COPIA-R7 retrotransposon into RPP7 co-opted the epigenetic transposon silencing signal H3K9me2 to a new function promoting expression of this Arabidopsis thaliana NLR gene. Recruitment of the histone binding protein EDM2 to COPIA-R7-associated H3K9me2 is required for optimal expression of RPP7. By profiling of genome-wide effects of EDM2, we now uncovered additional examples illustrating effects of transposons on NLR gene expression, strongly suggesting that these mobile elements can play critical roles in the rapid evolution of plant NLR genes by providing the “raw material” for gene expression mechanisms. We further found EDM2 to have a global role in NLR expression control. Besides serving as a positive regulator of RPP7 and a small number of other NLR genes, EDM2 acts as a suppressor of a multitude of additional NLR genes. We speculate that the dual functionality of EDM2 in NLR expression control arose from the need to compensate for fitness penalties caused by high expression of some NLR genes by suppression of others. Moreover, we are providing new insights into functional relationships of EDM2 with its interaction partner, the RNA binding protein EDM3/AIPP1, and its target gene IBM1, encoding an H3K9-demethylase.Plant NLR-type receptors serve as sensitive triggers of host immunity. Their expression has to be well-balanced, due to their interference with various cellular processes and dose-dependency of their defense-inducing activity. A genetic “arms race” with fast-evolving pathogenic microbes requires plants to constantly innovate their NLR repertoires. We previously showed that insertion of the  Arabidopsis thaliana PHD-finger protein EDM2 to serve as a chromatin-associated factor controlling expression of the NLR-type immune receptor gene RPP7. EDM2 binds to the transposon-silencing signal H3K9me2 and affects levels of this epigenetic mark at various loci. By genome-wide profiling of transcript- and H3K9me2-levels we now found EDM2 to have a broader role in controlling NLR gene expression. In order to mitigate fitness costs caused by its promoting effects on RPP7 expression and that of several other NLR genes, EDM2 seems to suppress expression of many additional members of this gene family. This observation is in line with multiple reports demonstrating the need for balanced expression of NLRs, which can substantially reduce overall plant fitness, but need to be present at certain minimal levels to confer sufficient immune protection. Our previous results demonstrated that the influence of EDM2 on RPP7 expression was co-opted to this immune receptor gene by the insertion of an EDM2-controlled transposon. Here, we are providing additional examples for transposon-associated effects on NLR gene expression, suggesting that these mobile elements play an important role for NLR genes by equipping members of this rapidly evolving gene family with regulatory mechanisms needed for balanced expression.We previously found the  AVR) gene products, these receptors induce a set of strong defense reactions, such as the hypersensitive response (HR), a form of local programmed cell death. A genetic “arms race” with fast-evolving pathogenic microbes requires plants to constantly innovate their NLR repertoires enabling them to maintain the capacity to recognize their microbial foes. Proper homeostasis of NLR activity is critical for their function . Le. LeRPP7/RPP7) is affected by EDM2. In edm2-2 compared to WT a total of 59 of 165 annotated Arabidopsis NLR loci show a significant change of their H3K9me2 and/or transcript levels  reduction of transcript levels in edm2-2 , while in most other cases, EDM2 may limit their expression to levels that are non-detrimental for proper plant development and fitness, thereby serving as a suppressor of constitutive defense activation.Collectively our observations show that EDM2 plays a role in balancing transcript levels of NLRs. In the case of edm2-2 compared to WT, while 612 TEs show the opposite behavior. The vast majority of EDM2-affected TEs are retrotransposons, such as Gypsy (63.94%), Copia (7.86%) or non-LTR/LINE (3.65%) elements . RPP7-mediated resistance against HpaHiks1 and wild-type RPP7 mRNA levels are restored in this line [RPP7 and RPP4 as well as other loci of interest for this study  and COPIA4 (AT4TE42860). Among those, we also observed direct association of HA-EDM2 with the retrotransposons AT1TE12295 and AT3TE06550  and feature high levels of EDM2-dependent H3K9me2  compared to WT  reduction of disease resistance in Arabidopsis cotyledons against the RPP4 cognate Hpa isolate Emoy2 (RPP4 in edm2 mutants and not due to some unspecific (or pleiotropic) effects. This view is supported by the fact that the NLR genes RPP2A and RPP2B, which are not affected in their expression in edm2-2, remain fully functional in this mutant. The NLR pair RPP2A and RPP2B is know to mediate in Col accessions of Arabidopsis immunity against the Hpa isolate Cala2 [RPP2A and RPP2B significant differences of reads mapped to their transcriptional units between edm2-2 and Col-0. Neither does close inspection of genome browser views of RNA-seq data for both genes hint at minor local differences in RNA expression/processing  and one member of RPP1 cluster (AT3G44400)  and AT5G44870  . Our HA-T1G59218 . In eachT5G44870 , for whiT5G44870  immediatedm2-2 plants compared to WT. Our genome profiling analysis further uncovered effects of EDM2 on H3K9me2 and the expression of numerous Arabidopsis NLR genes. As for RPP7, the effect of EDM2 on these genes seems, at least in some cases, linked to roles of EDM2 in controlling silencing states of nearby TEs. TEs have long been suspected to be critically important for the fast evolution of structural NLR diversity by facilitating non-homologous recombination events in NLR gene clusters [This study demonstrates a broad role of EDM2 in controlling silencing marks at TEs. Over 2,000 TEs, predominantly Copia and Gypsy retrotransposons, exhibit altered H3K9me2 and/or transcript levels in clusters , 40. Togclusters , 29–31 osuvh456 mutant . We identified 6,082 genes and 2,706 TEs with significant changes in H3K9me2 levels in ibm1-4 compared to WT  and ibm1-6 [edm2-2. As shown in supplementary figures  EDM2 has IBM1 independent functions and (2) interactive relationships between IBM1 and EDM2 are complex and variable.Feedback mechanisms may further be responsible for reciprocal effects of d ibm1-6  mutants  figures  Figs varRPP7/IBM1 regulation. Through regulation of IBM1, EDM2 has also been implicated in global suppression of CHG methylation in genic regions associated with heterochromatin. In this study, we expanded on previous findings by genome-wide H3K9me2 ChIP-seq, RNA-seq and HA-EDM2 ChIP-seq analysis. Our new data show that EDM2 controls genome-wide expression of NLR genes and TEs, as we observed in edm2-2 plants effects on H3K9me2 and/or transcript levels at 59 NLR genes and over 2,000 TEs.EDM2 was previously shown to be a chromatin-associated protein controlling expression of the RPP7 immune receptor and the histone H3K9 demethylase IBM1 by promoting the synthesis of full-length transcripts encoding these proteins. EDM2 has also been reported to control silencing states of some TEs, and its physical association with heterochromatic TE/repeat sequences was found to be critical for its function in RPP7 expression. EDM2 is physically associated with chromatin of the Columbia RPP5 cluster members RPP4 and AT4G16900 as well as TEs directly adjacent to these NLR genes. For both RPP4 and AT4G16900, H3K9me2 seems to have spread from the adjacent TEs into genic regions of both NLRs. As with RPP7, EDM2 promotes the synthesis of full-length transcripts of RPP4 and suppresses its proximal transcript polyadenylation/termination. This effect is also dependent on high H3K9me2 levels at the respective proximal polyadenylation site.In some cases, the involvement of EDM2 in NLR gene regulation resembles its role in controlling RPP4 expression are complex, as we found it also to increase expression of the TIR domain-encoding exon 1 of this NLR. If this observation is related to the use of alternative transcription start-sites identified downstream from exon 1 [RPP4 expression, EDM2 is required for maximal function of RPP4 in mediating resistance against the H. arabidopsidis isolate Emoy2 in Arabidopsis cotyledons. Highlighting the critical importance of COPIA4 in this context, mutation of this retrotransposon by a T-DNA insertion or silencing of its expression resulted in reduced cotyledon resistance against another RPP4-recognized Hpa isolate, Emwa1 [Effects of EDM2 on m exon 1  remains m exon 1 , 47. Cone, Emwa1 .RPP7, RPP4 and At4g16900), it acts as a suppressor of a significantly larger set of NLR genes . Thus, mechanistic details, of the suppressive effect of EDM2 on these 40 NLR genes may vary from case to case and they are likely indirect targets of EDM2.While EDM2 promotes expression of a small number of NLR genes  almost four-times more NLR genes are up-regulated than down-regulated and which, therefore, likely exhibit a strong net-increase of NLR background activity. Up-regulation of NLR expression has been linked to reduced fitness and constitutive immunity in several Arabidopsis mutants before. For example, the bal mutant contains an extra copy of the NLR gene SNC1 and, consequently, exhibits elevated expression of this gene as well as substantially stunted growth, activated defense response mediated by salicylic acid and enhanced immunity against Pseudomonas syringae bacteria [cpr1 mutant is deficient in an F-box protein mediating degradation of the NLRs SNC1 and RPS2. Higher levels of these NLRs are correlated with reduced growth and enhanced immunity against various pathogens in this mutant [edm2-2 is a consequence of some disturbance of cellular homeostasis associated with reduced fitness and an indirect pleiotropic effect, rather than the cause of fitness costs. However, we consider this as unlikely, given that causality between NLR overexpression and reduced fitness has been established in several cases [However, the observation that such a large number of NLR genes is suppressed by EDM2, while the sets of all genes it acts on as a positive and negative regulator are equally large, appears compelling to us. Clearly, EDM2 has evolved into a role as a broad suppressor of NLR genes in Arabidopsis. Such a role is consistent with the enhanced defense against bacteria , 8. The s mutant , 49. Nonal cases , 49.EDM3 mutant aipp1 [edm2 , transposons also seem to nucleate the formation of H3K9me2, which spreads into the neighboring genes affecting their expression. Interestingly, this heterochromatic transposon silencing signal does not silence the genes’ transcription (promoter silencing), but serve the different function to suppress proximal genic transcript polyadenylation. As we observed for RPP7, RPP4, AT4G16900, AT1G11270 and AT3G05410, EDM2-mediated effects on H3K9me2 and/or transcript levels are likely recruited by insertions of TE insertions to these sites.In addition to its role in controlling NLR gene expression we found EDM2 to control silencing states of a large number of transposons. Our results further show that the influence of EDM2 on the expression of some NLR genes seems recruited into the context of these genes by insertions of TEs. A large body of literature supports a critical function of TEs in the evolution of structural and functional diversity of plant NLR genes. NLR genes are one of the fastest evolving and structurally diverse gene families in plants. Their association with TEs and organization in complex gene clusters is believed to be critical for the fast pace of their structural and functional diversification , 51. Frensposons , 56. Takof genes , 57, 58.of genes  and currCOPIA-R7, additional TE sequences affect the complex regulation of RPP7. Several TEs and other repeat sequences in its 5’UTR seem to attract massive cytosine methylation to a region directly upstream of the transcription start-site [SimpleHAT DNA transposon in the distal promoter of RPP7 to provide a docking site for an unknown DNA binding activity and to serve as a pathogen-responsive cis-element [COPIA-R7 and COPIA4) illustrating the ability of TEs to equip NLR genes with regulatory mechanisms include the AtCOPIA93-derived solo LTR upstream from RPP4, which mediates pathogen-responsive transcriptional upregulation of this NLR gene [Renovator which is inserted upstream of the rice NLR gene Pit and provides promoter sequences directing high transcript levels of this gene [N, which contains the miniature inverted-repeat transposable element MiS1-1, that serves as a differentially expressed alterative exon of critical importance for the function of this disease resistance gene [Besides art-site . However-element . AdditioNLR gene , the LTRhis gene , as wellnce gene , 30.While recruitment of regulatory mechanisms by TEs is observed for many types of genes , it is lWhile our study provides examples related to EDM2-controlled NLRs, the general statistical association of TEs with NLR genes suggests that this role extends to a wide variety of different TEs. From a broader perspective, TEs are also emerging as important factors for the function and evolution of pathogen effector genes and proteins that promote virulence or are recognized as avirulence proteins by NLR receptors , 64. ThuRPP7, which requires EDM2 for optimal levels of expression, causes substantial loss of fitness in Arabidopsis. Loss-of-function mutants of RPP7 are clearly more vigorous than wild type plants . Based on these observations, it is tempting to speculate that the role of EDM2 in global NLR gene suppression serves the purpose to compensate for fitness penalties caused by high expression of RPP7 and possibly other NLRs, whose expression is promoted by EDM2. Cooption of EDM2 to roles in promoting expression of certain NLRs may not have been possible without suppressing at the same time expression of other members of this immune receptor gene family.The dual nature of EDM2 functions in NLR expression control  is unlikely coincidental and both functions may be causally linked. Numerous studies have implied that plants have limited tolerance for NLR expression, and that certain thresholds levels for NLR transcripts cannot be exceeded , 65, 66.E2pro:HA-E2c plants is undetectable by Western Blotting). Thus, in vivo-detection of this protein by ChIP appears only possible at a limited set of strong target sites. While our ChIP-seq experiments allowed us to clearly establish certain direct target sites of EDM2, we have almost certainly missed many loci bound by this protein, limiting our ability to draw conclusions about its general mechanistic roles in gene regulation. Only at a small number of loci, such as RPP7 and IBM1, such mechanistic details have been uncovered. In each of these cases, EDM2 serves as a chromatin binding protein, recruiting the RNA binding proteins ASI1/IBM2 and EDM3/AIPP1 to intronic heterochromatic regions [RPP7 and IBM1, this complex promotes the synthesis of full-length transcripts, by suppressing proximal polyadenylation, the outcome at other loci may be different and affected by differences of the stoichiometry and function of the various complex components.While our results provided substantial new insight into genome-wide roles of EDM2, they leave room for speculation regarding EDM2’s mechanisms of action at its target genes. We previously observed that gene regulatory roles of EDM2 are context dependent and vary from locus to locus , 22. Our regions , 67, 68. regions  the EDM2IBM1, we also found 299 transcription factor genes to be affected by EDM2. These regulators likely cause additional indirect effects and may be responsible for many EDM2-dependent transcript level changes not associated with changes of H3K9me2 levels.Most effects observed in our study are likely indirect and mediated secondarily by EDM2 controlled regulators. Some of these are mediated via the H3K9-demethylase IBM1 . Our obsedm2-2, EDM2-controlled NLR genes vary regarding changes of H3K9me2 and their dependency on IBM1  were obtained from the Arabidopsis Biological Resource Center . The edm2-2 (SALK_014520), edm2-3 (SALK_114312) [E2pro:HA-E2c) [suvh4 suvh5 suvh6 triple mutant (suvh456) was kindly provided by Dr. Judith Bender . All Arabidopsis mutants used in this study are in Col-0 background. All Arabidopsis seedlings were grown on soil or half-strength Murashige and Skoog (1/2 MS) solid medium containing 1% (w/v) sucrose in a growth chamber .The Arabidopsis ecotype Columbia (Col-0) and -2 SALK_0520, edm2:HA-E2c)  and Oy1 :HA-E2c)  were desTM kit . Reverse transcription was conducted by Maxima reverse transcriptase  with 100 pmol of oligo (dT)18. qRT-PCR was performed with the CFX Connect detection system (Bio-Rad) using iQ SYBR Green Supermix . Actin8 was served as an internal control. All the primers used for qRT-PCR are listed in Aerial parts of 2-week-old plants were harvested from 1/2 MS solid plates and used for total RNAs isolation. Total RNA was isolated using TRIzol reagent  and treated with TURBO DNA-freeH. arabidopsidis isolate Hpa-Emoy2 were described previously [4 spores/mL) using Preval sprayers . The extent of infections was determined by counting visual sporangiophores at 7 dpi.The growth condition, propagation and application of eviously . Two-weeTM kit , following the manufacturer’s instruction. All primers used for RACE are listed in The 3’ RACE were carried out using the GeneRacerAerial parts of 2-week-old plants were harvested from 1/2 MS solid plates and two grams of seedlings per sample were used for Chromatin Immunoprecipitation (ChIP) assays. ChIP was performed as descried previously  using anTM II DNA library prep kit for Illumina  according to the manufacturer’s instruction. Libraries were sequenced on the Illumina NextSeq500 generating 75 bp -single-end sequence reads.ChIP-seq libraries were prepared using NEBNext Ultrahttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and any base with a quality score below 25 or N was trimmed using Sickle (https://github.com/najoshi/sickle). Trimmed reads were then mapped to the A. thaliana genome (TAIR 10) using BWA 0.7.15-r1140 with mem option default parameters [edm2-2 samples, non-expressed transcripts with coverage value below 1 in all libraries were removed. Transcripts representing differentially methylated regions were determined using DEseq2 in R [P-adjusted value of 0.05 and a 1.2 fold change.For each Chip-seq library, raw reads quality was first analyzed using FastQC  to eliminate the genomic DNA contamination. Five micrograms of DNA-free total RNA was treated with Ribo-Zero rRNA Removal Reagents (Plant Leaf) to remove Ribosomal RNA. RNA-seq libraries were prepared using NEBNext UltraTM Directional RNA library prep kit for Illumina  according to the manufacturer’s instructions. Libraries were sequenced on the Illumina NextSeq500 platform generating 2×75 bp pair-end sequence reads.Total RNAs were isolated from two-week-old plants grown in 1/2 MS solid plates using TRIzol reagent . Ten micrograms of total RNA were treated with TURBO DNA-freehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The first 10 bases and the last base were trimmed. Contaminating adaptor reads, reads that were unpaired, bases below 25 and Ns, and reads shorter than 18 bases are also filtered using Sickle (https://github.com/najoshi/sickle). All trimmed reads were mapped to A. thaliana genome (TAIR 10) using HISAT2 v2.1.0 [P-adjusted value of 0.05 and a 1.2 fold change. RNA-seq raw data for ibm1-6 and the corresponding WT were obtained from NCBI (GEO: GSE93024) and processed using the same parameters described above.After sequencing, the quality of raw reads was analyzed using FastQC  [For genome browser tracks, read coverage per nucleotide is calculated using BEDTools. Coverage values were then normalized per million mapped reads. Genome tracks were displayed in IGV (Integrative Genome Viewer) . For HA-E111609) .P-value for all re-sampling in bedmap, we calculated the cumulative length of TE annotation (from TAIR10) present in the surrounding area of the genic mRNA from 0 to 1,000 bp with a step of 100 bp. Statistical significance was tested using an ANOVA test by randomly re-sampling 1,000 time 165 non- NLR genes and reported the average P-value.The proportion of TE distribution was calculated based on the ratio of TE family size divided by the total size of TEs present in each category. Significant differences between the proportion of EDM2-controlled TEs and the proportion of TEs within the chromosome arms or pericentromeric regions was determined using annotation from Kawabe et al.  and the Pst DC3000 suspension containing 2 × 108 cfu/mL in 10 mM MgCl2 with 0.04% silwet L-77. Leaves were harvested at 3 hrs, 2 dpi and 3 dpi. Each leaves sample was weighed, surface sterilized in 70% ethanol for 30 s and washed in sterile distilled water for 30 s before ground in 10 mM MgCl2. Serial dilutions were plated on King’s B plates with appropriate antibiotics. Each data point represents the average of three replicates. Each replicate contains the bacterial titer of average of three individual pots with a density of 20 seedlings.Bacterial growth and inoculation were performed as previously described . Two-weeAnti-HA beads were used to pull-down HA-EDM2 fusion protein after chromatin precipitation. The protein samples were trypsin-digested and analyzed with a MudPIT method described previously  with somS1 Figedm2-2 are shown in each tracks. The y-axis represents coverage values .H3K9me2, H3C and input ChIP-seq for WT and (TIF)Click here for additional data file.S2 FigSpearman correlation for replicates of ChIP-seq (A) and RNA-seq (B) analyses.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig-axis represents coverage values .The y(TIF)Click here for additional data file.S5 FigRPP4 locus(A) Genome browser view of HA-tagged EDM2 ChIP-seq, H3K9me2 ChIP-seq and RNA-seq data at . The y-axis represents coverage values . (B) Genome browser view of WT and ibm1 RNA-seq data at RPP4 locus with different data ranges.(TIF)Click here for additional data file.S6 FigAll detected y-series as well as b-series fragment ions are labeled. Many neutral-loss fragments are also detected but not labeled here. Both Sequest HT and Mascot search engines matched this spectrum to an EDM2 peptide, 395—EISFEDIEDEDILTR—409, with high confidence. Summary for two mass spectrometry identified EDM2 peptides are shown in the bottom.(TIF)Click here for additional data file.S7 FigEDM2 (A) and IBM1 (B) loci.(A and B) Genome browser view of HA-tagged EDM2 ChIP-seq, H3K9me2 ChIP-seq and RNA-seq data at the  The y-axis represents coverage values .(TIF)Click here for additional data file.S8 Fig(A and B) Genome browser view of HA-tagged EDM2 ChIP-seq, H3K9me2 ChIP-seq and RNA-seq data at AT1G11270 (A) and AT3G05410 (B) loci. The y-axis represents coverage values .(TIF)Click here for additional data file.S9 Fig(A) Schematic representation of RPP4 with two alternative RNA transcript isoforms. (B) Nucleotide sequence of RPP4 exon 6. Coding nucleotides of exon 6 are in upper case black letters. Lower case letters in red are 3’UTR. The 130 bp of 5’LTR sequence for COPIA4 are shown in bold. Polyadenylation sites determined by 3’RACE are labeled by numbers 1–8. Blue underlined sequences between areas a (#5- #6) and b (#6- #8) indicate regions used for read counts shown in (C) Amino acid sequence of protein isoforms encoded by RPP4. Purple: TIR domain; Blue: NB-ARC domain; Green: Leucine-rich repeat (LRR) domain, each one of 18 putative LRRs (predicted by Uniprot: F4JNA9) is underlined in pink. (D) Genome browser view of edm2, suvh456 and respective WT RNA-seq data at RPP4 3’UTR. Red arrows indicate polyadenylation sites 5–8 shown in (B). Blue underlined areas a (#5- #6) and b (#6- #8) indicate regions used for read counts shown in (E) Genome browser view of TSSs at RPP4. Genome tracks of two biological replicates for Col-0 were shown. TSS-seq data were obtained from NCBI (GEO: GSE113677)[(F and G) Genome browser views of edm2 and WT RNA-seq data at RPP2A (F) and RPP2B (G).(TIF)Click here for additional data file.S10 Fig-axis represents coverage values .The y(TIF)Click here for additional data file.S11 FigGenome browser view of HA-tagged EDM2 ChIP-seq, H3K9me2 ChIP-seq and RNA-seq at AT3G44630 and AT3TE65615 (A) and AT5G44870 and AT5TE65325 (B) loci. The y-axis represents coverage values .(TIF)Click here for additional data file.S12 FigGenome browser view of AT5G41740 and AT5G41750 (A) and AT5G47260 and AT5G47280 (B). The y-axis represents coverage values .(TIF)Click here for additional data file.S13 FigThe AGI numbers for these loci are AT1G17600, AT1G17610, AT1G58390, AT3G07040, AT3G25510, AT3G44400, AT5G18360, AT5G46260, AT5G46520 and AT5G66630. The y-axis represents coverage values .(TIF)Click here for additional data file.S14 Fig-axis represents coverage values .The AGI numbers for these loci are AT1G12290, AT1G33560, AT1G56510, AT1G56540, AT1G63860, AT1G63880, AT1G72860, AT1G72870, AT3G50950, AT3G51560, AT3G51570, AT4G16960, AT4G27220, AT5G05400, AT5G17680, AT5G18370, AT5G22690, AT5G38340, AT5G41550, AT5G45240, AT5G46450, AT5G46510 and AT5G48770. The y(TIF)Click here for additional data file.S15 Fig-axis represents coverage values .The AGI numbers for these loci are AT1G58400, AT1G63750, AT4G14370, AT4G33300 and AT5G58120. The y(TIF)Click here for additional data file.S16 Fig-axis represents coverage values .The AGI numbers for these loci are AT3G14460 and AT3G14470. The y(TIF)Click here for additional data file.S17 Fig-axis represents coverage values .The AGI numbers for these loci are AT1G59124 and AT4G16920. The y(TIF)Click here for additional data file.S18 Fig-axis represents coverage values .The AGI numbers for these loci are AT1G58602, AT1G59620, AT1G62630, AT1G72910, AT2G14080, AT4G16860 and AT5G43740. The y(TIF)Click here for additional data file.S19 Figχ2 test of independence showed significant differences between actual and expected equal distribution (50% up-regulated and 50% down-regulated genes). *: P-value <0.05. ***: P-value <0.001.(TIF)Click here for additional data file.S20 Figedm2-2, aipp1 and ibm1-6 transcriptionally up-regulated (A) and down-regulated (B) NLR genes.Venn diagram showing overlaps between (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(A) and transcript levels (B) in edm2. (C) Arabidopsis defense-associated genes showing up-regulated changes of transcript levels in edm2. (D) Arabidopsis defense-associated genes showing down-regulated changes of transcript levels in edm2. (E) Arabidopsis NLRs show significant changes of H3K9me2 levels in ibm1.Arabidopsis NLRs show significant changes of H3K9me2 levels (XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(A) and TEs (B) showing significant association with HA-EDM2 identified by mapping unique (A) or non-unique (B) reads.List of genes (XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(A) and TEs (B) showing significant changes of H3K9me2 and/or transcript levels in edm2 and/or ibm1.List of HA-EDM2 associated genes (XLSX)Click here for additional data file.S10 Table(XLSX)Click here for additional data file.S11 Table(XLSX)Click here for additional data file.


The enhancing effect on reverse transcription by IP6 and the consequences of interactions between CA and nucleotides were corroborated using atomic force microscopy, transmission electron microscopy, and virological assays. Collectively, our results provide an atomistic description of the permeability of the HIV-1 capsid to small molecules and reveal a novel mechanism for the involvement of metabolites in HIV-1 capsid stabilization, nucleotide import, and reverse transcription.Reverse transcription, an essential event in the HIV-1 life cycle, requires deoxynucleotide triphosphates (dNTPs) to fuel DNA synthesis, thus requiring penetration of dNTPs into the viral capsid. The central cavity of the capsid protein (CA) hexamer reveals itself as a plausible channel that allows the passage of dNTPs into assembled capsids. Nevertheless, the molecular mechanism of nucleotide import into the capsid remains unknown. Employing all-atom molecular dynamics (MD) simulations, we established that cooperative binding between nucleotides inside a CA hexamer cavity results in energetically favorable conditions for passive translocation of dNTPs into the HIV-1 capsid. Furthermore, binding of the host cell metabolite inositol hexakisphosphate (IP This study shows that the HIV-1 capsid protein, in addition to its structural role, regulates reverse transcription, an essential metabolic process of the virus, by mediating the import of nucleotides. In addition, host cell metabolites such as inositol phosphates are recruited by the capsid to regulate viral DNA synthesis. Fusion between HIV-1 virions and target cells engenders the release of the viral capsid into the host cell cytoplasm. The capsid, a cone-shaped protein assembly composed of approximately 250 capsid protein (CA) hexamers and 12 CA pentamers, encapsulates 2 copies of the viral single-stranded RNA (ssRNA) genome and viral proteins, including reverse transcriptase (RT) and integrase (IN) . Success6) have been reported to bind to the HIV-1 Gag polyprotein and CA hexamers, affecting virion assembly and reverse transcription [6 stabilizes the core by interacting with positively charged residues located near the central axis of a CA hexamer. Nevertheless, CA hexamers have been shown to be stable in the absence of IPs. In addition, we and others have proposed that reverse transcription mechanically induces changes in capsid morphology and triggers uncoating [Inositol phosphates (IPs) are abundant cellular metabolites that play crucial roles in fundamental cellular processes . Interescription ,12–16. Incoating ,18. To sncoating ,4,8.6 on HIV-1 capsid stability and reverse transcription and determined the molecular mechanism that regulates translocation of dNTPs through the capsid.To test the hypothesis that IPs and other metabolites modulate the translocation of dNTPs through CA oligomers, we have employed a multipronged approach combining all-atom molecular dynamics (MD) simulations , atomic The CA hexameric cavity is surrounded by 6 copies of a β-hairpin, helix 1, and a short loop connecting them . Its rad6 interacting with the central cavity , and monocyte-derived macrophages were infected with normalized amounts of WT or K25N HIV-1NL4-3 that encoded the luciferase reporter gene. While WT HIV-1 infected all cell types tested, K25N was noninfectious , and IP6 binding increased their stiffness by nearly 2-fold to an average value of 0.245 ± 0.021 N/m (n = 36)  and IP6 increased this value to 0.258 ± 0.035 N/m . However, in contrast to untreated WT capsids, IP6-treated capsids progressively lost their stiffness over time, reaching a minimum of 0.078 ± 0.013 N/m (n = 7) at 7 h; the minimum stiffness value was reached earlier for capsids treated with IP6 than for capsids without IP6 (5 h compared to approximately 20 h) [6 initially stiffens the capsids, then induces faster softening after initiation of reverse transcription. In contrast, addition of the synthetic molecule BHC resulted in a smaller initial increase in stiffness  . Statist6 on reverse transcription, we analyzed the morphologies of IP6-treated WT capsids during reverse transcription by AFM operated in the quantitative imaging mode. Prior to reverse transcription, capsids had a well-defined conical appearance. A representative capsid  is shown in 2 to induce reverse transcription, openings at various sizes in the capsid appeared . Analysis of the reactions beyond 7 h of reverse transcription revealed [6 accelerated capsid disassembly during reverse transcription.To further characterize the effects of IPappeared . Similarrevealed  mostly f6 did not affect CA retention of K25N/E45A capsids  remained unchanged over the experimental time course . Although addition of IP capsids , we nonee course , which i6 within the channel provided the required energy to release the initial dNTP within the capsid lumen.Reverse transcription and the consequent disassembly of the HIV-1 capsid are fueled by the ability of the capsid to import dNTPs. Free energy calculations revealed a molecular dynamic view of the interaction between a freely diffusing nucleotide and the HIV-1 CA hexamer . First, 6 but are unstable in cells where IP6 concentrations are high. Thus, capsid instability can be independent of IP6, which we tested here by combining the hyperstable E45A change with K25N. The lack of IP6 stabilization of K25N capsids may be due to a similar general hypostability. Alternatively, the source of K25N hypostability could stem from the interaction between IP6 with Lys25 in CA pentamers, thereby reducing the IP6 stabilizing effect. To investigate this possibility, we performed MD simulations of IP6 bound to CA pentamers, which revealed 100% occupancy of Lys25 by IP6  stabilizing capsid pentamers by interacting with K25; and (2) regulating nucleotide import.Hypostable capsid mutants, such as K203A, likely bind IP5 by IP6 . The obs6 promotes dNTP import while BHC inhibits it.Ion channels and transporters have evolved to employ a cooperative translocation mechanism to displace cargos between distinct environments ,34. Our 6 enhances dNTP import for efficient reverse transcription. The discovery of metabolite-dependent nucleotide-specific import provides a unique target for development of new therapies against HIV-1.Recent findings indicate that HIV-1 reverse transcription terminates in the nucleus where dNTP concentrations are comparatively high ,35,36. PNL4-3 wild-type sequence, and the complete pentamer was then rigid body docked, using Chimera, into the electron microscopy density of the pentamer obtained from intact particles (EMD accession code EMD-3466). To obtain an atomistic model of the 5MCY pentamer, flexible fitting was performed employing the MDFF method as follows [The initial coordinates of the cross-linked pentamer, P17C/T19C and N21C/A22C, model were directly derived from the crystal structure (PDB accession code 3P05), missing loops were added using Modeler . Subsequ follows . Ions ne follows . Bulk wa follows . MDFF wa follows  with the follows . The resThe initial coordinates of the HIV-1 CA hexamer  were gencoordnum in Colvars [Parameters for the negatively charged small molecules , except  Colvars .6/BHC were placed in the central cavity of the CA hexamer model. The models were then solvated using the TIP3P water model [Small charged molecules such as NTP/IPer model . Addition in VMD , and then in VMD . The tot6/BHC molecules were fixed. In the second stage, the backbone atoms of the CA protein were restrained with a force constant of 10.0 kcal mol−1 Å−2. Convergence of the minimization procedure was confirmed once the variance of the gradient was below 0.1 kcal mol−1 Å−1. Following minimization, the systems were tempered from 50 K to 310 K in increments of 20 K over 1 ns. Subsequently, the systems were equilibrated at 310 K for 100,000 steps, while the protein backbone atoms were restrained. Equilibration of the systems was assessed using the RMSD trace versus time plateauing at 2 Å  of interest. The origin of the progress variable was set to the center of mass of Cα atoms in the N-terminal domain of the CA hexamer (−1 Å−2 using Colvars [Progress variables (PVs), akin to reaction coordinates, for all free-energy molecular dynamics calculations were chosen as the location on the  hexamer . One- an Colvars . The wid6, or BHC in the CA cavity. The initial configurations for the 2D simulations were derived by pulling dATP using constant velocity steered MD [6/BHC was restrained with a harmonic force constant of 2.5 Kcal mol−1 Å−2 using Colvars  ,54. In Wk-th slice was calculated using the equation:Nbins), Gi, k and Gi, reference are the free energies at the i-th PMF bin for the k-th and last slice, respectively. Convergence of 2D PMF was established once the RMSE was not larger than 3.0 kcal/mol. The bins before convergence were discarded. Subsequently, utilizing the converged slices, the 2D PMF surfaces  . Each 2Dsurfaces  and theisurfaces  were comsurfaces .2. Human PBMCs were isolated from leukopheresis packs obtained from the Central Blood Bank  via Ficoll-Paque Plus  density gradient centrifugation, following manufacturer’s instructions, and stimulated with 50 U/mL recombinant interleukin-2  and 5 μg/mL phytohaemagglutinin  for 48 to 72 h prior to infection. CD14+ monocytes were purified from PBMCs via CD14 MicroBeads  magnetic cell sorting, following manufacturer’s instructions, and differentiated into macrophages with 50 ng/mL granulocyte macrophage colony-stimulating factor  for 6 d prior to infection.HEK 293T, GHOST, and HeLa cells were maintained at 37°C in Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated bovine serum, 1% penicillin-streptomycin, and 1% glutamine in 5% CO6 human embryonic kidney (HEK) 293T cells were transfected with 2.5 μg of ΔEnv IN- HIV-1 plasmid (DHIV3-GFP-D116G) [HIV-1 pseudovirions were used to isolate capsids as previously described ,56. BrieP-D116G)  using 10Capsids were isolated from concentrated virus-containing material using a previously described protocol  with mod6 was provided from the James laboratory as a generous gift. Measurements were carried out with a JPK Nanowizard Ultra-Speed atomic force microscope  mounted on an inverted optical microscope  using silicon nitride probes . Height topographic and mechanical map images were acquired in quantitative imaging (QI) mode, at a rate of 0.5 lines/s and a loading force of 300 pN. All measurements were carried out in MOPS buffer which contained 100 μM IP6 or mellitic acid (Sigma) when relevant.AFM measurements and analysis were performed as previously described,28. BrieCapsid stiffness was obtained by the nanoindentation method as previously described ,28,57. BThe maximum indentation of the sample was 4 nm, which corresponds to a maximum loading force of 0.2 to 1.5 nN. Prior to analysis, each curve was shifted to set the deflection in the noncontact section to zero. The set of force distance curves was then averaged . From thNL4-3 (pNLdE-luc) [Mutations were generated in previously described single-round plasmid derivatives of HIV-1LdE-luc)  that encLdE-luc)  and, forLdE-luc)  using LiLdE-luc) .2 in 100 mM MOPS buffer (pH 7.0)) [Reverse transcription was induced in isolated capsids attached to HMDS-coated microscope glass slides. To initiate reverse transcription, MOPS buffer was replaced with reverse transcription buffer (100 μM dNTPs and 1 mM MgClpH 7.0)) . All mea6 and adhered to 35 mm glass-bottom dishes  coated with Cell-Tak (Corning) for 30 min at 37°C following manufacturer’s instructions. Samples were washed with PBS and briefly permeabilized with 0.02% Triton X-100  for 30 s, then again washed with PBS and fixed with 2% paraformaldehyde (PFA). For IP6 studies, samples that had been adhered to glass and briefly permeabilized as above were additionally incubated for 2 h at 37°C in STE buffer with or without 100 μM IP6. To address the possibility that the preincubation brief permeabilization was transient and the virus membrane had resealed during the 2-h incubation (thereby trapping loose CA within the virus membrane), samples were briefly permeabilized again after incubation to ensure loose CA was able to disperse, prior to washing and fixation. Fixed samples were permeabilized with 0.1% Triton X-100, blocked with normal donkey serum, immunostained for CA/p24 using mouse anti-CA monoclonal antibody 24–4  and donkey anti-mouse IgG-Cy5, and mounted with coverslips. Confocal imaging was performed on a Nikon A1 laser scanning confocal microscope, with 6 fields imaged per sample. CA staining and mRuby3-IN-containing particles were modeled and enumerated with Imaris imaging analysis software . The number of stained CA puncta per field was normalized to the number of mRuby3-IN particles, which were taken as representing intact capsids. The normalized number of CA puncta was reported for each condition (with or without permeabilization) or reported as the percentage of non-permeabilized puncta retained upon permeabilization.Equal p24 amounts of virus containing mRuby3-IN packaged in trans were diluted in STE buffer ) supplemented with 100 μM IPCells were plated overnight in 24-well or 96-well plates and challenged with virus at equal amounts of p24. Virus infectivity was determined by luciferase production (Promega) after 48 h using a Synergy2 Multi-Detection Microplate Reader HeLa cells were plated in 6-well plates and infected with virus at equal amounts of p24 after treatment with DNaseI  for 30 min at 37°C. Control infections were performed in the presence of 25 nM rilpivirine. After 24 h, cells were washed with PBS, trypsinized, and pelleted, and DNA was extracted using the Blood Mini Kit . Early (RU5) and late (gag) HIV-1 reverse transcripts and 2-LTR circles were measured by qPCR as previously described . Values t test (infectivity assays and quantitative PCR) or 1-way ANOVA with Tukey multiple comparisons test (capsid stability assay) with Prism software . A p-value of less than or equal to 0.05 was used to indicate statistical significance.Results were analyzed for statistical significance by 2-sided Student Virus produced from HEK 293T cells was centrifuged to remove cells, filtered through a 0.45 μm Polyethersulfone (PES) syringe filter , transferred into 25 × 89 mm polyallomer ultracentrifuge tubes , and ultracentrifuged in an Optima XL-100K ultracentrifuge (Beckman Coulter) using an SW28 rotor at 25,500 rpm  for 2.5 h at 4°C. Following aspiration of supernatant, the pellet was fixed in FGP fixative ) for 2 h at room temperature and stored at 4°C. Ultrathin sections (60 nm) were cut on a Reichert Ultracut-S microtome, transferred to copper grids stained with lead citrate, and observed using a JEOL 1200EX microscope with an AMT 2k charge-coupled-device camera. Images captured at 30,000× magnification were visually inspected to classify viral particles as mature, immature, eccentric, or containing 2 apparent capsids, and over 100 particles were counted per virus preparation; representative TEM images are shown in 13C,15N labeled CA K25N and K25A mutants were isolated and purified using the protocol reported previously with minor modifications [6) (pH 6.0). CA assemblies were pelleted after the incubation for 1 h and stored at 4°C.U-ications . Pure prications  with minProtein was purified and assembled as previously described by the authors .After assembly, a small aliquot of each sample was removed and immediately stained with 2% uranyl acetate on copper grids. Images were collected using the TALOS F200C at the Keck Center for Advanced Microscopy and Microanalysis of Interdisciplinary Science and Engineering (ISE) Laboratory of the University of Delaware.1H, 13C, and 15N were 850.4, 213.8, and 86.2 MHz, respectively.MAS NMR experiments were performed on a Bruker 20.0 T narrow bore AVIII spectrometer. The Larmor frequencies of 13C, 15N-CA WT and K25A conical assemblies were packed into 3.2 mm thin wall MAS NMR rotors, and 13C-13C CORD data sets were acquired using a 3.2 mm E-Free HCN probe. The sample temperature was 5 ± 1°C controlled by the Bruker VT controller. The MAS frequency was 14.000 ± 0.001 kHz controlled by the Bruker MAS-3 controller. The 90° pulse lengths were 2.9 μs (1H) and 4.3 μs (13C). The cross-polarization contact time was 1 ms; a linear 90% to 110% amplitude ramp of was applied on 1H; the center of the ramp was at 82 kHz and Hartmann-Hahn matched to the first spinning sideband; ωRF = 54 kHz was applied on the 13C channel. SPINAL-64 1H decoupling [RF = 86 kHz) was applied during t1 and t2 periods. The CORD mixing time was 50 ms. The spectra were acquired as a 2,048 × 864 (t2 × t1) points complex matrix to the final acquisition times of 15.98 and 10.29 ms, respectively. States-TPPI protocol [The U-coupling   and 2.85 μs (13C), with TCP of 1 ms; a linear 90% to 110% amplitude ramp of was applied on 1H; the center of the ramp was at 100 kHz and Hartmann-Hahn matched to the first spinning sideband; ωRF = 20 kHz was applied on the 13C channel. TPPM 1H decoupling [RF = 10 kHz) was applied during t1 and t2 periods. The spectrum was acquired as a 2,048 × 800 (t2 × t1) points complex matrix to the final acquisition times of 15.98 and 9.84 ms, respectively. States-TPPI was used for frequency discrimination in the indirect dimension. A total of 256 transients were added up for each FID; the recycle delay was 2 s.The U-coupling   1D free energy landscapes of dATP binding to WT CA and indicated mutants. Compared to WT, K25A should result in leaky capsids, in contrast to K25N, K25E, and K25E/E28K, which each should result in blocked capsid cavities. Numerical data for panels A, B, C, and D can be found in 6, inositol hexakisphosphate; rNTP, ribonucleoside triphosphate; WT, wild-type.(A) Binding profiles of rNTPs show different binding affinities and specifics compared to dNTPs. (B) Binding profiles of nucleotide monophosphates, which are weaker compared to rNTPs and dNTPs. (C) Binding profile of BHC and IP(TIF)Click here for additional data file.S2 FigThe radius of the spheres represent the van der Waals radii for each nuclei, hydrogens are shown in white, phosphates are shown in yellow, oxygen in red, nitrogen in dark blue, and carbon in cyan. The NTD domain of CA is shown in yellow, illustrating helix 1 and the beta-hairpin region. (A) Two dATP molecules near R18 are shown. (B) Two dATP molecules one above R18 and the second below, interacting with K25 are shown. dATP, deoxyadenosine triphosphate; NTD, N terminal domain.(TIF)Click here for additional data file.S3 Fig6 and dNTPs also creates a gradient toward the interior, but almost twice as strong compared to dNTPs alone. (D) BHC inside of the CA cavity facilitates dNTP binding but not translocation. Numerical data for panels A, B, C and D can be found in 6, inositol hexakisphosphate.(A) Free energy profile for a single dATP interacting with CA. The profile indicates dNTP binding, but not translocation as the energy barrier is too high to be overcome by simple thermal fluctuations. (B) Cooperation between multiple dATP molecules shifts the free energy profile from binding to a gradient pointing toward the interior of the capsid. (C) Cooperation between IP(TIF)Click here for additional data file.S4 FigNumerical data for the plot can be found in (TIF)Click here for additional data file.S5 Fig(A) A significant dewetting of dNTPs is observed near Arg18, indicating a loss of conformational entropy. (B) Similarly, loss of water molecules is observed for rNTPs. The loss of entropy is compensated by the formation of salt-bridges between Arg18 and dNTPs (C) and rNTPs (D). (E) Correlation analysis between loss of solvation molecules and formation of bonds with Arg18 and NTPs. Importantly, solvation of the dNTP/rNTP as it moves toward the interior of the capsid is assisted by several charged or polar residues including K25/30 and E28/29. Numerical data for panels A, B, C, D, and E can be found in (TIF)Click here for additional data file.S6 Fig6 and (D) BHC to the ring of R18. (E) The angle of inclination is defined as the angle between the plane defined by the 6-membered rings and the plane of the CA-hexamer. Time evolution of the inclination angle for IP6 and BHC. The 2 molecules exhibit different inclination angles. (F) NCFGs within 3.4 Å of R18. (G) Number of R18 within 3.4 Å of IP6 and BHC. Numerical data for panels E, F, and G can be found in 6, inositol hexakisphosphate; NCFG, number of negatively charged functional group.(A) Molecular architecture of the R18 ring in hexamers as observed in the crystal structure (PDBID: 4xfx). (B) Molecular structure of BHC and myo-IP6. (C) Binding of IP(TIF)Click here for additional data file.S7 Fig6. (G–J) Negatively stained TEM images of WT  K25N (I) and K25N (J) CA show virus-like particles of mature morphology, namely tubes and cones. Assemblies in panel (G) contain 20 mg/ml CA in 50 mM Tris-HCl (pH 8.0), 2.4 M NaCl. Assemblies in panels H, L, and J contain 20 mg/ml of the respective protein in 50 mM MES (pH 6.0), 9 μM IP6. TEM, transmission electron microscopy; WT, wild-type.(A–F) Negatively stained TEM images of WT , K25N  and K25A  CA-SP1-NC. The mutants produce virus-like particles with immature morphologies. Assemblies in  contain 2 mg/ml of the respective protein in 50 mM Tris-HCl (pH 8.0), 100 mM NaCl. Assemblies in  contain 10 mg/ml of the respective protein in 50 mM MES (pH 6.0), 10 μM IP(TIF)Click here for additional data file.S8 FigThe assemblies contain 20 mg/ml of the respective protein in 50 mM MES (pH 6.0), 0.9 μM IP6. The similar chemical shifts indicate that K25N mutant is folded and its overall structure is the same as in the WT CA conical assemblies. CORD, combined R2vn-driven spin diffusion; IP6, inositol hexakisphosphate; MAS, magic angle spinning; RFDR, homonuclear radio-frequency driven recoupling; WT, wild-type.(TIF)Click here for additional data file.S9 FigP < 0.01; *** P < 0.001; **** P < 0.0001. Numerical data for panels A and B can be found (A) Schematic of the in vitro capsid stability assay. An intact virus membrane  traps loose CA, such that a stable and unstable capsid have similar stained CA fluorescence signals. Upon light permeabilization of the virus membrane (right), loose CA can diffuse from the virus particle and does not contribute to the stained CA fluorescence signal. Fluorescently labeled IN associated with the RNA genome remains trapped within the lightly permeabilized virus membrane. Comparing the IN-normalized CA staining retention upon light permeabilization permits differentiation of stable and unstable capsid. (B) Viruses packaging mRuby3-IN in trans with WT CA or CA bearing the indicated mutations were captured on glass either with or without prefixation virus membrane permeabilization, immunostained for CA, and imaged. The number of CA particles per IN particles is shown for each virus +/− permeabilization. E45A and K203A HIV-1 are included as examples of hyperstable and hypostable CA mutants, respectively. Error bars indicate SEM for 2 experiments. (C) The mean fluorescence intensity of CA staining for each imaged virus particle is shown, with the overall mean intensity for the population indicated by the purple bar. Representative results are shown from 1 experiment. ** (TIF)Click here for additional data file.S10 Fig6. Viruses were lightly permeabilized again, fixed, immunostained for CA, and imaged. (A) The number of CA particles per IN particles is shown for each virus +/− IP6. Error bars indicate SEM for 2 experiments. (B) The data from panel A are shown expressed as the fold change of CA staining retained for each virus with the addition of IP6 during incubation. (C) The mean fluorescence intensity of CA staining for each imaged virus particle is shown, with the overall mean intensity for the population indicated by the purple bar. Representative results are shown from 1 experiment. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Numerical data for panels A, B, and C can be found in 6, inositol hexakisphosphate; WT, wild-type.WT or mutant viruses containing mRuby3-IN were captured on glass, lightly permeabilized, and incubated for 2 h in STE buffer with or without 100 μM IP(TIF)Click here for additional data file.S11 Fig(A) Models based on the crystallographic cross-linked pentamer, and the cryoEM-derived model from intact HIV-1 particles. The figures illustrate the structures of IP6-bound CA pentamers before and after MD simulations. (B) Contact occupancy between CA pentamer residues and IP6. (C) Traces of the root mean squared deviations for alpha carbons in the CA pentamer models. The low RMSD illustrate that IP6 stabilizes both structures. Numerical data for panel C can be found in (TIF)Click here for additional data file.S12 Fig(A) The structure of an HIV-1 CA monomer and (B) hexamer. (C) The CA hexamer in a hexagonal water box with 150 mM NaCl and dATP. (D) A flat CA hexamer lattice formed by applying periodic boundary conditions.(TIF)Click here for additional data file.S13 Fig6, BHC, and dNTP and rNTP bases. BHC, benzenehexacarboxylic acid; dNTP, deoxynucleotide triphosphate; rNTP, ribonucleoside triphosphate.dNTP, rNTP, myo-IP(TIF)Click here for additional data file.S14 Fig(A) RMSD trace as a function of time during the equilibration phase of the systems. (B) RMSF of CA monomers during equilibration of the systems. (C) Convergence of the radius of the CA hexamers pores. Numerical data for panels A, B, and C can be found in the file (TIF)Click here for additional data file.S15 Fig6 and (C) BHC model, through 3 cycles of 5 ns HREX/US simulations. In each cycle, new US windows were generated to increase the sampling in 2D space. The initial conformations of the first cycles were extracted from SMD simulations pulling dATP through the hexamer central pore. After that, the initial conformations for new US windows (shown as yellow dots) were copied from the last frame of nearest previous US windows (shown as white dots). After 3 cycles, the generated seeds were used in 30 ns production runs. Numerical data for panel A, B and C can be found in the file 6, inositol hexakisphosphate; SMD, steered MD.(A) Generation of initial seeds for 2D HREX/US simulations of the 2 dATP models, (B) a dATP with IP(TIF)Click here for additional data file.S16 FigSequential changes of PMF from every 10 ns HREX/US simulations are shown. After 10 ns, the PMF profiles were used to compute the 1D PMF for NTP translocation. Numerical data for all panels can be found in the file (TIF)Click here for additional data file.S17 Fig6 and (D) dATP with BHC. Numerical data for panels A, B, C, and D can be found in the file 6, inositol hexakisphosphate; PMF, potential of mean force.(A) Root mean squared errors of three 2D HREX/US simulations with 0.5 ns. Standard deviations of the 2D PMF surface of panel (B) the 2 dATP models, (C) dATP with IP(TIF)Click here for additional data file.S18 Fig6 and BHC translocation simulations, and (C) the three 2D dATP translocation simulations. Numerical data for panels A, B, and C can be found in the file 6, inositol hexakisphosphate; rNTP, ribonucleoside triphosphate.(A) 1D rNTP translocation simulation, (B) 1D IP(TIF)Click here for additional data file.S19 Fig(A) Typical averaged force-distance curve of a core attached to an HMDS pretreated glass slide. For each experiment, approximately 400 such curves were acquired and averaged. Stiffness values were calculated by fitting a linear function to the force curve region bounded by 3- and 4-nm indentation depths. The corresponding line fit is plotted in red. (B) Histogram and Gaussian-fitted curves of the individual measured point stiffness values derived from the consecutive force-distance curves of a single core attached to a glass slide. (C) The individual measured point stiffness values obtained for the core shown in panel A, during a single experiment against the experiment number (count). The point stiffness measurements plot together with an analysis of the narrow distribution of the individual measured spring constants demonstrate that the core did not undergo a significant irreversible deformation during the indentation measurements. Numerical data for panels A, B, and C can be found in the file (TIF)Click here for additional data file.S20 FigMagnification is ×30,000 . The total number of WT, K25N, E45A, and K25N/E45A particles counted in each TEM experiment is shown in (TIF)Click here for additional data file.S21 FigMagnification is ×30,000 . The total number of WT, K25N, E45A, and K25N/E45A particles counted in each TEM experiment is shown in (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S1 Movie(MP4)Click here for additional data file.S2 Movie(MP4)Click here for additional data file.S1 DataNumerical data for panel D in (XLSX)Click here for additional data file.S2 DataNumerical data for panels A, C, and E in (XLSX)Click here for additional data file.S3 DataNumerical data for panels A, B, C, D, and E in (XLSX)Click here for additional data file.S4 DataNumerical data for panels A and B in (XLSX)Click here for additional data file.S5 DataNumerical data for panels A, B, C, and D in (XLSX)Click here for additional data file.S6 DataNumerical data for panels A, B, C, and D in (XLSX)Click here for additional data file.S7 DataNumerical data for the plot in (XLSX)Click here for additional data file.S8 DataNumerical data for panels A, B, C, D, and E in (XLSX)Click here for additional data file.S9 DataNumerical data for panels E, F, and G in (XLSX)Click here for additional data file.S10 DataNumerical data for panels A and B in (XLSX)Click here for additional data file.S11 DataNumerical data for panels A, B, and C in (XLSX)Click here for additional data file.S12 DataNumerical data for panel C in (XLSX)Click here for additional data file.S13 DataNumerical data for panels A, B, and C in (XLSX)Click here for additional data file.S14 DataNumerical data for panels A, B, and C in (XLSX)Click here for additional data file.S15 DataNumerical data for all panels of (XLSX)Click here for additional data file.S16 DataNumerical data for panels A, B, C, and D in (XLSX)Click here for additional data file.S17 DataNumerical data for panels A, B, and C in (XLSX)Click here for additional data file.S18 DataNumerical data for panels A, B, and C in (XLSX)Click here for additional data file.


Glycine max) is an important oil provider and ecosystem participant. The protein phosphatase 2C (PP2C) plays important roles in key biological processes. Molecular evolution and functional analysis of the PP2C family in soybean are yet to be reported.Soybean (The present study identified 134 GmPP2Cs with 10 subfamilies in soybean. Duplication events were prominent in the GmPP2C family, and all duplicated gene pairs were involved in the segmental duplication events. The legume-common duplication event and soybean-specific tetraploid have primarily led to expanding GmPP2C members in soybean. Sub-functionalization was the main evolutionary fate of duplicated GmPP2C members. Meanwhile, massive genes were lost in the GmPP2C family, especially from the F subfamily. Compared with other genes, the evolutionary rates were slower in the GmPP2C family. The PP2C members from the H subfamily resembled their ancestral genes. In addition, some GmPP2Cs were identified as the putative key regulator that could control plant growth and development.A total of 134 GmPP2Cs were identified in soybean, and their expansion, molecular evolution and putative functions were comprehensively analyzed. Our findings provided the detailed information on the evolutionary history of the GmPP2C family, and the candidate genes can be used in soybean breeding. Reversible protein phosphorylation by protein kinases (PKs) and protein phosphatases (PPs) can regulate numerous biological processes . The PKs2+/Mn2+-dependent PP [Brachypodium distachyon [Medicago truncatula [The PP2C enzyme is a monomer Mgndent PP . In planndent PP , 6. Overndent PP , rice [8stachyon , Medicaguncatula , maize [uncatula , and cotuncatula . In addiuncatula , organ iuncatula , stem ceuncatula , seed douncatula , and celuncatula . Moreoveuncatula , wound [uncatula , droughtuncatula , salt [2uncatula , and coluncatula . PP2C meuncatula , jasmoniuncatula  and saliuncatula  pathwaysGlycine max) is an important oil provider and a natural nitrogen-fixing plant. The release of the soybean genome can help comprehensively characterize the important gene families and analyze their putative functions [Soybean  profile of the PP2C domain PF00481) was used as the query to identify the PP2C members in soybean, grape, Arabidopsis, and two sequenced basal angiosperms through the HMMER program. After confirming the presence of the PP2C domain using the CDD program, we found 134 GmPP2Cs in soybean, 62 VvPP2Cs in grape, 70 PP2C members in Arabidopsis, 43 AtrPP2Cs in  was usedata Fig. , 6.Fig.A. trichopoda, and N. colorata had the similar subfamily distributions. In addition, all of the subfamilies had more PP2C members in soybean than in other plants. The number of PP2C members in the other four eudicots was similar in the B, F, H, I and J subfamilies, but other subfamilies had more PP2Cs in grape and Arabidopsis.The phylogenetic analysis of the identified PP2C members was performed using FastTree, Mega, Phylip, and Bayesian Fig. , 7 and 8All PP2C members had the PP2C domain through the CDD program  to 610 genes (ivory)  Fig. . A totalFive subnetworks with GmPP2Cs were extracted in the major modules Fig. D. In blucis-regulatory elements are important in the biological functions and regulatory networks. In this study, the promoter regions of the GmPP2C members included six cis-regulatory elements -responsive elements existed in the GmPP2C promoters from the E, G and H subfamilies. Most of the promoter regions of the duplicated gene pairs contained the similar cis-regulatory elements in the GmPP2C family.The nts Fig. . All GmP2H2 families. Meanwhile, six and five GmPP2Cs were target genes of Glyma.02G051100 and Glyma.15G232000, respectively  could regulate the stamen filaments in Arabidopsis [GmPP2C024, GmPP2C035 and GmPP2C091 directly co-expressed with certain plastid-related genes in chloroplast development, and AT4G27800, an orthologous gene of GmPP2C024 and GmPP2C035, reportedly played key roles in LHCII dephosphorylation [GmPP2C029, GmPP2C038 and GmPP2C117 might be very important to leaf development. The mutant of AT2G28890 and AT1G07630 (the GmPP2C117 orthologous genes) had abnormally shaped leaves [GmPP2C031, GmPP2C071 and GmPP2C131 might be involved in seed development, and an orthologous gene of GmPP2C071 (AT5G51760) could regulate seed germination in Arabidopsis [GmPP2C005, GmPP2C013, GmPP2C059 and GmPP2C118 might play important roles in the nodulation development stage, and LjNPP2C1 (a GmPP2C005 orthologous gene) in Lotus japonicus was related to both early and late stages of nodule development [cis-regulatory element analysis .A total of 134 GmPP2C members with 10 subfamilies were found in the present study. The number of duplication events from the legume-common duplication event and soybean-specific tetraploid led to the GmPP2C expansion. All of the duplication events were the segmental duplication events. Sub-functionalization was the main evolutionary fate of duplicated PP2C members in soybean. Meanwhile, the GmPP2C family lost massive genes, especially in the F subfamily. The GmPP2C members evolved more slowly than other genes, and the PP2C members from the H subfamily resembled their ancestral genes. In addition, some genes were considered as the putative key candidates that could control plant growth and development . The genome sequences of N. colorata were obtained from the Waterlily Pond (http://waterlily.eplant.org). The HMM profile of the PP2C domain (PF00481) from the Pfam database (https://pfam.xfam.org/) was used to identify the PP2C members in soybean, grape, Arabidopsis, A. trichopoda and N. colorata with hmmsearch tool in HMMER 3.0 program [https://www.ncbi.nlm.nih.gov/cdd). The conserved motifs of the PP2C family in soybean were identified by the MEME program (http://meme-suite.org/tools/meme). The parameters were set according to the previous report [GmPP2C genes were visualized by the TBtools software [The genome sequences of  program . Then, amain PF001 from thsoftware . “Intronsoftware . FurtherThe PP2C proteins were aligned using the ClustalX software with default configurations . The phySyntenic blocks were found by the MCScan software , and the2(FPKM+ 1) function [The RNA-seq transcriptome dataset of soybean in different tissues  were downloaded from the Phytozome database, and more details about experimental materials and RNA-seq data analysis are described in the previous report . The expfunction . The expG. max cv. Tianlong-1 was used to construct the expression profiles of the PP2C family in soybean. Roots, stems, and leaves were harvested from 4-week-old seedlings. RNA extraction and qRT-PCR analysis were performed on the basis of previous studies [GmHELIC gene was used as the reference gene, and gene-specific primers were designed on the basis of their coding sequences  in R . The expP value ≤0.01 in PlantRegMap (http://plantregmap.cbi.pku.edu.cn/). Meanwhile, the 1000 bp upstream of the initiation codon (ATG) were extracted from the Phytozome database genes in the GmPP2C family. The potential responsive-regulatory elements in the GmPP2C promoters were identified by the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The distributions of the responsive-regulatory elements were visualized by the TBtools software [GO enrichment was performed using the GO Term Enrichment tool with software .http://planttfdb.cbi.pku.edu.cn/index.php). The TF binding site database in soybean as the prior networks was obtained from the PlantRegMap database. The WGCNA module was set as the initial cluster assignment. The GRN networks were analyzed and visualized by the Cytoscape software.GRN was performed by the MERLIN+Prior method . The expAdditional file 1 Phylogenetic and sequence analysis of the PP2C members in soybean, Arabidopsis, grape, A. trichopoda and N. colorata. The inner circle was the phylogenetic tree using the FastTree method. The numbers in the clades were the FastTree bootstrap values. The PP2C subfamilies were labeled using different letters and were indicated using different colors. The outer circle was the sequence analysis of PP2Cs. The conserved PP2C domain was represented by the blue box. The location of each PP2C protein can be estimated using the scale at the end of the circle.Additional file 2 GmPP2C proteins identified in G. max.Additional file 3 VvPP2C proteins identified in V. vinifera.Additional file 4 AtPP2C proteins identified in A. thaliana.Additional file 5 AtrPP2C proteins identified in A. trichopoda.Additional file 6 NcPP2C proteins identified in N. colorata.Additional file 7. Phylogenetic relationship of the GmPP2C members. The neighbor-joining (NJ) tree was constructed through the MEGA program, and the numbers in the clades are the bootstrap values. The PP2C subfamilies are grouped by colors.Additional file 8. Phylogenetic analysis of the GmPP2C members. The neighbor-joining (NJ) tree was generated through PHYLIP package, and the numbers in the clades are the bootstrap values. The PP2C subfamilies are grouped by colors.Additional file 9. Percentage of GmPP2C numbers in the PP2C subfamily (upper) and its location distribution on each chromosome (bottom).Additional file 10. Locations of the conserved PP2C domain in GmPP2Cs, VvPP2Cs, AtPP2Cs, AtrPP2Cs and NcPP2Cs.Additional file 11. Motif information of GmPP2Cs through the MEME tool.Additional file 12. Motif distributions in the GmPP2C subfamily.Additional file 13. Sequence similarity of GmPP2Cs. (A) Sequence similarity of GmPP2Cs from the same and different subfamily. (B) Sequence similarity of duplicated GmPP2Cs in each subfamily.Additional file 14. Structural analysis of GmPP2C in this study.Additional file 15. Chromosomal locations of GmPP2Cs on all 20 chromosomes. The scale used is base pair (bp). Parentheses after the gene names show the PP2C subfamily.Additional file 16 Genomic locations of the GmPP2C genes.Additional file 17. Ka/Ks analysis for the duplicated PP2Cs in soybean.Additional file 18. Ka/Ks analysis for the duplicated PP2Cs in grape.Additional file 19. Ka/Ks analysis for GmPP2Cs and its corresponding orthologous genes in grape.Additional file 20. Number of VvPP2C orthologous genes in soybean.Additional file 21. Sequence similarity between GmPP2Cs and its corresponding AtrPP2Cs.Additional file 22. Sequence similarity between GmPP2Cs and its corresponding NcPP2Cs.Additional file 23. The FPKM values of GmPP2Cs in seven tissues.Additional file 24. PCC of the FPKM values for the duplicated GmPP2Cs pairs.Additional file 25. The constructed co-expression modules in soybean by hierarchical clustering and dynamic tree cut.Additional file 26. Module information of GmPP2C through WGCNA analysis.Additional file 27. Scatterplot of gene significance and module membership in certain significant modules.Additional file 28 Number of the responsive-regulatory elements in the promoter regions of GmPP2Cs.Additional file 29 Regulatory relationship between TFs and GmPP2Cs through the MERLIN+Prior method.Additional file 30 Regulatory relationship between TFs and duplicated GmPP2Cs through the MERLIN+Prior method.Additional file 31. PCR primers used in the study.