M. truncatula ('FIId') and M. pudica ('Release') datasets, displays a peculiar evolutionary pattern (see below and Fig. 4d ), this symbiotic stage also involved genes that are part of the ancestral transcriptomic response. Suppressors of plant defence in nodules, SymCRK  67  and RSD  73  , as well as the transcription regulator EFD required for both plant and bacteroid differentiation in M. truncatula  74, 75  participate in this ancestral response. Looking specifically at the M. pudica data, we found VAPYRIN, RPG, some flotillin and remorin genes and the syntaxin SYN, which are well-known infection-associated genes  41, [76] [77] [78].
Article https://doi. org/10. 1038/s41477-023-01441-w Papillionideae subfamily and the Mimosoid clade in the Fabales order  10. Our results support the hypothesis that evolutionary stability was acquired through the convergent evolution of symbiosome release and enhanced control of the bacterial symbiont gained by the expansion of putative antimicrobial peptide gene families. Besides providing an evolutionary perspective linked to multiple symbiotic traits and reconstructing a shared RNS transcriptomic response, our comparative transcriptomic approach has determined a list of conserved orthogroups that can be considered as targets for further spatial, temporal and biological characterization in multiple species. We believe that our dataset will help the community to integrate evolutionary perspectives in future studies.
The size and concentration of the final library were assessed using the Fragment Analyzer system (Agilent) and the Qubit fluorometer and Qubit dsDNA HS reagents assay kit (Thermo Fisher), respectively. Sequencing primer v3 and Sequel DNA Polymerase 3. 0 were respectively annealed and bound to the SMRTbell library. The library was loaded on 8 SMRTcells 1M and sequencing was performed on the Sequel I system with Sequel sequencing kit 3. 0, a run movie time of 600 min and Software v6. 0 (PacBio).
M. pudica genome assembly The genome was assembled in three steps from PacBio reads. In Step 1, Canu v1. 8 (ref. 89) was used to trim, correct and assemble the 5,815,198 subreads for a total size of 85 Gb, that is, an estimated coverage of 94X. Programme parameters were corOutCoverage=40, min-ReadLength=1000 and input genome size estimate=900 Mb. In Step 2, the raw data from PacBio Sequel bam files were aligned on the draft assembly. For this step, we used the wrapper of minimap2 (ref. 90), pbmm2, included in the SMRT Analysis Software v7. 0. 0 (https://www. pacb. com/products-and-services/analytical-software/smrt-analysis/). Programme parameters were « pbmm2 align-preset 'SUBREAD' -c 70 -l 500 ». In the final step (Step 3), we used this mapping result to polish the draft assembly and generate a high-quality final assembly.
For this step, we used the variantCaller command included in the SMRT Analysis Software v7. 0. 0, with the arrow algorithm. Programme parameters were -algorithm arrow -minConfidence 40 -minCoverage 70 -coverage 100 -minReadScore 0. 65. The final polished assembly produced had a total of 1,343 contigs with a total length of 842,189,795 bp, a largest contig of 24,170,175 bp and an N50 contig of 13,257,064 bp.
Data collection, optical mapping and genome scaffolding Labelled DNA was loaded on a Saphyr G1 chip according to the Saphyr System user guide (30247, Bionano Genomics). Data processing was performed using the Bionano Genomics Access software (https:// bionanogenomics. com/support-page/bionano-access-software/). Molecules (480 Gb) larger than 150 Kb with an N50 of 199 kbp were produced and represented 533X. This corresponded to 533X coverage of the 900 Mb estimated size of the M. pudica genome. These molecules were assembled using RefAligner with default parameters, producing 110 genome maps with an N50 of 16. 1 Mbp for a total genome map length of 833 Mbp. Finally, hybrid scaffolding was performed between the polished PacBio assembly and the optical genome maps using hybridScaffold pipeline with default parameters.
M. pudica genome structural annotation The M. pudica gene models were predicted by the eukaryotic genome annotation pipeline egn-ep (http://eugene. toulouse. inra. fr/Downloads/egnep-Linux-x86_64. 1. 5. 1. tar. gz) using trained statistical models adapted for plants (http://eugene. toulouse. inra. fr/Downloads/ WAM_plant. 20180615. tar. gz). This pipeline manages automatically probabilistic sequence model training, genome masking, transcript and protein alignments computation, alternative splice sites detection and integrative gene modelling by the EuGene software (release 4. 2a  91  ; http://eugene. toulouse. inra. fr/Downloads/eugene-4. 2a. tar. gz). Four protein databases were used to detect translated regions: (1) the proteome of M. truncatula A17 (v5 annotation release 1. 6; https:// medicago.
toulouse. inra. fr/MtrunA17r5. 0-ANR/), (2) the proteome of the previous M. pudica Illumina genome  16  , (3) Swiss-Prot, October 2016 and (4) the proteome of A. thaliana TAIR10 version. Proteins similar to REPBASE were removed from the three datasets (to avoid the integration of transposable element related proteins in the training steps). Chained alignments spanning less than 50% of the length of the database protein were removed. The proteome of M. truncatula (release 1. 6) was used as a training proteome by EuGene.
Article https://doi. org/10. 1038/s41477-023-01441-w Three input transcripts for EuGene were used. One transcriptome was predicted on the basis of the mapping of reads from the 136 RNA-seq samples generated in this study (Supplementary Table  1 ). To obtain these transcripts, the raw fastq paired-end reads were cleaned by removing the adapters and the low-quality sequences using cutadapt  92  (v2. 1) and TrimGalore (v0. 6. 5, https://github. com/FelixKrueger/ TrimGalore) with the -q 30-length 20 options. The cleaned reads were mapped against the M. pudica genome assembly using HISAT2 (ref. 93) (v2. 1. 0) with the -score-min L,-0. 6,-0. 6-max-intronlen 10000-dtarna-strandness RF options. Duplicated reads were removed using the SAMtools  94, 95  (v1. 9) markdup command. Transcripts were predicted using Stringtie  96 (v2.
1. 4) with -fr -f 0. 8 on each sample. All 80 gtf sample files were merged using stringtie-merge with standard options. Transcript fasta files were generated using gffread  96,97 (v0. 11. 6)  with the -w option. We also de novo predicted two transcriptomes from two batches of ten samples (one sample per condition) of our same RNA-seq data using DRAP pipeline  98  (v1. 92, http://www. sigenae. org/drap). runDrap was used on the 20 samples, applying the Oases RNA-seq assembly software  99. runMeta was used to merge assemblies without redundancy on the basis of predicted transcripts with fpkm 1. These transcriptomes were employed as a training transcriptome by EuGene. Finally, 73,541 protein-coding genes, 1,107 transfer RNAs, 114 ribosomal RNAs and 3,913 ncRNAs were annotated. Genome assembly, annotation file and gene models are publicly available through myGenomeBrowser  100  and through NCBI under BioProject PRJNA787464.
Differential gene expression analysis All RNA-seq libraries were mapped against their representative genome (Supplementary Table  1 ) using nextflow  105  (v20. 11. 0-edge) and run on nf-core/rnaseq 106 (v3. 0, 10. 5281/zenodo. 1400710) using the '-profile debug,genotoul-skip_qc-aligner star_salmon' options. The workflow used bedtools 107 (v2. 29. 2), bioconductor-summarizedexperiment (v1. 20. 0), bioconductor-tximeta (v1. 8. 0), gffread  97  (v0. 12. 1), picard (v2. 23. 9), salmon  108  (v1. 4. 0), samtools  94  (v1. 10), star  109  (v2. 6. 1d), stringtie  96 (v2. 1. 4) , Trimgalore (v0. 6. 6) and ucsc (v377). DEGs for the different species and experiments were estimated using 'edgeR' 110 in R  111 (v4. 1. 2). Template script to estimate and identify DEGs is stored in GitHub at https://github.
Statistical analyses The different scripts used to cross-reference orthogroups and DEGs to estimate differentially expressed orthogroups (DEOGs), reconstruct the ancestral transcriptomic symbiotic response, identify symbiosome accommodation proteins and all statistical related analyses are freely available at the dedicated GitHub repository 'Universal_nodulation_transcriptomic_response'. Briefly, to determine at which evolutionary node each orthogroup was probably transcriptionally recruited for RNS, we used the process described below: (1) DEGs to DEOGs: if at least one gene was up/downregulated in an orthogroup (HOG), the orthogroup was coded as 1 for the species in which the gene was differentially regulated. This process was done for the nine species in the 56,508 orthogroups. (2) On the basis of a simplified tree with only the nine species, we used the 'asr_mk_model' function from 'castor' package in R with the following options: 'Nstates = 2, include_ancestral_likelihoods = TRUE, rate_model = 'ER'', giving a likelihood between 0 and 1 at each node.
Acknowledgements We thank  O. Valdes-Lopez ,  P. Gamas  and the  ENSA consortium  for discussions and feedback on the manuscript,  B. Perret  for the Lupinus albus seeds and the genotoul bioinformatics platform  Toulouse Occitanie  ( Bioinfo Genotoul , https://doi. org/10. 15454/1. 5572369328961167E12 ) for providing computing resources. This study was supported by grants from the  Fédération de Recherche Agrobiosciences, Interactions et Biodiversité  to P. -M. D. and P. R. , and from the  French National Research Institute for Agriculture, Food and Environment (INRAE, Plant Health and Environment Division)  to D. C. Research performed at LRSV and LIPME was also supported by the  'Laboratoires d'Excellence (LABEX) '  TULIP  ( ANR-10-LABX-41 ) and the  'École Universitaire de Recherche (EUR) '  TULIP-GS  ( ANR-18-EURE-0019 ).
J. K. , C. L. and P-M. D were supported by the project  Engineering Nitrogen Symbiosis for Africa (ENSA)  currently funded through a grant to the  University of Cambridge  by the  Bill and Melinda Gates Foundation  ( OPP1172165 ) and the  UK Foreign, Commonwealth and Development Office as Engineering Nitrogen Symbiosis for Africa  ( OPP1172165 ). This project received funding from the  European Research Council (ERC)  under the European Union's  Horizon 2020 research and innovation programme  (grant agreement no. 101001675 -ORIGINS ) to P. -M. D. P. R. received funding from the  EU  in the framework of the  Marie-Curie   FP7 COFUND People Program , through the award of an  AgreenSkills+ fellowship  (under grant agreement no. 609398 ) and from the  European Union 's  Horizon 2020 research and innovation programme  under the  Marie Skłodowska-Curie  grant agreement no.
*the number of matched peptides was obtained from the incomplete protein sequences before identi cation of the complete sequence by 5'-RACE-PCR (PTP2) or RNAseq sequencing data assembly (putative PTP3). Both nucleic and proteic sequences are listed in the Table  S2. kDa: kiloDalton. Sequence alignment of PTP3 and PTP3b proteins revealed that these two proteins present 29. 1% of identity (see Supplementary Fig  S2 ). However, these PTP3 proteins present conserved biochemical characteristics including a similar high molecular weight (121 vs 127 kDa), an acidic isoelectric point close to 5 and a richness in alanine residues (Table  1 ). To con rm that PTP3b is a new polar tube component, antibodies were produced against a recombinant partial protein expressed in E. coli. As expected, these antibodies reacted with a band near 180 kDa but higher than the PTP3.
6b  and see Supplementary Table  S2 ). More interestingly, 26 hypothetical proteins with unknown function were identi ed and 14 of them present a predicted signal peptide suggesting they can be secreted (Table  3 ). Six of these potentially secreted proteins were expressed in E. coli and injected in mice to produce speci c antibodies. One of them, for which the complete CDS was obtained through RNAseq data assembly and corresponding to a threoninerich protein of 417 aa, was shown to localize at the terminal end of the extruded polar tube, a labelling similar to that observed with anti-PTP4 antibodies (Fig. 7a ). In Western blot, these antibodies detected a single protein band around 60 kDa only in protein extracts containing reducing agents (Fig. 7b ). This new polar tube component named PTP6 had no homology with other PTPs and proteins from databases.
Thus, identi cation of PTPs often requires a combination of sequence similarity searches in databases and comparison of both biochemical properties and gene organization. For example, the syntenic relationship of ptp1 and ptp2 genes facilitated their identi cation despite their strong sequence divergences in the insect microsporidia Antonospora locustae and Paranosema grylli  22. In the present study, we succeeded in the identi cation of the genes coding for orthologous proteins belonging to the 5 PTP families in Anncaliia algerae. As expected, these proteins in particular the major PT component PTP1 are characterized by a high degree of amino acid sequence divergence when compared to their orthologs in other microsporidian species. Although ptp1 and ptp2 genes were found at the same genomic locus in numerous microsporidian genomes including those of Encephalitozoon spp.
genome PTPs sequences described in other microsporidian species and available in NCBI and/or MicrosporidiaDB databases (https://microsporidiadb. org/micro/app) were used as query. Using these sequences, a search by sequence homology was realized against the genome of A. algerae using BLAST with default settings. Sequences identi ed in the A. algerae genome were then characterized with different bioinformatic tools. N-terminal signal peptides were predicted using a combination of SignalP-5. 0 (http://www. cbs. dtu. dk/services/SignalP/), TargetP-2. 0 (http://www. cbs. dtu. dk/services/TargetP/) and Phobius (https://phobius. sbc. su. se/) tools. Theoretical isoelectric point, molecular weight and amino acid composition were determined with ExPASy ProtParam tool (https://web. expasy. org/protparam/).
Potential O-glycosylation sites were predicted with NetOGlyc 4. 0 server (http://www. cbs. dtu. dk/services/NetOGlyc/). Functional domains and gene ontology terms were determined with InterPro 86. 0 tool (https://www. ebi. ac. uk/interpro/). Multiple sequence alignments were performed using the ClustalOmega software (https://www. ebi. ac. uk/Tools/msa/clustalo/).
Slides were observed under a Leica DM IRB epi uorescence microscope and images were superimposed with GIMP 2. 10. 8 software. Protein identi cation by Nano-LC-MS/MS The preparation of protein bands was carried out according to Zhu et al. 40   ) and a minimum of 2 K-mers). A total of 2793 and 4833 contigs were obtained with these two softwares, respectively. Using these two sequences set as databases, genes coding for PTPs were identi ed by a local BLASTn approach with a word size of 11 nucleotides using our previously identi ed A. algerae ptp sequences as queries. Declarations raw data from A. algerae-infected HFF cells have been submitted to NCBI with BioProject accession number PRJNA927239. Full amino acid sequences of proteins identi ed by mass spectrometry are available in the supplementary Table  S2.
PTPs and proteins with unknown function identi ed by mass spectrometry analysis in puri ed polar tube protein extracts. proteins with unknown function selected to produce antibodies. The newly identi ed PTP6 is indicated in red. *Sequence incomplete in C-terminal or in N-terminal. The number of amino acids is indicated for complete proteins and the predicted size for mature proteins. The number of corresponding peptides is the higher number identi ed in at least one extract. (+) Presence of a predicted signal peptide. (-) No predicted signal peptide. Identi cation of potential orthologs was performed by BLASTp search against NCBI and MicrosporidiaDB databases. (nd) not determined because of partial sequence.
39The peptides extract adjusted exactly to 30 µL with a recovery solution (H 2 O/ ACN/TFA - 94. 95/5/0. 05) was analysed by nano-LC-MS/MS using an Ultimate 3000 system coupled to a QExactive HF-X mass spectrometer (MS, Thermo Fisher Scienti c). One µL of hydrolyzate was rst preconcentrated and desalted during 6 min on a C18 pre-column (equilibration Tri uoroacetic Acid 0. 05% in H 2 O -ow rate 30 µl/min) then analysed on a C18 Raw data processing was carried out with the Proteome Discoverer Sofware v1. 4 and MS/MS ion search was performed with Mascot v2. 5. 1 (http://www. matrixscience. com)againstthelocaldatabankLocal_annc_alg database39with the following parameters: precursor mass tolerance of 10 ppm, fragment mass tolerance of 0. 05 Da, maximum of two missed cleavage sites of trypsin, carbamidomethylation (C), oxidation (M) and deamidation (NQ) set as variable modi cations.
72) and reads were then mapped against the human genome (version HG38 NCBI 18. 05. 21) with HISAT2 software (Galaxy version 2. 1. 0) to remove mRNAs corresponding to host cells. Two de novo assemblies of the 252 x 10 6 remaining reads were done with Megahit (Galaxy version 1. 1. 3. 5; default settings; minimum multiplicity set to 2 for ltering) and Trinity (Galaxy version 2. 8. 4; default settings (k value = 25.
Acknowledgments Maurine Fayet  was by grant from  « Ministère  de l'éducation, de la recherche et de la technologie ». We thank  Mohamed Dorbani  for his involvement in the polar tube puri cation protocol development as others trainees at LMGE for their involvement in the implementation of several experiments. We also thank  G. Junion  for western blot analysis with ChemiDoc system and both  D. Viala  and  C Chambon  for providing the mass spectrometry analysis at PFEM cp protéomique ( INRA Theix, France ).
Data availability The assembled contig sequences for A. algerae are available in the NCBI Assembly database (www. ncbi. nlm. nih. gov/assembly/) under the accession number GCA_000313815. 1. Accession numbers for complete A. algerae PTPs are WAQ68434. 1 (PTP1), WAQ68435. 1 (PTP2), WAQ68436. 1 (PTP3), WAQ68437. 1 (PTP3b), WAQ68438. 1 (PTP4), WAQ68439. 1 (PTP5) and WAQ68440. 1 (PTP6). RNA-seq.
72) and reads were then mapped against the human genome (version HG38 NCBI 18. 05. 21) with HISAT2 software (Galaxy version 2. 1. 0) to remove mRNAs corresponding to host cells. Two de novo assemblies of the 252 x 10 6 remaining reads were done with Megahit (Galaxy version 1. 1. 3. 5; default settings; minimum multiplicity set to 2 for ltering) and Trinity (Galaxy version 2. 8. 4; default settings (k value = 25 Acclaim PepMap 100 column (equilibration 96% solvent A (99. 9% H 2 O, 0. 1% formic acid) -ow rate 300 nl/min) with a gradient of solvent B (99. 9% acetonitrile, 0. 1% formic acid) of 5 to 32% in 50 min. Eluted peptides were electrosprayed through a nanoelectrospray ion source in the mass spectrometer operating in top N data dependent mode (DDA).