# The Natural Antisense Transcript DONE40 Derived from the lncRNA ENOD40 Locus Interacts with SET Domain Protein ASHR3 During Inception of Symbiosis in Arachis hypogaea

Pritha Ganguly, Dipan Roy, Troyee Das, Anindya Kundu, Fabienne Cartieaux, Zhumur Ghosh, Maitrayee Dasgupta

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Pritha Ganguly, Dipan Roy, Troyee Das, Anindya Kundu, Fabienne Cartieaux, et al.. The Natural Antisense Transcript DONE40 Derived from the lncRNA ENOD40 Locus Interacts with SET Domain Protein ASHR3 During Inception of Symbiosis in Arachis hypogaea. Molecular Plant-Microbe Interactions, 2021, 34 (9), pp.1057-1070. 10.1094/MPMI-12-20-0357-R. hal-03597062

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# The Natural Antisense Transcript *DONE40* Derived from the IncRNA *ENOD40* Locus Interacts with SET Domain Protein ASHR3 During Inception of Symbiosis in *Arachis hypogaea*

Pritha Ganguly,<fnref n="1" /> Dipan Roy,<fnref n="1" /> Troyee Das,<fnref n="2" /> Anindya Kundu,<fnref n="1" /> Fabienne Cartieaux,<fnref n="3" /> Zhumur Ghosh,<fnref n="2" /> and Maitrayee DasGupta<fnref n="1" />,†

Accepted 29 April 2021.

The long noncoding RNA *ENOD40* is required for cortical cell division during root nodule symbiosis (RNS) of legumes, though it is not essential for actinorhizal RNS. Our objective was to understand whether *ENOD40* was required for aeschynomenoid nodule formation in *Arachis hypogaea*. *AhENOD40* express from chromosome 5 (chr5) (*AhENOD40-1*) and chr15 (*AhENOD40-2*) during symbiosis, and RNA interference of these transcripts drastically affected nodulation, indicating the importance of *ENOD40* in *A. hypogaea*. Furthermore, we demonstrated several distinct characteristics of *ENOD40*. (i) Natural antisense transcript (NAT) of *ENOD40* was detected from the *AhENOD40-1* locus (designated as NAT-*Ah DONE40*). (ii) Both *AhENOD40-1* and *AhENOD40-2* had two exons, whereas NAT-*Ah DONE40* was monoexonic. Reverse-transcription quantitative PCR analysis indicated both sense and antisense transcripts to be present in both cytoplasm and nucleus, and their expression increased with the progress of symbiosis. (iii) RNA pull-down from whole cell extracts of infected roots at 4 days post-infection indicated NAT-*Ah DONE40* to interact with the SET (Su(var)3-9, enhancer of Zeste and Trithorax) domain containing absent small homeotic disc (ASH) family protein AhASHR3 and this interaction was further validated using RNA immunoprecipitation and electrophoretic mobility shift assay. (iv) Chromatin

immunoprecipitation assays indicate deposition of ASHR3-specific histone marks H3K36me3 and H3K4me3 in both of the *ENOD40* loci during the progress of symbiosis. ASHR3 is known for its role in optimizing cell proliferation and reprogramming. Because both ASHR3 and *ENOD40* from legumes cluster away from those in actinorhizal plants and other nonlegumes in phylogenetic distance trees, we hypothesize that the interaction of *DONE40* with ASHR3 could have evolved for adapting the nodule organogenesis program for legumes.

Keywords: *ENOD40*, *DONE40*, ASHR3, long noncoding RNA, natural antisense transcript, root nodule symbiosis

In nitrogen-fixing root nodule symbiosis (RNS), a compatible rhizobia–legume interaction activates the Nod-factor-dependent SYM pathway that leads to a local trigger of complex phytohormonal signaling. Together with the SYM pathway, these phytohormonal signals reprogram the cortical cells and regulate their division, ultimately building a nodule meristem for the endocytic accommodation of the symbionts and generation of the nodule primordium (Limpens and Bisseling 2003; Stougaard 2001). During the inception of symbiosis, downstream to the SYM pathway, phytohormonal signaling and nitrate sensing (Mathesius et al. 2000), the long noncoding RNA (lncRNA) *ENOD40* is induced to initiate the proliferation of root cortical cells (Crespi et al. 1994). The *ENOD40* genes encode polyA containing RNAs of 0.7 kb that contain two short open reading frames (ORFs) and are also present in nonlegumes (Larsen 2003; Sousa et al. 2001). The 10- to 13-mer oligopeptide encoded by ORF1 is conserved among all species (Compaan et al. 2001; Varkonyi-Gasic and White 2002), except for *Casuarina glauca* (Santi et al. 2003) (Supplementary Fig. S2A and B). ORF2 possibly does not encode for peptides but may contribute to the folding of the RNA into a highly structured form (Compaan et al. 2001; Girard et al. 2003; Sousa et al. 2001). The region encompassing ORF1 and ORF2 (inter-ORF) shows a high degree of conservation, and this region of *ENOD40* RNA sequences tends to form particularly stable secondary structures, indicating these regions to be functionally essential for *ENOD40* (Wan et al. 2007). Thus, the *ENOD40* RNA forms a special class of RNA in plants known as bifunctional RNAs, with both coding and noncoding characteristics (Kouchi and Hata 1993; Röhrig et al. 2002).

The possible functions of legume *ENOD40* have been argued mainly in favor of the induction of cortical cell divisions that lead

P. Ganguly and D. Roy contributed equally to this work.

Current address of D. Roy: Department of Biosciences, Durham University, Durham, DH1 3LE, U.K.

Current address of A. Kundu: NIAB, Cambridge, U.K.

†Corresponding author: M. DasGupta; maitrayee_d@hotmail.com

**Funding:** This work was funded by grants from the Government of India: DST (EMR/2015/001006), CEFIPRA (IFC-6303-2), J. C. Bose Fellowship (JCB/2019/000003), and SERB-NPDF (PDF/2016/001449); and fellowships to P. Ganguly (DBT-BBSRC, BT/IN/UK-VNC/41/DLN/2015-16), D. Roy (PDF/2016/001449), A. Kundu (Council of Scientific and Industrial Research, CSIR-09/028[0756]/2009-EMR-I), and T. Das (Council of Scientific and Industrial Research, CSIR-09/015[0517]/2017-EMR-I).

*The e-Xtra logo stands for “electronic extra” and indicates that supplementary materials and supplementary tables are published online.

The author(s) declare no conflict of interest.

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Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

<footnote n="1">¹Department of Biochemistry, University of Calcutta, Kolkata, West Bengal, 700019, India</footnote>

<footnote n="2">²Division of Bioinformatics, Bose Institute, Kolkata, West Bengal, 700054, India</footnote>

<footnote n="3">³LSTM, Université de Montpellier, CIRAD, INRA, IRD, SupAgro, Montpellier, France</footnote>
to the initiation of nodule primordia and appropriate differentiation and development of nodules (Charon et al. 1997, 1999; Mylona et al. 1995). Expression of infection-related genes is independent of *ENOD40* activation, indicating rhizobial invasion to be independent of *ENOD40* (Kumagai et al. 2006). Ectopic expression of *ENOD40* induces dedifferentiation and extensive division of cortical cells, leading to increased nodules at early time points (Charon et al. 1997). On the other hand, the transcription factor nodule number control 1 directly binds to the *ENOD40* promoter to repress it for restricting nodule numbers (Wang et al. 2014). Identification of the interactors has also provided leads in understanding *ENOD40* action. For example, *ENOD40* interacts with MtRBP1, a homolog of nuclear speckle RNA binding protein (RBP) in *Medicago truncatula*, where *ENOD40* is believed to relocalize RBP1 to the cytoplasm from nucleus speckle (Campalans et al. 2004). This appears similar to the *ASCO* lncRNA in *Arabidopsis*, which binds to nuclear speckle RBPs and thereby regulates plant root development (Bardou et al. 2014). The peptide from ORF1 of *ENOD40* from *Glycine max* binds via S-thiolation to sucrose synthase, an important enzyme in sucrose utilization, suggesting a role for *ENOD40* peptides in photosynthate accumulation in sink regions (Röhrig et al. 2002; Winter and Huber 2000). The pronounced impact of *ENOD40* in the developmental program of nodule organogenesis involving initiation of dedifferentiation and division of cortical cells have been extensively documented. However, the intricate molecular mechanisms associated with the process remain to be understood.

In this study, we analyzed *ENOD40* genes in a dalbergoid legume, *Arachis hypogaea*. In these plants, rhizobia invade through natural “cracks” to directly access the cortical cells that divide and develop the characteristic aeschynomenoid nodules with uniformly infected central tissue and loss of meristematic activity (Fedorova et al. 2007; Lavin et al. 2001; Tajima et al. 2008). Herein, we show *ENOD40* to be encoded from both chromosome 5 (chr5) (designated as *AhENOD40-1*) and chr15 (designated as *AhENOD40-2*). Additionally, a natural antisense transcript (NAT) of *ENOD40* was detected from the *AhENOD40-1* locus at chr5, and was designated as NAT-*Ah DONE40*. We demonstrated the interaction of NAT-*Ah DONE40* with AhASHR3 (ABSENT SMALL HOMEOTIC DISC1 RELATED3), a member of the Trithorax group of proteins, and have observed significant enrichment of activating histone modification marks at the *ENOD40* locus from both chr5 and chr15 of *A. hypogaea* during the progress of rhizobial infection. Moreover, with *ENOD40* RNA being required for initiation of dedifferentiation and division of cortical cells during nodule organogenesis (Charon et al. 1997), and the ASHR3 protein being required for the control of cell division at the root meristem and quiescent center in *Arabidopsis* (Kumpf et al. 2014), the interaction between AhASHR3 and *AhENOD40* may have implications in the regulation of rhizobia-induced cortical cell division at the nodule primordia in legumes.

## RESULTS

**ENOD40 is essential for symbiosis in *A. hypogaea*.

*ENOD40* is yet to be annotated in the cultivated peanut genome (tetraploid) database or either of its two parental genomes (diploid), *A. duranensis* (AA) and *A. ipaensis* (BB). All *ENOD40* sequences reported thus far contain two short conserved regions, ORF1 and ORF2, embedded within nonhomologous sequences (Compaan et al. 2001). BLAST search was done against the cultivated peanut genome database (arahy.Tifrunner.gnm1) with *M. truncatula* *ENOD40* sequences (X80262), whereby putative orthologs of *ENOD40* in *A. hypogaea* were identified in chr5 (AA) and chr15 (BB). Additionally, BLAST search using *Lupinus luteus* *ENOD40* sequences (AF352375) identified putative orthologs of *ENOD40* in chr3 (AA) and chr13 (BB) although, in this

case, only sequences homologous to ORF2 were identified (Supplementary Fig. S1). Alignment of these putative *ENOD40* sequences from *A. hypogaea* indicated the core sequences of ORF1 and ORF2 to be conserved in all four loci (Supplementary Fig. S2A and B). Microsynteny analysis of *AhENOD40* loci revealed strongly conserved gene collinearity with other *ENOD40s* from legumes, indicating them to be orthologous (Fig. 1A). In all legumes, *ENOD40* was convergent with *YELLOW LEAF SPECIFIC GENE 7-like* (*YLS7*), a member of Trichome birefringence family protein within 2Kb. Intriguingly, in *Aeschynomene evenia*, a Nod-independent crack entry legume, *AeENOD40* loci from chr5 was found to be in synteny with other legumes but, in chr3, *YLS7* was absent. Within nonlegumes, synteny of *ENOD40* with *YLS7* was only conserved in actinorhizal plants although, in most members, only ORF2 sequences were conserved (Supplementary Table S1). In contrast, in nonlegumes such as *Populus tricocarpa*, *Vitis vinifera*, and *Solanum lycopersicum*, only ORF2 sequence was detectable, but it was not syntenic with *YLS7*. Similarly, in *Zea mays* and *Oryza sativa*, *ENOD40* and *YLS7* were located in different chromosomes and, in *Arabidopsis thaliana* and *Brassica rapa*, *ENOD40* was completely undetectable, like other SYM gene homologs (Griesmann et al. 2018).

Our first objective was to understand whether *ENOD40* was important for RNS in *A. hypogaea*. For this, RNA interference of *AhENOD40* was done targeting the region encompassing ORF1 and ORF2 that was conserved in all four loci detected in *A. hypogaea*. Quantitative reverse-transcription PCR (RT-qPCR) for *AhENOD40* revealed approximately 66 to 83% reduction of the *AhENOD40* transcripts in transgenic hairy roots (n = 20) (Fig. 1B). Both RNA interference (RNAi) and empty-vector-transformed composite plants were scored for nodulation at 28 days postinfection (DPI) (Fig. 1C). The downregulation of *AhENOD40* resulted in approximately 80% reduction in nodule number in RNAi as compared with empty-vector-transformed hairy roots. The infection zone (IZ) of these matured empty-vector-transformed control nodules showed the presence of uniformly infected plant cells filled with spherical symbiosomes (Fig. 1D). In contrast, the *AhENOD40*-RNAi nodules appeared to be whitish, and the IZ showed the presence of undifferentiated rod-shaped bacteria. We concluded *ENOD40* to have a role in bacteroid differentiation in addition to its known role in cortical cell division during nodule organogenesis. This is similar to what we observed in *AhHK1*-RNAi and *AhCYCLOPS*-RNAi conditions, where undifferentiated rhizobia failed to differentiate into spherical symbiosomes (Das et al. 2019; Kundu and DasGupta 2018). For the readout of the symbiotic signaling, we chose to monitor the symbiotic genes such as *CYCLOPS* and *NIN*, whose expressions were previously reported to be induced during the progress of symbiosis in *A. hypogaea* (Karmakar et al. 2019). *CYCLOPS* acts as a transcription factor specifically binding to the CYC-box element present in the promoter of *NIN* to drive its expression in *Lotus japonicus* during nodulation (Singh et al. 2014). Expression of *AhCYCLOPS* was approximately fivefold lower in the *AhENOD40*-RNAi nodules, whereas expression of *NIN* was unaffected (Fig. 1E). The decrease in *AhCYCLOPS* could be due to a feed-forward positive effect of *ENOD40* on *CYCLOPS* expression, and the absence of any change in *AhNIN* expression indicates that *AhENOD40* is activated downstream from *AhNIN* during organogenesis, as noted earlier (Liu et al. 2019).

**Both sense and antisense *ENOD40* is expressed during symbiosis in *A. hypogaea*.

Transcriptome analysis indicated a significant change of expression of *AhENOD40* from chr5 and chr15 with the progress of symbiosis, whereas expression from chr3 and 13 showed no change (Supplementary Fig. S3A). Therefore, we focused our investigation
on *AhENOD40* alleles encoded from chr5 and 15. Transcript assembly by Cufflinks followed by the screening of intergenic lncRNAs (as described in Materials and Methods) revealed that both of the alleles for *AhENOD40* in chr5 and 15 coded for lncRNA

where the nearest protein-coding gene (pcGene) was YLS7 (Fig. 1A). The coding potential for the *AhENOD40* sequences was calculated using the Coding Potential Calculator and is shown in Supplementary Figure S3B. *AhENOD40* was transcribed from the

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** *ENOD40* is essential for symbiosis in *Arachis hypogaea*. **A**, Syntenic relationship of *ENOD40*. YLS7-*ENOD40* enclosed in a dashed box; YLS7 = pink and *ENOD40* = yellow (indicated by a red star). Gene IDs from *Lotus japonicus*, *A. hypogaea*, *Glycine max*, *Medicago truncatula*, *Phaseolus vulgaris*, and *Aeschynomene evenia* are shown on the left. **B**, Quantitative reverse-transcription PCR (RT-qPCR) analysis of *AhENOD40* in *AhENOD40*-RNA interference (RNAi) roots relative to empty-vector-transformed roots (control) at 28 days postinfection (DPI). Primers *AhENOD40F*-RT and *AhENOD40R*-RT were used for the RT-qPCR. *AhActin* was used as a reference gene. The histogram represents an average of three biological replicates, each having *n* > 4 root systems, and the error bar represents standard deviation. **C**, Histogram shows the average number of nodules in transformed (gray) and nontransformed (black) hairy roots in control and *AhENOD40*-RNAi plants at 28 DPI. Error bar represents the standard deviation (*n* = 30 for control and *AhENOD40*-RNAi plants). **D**, Section of nodule in control and *AhENOD40*-RNAi roots at 28DPI. Image shows propidium iodide (red) and calcofluor (blue) merged. Scale bar = 10 µm. Scanning electron microscopy image of the infection zone in control and *AhENOD40*-RNAi nodule. Scale bar = 2 µm. **E**, RT-qPCR analysis of symbiotic marker *AhNIN* and *AhCYCLOPS* in *AhENOD40*-RNAi roots relative to the control at 28 DPI. *AhActin* was used as a reference gene. The histogram represents an average of three biological replicates, each having *n* > 4 root systems, and the error bar represents standard deviation.</figcaption></figure>
negative strand in the loci arahy.Tifrunner.gnm1.Arahy.05:33566965-33568044 and from the positive strand in the loci arahy.-Tifrunner.gnm1.Arahy.15:24083907-24084972. Sequences of the reads mapped to these loci are shown in Figure 2A, with their detail in Supplementary Table S2. A distinguishing feature of *AhENOD40* transcribed from chr5 was an insertion of 28 bp (position 33,567,419 to 33,567,446) within the ORF2, which was absent in *ENOD40* transcribed from chr15 (Fig. 2A; Supplementary Fig. S3C). We designate *ENOD40* from chr5 as *AhENOD40-1* and from chr15 as *AhENOD40-2*. Such multiple copies of *ENOD40* were previously noted in both legumes and nonlegumes (Crespi et al. 1994; Fang and Hirsch 1998; Flemetakis et al. 2000; Roussis et al. 1995). The alignment of mapped reads on the *A. hypogaea* genome suggested both *AhENOD40-1* and *AhENOD40-2* to have two exons (Fig. 2A). To verify, the complementary DNAs (cDNAs) for *AhENOD40* were amplified using a gene-specific primer and oligdT-adapter primer from messenger RNA isolated from nodulated roots, where *AhENOD40-1* and *AhENOD40-2* contained 719 bp (accession number MW048740) and 688 bp (accession number MW048741), respectively (Fig. 2B). We verified the presence of a 361-bp intron in *AhENOD40-1* and a 377-bp intron in *AhENOD40-2* by PCR amplification on genomic DNA (gDNA) and cDNA by exon-specific and chr-specific primers (Fig. 2B). The length of exon1 and exon2 in *AhENOD40-1* was 22 and 697 bp, respectively, and in *AhENOD40-2* was 69 and 619 bp, respectively (Fig. 2C). This exercise allowed us to verify the exon intron boundary, and the splice sites were identified. The terminal exon 3' acceptor site is conserved for both *AhENOD40-1* and *AhENOD40-2*, though the 5'donor site varies (Fig. 2D).

The most distinguishing feature of *ENOD40* loci in *A. hypogaea* was the detection of natural antisense *ENOD40* transcripts from our strand-specific transcriptome. Sequences of these antisense read mapped to arahy.Tifrunner.gnm1.Arahy.05:33567054-33567703 overlapping with exon2 and partially with the intron of *AhENOD40-1* (Fig. 2A). We have designated the antisense transcript as NAT-Ah DONE40. The antisense transcript was amplified from gDNA and cDNA from nodulated roots and was confirmed to be monooxonemic of 649 bp (accession number MW048742) (Fig. 2C). Additionally, NAT-Ah DONE40 was also predicted to be an lncRNA (Supplementary Fig. S3B). Together, these findings indicate the existence of a natural antisense transcript for *AhENOD40-1*, where it may function as a sense-antisense (SAS) lncRNA pair. It may be noted that, in *ENOD40* RNAi roots, there was significant downregulation of both the sense (*AhENOD40-1*; approximately 930-fold, *AhENOD40-2*; approximately 40-fold) and antisense (NAT-*Ah DONE40*; approximately 30-fold) transcripts, indicating that the observed phenotypic effect could be contributed by either or both of the sense and antisense transcripts (Supplementary Fig. S3D). Previously, we have analyzed the transcriptome of five distinct stages of symbiosis in *A. hypogaea* as follows: 1 DPI = recognition and invasion, 4 DPI = primordia formation, 8 DPI = nodule-like structure, 12 DPI = immature nodules with rod-shaped rhizobia, and 21 DPI = mature nodules with spherical symbiosomes (Karmakar et al. 2019). To detect the sense (*AhENOD40-1* and *AhENOD40-2*) or antisense (NAT-*Ah DONE40*) transcripts and validate their symbiotic pattern of expression revealed by the transcriptomic data (Supplementary Fig. S3A), we performed strand- and chromosome-specific RT, and the relative levels of all of the RNAs were quantified by qPCR (Fig. 2B and E). The RT-qPCR estimation of these transcripts follows the same expression pattern as was revealed by the raw count of the transcripts (Fig. 2E; Supplementary Fig. S3A) and indicated *ENOD40* transcripts (both sense and antisense) to increase from the primordia formation stage at 4 DPI. This symbiosis-associated expression of the NAT-*Ah DONE40* strongly suggest its importance during the process. We then purified nuclear RNA and, using the same strategy,

showed that both sense and antisense transcripts were also detected in the nucleus (Fig. 2E). These data are in accordance with the previous reports where *ENOD40* was noted to shuttle between cytoplasm and nucleus (Campalans et al. 2004). The dynamic nature of expression and distribution of both sense and antisense transcripts of *AhENOD40* during symbiosis indicate them to be functionally important during nodule organogenesis in *A. hypogaea*.

**RNA pull-down indicates NAT-Ah DONE40 RNA to interact with AhASHR3.**

Previous investigations have identified RBP1 from nodular extracts to be an interactor of sense *ENOD40* (Campalans et al. 2004). To understand the biological significance of NAT-Ah DONE40, we explored the use of RNA pull-down assays coupled with mass spectrometry (MS) analysis to identify the potential interactor proteins of NAT-Ah DONE40. We attempted to identify such interactors in whole-cell extracts of infected roots at 4 DPI because the expression of both sense and antisense *AhENOD40* significantly increases from 4 DPI onward (Fig. 2E). Moreover, our previous investigation revealed an onset of a major transcriptional program at 4 DPI during the primordia formation (Karmakar et al. 2019).

For RNA pull-down, we used the ORF1-interORF-ORF2 region (298 bp) because earlier reports have indicated that both ORF1 and ORF2 and the interORF region are involved in the regulation of *ENOD40* activity in *Medicago* roots (Sousa et al. 2001). Additionally, the interORF region of *ENOD40s* was previously noted to have conserved secondary structure (Sousa et al. 2001), and our analysis indicated that both sense (*AhENOD40-1* and *AhENOD40-2*) and antisense (NAT-*Ah DONE40*) RNA has that secondary structure conserved in the interORF region (Supplementary Fig. S4). Following RNA pull-down assays, matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) analysis was performed, where we considered proteins having at least two unique peptides with a Mascot Ion Score above 20 as potential interactors of *Ah DONE40* (Supplementary Table S3). As a control, MALDI-TOF analysis was also performed using *AhENOD40-1* as bait to parallelly profile the potential interactor proteins of *AhENOD40-1* (Supplementary Table S4). The best hit with *Ah DONE40* was a putative Su(var)3-9, enhancer of Zeste and Trithorax (SET) domain containing protein of the Trithorax family from *Arabidopsis thaliana*, which was not found among the interactors of *AhENOD40-1*. Taking this as a clue, the RNA pull-down experiment was repeated, where the interacting proteins were analyzed using liquid chromatography tandem MS (LC-MS/MS). In this case, the peak lists were searched against a forward and reverse *Arabidopsis thaliana* UniProt database appended with all 50 SET domain-containing proteins in *A. hypogaea*. We could detect three spectra representing unique peptides from AhXD33H1 (chr1, AA) and AhBQLE9W (chr11, BB) (Fig. 3A). We developed a distance tree with all of the SET domain-containing proteins from *A. hypogaea* and *Arabidopsis thaliana*, where AhXD33H1 and AhBQLE9W clustered with AtASHR3 within the ASH group and, therefore, was predicted to be an ortholog of AtASHR3 (Supplementary Fig. S5A). We profiled the relative (log-twofold) expression values of all 50 SET domain-containing proteins of *A. hypogaea* from our symbiotic transcriptome (GSE98997), where the expression of *Aradu.4C25F* (*AhXD33H1*, chr1) and *Araip.VL7Z8* (*AhBQLE9W*, chr11) was significantly high at 4 and 8 DPI (Fig. 3B). However, the absolute values of expression indicated only *AhXD33H1*- but not *AhBQLE9W*-encoded *AhASHR3* to be highly expressed during the progress of symbiosis (Fig. 3C). RT-qPCR analysis then confirmed the expression of *AhXD33H1*-encoded *AhASHR3* to be maximum at 4 DPI (Fig. 3D). Interestingly, in *AhENOD40*-RNAi, there was no significant change in the expression of *AhASHR3*, which shows that *ENOD40* transcripts may not be
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<figure><img src="image_7.png" /><figcaption>**Fig. 2.** Both sense and antisense *ENOD40* is expressed during symbiosis in *Arachis hypogaea*. A, Alignment of the Sequence Read Archive contigs with the genome sequence of Arahy.05 (33,566,964 to 33,568,044) and Arahy.15 (24,083,907 to 24,084,972). Contigs from positive-strand coding for *AhDONE40* are marked with green (33,567,054 to 33,567,703). Contigs from negative-strand coding for *AhENOD40-1* are marked with red (33,566,964 to 33,567,661 and 33,568,022 to 33,568,044). Contigs from positive-strand coding for *AhENOD40-2* are marked with green (24,083,907 to 24,083,976 and 24,084,353 to 24,084,972). All contigs are aligned in the 5' to 3' direction. Conserved boxes are aligned to the 5' to 3' direction of the peptides translated. Open reading frame 1 (ORF1) is marked with yellow and ORF2 with orange in both *AhENOD40-1* and *AhENOD40-2*. The blue shaded region represents the 28-nucleotide insertion in Arahy.05. B, Schematic representation of the primer positions in *AhDONE40*, *AhENOD40-1*, and *AhENOD40-2* transcripts. Exons are marked with black boxes, and ORF1 and ORF2 are denoted as in A. The 28-bp insertion in Arahy.05 is denoted by a blue box and the region of insertion in Arahy.15 by a blue triangle. The transcriptional start site is denoted by a black arrow. Primer positions are marked above each transcript. C, PCR amplification of *AhDONE40*, *AhENOD40-1*, and *AhENOD40-2* from both genomic DNA (gDNA, marked with navy blue bar) and complementary DNA (cDNA, marked with cyan red bar). Primer combinations are marked on each lane. D, Alignment of splicing boundaries of *AhENOD40-1* and *AhENOD40-2* with splice sites highlighted in blue. The black box represents exon position. E, Relative transcript abundance of *AhENOD40-1*, *AhENOD40-2*, and *AhDONE40* in the total RNA and nuclear RNA in different time points (1, 4, 8, 12, and 21 days postinfection). Transcript levels were normalized relative to the histone H3 gene. Error bars represent the standard deviation for three biological replicates. Two-way analysis of variance was used to assess significant differences, where *P* < 0.05.</figcaption></figure>
A

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B

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C

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D

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E

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F

<figure><img src="image_13.png" /><figcaption>**Fig. 3.** RNA pull-down indicates that *Ah DONE40* RNA interacts with *Ah ASHR3*. **A**, Product ion spectrum of indicated peptides of *Ah ASHR3*. **B**, Phylogenetic analysis of putative *Su(var)*3-9, enhancer of Zeste and Trithorax (SET) domain-containing proteins from *Arachis hypogaea* database. The Atx, E(z), SMYD, Ash, and SUV groups are marked. Expression profiles from transcriptome data corresponding to the proteins are mentioned as log-twofold change at indicated time points of rhizobial infection. **C**, Absolute counts of *Ah XD33H1* and *Ah BQLE9W* transcripts are plotted. **D**, Relative transcript abundance of *Ah ASHR3* was determined by quantitative reverse-transcription PCR at different time points post rhizobial infection. Transcript levels were calculated relative to the expression of histone H3. Error bars represent standard errors for three biological replicates. **E**, Schematic representation of the *Ah ASHR3* protein (accession number MW167779) with its respective domains. **F**, Phylogenetic analysis of ASHR3 and ENOD40 genes. Symbiotic leguminous plants are marked in green, actinorhizal plants in red, and the nonsymbiotic plants in blue.</figcaption></figure>
required for *ASHR3* expression (Supplementary Fig. S5B). The 1,563-bp coding sequence of putative AhASHR3 (GenBank accession number MW167779) was generated by PCR amplification from cDNA prepared from infected roots at 4 DPI. AhASHR3 contains dual PHD domains at the N-terminal region (residues 145 to 193 and 194 to 239), an associated-with-SET (AWS) domain (residues 306 to 351), SET domain (residues 350 to 473), and post-SET domain (residues 473 to 489) (Fig. 3E).

Unlike legumes, *ENOD40* expression does not increase during actinorhizal symbiosis and, in both actinorhizal plants and nonlegumes, *ENOD40* expression is restricted to vascular bundles (Kouchi et al. 1999; Santi et al. 2003). Again, actinorhizal nodules appear to be modified lateral roots with central vascular tissue, whereas legume nodules represent stem-like organs with a peripheral vascular system (Pawlowski et al. 2003). It may be noted that, in distance trees, both *ENOD40* and ASHR3 were completely segregated between legumes, nonlegumes, and actinorhizal plants (Fig. 3F). Such congruence between phylogeny and phenotype indicates the biological implication of *ENOD40*-ASHR3 interaction during the origin of nodule primordia in legumes.

**AhASHR3 directly binds NAT-*Ah DONE40*.$$

Our next objective was to validate *AhENOD40*-AhASHR3 interaction in vivo by RNA immunoprecipitation (RIP) assays. For this, we overexpressed the conjugate protein AhASHR3-green fluorescent protein (GFP) in hairy roots of *A. hypogaea*, and interacting RNAs were precipitated using an anti-GFP antibody. Because both sense and antisense transcripts of *AhENOD40* were noted in the nucleus and cytoplasm, we used total cellular extracts of 35S::AhASHR3-GFP-transformed roots at 4 DPI for the RIP assay. As a control, empty-vector-transformed roots expressing GFP were analyzed under identical conditions.

Interestingly, only NAT-*Ah DONE40* was significantly enriched by immunoprecipitation of AhASHR3-GFP, whereas detection of sense transcripts *AhENOD40-1* and *AhENOD40-2* was insignificant (Fig. 4A). It may be noted that, in vector-transformed roots, there was no enrichment of both sense and antisense *ENOD40* transcripts in anti-GFP immunoprecipitate, confirming the specificity of the observed NAT-*Ah DONE40*-ASHR3-GFP interaction.

Finally, our objective was to check whether AhASHR3 directly binds to NAT-*Ah DONE40*. For this, electrophoretic mobility shift assays (EMSAs) were employed using the ORF1-interORF-ORF2 region (298 bp) of NAT-*Ah DONE40* that was used for our pull-down experiment. For the AhASHR3 protein, we used the AWS-SET-postSET region (184 amino acids) because several factors indicated that the preSET-SET-postSET boundaries in the SET family proteins contain motifs that tightly bind single-strand DNA and RNA (Krajewski and Vassiliev 2011; Krajewski et al. 2005) (Fig. 3E; Supplementary Fig. S6). The representative gel in Figure 4B shows the change in NAT-*Ah DONE40* migration on the native gel in the presence of AhASHR3. The free RNA migrates as multiple slow-moving bands and a single fast-moving band, indicating different conformers of NAT-*Ah DONE40*. In the presence of AhASHR3, the mobility shift of these bands indicated that NAT-*Ah DONE40* could directly bind with AhASHR3. Unlabeled NAT-*Ah DONE40* in 50-fold molar excess successfully competed for the binding and release of the free probe, indicating the specificity of the interaction. Unlabeled *AhENOD40-1* could also compete for the binding at 200-fold molar excess but the release of the free RNA probe was not noted, further indicating the specificity of interaction with NAT-*Ah DONE40* and AhASHR3 (Fig. 4B). AhASHR3 does not interact with a highly structured 18S rRNA, indicating the specificity of the observed interaction between AhASHR3 and NAT-*Ah DONE40* transcripts (Supplementary

<figure><img src="image_14.png" /><figcaption>**Fig. 4.** AhASHR3 directly binds *Ah DONE40*. A, RNA immunoprecipitation was performed using anti-green fluorescent protein (GFP) antibody on extracts from 4 days postinfection AhASHR3-GFP overexpressed and empty-vector-transformed roots. Enrichment of *Ah DONE40*, *AhENOD40-1*, and *AhENOD40-2* was normalized to the input. Error bars represent the standard deviation for three biological replicates. Two-way analysis of variance was used to assess significant differences; an asterisk (*) indicates *P* < 0.05 whereas “ns” indicates statistically not significant. B, Electrophoretic mobility shift assays were performed with a recombinant chimera of AhASHR3 protein comprising the Su(var)3-9, enhancer of Zeste and Trithorax (SET), associated-with-SET (AWS), and postSET domains with *Ah DONE40* RNA at concentrations of the protein of 0.5 and 3 µM for binding and 3 µM for competition assays. α-32P-labeled RNA (1.5 fmol) was used as radiolabeled probe and 10-, 25-, 50-, 100-, and 200-fold molar excess of the unlabeled RNA for competition assays. The specifically bound RNA protein complexes and free probe are indicated by the solid bracket and dashed bracket, respectively.</figcaption></figure>
Fig. S7A). When we tested the direct interaction of AhASHR3 with *AhENOD40-1*, we could detect shifted bands that were outcompeted by both unlabeled *AhENOD40-1* and NAT-*Ah DONE40* (Supplementary Fig. S7B). It may be noted that NAT-*Ah DONE* and *AhENOD40* have approximately 34% sequence identity, which may explain the interaction of *AhENOD40-1* in vitro (Supplementary Fig. S7C). Overall, our results indicate that both sense and antisense transcripts of *AhENOD40* have the potential to interact with AhASHR3 but only the interaction between the antisense transcripts could be biologically significant.

**ENOD40 loci from both chr5 and chr15 undergo dynamic histone H3 modifications during the progress of symbiosis in A. hypogaea.**

To understand whether there is any link between the NAT-*Ah DONE40*-AhASHR3 interaction and epigenetic regulation of the expression of *ENOD40*, we followed the dynamic changes in the histone modification landscape of the *ENOD40* loci. We chose to monitor the levels of H3K36me3 and H3K4me3, which are specific marks associated with ASH1 homologs (Cartagena et al. 2008). Additionally, we monitored H3K27ac that is linked to Trithorax group (TrxG)-mediated activation (Geisler and Paro 2015; Schuettengruber et al. 2007) and H3K9ac as a general activation marker (Schuettengruber et al. 2011). A repressive H3K27me3 mark linked to Polycomb complex group (PcG)-mediated silencing (Mikulski et al. 2017) was also monitored. We used the same set of primers to distinguish between the *ENOD40* locus from chr5 and chr15 and monitored the above marks in both loci during the early stages of symbiosis in *A. hypogaea* roots, where

AhASHR3 expression was maximally upregulated (Fig. 5A). At 4 DPI in both *ENOD40* loci, there was a significant enrichment of H3K36me3, the ASHR3-specific mark (Lee et al. 2020; Zhang and Ma 2012), over the other TrxG-mediated activation marks H3K4me3 and H3K27ac (Fig. 5B). At 8 DPI, all of these activation marks were similarly enriched, though enrichment of H3K27ac remained insignificant at the *ENOD40* loci in chr15. The general activation mark H3K9ac was also significantly upregulated at both loci at 8 DPI, and the repressive mark H3K27me3 was absent in all of the stages. Our results reveal a robust enrichment of H3K36me3 at both *ENOD40* loci that is associated with the significant increase in expression of *AhENOD40-1*, *AhENOD40-2*, and NAT-*Ah DONE40* in *A. hypogaea* roots during the onset of symbiosis. This evidence further indicates the functional implications of the interaction between AhASHR3 and NAT-*Ah DONE40*, where the interaction may facilitate a feedforward loop that promotes the expression of *ENOD40* via the recruitment of ASHR3 to the *ENOD40* loci.

DISCUSSION

In dalbergoid legumes such as *A. hypogaea* and *A. evenia*, rhizobia directly invade cortical cells through epidermal cracks to generate the aeschynomenoid nodules (Sprent and James 2007). Our objective was to functionally characterize *ENOD40* during the aeschynomenoid nodule development in *A. hypogaea*. During symbiosis, *ENOD40* is expressed from chr5 (*AhENOD40-1*) and chr15 (*AhENOD40-2*), which differ by insertion of 28 bp in the ORF2 region of *AhENOD40-1* (Fig. 2A; Supplementary Fig.

<figure><img src="image_15.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_16.png" /></figure>

**Fig. 5.** Chromatin immunoprecipitation (ChIP) assays indicate that *ENOD40* loci from chromosome 5 (chr5) and chr15 undergo dynamic histone H3 modifications during symbiosis in *Arachis hypogaea*. **A**, Primer positions are indicated in the genomic sequence of Arahy.05 and Arahy.15, encoding *Ah DONE40*, *AhENOD40-1*, and *AhENOD40-2*. **B**, ChIP analysis indicating the dynamic changes in the levels of the different H3 modifications during indicated days of rhizobial infection at *ENOD40* locus from chr5 and chr15. Error bars represent standard errors from three independent experiments (n = 3). Paired two-tail Student's *t* test analyzed the significance of the results; an asterisk (*) indicates *P* ≤ 0.05, whereas “ns” indicates statistically not significant.
S3C). As in all other legumes, *ENOD40*-RNAi drastically reduced the nodule numbers in *A. hypogaea* (Fig. 1C), which is in accordance with its role in nodule primordia formation (Kumagai et al. 2006). Thus, the function of *ENOD40* appeared to be conserved in legumes. In all tested legumes, *ENOD40* was found to be in synteny with *YLS7*, a *Trichome birefringence-like* gene, in a convergent manner (Fig. 1A). *Trichome birefringence* genes have a role in plant-pathogen interaction by acetylation of the cell wall and can also play a role in rhizobial accommodation during symbiosis (Escudero et al. 2017; Sun et al. 2020). Because sense and antisense lncRNAs are defined according to the nearest pcGene position (RIKEN Genome Exploration Research Group et al. 2005), sense *AhENOD40* appears to be an antisense lncRNA for *YLS7* because their transcriptions are convergent. The only exception was chr3 of *A. evenia*, where *YLS7* was not detectable, and *ENOD40* was convergent with a *leucine-rich repeat receptor-like kinase* gene. Transcriptomic analysis of *A. evenia* during symbiosis revealed *AeENOD40* to be expressed from chr3 and not chr5, where *ENOD40* was syntenic with *YLS7* (Supplementary Fig. S8). It may be noted that, similar to actinorhizal plants, *A. evenia* was a nod-factor-independent legume. Though the significance of proximity of *YLS7* and *ENOD40* is not understood, its association with nod factor dependence is intriguing.

Our results reveal the presence of a natural antisense transcript of *ENOD40* (NAT-*Ah DONE40*) encoded from the *Ah ENOD40-1* locus in chr5, which is unique and has never been reported earlier. This makes *AhENOD40-1*-*NAT-Ah DONE40* a SAS gene pair, where opposite genomic strands within the same locus get transcribed (Galante et al. 2007). Because *AhENOD40-1* and *AhENOD40-2* differ by a 28-bp insertion, *Ah DONE40* may also act as a *trans*-NAT for *AhENOD40-2*. Transcriptome analysis of numerous model organisms reveals that a significant subset represents SAS partners involving both pcGenes and lncRNA, most often one coding and one noncoding in each pair (Babak et al. 2007; Georg and Hess 2011; Grinchuk et al. 2010; Hongay et al. 2006; Li et al. 2008). In plants, the *CDF5 LONG NONCODING RNA* is a circadian-regulated lncRNA that is a NAT of CYCLING DOF FACTOR 5 (Henriques et al. 2017), the heat-inducible lncRNA *asHSFB2a* is a NAT of heat-shock factor HSFB2a (Wunderlich et al. 2014), the germination-promoting lncRNA *asDOG1* is a NAT of *delay of germination 1* gene (Fedak et al. 2016), and the phosphate-deficiency-induced lncRNA *cis-NAT PHO1;2* is a NAT of phosphate exporter PHO1;2 (Jabnoune et al. 2013). Another example is *COOLAIR*, a collection of antisense lncRNA of *flowering locus C (FLC)*, where the transcription of sense and antisense strands are mutually exclusive (Whittaker et al. 2017). Unlike these examples, *AhENOD40-Ah DONE40* is a SAS pair between two lncRNAs. SAS pairs between lncRNAs such as *Xist-Tsix* and *MALAT1-TALAM1* are widely studied in the animal system (Gomes et al. 2019; Jeon et al. 2012). Expression of NAT-*Ah DONE40* was less abundant than *AhENOD40-1*, which is similar to the noted low abundance of *TALAM1* compared with *MALAT1* (Gomes et al. 2019). In both *Xist-Tsix* and *MALAT1-TALAM1*, the sense and antisense expression levels were inversely correlated with their unique biological roles in cell differentiation and development (Gomes et al. 2019; Loos et al. 2016) although, in the case of *AhENOD40-Ah DONE40*, we found both RNAs to express together during symbiosis.

Both *AhENOD40* and NAT-*Ah DONE40* were found in nucleus and cytoplasm, indicating that nucleocytoplasmic transport could be important for their function as positive or negative regulators of gene expression (Fig. 2E) (Lipovich et al. 2010; RIKEN Genome Exploration Research Group et al. 2005). Earlier reports demonstrate *ENOD40* to physically interact with RBP1, which has a role in alternative splicing (Bardou et al. 2014; Campalans et al. 2004). We could not detect RBP1, possibly because we looked for interactors during the early stages of rhizobial infection instead of

mature nodules from which RBP1 was identified (Campalans et al. 2004). Interactions with both RBP1, a nuclear speckle protein, and ASHR3, a chromatin modifier, highlighted the importance of nuclear localization of *ENOD40*. It may be noted that we could detect the association of both *AhENOD40* and NAT-*Ah DONE40* with the translation machinery (Supplementary Tables S3 and S4), which is similar to what has been reported before (van de Sande et al. 1996). This is in accordance with *ENOD40* encoding small peptides (Röhrig et al. 2002) and justifies the cytoplasmic localization of *ENOD40*. Notably, the SAS pair of *PHO1;2-cis-NAT PHO1;2* is associated with their shuttle to the cytoplasm and recruitment in the polysome for *PHO1;2* translation (Jabnoune et al. 2013).

From a functional standpoint, both *ENOD40* RNA and ASHR3 have been previously shown to be implicated in the control of cell division in different independent studies (Charon et al. 1997; Kumpf et al. 2014). Mutations in ASHR3 in *Arabidopsis* disrupts the pattern of coordinated DNA replication and cell division and increase the cell division rate in the quiescent center of roots (Kumpf et al. 2014), whereas overexpression of *ENOD40* in nitrogen-deprived transgenic *Medicago* plants resulted in extensive cortical cell division in the roots (Charon et al. 1997). Considering the involvement of both ASHR3 and *ENOD40* RNA in the control of cell division in roots, it can be envisaged that the observed interaction of NAT-*Ah DONE40* and ASHR3 may act as a mechanism for the regulation of cortical cell division during RNS. Regardless of the downstream functional implications, the interaction between AhASHR3 and NAT-*Ah DONE40* connects, for the first time, the antisense transcripts of an lncRNA implicated in the development of a novel organ (nodule) with master epigenetic controllers of developmental processes (PcG/TxG complexes) that are ancient and evolutionarily conserved across kingdoms. Such interactions between lncRNA and chromatin modifying complexes are well documented in both plant and animal systems. For example, in plants, the lncRNAs *COLDAIR* and *COLDWRAP* generated from the *FLC* locus, and *AG-incRNA4* transcribed from the second intron of *AGAMOUS* (*AG*), interact with CURLY LEAF, a plant E(z) homolog in PcG (Berry and Dean 2015; Heo and Sung 2011; Kim and Sung 2017; Wu et al. 2018). Another example is the interaction of lncRNA *AUXIN-REGULATED PROMOTER LOOP RNA* with LIKE HETEROCHROMATIN PROTEIN 1, a PcG protein that regulates the auxin response in roots (Ariel et al. 2014). The MAF4 antisense RNA (*MAS-NAT*) produced from the *MADS AFFECTING FLOWERING 4 (MAF4)* locus interacts with WDR5a, a TrxG member that activates the *MAF4* locus (Zhao et al. 2018). In the animal system, the *Xist* and *HOTAIR* lncRNA interact with the PcG protein EZH2 (Rinn et al. 2007; Zhao et al. 2008), and the lncRNA *DBE-T* interacts with the TrxG protein ASH1-like (Cabianca et al. 2012).

lncRNA interactions with the TrxG and PcG complexes are associated with epigenetic regulation. For example, the lncRNAs *COOLAIR* and *AG-incRNA4* interaction with CURLY LEAF leads to deposition of the repressive mark H3K27me3 in the *FLC* and *AG* locus, respectively (Csorba et al. 2014; Wu et al. 2018). Both *COOLAIR* and *TWISTED LEAF* cause synchronized replacement of activating histone marks (H3K36me3 and H3K4me3) with the repressive ones (H3K27me3), highlighting the role of lncRNAs in coordinating the switching of chromatin states (Csorba et al. 2014; Liu et al. 2018). On the other hand, *MAS-NAT* interaction with a TrxG member (WDR5a) leads to deposition of activation mark (H3K4me3) at the *MAF4* locus (Zhao et al. 2018). This is similar to our results, where we demonstrate a significant increase in deposition of activation marks (H3K36me3, H3K4me3, H3K27ac, and H3K9ac) on *ENOD40* loci. Among these modifications, H3K36me3 and H3K4me3 are considered to be the signature H3 modification marks deposited
by ASHR3 (Cartagena et al. 2008; Lee et al. 2020; Zhang and Ma 2012).

Because AhASHR3 interacts with NAT-*Ah DONE40* during the early stages of rhizobial infection, we speculate that this interaction facilitates the recruitment of AhASHR3 to the *AhENOD40* locus that results in the activation of expression of all of the *AhENOD40* transcripts, including NAT-*Ah DONE40*, by changing its epigenetic landscape. This is similar to the existing model where, lncRNA and PcG/TxG complexes function together to coordinate the activation or repression state of any locus (Hekimoglu and Ringrose 2009). The Polycomb/Trithorax response elements (PRE/TRE) get transcribed into lncRNAs that bind to PcG and TxG component proteins for recruiting them by DNA-RNA pairing, thereby acting as a bistable switch for preserving the transcriptional state of their associated genes over cell generations (Hekimoglu and Ringrose 2009; Steffen and Ringrose 2014). Intriguingly, the *ENOD40* loci in both chr5 and chr15 were enriched in plant PRE/TRE cis motifs; for example, presence of GA repeats (Xiao et al. 2017) and also binding sites for FERTILIZATION INDEPENDENT ENDOSPERM (Deng et al. 2013) and ULTRAPETALA 1 (Roy et al. 2019), which may also have a similar role in *ENOD40* locus opening (Supplementary Fig. S9).

In summary, our investigation provides significant clues toward understanding the mode of action of *ENOD40* during the initiation of cortical cell division by demonstrating the interaction of NAT-*Ah DONE40* with AhASHR3 and dynamic alterations of *ENOD40* locus that may be required for cell fate reprogramming. Further experimentation is required for understanding the individual roles of sense and antisense *ENOD40s* and their mechanistic relationships. It remains to be seen whether NAT-* DONE40* expression is universal in legumes and whether they function as the *ENOD40*--NAT-* DONE40* pair for nodule organogenesis.

## MATERIALS AND METHODS

### Plant material and rhizobial strain.

A. hypogaea seed JL-24 were acquired from ICRISAT, Telangana, India. The seed were germinated and inoculated with SEMIA 6144 according to Karmakar et al. (2019) and Sinharoy et al. (2009).

### RNAi of AhENOD40.

The *AhENOD40-RNAi* construct was generated by amplification of *ENOD40* from A. hypogaea root cDNA using primers 5'-CACCATGAAGCTTCTTTGTTGGC-3' and 5'-GCAAGTT GACCAAGTAAATTCTCCAC-3'. Amplified fragments were cloned into pENTR/D-TOPOR (Life Technologies) and then into binary vectors pK7GWIWG2D(II) (Karimi et al. 2002) by gateway technology through the LR clonase reaction kit (Life Technologies). Plant transformation and analysis was done according to Kundu and DasGupta (2018) and Sinharoy et al. (2009).

### Transcriptome analysis and filtering out intergenic lncRNAs.

Sequence Read Archives of A. hypogaea from previously published strand-specific RNA transcriptome GEO data having accession ID GSE98997 have been analyzed (Karmakar et al. 2019). The data, corresponding to six time points in triplicate, were mapped to the A. hypogaea whole genome downloaded from Pea-nutBase. Alignment was performed with Hisat2 using the option "-rna-strandness R" (Pertea et al. 2016). Transcript assembly was performed with Cufflinks v2.1 (Trapnell et al. 2012) followed by Cuffcompare (Trapnell et al. 2012) using the A. hypogaea genome annotation file, and intergenic lncRNAs were filtered out. (i) Intergenic lncRNAs were initially filtered out using the -u class code option of Cuffcompare. In order to check whether the

transcripts obtained actually lie within the intergenic region of A. hypogaea, intergenic positions were obtained by bedtools intersect (Quinlan and Hall 2010) using the A. hypogaea genome annotation file. (ii) At least three reads getting mapped to transcript had to be represented in read count. (iii) Transcript coverage of the mapped reads should be more than 70% (both read counts and read coverage are checked with bedtools coverage (Quinlan and Hall 2010). (iv) Transcripts should be at least 500 bp away from nearest coding gene (checked by in house perl script). Finally, the sequence was obtained for the filtered set of transcripts using bedtools getfasta (Quinlan and Hall 2010) and coding potential scores were calculated with the CPC2 tool (Kang et al. 2017). Transcripts whose CPC2 score indicated noncoding were chosen finally.

### Sequence analyses.

Multiple sequence alignment was performed using MUSCLE with default parameters (Edgar 2004). The phylogenetic maximum-likelihood tree was generated using MEGA X (Kumar et al. 2018) with the bootstrap test of 1,000 replicates. The synteny analysis was done using PeanutBase. The distance and position of *ENOD40* were manually analyzed and aligned.

### Detection of AhENOD40 sense and antisense transcripts by RT-qPCR-based assay.

Total RNA was prepared using the Macherey Nagel Nucleo-Spin RNA plant kit according to the manufacturer's protocol. For nuclear RNA isolation, the nucleus was first isolated using the Cell Lytic Plant Nuclei Isolation/Extraction Kit (Sigma), and RNA was isolated using Trizol/chloroform (Simms et al. 1993). The purified total RNA and nuclear RNA were reverse transcribed separately with the forward primer (*ENOD40* F RT) designed to hybridize specifically with NAT-*Ah DONE40*, yielding F-cDNA, and the reverse primer (*ENOD40* R RT) that specifically hybridizes with both *AhENOD40-1* and *AhENOD40-2*, yielding R-cDNA. Both RT reactions were performed at 50°C for 1 h using the Superscript III RT (Invitrogen) and, for each sample, a negative control reaction without Superscript III RT was also performed. During RT-qPCR analysis, to distinguish between chr5 and chr15, the 28-bp insertion in chr5 was used as a marker. Primers were designed for chr5 encompassing the 28-bp and were marked as L1F and L1R. Similarly, the flanking regions of the 28-bp insertion in chr5 was selected for chr15-specific primer L2R which, in the case of chr5, will not bind to it. To assess the presence of *AhENOD40-1* and *AhENOD40-2*, the R-cDNA was amplified using primers *ENOD40* R RT and L1R for chr5 and L2F for chr15. Similarly, to assess the presence of NAT-*Ah DONE40*, the F-cDNA was amplified using primers *ENOD40* F RT and L1F (Røsok and Sioud 2004). Quantification of the respective amounts of *AhENOD40-1*, *AhENOD40-2*, and NAT-*Ah DONE40* from the uninfected and the infected samples was done by the absolute quantification method using standard curves generated for the amplicon corresponding to the region amplified by the respective primer pairs (Boulter et al. 2016).

### In vitro transcription reaction.

The *AhENOD40-1*, *AhENOD40-2*, and NAT-*Ah DONE40* clones were used as the template for the PCR-based amplification of *ENOD40* with the forward primer harboring the T7 polymerase binding site at the 5' end for the generation of sense and antisense transcripts. PCR product (1 µg) was mixed with 10× transcription buffer, rNTP mix, radiolabeled or biotinylated UTP, RNase inhibitor (Promega Corp.), and T7 RNA polymerase (New England Biolabs) according to the supplier's instructions, and the reaction mixture was incubated at 37°C for 1 h followed by DNasel digestion for 15 min. The reaction mixture was separated in 5% native
polyacrylamide gel electrophoresis, and the *ENOD40* RNA was extracted.

### RNA pull-down assay.

Total cellular protein extracts from *A. hypogaea* root tissues were prepared by homogenization of the root tissues in binding buffer containing 50 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0), 50 mM KCl, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 1% Nonidet P40. The homogenate was vortexed intermittently for 45 min and kept on ice between the pulses. The homogenate was centrifuged at 13,000 rpm for 30 min at 4°C, and the supernatant was collected. The supernatant (1 ml) representing 1 to 2 mg of the total protein extract was incubated overnight at 4°C with 50 µmol of in vitro transcribed biotinylated *AhENOD40* or NAT-*Ah DONE40* RNA (encompassing ORF1 to ORF2) in the presence of 1 mM dithiothreitol and 5 mM MgCl₂ on a rotation wheel at 10 rpm. Streptavidin-coated magnetic beads (Dynal) were washed according to the manufacturer's protocol, then equilibrated in binding buffer for 15 min. In all, 100 µl of the beads was added to the binding reaction mixture for 5 h to isolate the RNA protein complexes. Following incubation, the beads were washed twice with binding buffer and resuspended in 50 mM ammonium bicarbonate buffer; then, bead tryptic digestion, MALDI-MS/MS, and LC-MS/MS proceeded according to Fukuyama et al. (2012) and Kaiser et al. (2008). The generated peptide masses were searched against the UniProt protein sequence of AhASHR3 using the Progenesis QI search engine. The sequences of SET-domain-containing proteins were appended to the forward and reverse *Arabidopsis thaliana* UniProt database.

### EMSA.

The DNA fragment corresponding to the coding sequences of the AWS, SET, and postSET domains of the *A. hypogaea* homolog of ASHR3 protein, AhASHR3 (residues 306 to 489), was cloned in pET32a, expressed in BL21 (DE3) (Thorstensen et al. 2008). EMSA was done according to Roy et al. (2019) using 1.5 fmol of RNA and 0.5 and 3 µM recombinant AhASHR3 for binding and 3 µM for competition assays.

### RIP.

The AhASHR3 overexpression construct was generated by amplification from *A. hypogaea* cDNA template with primers 5'-CACCATGCCCCGATTTGGGGAATTTGTCTCTTTCCGAG-3' and 5'-TCATACATGTGTTTCTATGGCAGGACATGCAGCT G-3' complementary to the full length of AhASHR3. Amplified fragments were cloned into pENTR/D-TOPOR (Life Technologies) and then into binary vectors pK7GWF2 (Karimi et al. 2002) by gateway technology through the LR Clonase reaction kit (Life Technologies). Constructs are then transformed in *Agrobacterium rhizogenes* strain R1000. The full-length AhASHR3 was overexpressed in *A. hypogaea*, and the transformed plants were inoculated with SEMIA 6144. At 4 DPI, the transformed roots were crosslinked by 1% formaldehyde under vacuum and quenched by the addition of 0.125 M glycine. RIP was performed as described by Mermaz et al. (2018), with some modifications. Instead of GFP-Trap_MA beads from ChromoTek, we used Magna ChIP Protein A+G Magnetic Beads (16-663; Sigma-Aldrich, Merck). For precleaning, the protein sample was incubated with equilibrated beads, and the beads were collected using the magnet. The supernatant was collected and was allowed to mix with anti-GFP antibody (11814460001; Roche) overnight on a rotator at 4°C for the immunoprecipitation of GFP-tagged proteins. Equilibrated beads (20 µl) were added to the supernatant and mixed for 3 h at 4°C. The beads bound with antibody-AhASHR3-RNA were collected using a magnet and the process according to Mermaz et al. (2018). The immunoprecipitated

RNA was reverse transcribed to cDNA and was quantified with RT-qPCR for detection of enrichment of RNAs. The enrichment of *Ah DONE40*, *AhENOD40-1*, and *AhENOD40-2* was normalized to the input using the standard %Input method.

### Chromatin immunoprecipitation assays.

Chromatin immunoprecipitation (ChIP) assays were done according to Roy et al. (2014) with root tissue samples collected at three different stages of rhizobial infection (0, 4, and 8 DPI), using antibodies against unmodified histone H3 (number ab1791; Abcam, Cambridge), H3K27me3 (number ab6002; Abcam, Cambridge), H3K27ac (number ab4729; Abcam, Cambridge) H3K4me3 (number ab8580; Abcam, Cambridge), H3K9ac (number ab0812; Abcam, Cambridge), and H3K36me3 (number ab9050; Abcam, Cambridge). qPCRs were carried out using Luna Universal qPCR Master Mix (New England Biolabs) with primers specific for the conserved region of the *ENOD40* locus from chr5 and 15. The *AhActin* gene was used for normalization in each case. Respective amounts of the immunoprecipitated DNA in uninfected and rhizobia infected samples were quantified according to Pavangadkar et al. (2010), and the data were normalized as the levels of histone H3 modifications relative to unmodified H3 for each independent sample. Postnormalization, the data were represented as fold change over uninfected.

### ACKNOWLEDGMENTS

We thank IISER Kolkata, India for access to sequencing facility; the Central Instrument Facility, Bose Institute, India for access to MALDI-TOF and LC-MS facilities; S. Sinharoy and S. Chaudhuri for critical reading of the manuscript; and H. Dey for excellent technical support.

### AUTHOR-RECOMMENDED INTERNET RESOURCES

GEO data: https://www.ncbi.nlm.nih.gov/geo/

PeanutBase: https://www.peanutbase.org/

### LITERATURE CITED

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