dataesr/scientific-paragraphs-categorization huggingface dataset:
The yeast transformants were spotted on SD/-Ura-Trp plates with or without 20 mg/ml X-gal. pB42AD-p53 and pLacZi-p53 were used as positive controls. (C) Luciferase reporter assays of GT-3a-induced suppression of TOZ and RAD23C expression in N. benthamiana. LUC activity was measured by normalizing to the REN signal. Values are means ± SE (n = 4). Asterisks indicate significant differences according to a two-tailed Student's t-test, ***P < 0. 001. Similar results were obtained from three independent experiments (biological replicates). (D) qRT-PCR analysis of TOZ and RAD23C expression in wild-type (WT) A. thaliana and GT-3a-overexpressing A. thaliana lines. UBP22 (AT5G10790) was used as an internal control. Data represent the mean of three independent experiments ± SE (n = 3). Similar results were obtained from three independent experiments (biological replicates).
Nematode infection and gall sections A. thaliana seedlings month after transplant) were inoculated with pre-J2s. For nematode susceptibility assays, A. thaliana roots were inoculated with 200 pre-J2s per plant. The roots were collected, and galls and egg masses were counted under a dissecting microscope (Olympus, Japan) 35 days post inoculation (dpi). For gall collection, roots were inoculated with 500 pre-J2s per plant. Galls were collected at 3, 5, 7, 14, and 21 dpi and fixed as described previously  (Gavrilovic et al. , 2016). At least 10 galls were fixed for each A. thaliana line. Gall sections were stained with 0. 05% toluidine blue and photographed on a Zeiss AxioImager Z2 microscope (Zeiss, Germany). The areas of the GCs were measured with ImageJ software  (Schindelin et al. , 2012).
ACKNOWLEDGMENTS We thank  Dr. Panpan Yang  ( Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China ) for providing the CP516 vector and the p3301 vector,  Dr. Jinzhuo Jian  ( Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China ) for advice concerning gall sections, and  Dr. Qian Wei  (the  Core Facility Platform, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China ) for assistance with confocal microscopy. The authors declare no.
FUNDING This research was supported by the  Youth Innovation Program of the Chinese Academy of Agricultural Sciences  (grant no. Y2022QC06 ), the  National Natural Science Foundation of China  (grant nos. 32001878 ,  32172366 ), the  Natural Science Foundation of Beijing  (grant no. 6222054 ), the  China Agricultural Research System  ( CARS-23 ), and the  French Government (National Research Agency, ANR)  through '' Investments for the Future'' LabEx SIGNALIFE  (# ANR-11-LABX-0028-01 ),  IDEX UCAJedi  (# ANR-15-IDEX-0 ).
incognita pre-J2s. Total numbers of galls and egg masses were counted at 35 dpi. M. incognita inoculated WT A. thaliana inoculated with M. incognita was used as a control. Data are the average number per plant ± SD (n = 16). Different letters indicate significant differences (P < 0. 05, one-way ANOVA). See also Supplemental Figure 2D.
Bioinformatic analysis of metabarcoding data In total, 81,589,286 sequencing clusters were paired-end sequenced. Raw reads were analysed using the OBITools software suite  (Boyer et al. , 2016) , specifically dedicated to handling metabarcoding data. Pairedend reads were first assembled with the program illuminapairedend, keeping only sequences with an alignment score higher than 40. Aligned sequences were assigned to the corresponding sample using the program ngsfilter by allowing two and zero mismatches for primers and tags, respectively. After sequence dereplication using obiuniq, poor-quality sequences (i. e. , containing "N"), sequences whose length fell outside the expected size interval (shorter than 40 bp for Bact02 or 66 bp for Fung02) and sequences observed less than ten times in at least one PCR replicate were filtered out.
The remaining unique sequences were clustered into molecular operational taxonomic units (MOTUs) as follows. First, the Sumatra algorithm  (Mercier et al. , 2013)  was run to compute pairwise similarities between sequences based on the length of the longest common subsequence (LCS). Then, pairwise similarities were used to cluster the sequences with a similarity threshold of 97 % using Sumaclust  (Mercier et al. , 2013). Abundances of sequences belonging to the same cluster (i. e. , MOTU) were summed, and the cluster centre was kept as the MOTU representative of the cluster. The final datasets contained 2440 MOTUs and 7,130,286 sequences for Bact02 and MOTUs and 25,772,129 sequences for Fung02. Finally, the taxonomic assignment of each MOTU was performed using the program ecotag of OBITools, which compares the MOTU sequence to a database of fulllength reference metabarcodes.
Then, another selection was carried out to check the significance of the remaining variables. This process was repeated until there was no significant variable. The proportion of variance explained by the selected explanatory variables was indicated by an adjusted R 2  (Borcard et al. , 2018). All statistical analyses were performed using R software (version 4. 1. 0; R Development Core Team). The Kruskal-Wallis tests and multiple comparisons were performed by the "kruskal" function in the "agricolae" package. Set intersections were visualized by the "upset" function in the "UpSetR" package. PERMANOVA and tests for multivariate homogeneity were carried out by the "adonis" and the "betadisper" functions in the "vegan" package, respectively. The "ordiplot" and "ordiellipse" functions in the "vegan" package were used to carry out PCoA and draw confidence ellipses, respectively.
Trees from the two tree species groups host distinct fungal communities The previous datasets described above highlighted the occurrence of two tree family groups, namely, Group 1 (Basidiomycota-enriched samples from the Fagaceae, the Salicaceae and the Betulaceae) and Group 2 (Ascomycota-enriched samples from all the other tree families). The ecological functions of the fungal species in these two groups were further assessed using FunGuild  (Nguyen et al. , 2016) , and the abundance of DNA reads was calculated and expressed as a percentage of the total reads. Our analysis indicated that 74. 9 %, 72. 9 % and 49. 2 % of the specific fungal reads of Group 1 tree families were assigned to the ECMF guild for the Leforest, Carrières and Thann samples, respectively (Fig. 5 , Figs. S1  and S2 ).
Microbial communities of OCA, PTS and SAQ change as a function of the study site As described in the Materials and methods section, OCA, PTS and SAQ were planted at the three sites, which allowed us to assess the impact of site on microbial communities. The results indicated that the three tree species exhibited different bacterial communities, depending on the site  (Fig. 6, Figs. S4 and S5). The Venn diagram indicates that OTUs (22. 9 %) were common at Leforest, Carrieres and Thann for SAQ. For OCA, 291 OTUs (25. 8 %) were recorded, and 293 OTUs (24. 2 %) were recorded for PTS. Similarly, the dataset for SAQ (Fig. 6 ), PTS (Fig. S4 ) and OCA (Fig. S5 ) shows that the fungal communities were specific to the site where the trees were grown. Only 6 % of the fungal OTUs were shared by all three sites (106 OTUs for SAQ, 115 OTUs for PTS and 144 OTUs for OCA).
This work was supported by the French National Research Agency [PHYTOCHEM ANR-13-CDII-0005-01], the French Environment and Energy Management Agency [PROLIPHYT ADEME-1172C0053], the Région Franche-Comté [Environnement-Homme-Territoire 2014-069] and the Pays de Montbéliard Agglomération [13/070-203-2015]. C. Z. received a PhD grant from the French Ministry of Higher Education and Research. CRediT authorship contribution statementStéphane Pfendler: Conceptualization, field investigation, bioinformatic analysis, statistical analysis, visualization, writing -original draft preparation. Lisa Ciadamidaro: Conceptualization, field investigation, writing -original draft preparation. Shinji Ozaki: Statistical analysis, visualization, writing -original draft preparation. Aurélie Bonin: DNA metabarcoding analysis and data acquisition, writing -review and editing.
drummondii (supplemental Figure  1 ). This contrasts with the other non-nodulator, F. sylvatica, in which NIN is intact, as in other non-nodulating Fagales. We also sequenced new transcriptomes from 20 phylodiverse nodulating and non-nodulating NFNC species (supplemental Table  14 ; supplemental Figure  7 ), emphasizing root and nodule samples. We failed with some (non-model) species, particularly in nodule sample collection, because of difficulty in tissue culturing followed by RNA extraction (supplemental Table  14 ; supplemental Figure  7 ). However, we generated high-quality RNA-seq datasets from at least one representative nodulating species from each of the four orders in the NFNC, including Casuarina glauca (Fagales), Datisca glomerata (Cucurbitales), P. tridentata (Rosales), and Medicago truncatula (Fabales) (Figure  4A ).
We also built a complete treatment collection involving growth conditions with and without exogenous nitrate and with and without inoculation with symbiotic bacteria (B+/NÀ, BÀ/N+, B+/N+, BÀ/NÀ) in mature root tissues of two closely related comparison pairs: Alnus and Betula from Fagales (Figure  4C ) and Datisca and Begonia from Cucurbitales (supplemental Figure  8 ). Pairwise correlations indicated high-quality and consistent RNA-seq datasets within and between species (supplemental Figure  7B ).
Again, NIN showed the most significant association with the nodulating phenotype (Figure  3C  and supplemental Table  13 ). We reconstructed the phylogenetic tree for all detected NIN and NIN-like protein (NLP) family members in the genome and transcriptome datasets (including 1KP transcriptomes) (Figure  3B ) and obtained a tree that was largely consistent with a recent legume-focused study  (Zhao et al. , 2021)  and other recent studies (e. g. ,  Wu et al. , 2022). The origin of the conserved NLP-like gene family, whose members serve as nitrate-responsive master regulators, can be traced back to the common ancestor of green plants. The ancestral gene subsequently experienced at least three duplication events that resulted in four NLP subgroups. Consistent with previous observations  (Liu and Bisseling, 2020) , a duplication event within NLP group 3 (NLP3) occurred early in the divergence of eudicots (Figure  3C , Duplication 3) and produced two paralogous clades, one of which includes NIN (in NFNC members) and its orthologs (in other eudicots).
From the four representative nodulating species described above, we classified genes as follows: shared by all four orders; shared by species from three orders; shared by species from two orders where one species was from either Fabales or Fagales and the other was from either Cucurbitales or Rosales; shared by species from two sister orders; or unique to a single species (Figure  5A ). Genes shared by three to four orders or by species package hclust. Gene expression values (TPM) were calculated and compared on the basis of one-to-one orthologous symbiosis genes across species. N, nitrogen; B, N 2 -fixing bacteria; +, with; À, without. (B) Gene expression levels (TPM) of NIN and NLP-3 in nodules and roots of selected plant species under different growth conditions. (C) Detailed comparison between the nodulating plant Alnus and its closely related comparator species Betula in Fagales under various treatments (N+BÀ, N+B+, NÀB+, and NÀ).
'' In addition to M. truncatula, we selected eight representative genomes comprising one non-nodulator and one nodulator from each of the four orders, as well as one outgroup species (Populus trichocarpa) (supplemental Figure  12 ), and we anchored all the identified CNEs to the Medicago genome for comparison. In total, we predicted 931 454 high-quality putative CNEs (R5 bp; corresponding to 4. 16% of the Medicago genome) (Figure  6A  and supplemental Table  29 ), many more than detected by  Pereira et al. , 2022 (supplemental Figure 13) , who reported only 6729 CNEs. Furthermore, 84. 49% of RNS-specific CNEs and 84. 60% of NFNspecific CNEs detected by  Pereira et al. , 2022  were also present in our study, including the five experimentally validated CNEs in cis-regulatory regions (supplemental Figure  13 ), suggesting a high-quality CNE dataset as well as many new CNE candidates first discovered in our study (supplemental Figures 2 and 13).
, 2022) , a trait confined to the NFNC. To further evaluate the quality and function of the identified CNEs, we compared each category of CNEs with the PLACE and plant-PAN 3. 0 databases (supplemental Figure  2B ). We calculated the enrichment of motifs based on a Z-score for each CNE, retrieved their hits from the databases, and found that 52. 94% of the CNEs had at least one database hit, suggesting the potential functional roles of these CNEs. Notably, one nodulation-related motif, ''AAAGAT,'' originally identified in the soybean leghemoglobin lbc3 promoter  (Stougaard et al. , 1990; Fehlberg et al. , 2005) , ranked in the top 10 sorting by the Z-score based on the CNEs that were conserved in all nodulating plants (supplemental Figure  2B ). At least 4468 CNEs contained the ''AAAGAT'' motif located around symbiosis genes, suggesting that an amplification of this motif in nodulating plants might have contributed to emergence of the RNS (supplemental Figure  2B ).
Significance was calculated with GAT (**P < 0. 001). The ChIP/ATAC dataset was downloaded from https://medicago. toulouse. inra. fr/MtrunA17r5. 0-ANR. Expected: frequency distribution of the length of genomic regions overlapping with the ChIP/ATAC peaks in a simulation experiment in which we randomly selected the same number of CNEs from the whole genome (repeated 10 000 times). (D) The same analysis as in (C), but for NFNC-specific CNEs only. As controls, those CNEs with functional validations, i. e. , the functional NFNC-specific CNEs PACE, CE, and CRE1-5, were overlapped with ATAC peaks, and CRE1-5 was also overlapped with H3K9ac peaks. Plant Communications 5, 100671, January 8 2024 ª 2023 The Author(s). 11 Genomic landscape of nodulation.
RNA was sequenced using the Illumina SBS (sequencing by synthesis) technique on a HiSeq 2000 instrument in singleread 100-nt mode. Image analysis, base calling, and quality filtering were performed using Illumina software. For D. glomerata and B. fuchsioides, RNA isolations were performed using the Sigma Spectrum Plant Total RNA extraction kit (Sigma-Aldrich, St. Louis, MO); Polyclar AT (Serva, Heidelberg, Germany) was added to the extraction buffer (2%, w/v). On-column DNA digestion was performed with the RNase-free DNase set (QIAGEN), followed by treatment with Ambion TURBO DNase (Thermo Fisher Scientific) after isolation. RNA quality was determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). RNA was extracted from P. tridentata samples using either a QIAGEN RNeasy Plant Mini-Kit or, for a subset of samples, the Spectrum Plant Plant Communications 5, 100671, January 8 2024 ª 2023 The Author(s).
Plant Communications Total RNA Kit, followed by treatment with the Ambion Turbo RNase-free DNase kit (Thermo Fisher Scientific) after isolation. The Spectrum Total RNA kit was used to enable BGI to test whether Frankia transcripts could be detected in nodule tissue, in addition to measurement of plant gene expression. Genome sequencing, assembly, and annotation Plant cultivation, DNA extractions, and RNA extractions were performed as described in  Griesmann et al. , 2018. The genomes of P. tridentata, D. octopetala, and F. sylvatica were sequenced using Illumina technology (HiSeq 2000 and HiSeq 4000). A hierarchical DNA library strategy was employed, which included multiple paired-end libraries with insert sizes ranging from 170 to 800 bp and mate-pair libraries using large DNA fragments with insert sizes of 2-20 kb.
CNE presence/absence was analyzed as described above for gene presence/absence. The overlapping CNEs between nodulating and non-nodulating species were calculated using a series of Python scripts. The significance of CNE motif enrichment was calculated by Z score = x À m ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ffi 1 N P N i = 1 ðxi À mÞ 2 r , where x i is the number of CNEs that matched a specific R6-nt motif in the PLACE or plantPAN3. 0 database (http://plantpan. itps. ncku. edu. tw/) ( x i R 2), N is the total number of matched motifs, and m is the average of number of CNE matched motifs. To investigate the enrichment of two repressive histone marks (H3K27me1 and H3K9me2), one activating mark (H3K9ac), and ATAC-seq data in CNEs, we used GAT  (Heger et al.
, 2019). Reference-guided transcript assembly for each library was performed using StringTie  (Pertea et al. , 2015). Assembled transcripts from each library were merged, and transcripts were re-annotated on the genome using gffcompare. For species without sequenced genomes, transcripts were assembled de novo using Trinity  (Grabherr et al. , 2011). We calculated the expression level (TPM) of each gene using Salmon  (Patro et al. , 2017). Pairwise correlations between transcriptomes were calculated using gene expression levels (estimated as TPM) of 3987 one-to-one orthologs across the 21 species (including the public dataset for Medicago) (supplemental Figure  7 ). Genes that were differentially expressed in different treatments (+N-B, +N+B, ÀN+B, ÀNÀB) were identified with DESeq2 (adjusted p value <0.
0 0. 0 -0. 9 0. 8 0. 1 0. 1 0. 2 0. 3 0. 5 0. 7 0. 6 0. 3 0. 1 0. 4 0. 4 0. 2 0. 2 0. 0 0. 1 -0. 1 0. 0 0. 0 -0. 2 -0. 2 -0. 5 1. 5 -0. 4 -0. 1 -0. 3 -0. 2 -0. 5 -0. 9 -0. 9 0. 0 0. 2 0. 1 0. 2 0. 3 0. 5 1. 9 2. 0 -0. 1 -0. 1 0. 4 0. 2 0. 1 0. 0 -0. 1 0. 1 -0. 2 0. 0 -0. 6 -0. 2 -0. 2 2. 2 1. 2 -0. 5 -0. 8 -0. 5 -0. 2 0. 2 0. 0 0. 0 -0. 7 -0. 6 -0. 7 -0. 7 -0. 6 -0. 6 -0. 4 2. 8 -0. 3 -0. 6 -0. 6 -0. 6 -0. 6 -0. 7 0. 0 -0. 6 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 -0. 2 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 Protein sequences of 744 orthologs/paralogs of NIN-like genes were retrieved by hmmer search using the Pfam models of the RWP-RK (PF02042) and PB1(PF00564) domains with 1e-5 as cut-off. All protein sequences were aligned using MAFT. The phylogenetic tree (left panel) was constructed using iqtree and bootstrap 1000.
CEs around all genes CEs around symbiosis-related genes Upstream Downstream Gene region B 1 0 -1 5 k 5 -1 0 k 1 0 -1 5 k 1 5 -2 0 k 1 5 -2 0 k > 2 0 k > 2 0 k 5 -1 0 k In tr o n 3 ' U T R 0 -2 k 2 -5 k 0 -2 k 5 ' U T R 2 -5 k Upstream Downstream Gene region 1 0 -1 5 k 5 -1 0 k 1 0 -1 5 k 1 5 -2 0 k 1 5 -2 0 k Motif PLACE PlantPAN3. 0 MYBCORE MYB2CONSENSUSAT NODCON1GM -t=p -b4=2 -b5=a). ML tree was then constructed using IQ-TREE and branch supports were tested with 1000 bootstraps (version 2. 0. 3) (parameter: -mset LG,JTT -mrate Reference G,I,G+I -bb 1000). Finally we used R package ggtree to visualize each phylogenetic tree, marked with additional information, such as genes from rhizobia nodulator are marked in red, genes from frankia nodulators are marked in yellow, genes from non-nodulators are marked as black and reference genes are marked in blue.
Significance was calculated with GAT (**P < 0. 001). The ChIP/ATAC dataset was downloaded from https://medicago. toulouse. inra. fr/MtrunA17r5. 0-ANR. Expected: frequency distribution of the length of genomic regions overlapping with the ChIP/ATAC peaks in a simulation experiment in which we randomly selected the same number of CNEs from the whole genome (repeated 10 000 times). (D) The same analysis as in (C), but for NFNC-specific CNEs only. As controls, those CNEs with functional validations, i. e. , the functional NFNC-specific CNEs PACE, CE, and CRE1-5, were overlapped with ATAC peaks, and CRE1-5 was also overlapped with H3K9ac peaks.
⑩ Annotate the identified conserved non-coding elements. ⑪ Integrate CNEs species matrix. Further analysis: ⑫ Association analysis with symbiosis-related genes. (B) Motifs found to be enriched (z score > 2) in the different category of CNEs. Numbers and coloring correspond to the fold enrichment compared to the permuted version of the motif (red, enrichment; blue, depletion). Motif annotations come from the PLACE and PlantPAN3. 0 database (D, G/A/T; B, G/T/C R, A/G; H, A/T/G; W, A/T; K, A/C; Y, C/T; N, any base).
32,000 bp 33,000 bp 34,000 bp 35,000 bp 36,000 bp 4,863 bp scaffold183_Putri:31,557-36,419 Dryas octopetala carries a 69 nt deletion compared to nodulators Dryas drummondii and Purshia tridentata in the NIN gene. B. Dryas drummondii NIN protein structure was predicted by Alphafold. 69nt / 23aa in green form a random coil. C. Nodules RNA seq data of Purshia tridentata shows the expression level of 69 nt region. Purshia tridentata Dryas drummondii Dryas octopetala Exons 69 nt deletion /23 aa deletion NSPKFPGSGQYSSLNMSDHSKQV Purshia tridentata Nodules RNA seq validation [0 -4145] Nodules. NIN. bam Coverage Nodules. NIN. bam Purshia_tridentata. gff Purtr183S11451_Putri The raw RNA-seq and DNA sequencing data, as well as the new Figure S1. A. Non-nodulator genome assemblies and annotations, have been deposited at the CNGB Sequence Archive (CNSA) of the China National Gene Bank DataBase (CNGBdb) under accession number CNP 0004055.
Analysis of spacers and identification of M. gallisepticum CRISPR targets We compared the set of spacers inferred from the M. gallisepticum CRISPR locus across all our M. gallisepticum isolates (Figure  S1 , Table  S4 ), and compiled a dataset of 490 non-redundant spacers (Table  S4C ). As a result, we identified a total of 438 unique spacers from 19 poultry strains and 55 unique spacers from 85 house finch isolates (Table  S4C ). Across the M. gallisepticum poultry strains, we observed a wide diversity of spacers, with no spacer found conserved across all strains (Figure  S1 ). Indeed, 66% of spacers from the 19 poultry strains were present in only 1-2 genomes, while 2% were found in 9-10 genomes. In contrast, we found a lower diversity of spacers across the CRISPR arrays of house finch isolates.
When considering the pattern of spacer acquisition over time in house finches, we found that most new spacers (i. e. 46 out of 55; >80%) had been integrated before 2000 and were therefore present in the early years of M. gallisepticum emergence in house finches. Surprisingly, despite the inactivation of MgCas9 from 2007, we also found evidence for the acquisition of six new spacers between 2009 and 2015 (Table  S4C ). We investigated the potential origin of M. gallisepticum spacers using Blast queries against PHASTER, GenBank and IMG databases (Table  S4D -S4G), as described in  (25). Out of 490 spacers, 431 (88%) showed no hits in the explored databases. The PHASTER database returned hits on known bacterial phages for 34 spacers. GenBank returned hits on bacterial genomes (11 spacers) and phages (4 spacers).
Acknowledgements We are very thankful to  Dr. Gasiunas  from  Caszyme  for sharing the 7N PAM-degenerated pTZ57-7n library, the pET28ID116 plasmid encoding M. gallisepticum CA06 Cas9 and for appreciated advices on the protocol used for in vitro cleavage. We also want to thank  Patrice Mora  for helping in the constitution of the M. gallisepticum spacers database,  Mammadou Sall  for his contribution to the study of spacers and  Alain Blanchard  for much appreciated discussions on the work and manuscript.
At the individual cow level, suboptimum fertility, mastitis, negative energy balance, and ketosis are major issues in dairy farming. These problems are widespread on dairy farms and have an important economic impact. The objectives of this study were (1) to assess the potential of milk mid-infrared (MIR) spectra to predict key biomarkers of energy deficit (citrate, isocitrate, glucose-6 phosphate [glucose-6P], free glucose), ketosis (β-hydroxybutyrate [BHB] and acetone), mastitis (N-acetyl-β-d-glucosaminidase activity [NAGase] and lactate dehydrogenase), and fertility (progesterone); (2) to test alternative methodologies to partial least squares (PLS) regression to better account for the specific asymmetric distribution of the biomarkers; and (3) to create robust models by merging large datasets from 5 international or national projects.