A GmNINa-miR172c-NNC1 Regulatory Network Coordinates the Nodulation and Autoregulation of Nodulation Pathways in Soybean

Molecular Plant Research Article

A GmNINa-miR172c-NNC1 Regulatory Network Coordinates the Nodulation and Autoregulation of Nodulation Pathways in Soybean Lixiang Wang1,2, Zhengxi Sun1, Chao Su1,3, Yongliang Wang1, Qiqi Yan1, Jiahuan Chen1, Thomas Ott3,4 and Xia Li1,* 1

State Key Laboratory of Agricultural Microbiology, College of Plant Science and Technology, Huazhong Agricultural University, No. 1 Shizishan Road, Hongshan District, Wuhan, Hubei 430070, P.R. China

2

College of Biological Science and Engineering, Panzhihua University, No. 10 Airport Road, Eastern District, Panzhihua, Sichuan, China

3

€nzlestrasse 1, 79104 Freiburg, Germany University of Freiburg, Faculty of Biology, University of Freiburg, Scha

4

CIBSS - Centre for Integrative Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany

*Correspondence: Xia Li ([email protected]) https://doi.org/10.1016/j.molp.2019.06.002

ABSTRACT Symbiotic root nodules are root lateral organs of plants in which nitrogen-fixing bacteria (rhizobia) convert atmospheric nitrogen to ammonia. The formation and number of nodules in legumes are precisely controlled by a rhizobia-induced signal cascade and host-controlled autoregulation of nodulation (AON). However, how these pathways are integrated and their underlying mechanisms are unclear. Here, we report that microRNA172c (miR172c) activates soybean (Glycine max) Rhizobia-Induced CLE1 (GmRIC1) and GmRIC2 by removing the transcriptional repression of these genes by Nodule Number Control 1 (NNC1), leading to the activation of the AON pathway. NNC1 interacts with GmNINa, the soybean ortholog of Lotus NODULE INCEPTION (NIN), and hampers its transcriptional activation of GmRIC1 and GmRIC2. Importantly, GmNINa acts as a transcriptional activator of miR172c. Intriguingly, NNC1 can transcriptionally repress miR172c expression, adding a negative feedback loop into the NNC1 regulatory network. Moreover, GmNINa interacts with NNC1 and can relieve the NNC1-mediated repression of miR172c transcription. Thus, the GmNINa-miR172c-NNC1 network is a master switch that coordinately regulates and optimizes NF and AON signaling, supporting the balance between nodulation and AON in soybean. Key words: nodule number, autoregulation of nodulation, miR172c-NNC1, negative feedback loop, nodule inception Wang L., Sun Z., Su C., Wang Y., Yan Q., Chen J., Ott T., and Li X. (2019). A GmNINa-miR172c-NNC1 Regulatory Network Coordinates the Nodulation and Autoregulation of Nodulation Pathways in Soybean. Mol. Plant. 12, 1211– 1226.

INTRODUCTION Legumes maintain the unique ability to undergo a symbiotic association with soil-borne rhizobia, which are hosted in a newly developed and highly specialized plant organ, the root nodule (Desbrosses and Stougaard, 2011; Oldroyd et al., 2011). Root nodules are formed simultaneously with onset of root hair infection in a process that requires the perception of specialized lipochito-oligosaccharide signals (nodulation [Nod] factors [NFs]) by plant root cells (Yang et al., 1994; Ferguson and Mathesius, 2003; Marsh et al., 2007; Kouchi et al., 2010; Guan et al., 2013). NFs are perceived by LysM-type receptor kinases (NF receptors), including NF RECEPTOR 1 and 5 (NFR1/5) in Lotus japonicus (Madsen et al., 2003; Radutoiu et al., 2003),

NOD FACTOR PERCEPTION (NFP) in Medicago truncatula (Arrighi et al., 2006), and GmNFR1a and GmNFR5a in soybean (Glycine max) (Indrasumunar et al., 2010, 2011). Upon recognition of NFs by NF receptors, legumes initiate a series of biochemical cascades, such as regular calcium oscillations in and around the nuclei of root epidermal cells (Oldroyd and Downie, 2008), to trigger the activation of downstream signaling components, e.g., calcium/calmodulin-dependent protein kinases and multiple transcription factors, including NODULATION-SIGNALING PATHWAY 1 (NSP1) and NSP2,

Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

Molecular Plant 12, 1211–1226, September 2019 ª The Author 2019. 1211

Molecular Plant ETHYLENE RESPONSE FACTOR REQUIRED FOR NODULE DIFFERENTIATION (EFD), ERF Required for Nodulation 1 (ERN1), and Nodule Inception (NIN), which activate nodulationrelated genes (e.g., ENOD40) (Papadopoulou et al., 1996; Fang and Hirsch, 1998; Schauser et al., 1999; Heckmann et al., 2006; Oldroyd and Downie, 2008; Vernie´ et al., 2008). Activation of these genes results in the initiation of root hair deformation, infection thread formation, cortical/pericycle cell division, and nodule primordia formation (Oldroyd and Downie, 2008), which collectively result in nodule formation and symbiotic nitrogen fixation. However, establishing symbiotic nitrogen fixation is a highly energy-demanding process and comes with a significant cost for the host: it largely depends on the delivery of photoassimilates from the host shoot (Streeter, 1980). Consequently, establishment of an excess number of nodules (supernodulation) markedly affects plant growth and development (Matsunami et al., 2004). Therefore, the total root nodule number is tightly controlled via a negative feedback mechanism called autoregulation of nodulation (AON) to maintain an optimal nitrogen and carbon balance in the host (Kosslak and Bohlool, 1984; Suzuki et al., 2008; Okamoto et al., 2009; Mortier et al., 2012; Soyano et al., 2014). It has been shown that AON is activated in root cortical cells during rhizobial infection and remains active during nodule primordium formation and nodule maturation (Lim et al., 2011). It starts with the production of root-derived nodulation-specific CLAVATA/ESR-related (CLE) peptides including LjCLE-RS1 and LjCLE-RS2 in L. japonicus (Okamoto et al., 2009), MtCLE12 and MtCLE13 in M. truncatula (Mortier et al., 2010), and rhizobiainduced CLE (GmRIC1) and GmRIC2 in soybean (Mortier et al., 2010) following the first induced cortical cell divisions during rhizobial infection, nodule development, and the onset of nodule functionality (Caetano-Anolle´s and Gresshoff, 1990; Li et al., 2009). These small functional CLE peptides can be transported from the root to shoot through the xylem (Matsubayashi, 2011), where they are specifically perceived by specific receptors: HYPERNODULATION ABERRANT ROOT FORMATION 1 (HAR1) in L. japonicus (Krusell et al., 2002; Nishimura et al., 2002; Okamoto et al., 2013), SUPER NUMERIC NODULES (SUNN) in M. truncatula (Schnabel et al., 2005) and NODULE AUTOREGULATION RECEPTOR KINASE (GmNARK) in soybean (Searle et al., 2003). These receptors then activate a still unknown signaling cascade to generate shoot-derived signals (SDIs) (e.g., cytokinin) in shoots, which then travel to the roots to suppress further nodule development, possibly by reducing the activity of NIN (Mathews et al., 1989; Krusell et al., 2002; Nishimura et al., 2002; Searle et al., 2003; Schnabel et al., 2005; Takahara et al., 2013; Sasaki et al., 2014; Soyano et al., 2014; Tsikou et al., 2018). The CLEs belong to a family of peptide hormones that are involved in intercellular, intertissue and interorgan communication in plants (Matsubayashi, 2011). The expression of the CLE genes is crucial for their precise regulatory functions. In legumes, alterations in expression of nodulation-specific CLEs greatly affect nodulation in a systemic manner (Okamoto et al., 2009; Reid et al., 2011). These CLE genes are specifically induced upon Rhizobium inoculation, but variation between homologous genes has been reported. For example, GmRIC1, LjCLE-RS1/2, and MtCLE13 are quickly induced by Rhizobium inoculation and NFs in soybean,

A GmNIN-miR172c Network in Nodulation L. japonicus, and M. trunctula, respectively (Okamoto et al., 2009; Mortier et al., 2010; Reid et al., 2011), whereas GmRIC2 expression is largely activated at a later stage of nodulation (Reid et al., 2011). Recently, it has been shown that Lotus NIN (LjNIN), an RWP-RK domain-containing transcription factor, can bind directly to NIN binding sites (NBSs) and activate LjCLE-RS1/2 expression and production of small functional LjCLE-RS1/2 in roots (Soyano et al., 2014). CLE-RS-HAR1 negatively regulates the expression of LjNIN, which in turn leads to downregulation of CLE-RS, thus forming a negative feedback loop. However, temporal and cell-/tissue-specific expression patterns of these CLE genes during nodulation have not been sufficiently addressed. Consequently, regulatory networks precisely controlling CLE gene expression remain largely unknown. MicroRNAs (miRNAs) are small non-coding RNAs (
22 nt in length) that regulate eukaryotic development by repressing the expression of target genes, particularly at the post-transcriptional level (Bartel, 2004). To date, a very limited number of miRNAs (e.g., miR169, miR172, miR171, miR167, miR393, and miR2111) have been validated for their roles in legume nodulation (Combier et al., 2006; Luis et al., 2012; Yan et al., 2013; Wang et al., 2015; Lelandais-Brie`re et al., 2016; Cai et al., 2017; Tsikou et al., 2018). Among them, miR2111 can translocate from the shoot to root in a HAR1-dependent manner in L. japonicus to mediate susceptibility of the host root to rhizobial infection by suppressing TOO MUCH LOVE (TML), a negative regulator of nodule number, thereby balancing infection and nodulation (Tsikou et al., 2018). In addition, miR172s (miR172a in soybean and L. japonicus, miR172c in soybean and common bean) play a crucial role in fine-tuning rhizobial infection and nodule organogenesis (Yan et al., 2013; Holt et al., 2015; Nova-Franco et al., 2015). miR172s mainly function through cleavage of APETALA2 (AP2) mRNA targets, which encode AP2 transcription factors (TFs) (Sakuma et al., 2002). These AP2 TFs activate or repress the expression of the downstream genes through direct binding to multiple cis elements in the target gene promoters (Abdelaty and Montserrat, 2003) to mediate various biological processes in plants (Licausi et al., 2013). Several members (e.g., AP2-1, AP2-2, and AP2-3 in soybean; AP2-1 in common bean; RAP27-like 1, AP2-like 1, and AP2-like 2 in L. japonicus) have been identified as the main targets of miR172s in nodulation signal transduction (Yan et al., 2013; Holt et al., 2015; Nova-Franco et al., 2015). It remains largely unclear how these AP2 TFs modulate rhizobial infection and nodule number in legumes. Previously, we identified miR172c as an endogenous miRNA in soybean that targets Nodule Number Control 1 (NNC1) encoding an AP2 transcriptional repressor (Wang et al., 2014). MiR172c is upregulated during rhizobial infection and positively regulates nodule number by cleaving NNC1 mRNA. Consequently, transcriptional repression of ENOD40 by NNC1 is removed and ENOD40-mediated nodule formation is activated. Interestingly, miR172c expression is negatively regulated by GmNARK, and aberrant hyperactivation of miR172c causes supernodulation of soybean. These observations suggest a crucial regulatory role for the miR172c/NNC1 module in AON and nodule number control. The molecular mechanisms by which the miR172c/ NNC1 module activates AON and in turn is suppressed remain largely unknown.

1212 Molecular Plant 12, 1211–1226, September 2019 ª The Author 2019.

Molecular Plant

A GmNIN-miR172c Network in Nodulation

Figure 1. NNC1 Functions as an Upstream Regulator of GmRIC1 and GmRIC2. (A) qPCR analysis of GmRIC1and GmRIC2 expression in transgenic roots carrying the empty vector (EV), 35S:miR172c, STTM172-48, 35S:NNC1m6, or RNAi-NNC1. Hairy roots were collected at 7 days after B. diazoefficiens USDA110 inoculation (DAI). Expression levels were normalized to that of GmELF1b. Data are presented as mean ± SD from three biological repeats. Letters indicate significant differences from the empty vector controls according to the Student–Newman– Keuls test (P < 0.05). (B) Number of nodules per hairy root expressing empty vector 1 (EV1), 35S:NNC1m6, 35S:GmRIC1, 35S:NNC1m6/35S:GmRIC1, EV2, 35S:NNC1m6/ EV2, 35S:GmRIC2, or 35S:NNC1m6/35S:GmRIC2. Nodule numbers were determined at 28 DAI. Data are presented as mean ± SD. More than 20 hairy roots were analyzed in each individual experiment. Letters indicate significant differences from the empty vector controls according to the Student– Newman–Keuls test (P < 0.05). (C) Representative images of nodulation phenotypes in (B).

Here, we show that miR172c activates GmRIC1 and GmRIC2 by removing the repressive effect of NNC1 to facilitate AON. NNC1 interacts with GmNINa, the soybean ortholog of LjNIN, reducing its transcriptional activation of GmRIC1 and GmRIC2. Notably, we found that GmNINa is a transcriptional activator of miR172c. Intriguingly, the expression of miR172c is negatively regulated by NNC1. The inhibition of miR172c expression by its own target gene constitutes a mutual inhibitory feedback loop. Importantly, we found that NNC1 interacted with GmNINa and predominantly inhibited the transcriptional activation of GmRIC1, GmRIC2, and miR172c by GmNINa. Therefore, we have identified a GmNINa-miR172c/NNC1 regulatory module that integrates the nodulation signaling pathway and AON, and revealed a unique regulatory mechanism that controls the activation threshold of genes controlling nodule number.

RESULTS The MiR172c/NNC1 Module Regulates GmRIC1 and GmRIC2 Expression To test whether the miR172c/NNC1 module regulates GmRIC1 and GmRIC2, we analyzed the effects of miR172c and NNC1 on the expression of GmRIC1 and GmRIC2. We generated transgenic hairy roots with altered expression of miR172c or NNC1, and root samples were collected at 7 days after Bradyrhizobium diazoefficiens USDA110 inoculation (7 DAI). Overexpression of miR172c or knockdown of NNC1 dramatically increased the expression of GmRIC1 and GmRIC2 in transgenic roots (Figure 1A). In contrast, downregulation of miR172c activity by a short tandem target mimicry (Yan et al., 2012; Wang et al., 2014) or overexpression of cleavage-resistant NNC1m6 markedly decreased GmRIC1 and GmRIC2 transcription (Figure 1A). To

further investigate the genetic relationship between miR172c/ NNC1 and GmRIC1 and GmRIC2, we performed phenotypic analyses of transgenic roots transformed with a single or two transgenes. In agreement with previously published data (Reid et al., 2011; Wang et al., 2014), downregulation of miR172c or overexpression of NNC1m6 markedly decreased the number of nodules compared with controls (Figure 1B and 1C; Supplemental Figure 1A and 1B). GmRIC1 and GmRIC2 overexpression (35S:GmRIC1 and 35S:GmRIC2, respectively) resulted in few (or, in some cases, no) nodules (Figure 1B and 1C; Supplemental Figure 1). In addition, simultaneous reduction of miR172c and overexpression of GmRIC1 resulted in the formation of only a few nodules, mimicking the nodule phenotype of 35S:GmRIC1 roots (Supplemental Figure 1A–1C). Thus, GmRIC1 and GmRIC2 function downstream of miR172c in nodule number control. Similarly, we tested the relationship between NNC1 and GmRIC1 and GmRIC2. As expected, roots overexpressing a mutated variant of NNC1 (NNC1m6) had significantly fewer nodules compared with those expressing the empty vector controls (Figure 1B and 1C; Supplemental Figure 2). Consistent with our previous results (Wang et al., 2014), nodule numbers were significantly increased in NNC1RNAi roots, while there were much fewer nodules in roots overexpressing GmRIC1 or GmRIC2 than in those overexpressing NNC1m6 (35S:NNC1m6) (Figure 1B and 1C). When 35S:NNC1m6/NNC1-RNAi and 35S:GmRIC1 or 35S:GmRIC2 were simultaneously overexpressed, the transgenic roots exhibited nodule phenotypes similar to those of 35S:GmRIC1 or 35S:GmRIC2 hairy roots but not those of 35S:NNC1m6 or RNAiNNC1 roots (Figure 1B and 1C; Supplemental Figures 2 and 3). These results suggest that both GmRIC1 and GmRIC2 act downstream of NNC1 and that the miR172c/NNC1 module regulates nodule number in soybean via GmRIC1 and GmRIC2.

Molecular Plant 12, 1211–1226, September 2019 ª The Author 2019. 1213

Molecular Plant

A GmNIN-miR172c Network in Nodulation

Figure 2. NNC1 Directly Targets GmRIC1 and GmRIC2. (A and B) Diagram of the GmRIC1/2 promoters. AP2 binding site (TTAAGGTT) shown as black boxes is a binding sequence for AP2 transcription factors, while the NBS sequence for GmNINa is shown as gray boxes. The fragments marked by the letters A to H indicate the regions examined in ChIP assays. (C and D) ChIP assays showing binding of NNC1 to the RIC1/2 promoters. DNA fragments were co-immunoprecipitated with polyclonal anti-GFP antibodies from chromatin suspensions prepared from 35S:NNC1m6-GFP or control (empty vector) samples. DNA fragments corresponding to the regions indicated in (A) and (B) were analyzed by qPCR. The DNA fragments were normalized to the input data. Data are presented as mean ± SD of three biological repeats. Asterisks indicate significant differences relative to the empty vector control according to Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. (E) NNC1 binding to oligo-DNAs containing TTAAGGTT. Biotin-labeled probes were incubated with MBP-NNC1. NNC1-GmRIC1 and NNC-GmRIC2 represent probes with the NNC1 binding sites from the promoters of GmRIC1 and NNC1-GmRIC2, respectively; the NNC1GmRIC1 and NNC1GmRIC2 fragments are the ones identified in (C) and (D). Competition for binding was performed with 2003 excess competitive GmRIC1 and GmRIC2 probes; MBP was used as a negative control. (F) The constructs harboring GmRIC1pro:GFP and GmRIC2pro:GFP were transformed with the empty vector and 35S:NNC1m6, respectively, into N. benthamiana leaves. The fluorescence of GFP in the N. benthamiana leaf cells was observed at 48 h after transformation.

NNC1 Directly Targets and Represses the Expression of GmRIC1 and GmRIC2 Since NNC1 is an AP2 TF, we asked whether it directly regulates the transcription of GmRIC1 and GmRIC2. To address this, we first searched for NNC1 binding sites in the promoters of GmRIC1 and GmRIC2. Indeed, we identified a single cis-regulatory element (TTAAGGTT) in the 2-kb promoter regions of GmRIC1 and GmRIC2 (Figure 2A and 2B). We then conducted chromatin immunoprecipitation (ChIP) assays to test for NNC1 binding to these promoters in soybean hairy roots. To avoid cleavage of NNC1 by native miR172c, we expressed a mutated version of the protein, NNC1m6-GFP (Wang et al., 2014). NNC1m6 was highly enriched at the regions of the GmRIC1 and GmRIC2 promoters containing AP2 binding sites (Jose´ et al., 2014) (Figure 2C and 2D). Unexpectedly, we also detected enrichment of NNC1m6 at other sites in the promoters of GmRIC1 and GmRIC2 (Figure 2A–2D), suggesting the presence of alternative

binding motifs. Sequence analysis showed that these additional NNC1 binding sites overlap with the core sequence of NBSs, suggesting the ability of NNC1 to bind NBSs (Konishi and Yanagisawa, 2013; Soyano and Kawaguchi, 2014). We next performed a DNA electrophoretic mobility shift assay (EMSA) to test direct NNC1 binding to AP2 binding sites in vitro. For this experiment, we expressed and purified the fulllength NNC1 protein fused with a maltose binding protein (MBP), MBP-NNC1, in Escherichia coli. Indeed, the MBP-NNC1 fusion protein was able to bind DNA probes (NNC1GmRIC1 and NNC1GmRIC2) containing the AP2 binding sites in the GmRIC1 and GmRIC2 promoters (Figure 2E), which are the same as those shown in Figure 2B and 2D. In a competition assay, we found that an excess amount of unlabeled DNA probe effectively reduced the binding of NNC1-MBP to the biotin-labeled DNA probe, suggesting that the interactions

1214 Molecular Plant 12, 1211–1226, September 2019 ª The Author 2019.

Molecular Plant

A GmNIN-miR172c Network in Nodulation between NNC1 and the GmRIC1 and GmRIC2 promoter fragments were specific (Figure 2E).

motifs (Supplemental Figure 9), suggesting that GmNINa is a functional ortholog of LjNIN.

To verify the negative regulatory role of NNC1 in GmRIC1 and GmRIC2 transcription, we transiently expressed GmRIC1pro:GFP or GmRIC2pro:GFP, or co-expressed GmRIC1pro:GFP or GmRIC2pro:GFP with NNC1m6 in Nicotiana benthamiana leaf cells. Both GmRIC1 and GmRIC2 were actively transcribed in leaf epidermal cells of N. benthamiana. However, GFP intensity was markedly decreased when GmRIC1pro:GFP or GmRIC2pro:GFP was co-expressed with NNC1m6 (Figure 2F). Next, a western blot was used to validate transcriptional repression of GmRIC1 and GmRIC2 by NNC1 in these N. benthamiana leaves. Indeed, co-expression of GmRIC1pro:GFP or GmRIC2pro:GFP with 35S:NNC1m6 greatly reduced levels of the GFP fusion proteins (Supplemental Figure 4). These data suggest that NNC1 is a transcriptional repressor of GmRIC1 and GmRIC2. To confirm these results, we further analyzed the effects of NNC1 knockdown on the expression levels of both GmRIC1 and GmRIC2 (Supplemental Figure 5). In the presence of rhizobia, NNC1 knockdown resulted in dramatically higher expression levels of GmRIC1 and GmRIC2 at 5 DAI, suggesting an NNC1 inhibits the expression of both genes during nodulation.

Considering that both GmNINa and NNC1 can bind the GmRIC1 and GmRIC2 promoters, we investigated whether these proteins interact physically with each other. To this end, we used several methods to systematically analyze protein–protein interactions. A yeast two-hybrid (Y2H) analysis showed that GmNINa directly interacts with NNC1 (Figure 3A and Supplemental Figure 10). A bimolecular fluorescence complementation (BiFC) analysis was employed to validate the GmNINa–NNC1 interaction. In brief, NNC1 and GmNINa were fused to the N terminus of YFP (nYFP) and the C terminus of YFP (cYFP) to generate nYFPNNC1 and GmNIN1-cYFP, respectively, then both fusion proteins were co-expressed in N. benthamiana leaf cells. Strong reconstituted YFP fluorescence in the nuclei of N. benthamiana leaf cells indicated that GmNINa interacts with NNC1 in planta (Figure 3B). The in vivo interaction of GmNINa with NNC1 was verified by coimmunoprecipitation (Co-IP) assays (Figure 3C).

NNC1 Interacts with the LjNIN Ortholog GmNINa A previous study showed that LjNIN transcriptionally activates CLE-RS1 and CLE-RS2, the orthologs of GmRIC1 and GmRIC2 in Lotus (Soyano et al., 2014). Thus, we asked whether NNC1 counteracts the GmNIN-activated expression of GmRIC1 and GmRIC2. To this end we studied GmNINa, the putative orthologous gene of LjNIN in soybean (Libault et al., 2010), although functional analysis of GmNINa during nodulation is still lacking. We first performed alignment and domain analysis and found that GmNINa indeed shares the highest amino acid sequence identity with LjNIN and MtNINa and possesses conserved Phox and Bem1 (PB1) and RWP-RK domains, and a mutated nitrateresponsive region disabling the responsiveness to nitrate (Konishi and Yanagisawa, 2019) (Supplemental Figures 6 and 7). Next, we attempted to test the function of GmNINa in soybean nodulation by overexpressing or silencing GmNINa. For specifically silencing GmNINa, we applied chimeric repressor silencing technology (CRES-T), which can overcome the possible functional redundancy between GmNIN family proteins (Hiratsu et al., 2003). To achieve this, we made a construct in which the sequence LDLDLELRLGFA known as the C-terminal SUPERMAN repressor domain X (SRDX) was fused to the C-terminal end of GmNINa cDNA (GmNINa-SRDX) to convert the transcription activator GmNINa into a dominant repressor (Hiratsu et al., 2003). We then constitutively overexpressed GmNINa (GmNINaOE) and GmNINa-SRDX (GmNINa-SRDXOE) under the control of the 35S promoter in composite transgenic roots. qRT–PCR analyses showed that GmNINaOE and GmNINa-SRDXOE upregulated and downregulated the expression of a number of genes downstream of GmNINa (GmENOD40, GmNSP1, GmNF-YA1a, GmRIC1, and GmRIC2); moreover, both GmNINaOE and GmNINa-SRDXOE nearly completely abolished nodulation in transgenic roots (Supplemental Figure 8), consistent with the previous result (Soyano et al., 2014). Notably, EMSAs and ChIP assays revealed that GmNINa bound directly to the promoters of GmRIC1 and GmRIC2 at the NBS

To determine which protein domains of GmNINa and NNC1 are responsible for the interaction, we performed both Y2H and BiFC assays using full-length or truncated GmNINa and NNC1. Unexpectedly, full-length and all of the truncated versions of GmNINa except GmNINa RWP-RK (GmNINa-RWP) were able to interact with NNC1 (Figure 3D and Supplemental Figure 10). Full-length NNC1 and all truncated versions including the NNC1 N terminus with or without the AP2 domain(s), NNC1 AP2 domain (NNC1-AP2), NNC1 C terminus with or without the AP domain, and NNC1 missing the AP2 domains were able to interact with GmNINa (Figure 3E). Furthermore, we analyzed the interaction between the conserved domains of both proteins. All the domains of NNC1 were able to interact with the N terminus of GmNINa (GmNINa-NT) but not with the RWP-RK and PB1 domains (Figure 3F and Supplemental Figure 11), suggesting that the N terminus of GmNINa is mainly responsible for its interaction with NNC1.

NNC1 Inhibits the GmNINa-Mediated Activation of GmRIC1 and GmRIC2 To investigate how GmNINa and NNC1 antagonistically regulate GmRIC1 and GmRIC2 transcription, we compared the binding sites for GmNINa and NNC1 in the promoters of GmRIC1 and GmRIC2. Interestingly, these binding sites have high similarity, indicating that GmNINa and NNC1 may bind to both sites (Supplemental Figure 12A). An EMSA showed that NNC1 could bind to the NIN binding cis element in GmRIC1 and GmRIC2, and, reciprocally, GmNINa was able to bind to the NNC1 binding site (Figure 4A). Given this result, we speculated that GmNINa and NNC1 antagonistically regulate GmRIC1 and GmRIC2 expression by competing for cis-element binding during nodulation. To test this prediction, we performed an in vivo ChIP assay to assess the binding of GmNINa and NNC1 to the promoters of GmRIC1 and GmRIC2. Significant enrichment was observed for GmNINa-FLAG and NNC1-FLAG at all DNA fragments of GmRIC1 and GmRIC2 containing NIN and AP2 binding sites (Figure 4B and Supplemental Figure 13A). The difference in fold enrichment between NNC1 and GmNINa suggests that NNC1

Molecular Plant 12, 1211–1226, September 2019 ª The Author 2019. 1215

Molecular Plant

A GmNIN-miR172c Network in Nodulation

Figure 3. NNC1 Interacts with the LjNIN Ortholog GmNINa. (A) Interactions between NNC1 and GmNINa were detected using a Y2H assay. Yeast cells co-transformed with pGADT7/pGBKT7-NNC1 and pGADT7/ pGBKT7-NINa (GmNINa) were selected and subsequently grown on selective media lacking Ade, His, Leu, and Trp (SD/-4) to test protein interactions. (B) BiFC assays to detect the interaction between NNC1 and GmNINa. NNC1 and GmNINa were fused to the N-terminal fragment of YFP (NNC1-nYFP) and C-terminal fragment of YFP (GmNINa-cYFP), respectively. The localization of the nucleus was detected by DAPI staining. Scale bars, 25 mm. (C) CoIP assays to verify the interaction of NNC1 with GmNINa. Protein was extracted from N. benthamiana leaves co-expressing NNC1-GFP with GmNINa-FLAG, then immunoprecipitated (IP) with FLAG antibody-bound agarose beads, and immunoprecipitated proteins were analyzed by using antiGFP and anti-FLAG antibodies. The experiments were repeated three times with similar results. (D–F) Mapping of the protein domains involved in the interaction between NNC1 and GmNINa using Y2H Assays. Based on the schematic protein structures of NNC1 and GmNINa, the interactions between GmNINa or its derivatives with NNC1 (D), between NNC1 or its derivatives with GmNINa (E), and between GmNINa derivatives with NNC1 derivatives were tested (F). Yeast cells co-transformed with pGBKT7-NNC1/GmNINa or pGBKT7- NNC1/ GmNINa derivatives (prey: For GmNINa-NT, we used pGADT7-GmNINa-NT as Prey) were selected and subsequently grown on selective media lacking Ade, His, Leu, and Trp (SD/-4) to test protein interactions.

has a greater binding affinity for promoter fragments containing either NIN binding or AP2 binding cis elements. Further expression analysis and western blotting revealed that NNC1 effectively suppressed GmNINa-induced GmRIC1 and GmRIC2 transcriptional activation in N. benthamiana leaf cells (Supplemental Figure 13B; Figure 4C and 4D). The inhibitory effects of NNC1 on the transcriptional activation of GmRIC1 and GmRIC2 by GmNINa were confirmed by overexpressing GmNINa or NNC1m6 or co-expressing these two transgenes in hairy roots (Figure 4E). Thus, NNC1 interacts with GmNINa and thereby hampers the transcriptional activation of GmRIC1 and GmRIC2. The immediate question was then how NNC1 represses the transcriptional activity of GmNINa. Given the fact that NNC1

does not interact with the RWP-RK DNA binding domain (Konishi and Yanagisawa, 2013), we speculated that NNC1 interaction with other domains of GmNINa may interrupt the binding activity of GmNINa. To test this, we performed EMSA assays and examined the effects of NNC1 on the binding of GmNINa to the promoter fragments of GmRIC1 and GmRIC2 containing NBS sequences. As shown in Figure 4A and 4F, GmNINa alone showed strong binding to both probes. However, addition of NNC1 completely abolished the binding of GmNINa to the probes (Figure 4F). Taken together, these results confirm that NNC1 interaction with other domains can sufficiently inhibit binding of GmNINa to DNA and its transcriptional transactivation activity. A detailed analysis of the promoters of functional orthologs of GmRIC1 and GmRIC2 in L. japonicus, Medicago sativa, and

1216 Molecular Plant 12, 1211–1226, September 2019 ª The Author 2019.

Molecular Plant

A GmNIN-miR172c Network in Nodulation

Figure 4. Competitive Inhibition of Specific Promoter Binding of GmNINa by NNC1. (A) GmNINa and NNC1 bind to the same sites in the GmRIC1/2 promoters. The Biotin-labeled NBS- containing probes and AP2 binding site-containing probes were incubated with NNC1-MBP or GmNINa-MBP. c-GmNINa is the C-terminus of GmNINa, and GmNINa GmRIC1/2 and NNC1 GmRIC1/2 represent the probes containing the GmNINa or NNC1 binding site in the GmRIC1/2 promoters. (B) ChIP assays showing the relative enrichment of NNC1-GFP and GmNINa-FLAG on the GmRIC1/2 promoters. DNA fragments were co-immunoprecipitated with polyclonal anti-GFP and anti-FLAG antibodies from chromatin suspensions prepared from transgenic or control (empty vector) hairy root samples. DNA fragments corresponding to the regions indicated in (Figure 2A and 2B) were analyzed by qPCR. The DNA fragments were normalized to the input data. Data are presented as mean ± SD of three biological repeats. Letters indicate significant differences from the empty vector controls according to the Student–Newman–Keuls test (P < 0.05). (C) Inhibition of GmNINa-mediated GmRIC1/RIC2 activation by NNC1. The constructs GmRIC1/2pro:GFP were transformed with 35S:NNC1m6, 35S: GmNINa, 35S:GmNINa-SRDX, or 35S:NNC1m6 and 35S:GmNINa into N. benthamiana leaves. GFP fluorescence was observed at 48 h after transformation. (D) The GFP levels were determined by western blot. 1, EV4 + GmRIC1/2pro:GFP; 2, 35S:NNC1m6 + GmRIC1/2pro:GFP; 3, 35S:GmNINa + GmRIC1/ 2pro:GFP; 4, 35S:GmNINa + 35S:NNC1m6 + GmRIC1/2pro:GFP; and 5, 35S:GmNINa-SRDX + GmRIC1/2pro:GFP. Fifteen independent plants were assessed. Similar trends were observed in three biological repeats. (E) Expression of GmRIC1 and GmRIC2 was analyzed in composite transgenic plants expressing empty vectors (EV1 or EV2), 35S:NNC1m6 in EV1, 35S:GmNINa in EV1, 35S:NNC1m6 in EV1, 35S:NNC1m6 in EV2 and 35S:GmNINa in EV2 at 6 DAI using qPCR. Data are presented as mean ± SD. More than 30 transgenic roots were analyzed in three independent biological repeats. Letters indicate significant differences from the empty vector controls according to the Student–Newman–Keuls test (P < 0.05). (F) NNC1 competes with GmNINa for binding to the promoters of GmRIC1 and GmRIC2. The biotin-labeled probes were incubated with MBP, NNC1AP2His, MBP-GmNINa, or NNC1AP2-His and MBP-C-GmNINa. GmNINaRIC indicates the GmNINa binding site-containing probes in promoters of GmRIC1/2; NNC1RIC indicates the NNC1 binding site-containing probes in promoters of GmRIC1/2.

Phaseolus vulgaris revealed that all of the genes contain AP2 and NBS cis elements in their promoter regions (Supplemental Figure 12B), suggesting that a conserved

mechanism exists for the antagonistic regulation of RIC genes by NIN and NNC1 or their orthologs during legume nodulation.

Molecular Plant 12, 1211–1226, September 2019 ª The Author 2019. 1217

Molecular Plant

A GmNIN-miR172c Network in Nodulation

Figure 5. GmNINa Functions as an Upstream Regulator of miR172c. (A) Nodule number per hairy root in composite transgenic plants expressing empty vectors (EV1, EV3 or EV2), STTM172-48 in EV3 and EV2, STTM172-48 in EV3 and 35S:GmNINa in EV2, STTM172-48 in EV3, 35S:NNC1m6 in EV1, 35S:GmNINa in EV2, 35S:NNC1m6 in EV1 and EV3, or 35S:NNC1m6 in EV1 and 35S:GmNINa in EV2 at 28 DAI. Data are presented as mean ± SD. More than 60 hairy roots were analyzed in three independent biological repeats. Letters indicate significant differences from the empty vector controls according to the Student–Newman–Keuls test (P < 0.05). (B) Representative nodulation phenotype of hairy roots transformed with the constructs presented in (A). (C) EMSA showing binding of MBP-GmNINa to the miR172c promoter fragments. GmNINa miR172-C/-E/-G/H denotes probes with GmNINa binding probes sites in the C/E/G/H promoter fragments of the miR172c. Competition for DNA binding was created using 250 3 excess unlabeled probe fragments. (D) The diagram of miR172c promoter. The pink box represents the NBS and the red box represents the AP2 binding site. (E) ChIP assay showing the binding sites of GmNINa to the miR172c promoter. DNA fragments corresponding to the regions indicated were analyzed by qPCR. The DNA fragments were normalized to the input data. All experiments had three biological replicates. Student’s t-test was performed. Asterisks indicate significant differences from the empty vector control. **P < 0.01.

MiR172c Is a Direct Target of GmNINa Our data indicated that miR172c activates AON, yet is reciprocally repressed by AON itself (Wang et al., 2014). This pattern was also reported for LjNIN (Soyano et al., 2014). Thus, we asked how miR172c and GmNINa are functionally related. To answer this question, we analyzed the genetic relationship between miR172c, NNC1, and GmNINa. Consistent with our previous results (Wang et al., 2014), STTM172 and 35S:NNC1m6 transgenic roots produced significantly fewer nodules compared with vector control roots. By contrast, 35S:GmNINa transgenic roots had almost no visible root nodules (Figure 5A and 5B). When STTM172 and NNC1m6 were co-expressed with GmNINa (Figure 5A and 5B; Supplemental Figure 14), the transgenic roots exhibited root nodule phenotypes that were comparable with those of transgenic roots expressing STTM172 and NNC1m6

only, suggesting that miR172c/NNC1 functions downstream of GmNINa. To validate this result, we analyzed the expression of miR172c and NNC1 in 35S:GmNINa and 35S:GmNINa-SRDX transgenic roots. MiR172c expression was markedly increased in 35S:GmNINa roots, whereas the transcription of NNC1 was substantially decreased in these roots (Supplemental Figure 15A). We then questioned whether GmNINa targets miR172c directly to moderate nodulation via the AON signaling pathway. Excitingly, we found three NBS cis elements in the 2-kb promoter region of miR172c (Figure 5D). EMSAs and ChIP assays demonstrated that GmNINa indeed specifically binds to promoter segments containing these and similar elements (Figure 5C and 5E). To confirm the effect of GmNINa on

1218 Molecular Plant 12, 1211–1226, September 2019 ª The Author 2019.

Molecular Plant

A GmNIN-miR172c Network in Nodulation miR172c transcription, we co-expressed 35S:GmNINa and miR172cpro:GUS in hairy roots of soybean. A subsequent histochemical analysis showed that GmNINa dramatically increased the GUS signal compared with the control with or without rhizobial inoculation (Supplemental Figure 15B). Thus, GmNINa targets miR172c directly to regulate its transcription.

NNC1 Directly Represses MiR172c Expression MiR172c regulates nodulation by repressing NNC1, and the overexpression of miR172c and NNC1m6 increased and reduced the number of nodules, respectively (Wang et al., 2014; Figure 6A and 6B). Simultaneous expression of miR172c and NNC1m6 resulted in a wild-type nodule phenotype (Figure 6A and 6B; Supplemental Figure 16), suggesting that miR172c is negatively regulated by NNC1. To assess this possibility, we analyzed miR172c expression in NNC1m6OE transgenic roots. qPCR analysis revealed that miR172c transcription was significantly reduced and increased in NNC1m6OE and NNC1-RNAi lines, respectively (Figure 6C). To explore whether a feedback loop exists between miR172c and NNC1, we analyzed the miR172c promoter and found a typical AP2 binding site, ‘‘CCTCGT’’ (Figure 5D). A ChIP assay of the NNC1-miR172c promoter complex in NNC1GFP roots inoculated with B. diazoefficiens USDA110 using an anti-GFP antibody showed that NNC1 indeed interacts with the miR172c promoter region containing the typical AP2 binding site in vivo (Figure 6D). Enrichment of NNC1 was also detected in two other regions of the miR172c promoter containing NBS, but not typical AP2 TF binding sites (Figure 6D). We then conducted EMSA as described above. The results obtained confirmed a direct interaction between NNC1 and the miR172 promoter (Figure 6E). Previously, we showed that NNC1 acts as a transcriptional repressor (Wang et al., 2014). To confirm the transcriptional repression of miR172c by NNC1, we transiently co-expressed 35S:NNC1m6 and miR172cpro:GFP in N. benthamiana leaf cells. The intensity of GFP fluorescence was dramatically reduced when miR172cpro:GFP was co-expressed with 35S:NNC1m6 (Supplemental Figure 17). Hence, there is reciprocal repression between miR172c and NNC1.

NNC1 and GmNINa Interfere with Each Other’s Transcriptional Regulation of MiR172c Expression The finding that NNC1 inhibits the transcriptional function of GmNINa in regulating GmRIC1 and GmRIC2 expression led us to speculate that NNC1 negatively affects the transcriptional function of GmNINa in regulating miR172c expression. In vivo ChIP assays revealed that NNC1 exhibited significantly greater enrichment at all tested regions of the miR172c promoter than GmNINa (Figure 6F). This may due to the fact that NNC1 could bind NBSs binding sites for GmNINa (Figure 6G), whereas GmNINa could not bind the AP2 motifs, which serve as NNC1 binding sites. It is likely that the pronounced differences in the binding activity of these TFs at other’s sites depend on the flanking sequences. (Supplemental Figure 18A). This result suggests that NNC1 may compete with GmNINa for the binding site in the miR172c promoter or interfere with GmNINa transcriptional activity. ubsequent expression analysis and western blotting revealed that NNC1 could interfere with GmNINa-activated miR172c transcription in N. benthamiana

leaf cells (Supplemental Figure 18B and 18C). The repressive effect of NNC1 on the GmNINa-mediated activation of miR172c expression was validated by analyzing pre-miR172c in hairy roots overexpressing GmNINa or NNC1m6 or co-expressing both genes (Supplemental Figure 19A). Furthermore, we also performed EMSA assays to analyze the effects of NNC1 on GmNINa binding to DNA and transcriptional transactivation activity. Indeed, NNC1 disrupted the binding of GmNINa to fragments of the miR172c promoter containing the NBS sequence (Supplemental Figure 19B). These data confirm a negative feedback loop where NNC1 represses miR172c expression by interacting with GmNINa and counteracting the transcriptional activation of miR172c by GmNINa.

GmNINa and NNC1 Function Downstream of GmNARK To further investigate the relationship between the transcription factors NNC1 and GmNINa and GmNARK, we analyzed NNC1 and GmNINa expression in the NARK loss-of-function mutant nts1007 in response to rhizobial infection. Expression of the NNC1 and GmNINa genes was repressed and elevated, respectively, in nts1007 roots during nodule development compared with wild-type soybean cultivar Bragg (Supplemental Figure 20A and 20B). To further clarify the genetic relationship between NNC1/GmNINa and GmNARK during nodulation, we generated composite Bragg and nts1007 plants overexpressing NNC1 and GmNINa. The number of nodules per transgenic root of nts1116 overexpressing NNC1 and GmNINa was dramatically decreased and almost completely complemented the supernodulating phenotype of the mutant (Supplemental Figure 20C–20F). Together, these results indicate that both NNC1 and GmNINa function downstream of GmNARK during nodulation.

DISCUSSION Among the various types of biological nitrogen fixation, symbiotic nitrogen fixation is the most efficient (Santi et al., 2013). However, the number of nodules at the roots is dynamically and precisely controlled to minimize excess loss of photosynthetic carbon to these symbiotically active organs. In the past three decades, several components of the molecular pathways that promote nodule organogenesis and control nodule number have been defined in various legumes. Transcriptional activation of CLE genes (e.g., GmRIC1/2, CLE12/13, and CLE-RS1/2) during nodule primordia formation is one of the hallmarks of the triggering of AON signaling, and the dynamic and precise regulation of transcription of these CLE genes is essential for modulating AON responses. In line with the rapid induction of CLE-RS1/2 and GmRIC1 upon rhizobial infection (Okamoto et al., 2009; Mortier et al., 2010), it has been suggested that AON is already triggered at very early stages of the symbiotic interaction (Suzuki et al., 2008). However, the molecular mechanism by which the transcription of CLE genes is repressed remains elusive. Here, we identified GmNINa as a functional ortholog of LjNIN (Figure 3 and Supplemental Figures 6–9). GmNINa is not only indispensable for transcriptionally activating the core genes involved in nodulation but also activating GmRIC1/GmRIC2 during AON (Supplemental Figure 9). Deactivation of CLE genes and attenuation of the AON signaling are equally important for regulation of optimal nodule number,

Molecular Plant 12, 1211–1226, September 2019 ª The Author 2019. 1219

Molecular Plant

A GmNIN-miR172c Network in Nodulation

Figure 6. NNC1 Directly Represses miR172c Expression by Reducing Transcriptional Function of GmNINa. (A) Nodule number per hairy root in transgenic plants expressing empty vector EV3, 35S:miR172c in EV3, empty vector EV1, 35S:NNC1m6 in EV1, 35S:miR172c in EV3 and EV1, 35S:NNC1m6 in EV1 and 35S:miR172c in EV3 at 28 DAI. Data are presented as mean ± SD from three independent experiments (n > 50). Letters indicate significant differences from the empty vector controls according to the Student–Newman-Keuls test (P < 0.05). (B) Representative nodulation phenotype of hairy roots transformed with the constructs presented in (A). (C) Relative expression levels of miR172c in hairy roots transformed with EV2, 35S:NNC1, 35S:NNC1m6, and RNAi-NNC1. The expression levels were normalized against the geometric mean of soybean miR1520d for miR172. Student’s t-test was performed. (D) ChIP assay showing the binding of NNC1 to the miR172c promoter. DNA fragments corresponding to the regions indicated in (C) were analyzed by qPCR. The DNA fragments were normalized to the input data. All experiments had three biological replicates. Student’s t-test was performed. Asterisks indicate significant differences from the empty vector control. *P < 0.05; **P < 0.01; ***P < 0.001. (E) EMSA showing binding of MBP-NNC1 to the miR172c promoter fragments. Competition for DNA binding was created using three different amounts of the unlabeled probe fragments; the last band was competed by mutated unlabeled probe in which the AP2 cis element was changed from CCTCGT into AAAAAA. The position of the cis element is shown in Figure 5D. (F) ChIP assays showing the relative enrichment of NNC1-GFP and GmNINa-FLAG binding to the miR172c promoter fragments C, E, F, and G as in Figure 5D. (G) NNC1 can bind to the GmNINa binding site in promoter of miR172c. The biotin-labeled NBS-containing probes were incubated with NNC1-MBP. GmNINamiR172c indicates the NIN binding site-containing probes from the promoter region of miR172c.

because constant activation and production of CLE peptide hormones will continuously activate AON signaling, resulting in nodulation inhibition. However, the molecular mechanism by which transcription of CLE genes is repressed remains elusive. Previously, we demonstrated that the miR172c/NNC1 module

positively regulates nodulation and that miR172c expression is regulated by GmNARK in soybean (Wang et al., 2014). However, the mechanism by which miR172c/NNC1 controls AON beyond its downregulation by GmNARK is unknown. Here, we provide several lines of evidence to support the notion

1220 Molecular Plant 12, 1211–1226, September 2019 ª The Author 2019.

Molecular Plant

A GmNIN-miR172c Network in Nodulation

differentially expressed during nodulation, and the CLE-RS3 gene is a new signaling component in the AON pathway in L. japonicus (Nishida et al., 2016), highlighting the complexity of AON in legumes. Thus, we cannot rule out the possibility that the miR172c/NNC1 module targets other early CLE genes involved in soybean nodulation and AON.

Figure 7. A Model for the Role of the GmNINa-miR172c/NNC1 Module in Balancing Nodulation and Autoregulation of Nodulation. Upon rhizobia infection, induction of GmNINa allows miR172c to be upregulated, leading to removal of transcriptional repression of ENOD40s and GmRIC1/2 by NNC1. GmNINa can also directly activate GmRIC1/2 expression. As a result, nodulation is initiated; in parallel, production of GmRIC1/2 turns on the autoregulation of nodulation (AON) pathway in the shoot. AON signaling then promotes production of shoot-derived inhibitors (SDIs) (e.g., cytokinin) that are transported to the roots to negatively regulate GmNINa expression and subsequent downregulation of miR172c. As such, an increase in NNC1 suppresses expression of miR172c and GmRIC1/2 to attenuate both the NF and AON signaling pathways and to maintain nodulation activity and an optimal nodule number in soybean.

that the miR172c/NNC1 module directly regulates the transcription of GmRIC1 and GmRIC2, which mediate AON activation and attenuation. First, the altered expression of miR172c or NNC1 caused substantial changes in GmRIC1 and GmRIC2 expression during nodulation (Figure 1A). Second, both miR172c and NNC1 function upstream of GmRIC1 and GmRIC2 in controlling nodule number (Figure 1B and 1C; Supplemental Figures 1–3). Third, and most importantly, NNC1 directly binds to the promoters of GmRIC1 and GmRIC2 to repress their transcription (Figure 2), and is indeed responsible for GmRIC1 and GmRIC2 repression during primordia formation (Supplemental Figure 5). These findings suggest that in the presence of rhizobia, the rapid upregulation of miR172c may remove the repressive effects of NNC1 on GmRIC1/ GmRIC2, turning on AON. Thus, the miR172c/NNC1 module temporally regulates nodule formation and systemic inhibition of nodulation in soybean. Nodulation inhibition is dynamically switched on or off at different stages of nodulation and in response to different environmental conditions, particularly nitrogen availability (Ligero et al., 1991; Soyano et al., 2014; Nishida et al., 2018). The interplay between nodulation and AON may occur at multiple levels and at different stages of nodulation. Since homologous genes, such as GmRIC1 and GmRIC2, exhibit different expression patterns during nodulation (Mortier et al., 2010; Wang et al., 2014), the repressive effects of NNC1 on GmRIC1 and GmRIC2 may occur at different stages of nodulation. It was shown that multiple CLE genes are

Thus far, we have identified GmNINa and NNC1 as the transcriptional activator and repressor, respectively, of GmRIC1/ GmRIC2 that antagonistically regulate the activation of these genes and the subsequent onset of AON. Our results demonstrate that NNC1 physically interacts with GmNINa and suppresses its transcriptional regulation of GmRIC1 and GmRIC2 (Figure 3), providing a novel mechanism for regulation of GmNINa by NNC1 during AON. It is known that the RWP_RK and PB1 domains in NIN and NIN-like proteins (NLPs) mediate DNA binding and interaction with other proteins, respectively (Chardin et al., 2014). Interestingly, NNC1 mainly interacts with GmNINa through the N terminus of GmNINa but not with the RWP_RK and PB1 domains (Figure 3). Thus, it is conceivable that inhibition of GmNINa transcriptional activity by NNC1 is not due to direct blocking of its DNA binding and/or protein interaction with other partners. It is likely that interaction with NNC1 results in a conformational change of GmNINa that hampers its DNA binding activity. Indeed, the AP2 domain of NNC1 greatly reduces the DNA binding activity of the GmNINa RWP_RK domain (Figure 4F), suggesting that NNC1 can outcompete GmNINa for binding to the target genes, which subsequently suppresses the transcriptional activity of GmNINa. This model is further supported by the stronger binding affinity of NNC1 to the GmRIC1 and GmRIC2 promoters, although NNC1 and GmNINa can bind to both the AP2 binding site and NBS cis elements (Figure 4). It is likely that upon rhizobial infection, NNC1 accumulation is reduced by repression of miR172c and that GmNINa takes over to activate the AON signaling (Figure 7). Considering that NIN is highly induced in response to rhizobial infection and during nodulation (Schauser et al., 1999; Marsh et al., 2007; Libault et al., 2010), the fact that NNC1 attenuates the activation of AON by GmNINa suggests that plants use this mechanism to avoid excess activation of AON by GmNINa, resulting in reduced nodulation and a decreased number of nodules. In soybean, several miR172s are responsive to rhizobial inoculation, and each has multiple target genes (Wang et al., 2014). Thus, it is plausible that nodulation-specific miR172s fine-tune nodulation and AON through NNC1 and NNC1-like proteins that can interact and interfere with key regulators such as GmNINa. We also found that miR172c is subject to transcriptional repression by its target NNC1 (Figure 6). The NNC1-miR172c negative feedback loop adds another branch to the NNC1 network, possibly acting to boost NNC1 protein levels in response to NNC1-repressing signals during nodulation. Since miR172c and NNC1 reciprocally repress each other, our work identifies an miR172c/NNC1 double-negative feedback loop during soybean nodulation. The miR172c/NNC1 feedback loop may fine-tune gene expression to control nodulation. Intriguingly, our study identified GmNINa as an additional transcriptional activator of miR172c (Figure 5), thereby placing miR172c downstream of GmNINa in the nodulation pathway. Previous studies have shown that LjNIN is a target of the long-distance AON feedback

Molecular Plant 12, 1211–1226, September 2019 ª The Author 2019. 1221

Molecular Plant loop in L. japonicus (Soyano et al., 2014) and that miR172c is negatively regulated by AON in soybean (Wang et al., 2014). Here we demonstrated that both GmNINa and NNC1 function downstream of GmNARK (Supplemental Figure 20). Thus, AON probably regulates transcription of miR172c and downstream genes through inhibiting GmNINa. Recently, it has been proposed that shoot-derived cytokinin can be transported to the root to modulate nodule number in both L. japonicus and M. truncatula (Sasaki et al., 2014; Azarakhsh et al., 2018). Considering the strong interconnection between NIN and cytokinin (Heckmann et al., 2011; Vernie´ et al., 2015), it is conceivable that AON inhibits nodulation by mainly targeting NIN, which then transcriptionally activates downstream genes including miR172c. The predominant role of NNC1 in regulating miR172c expression (Figure 6) may subsequently enable attenuation of the rhizobia-induced upregulation of miR172c to maintain the an appropriate level of miR172c during nodulation. This hypothesis is supported by the fact that NNC1 binds both AP2 and NBS motifs in the miR172 promoter, whereas GmNINa can only bind its own motif (Figure 6G and Supplemental Figure 16A). In summary, we propose that the GmNINa/ miR172c/NNC1 circuit is dynamically regulated at both temporal and spatial scales to ensure effective control of nodule number. The double-negative feedback loop within the GmNINa-miR172c/NNC1 network may function as a bistable switch that ensures effective control by enhancing the robustness of the system in complex cellular environments (Figure 7).

METHODS Plants and Rhizobium Strain Soybean (G. max cv. Williams 82, Bragg and the GmNARK mutant nts1007) plants were used in our experiments. B. diazoefficiens USDA110 was used for rhizobial inoculation and associated nodulation. The plant growth conditions and Rhizobium inoculation procedures were described previously (Wang et al., 2014).

Vector Construction Several plasmid backbones containing different selective markers were used in the study. Among them, pMDC32 (EV2), pMDC83, pMDC107, pMDC162, and pTCK303 (EV5) contain the hygromycin B (Hyg B) gene conferring plant resistance to hygromycin; pTF101-GFP (EV1), pEG100 carrying 33FLAG at the C terminus (EV4), and pEGAD (EV3) contain the Bar gene conferring glufosinate resistance in plants. The promoter:GFP reporter fusion constructs, the putative promoter regions 1883 bp upstream of the GmRIC1 (Glyma13g36831) start codon, 2075 bp upstream of the GmRIC2 (Glyma06g43681) start codon, and 2001 bp upstream of mature miR172c (MI0010727), were amplified from cv. Williams 82 genomic DNA and cloned into pDONR207 by BP reactions. Next, the plasmids (pDONR207 constructs harboring the GmRIC1, GmRIC2, and miR172c promoters) were used to generate the constructs pMDC107GmRIC1pro:GFP and pMDC107-GmRIC2pro:GFP by LR reactions. For the overexpression constructs of GmRIC1, GmRIC2, GmNINa, and GmNINa-SRDX (GmNINa fused with the SRDX motif: LDLDLELRLGFA, Hiratsu et al., 2003), the relevant coding sequences were amplified from cv. Williams 82 cDNA and inserted into the plant expression vector pMDC32 under the control of the CaMV35S (35S) promoter. For the GmNINa ChIP analysis construct, the GmNINa cDNA sequence was cloned into pDONR207 (Invitrogen) and then transferred into pEG100 carrying 33FLAG at the C terminus by LR clonase (Invitrogen). To construct plasmids for the expression of recombinant C-terminal GmNINa protein (amino acids 505–786 containing RWP-RK and the PB1 DNA binding region), NNC1 protein, and NNC1AP2 in E. coli BL21 cells, we amplified the full-length coding sequences of NNC1/NNC1AP2

A GmNIN-miR172c Network in Nodulation and C-terminal coding sequences of GmNINa and inserted them between the EcoRI and BamHI sites of pMAL-c2x. The construction of miR172c, STTM172-48, NNC1m6, and MBP-NNC1 was done as described previously (Wang et al., 2014). The primers used for plasmid construction are listed in Supplemental Table 1.

Soybean Hairy Root Transformation and B. diazoefficiens Inoculation Assay Agrobacterium rhizogenes strain K599 containing various constructs was used for soybean hairy root transformation; the transformation procedure is exactly the same as that described previously by Wang et al. (2014). For the nodulation phenotypic analyses, the putative transgenic composite plants were transplanted to pots (13 3 10 3 8.5 cm) containing vermiculite and grown for 1 week (16 h light/8 h dark, 25 C, 50% relative humidity). They were then inoculated with a suspension of B. diazoefficiens USDA110 (OD600 = 0.08) as described previously (Wang et al., 2014). At the indicated time points (DAI), individual roots and the nodules were harvested for molecular characterization of the transgenic roots and for nodule number quantification. When two constructs containing different genes were co-transformed, we cultured A. rhizogenes K599 strains containing individual genes as described above and mixed the strains at a 1:1 ratio for hairy root transformation. We have tested this method and found that hairy roots simultaneously expressing two genes can be obtained effectively.

DNA Extraction and Validation of Transgenes Hairy root samples harvested from the composite plants were extracted using the CTAB method as described by Porebski et al. (1997) with some modifications as described in our previous paper (Wang et al., 2014). Presence of the Bar or Hygromycin B gene (Hyg B) was was validated using PCR, and the hairy roots transformed with the corresponding empty vector were used as a negative control. For the hairy roots harboring two genes, the presence of both Bar and Hyg B genes was checked by PCR. The primers for the Bar and Hyg B genes are listed in Supplemental Table 1.

RNA Extraction and Expression Analysis Total RNA was extracted from wild-type or composite plants using TRIzol reagent according to the manufacturer’s protocol (Tiangen Biotech, Beijing, China). The removal of genomic DNA from the total RNA samples and first-strand cDNA synthesis from the total RNA were accomplished using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Tiangen Biotech). Stem-loop-specific reverse transcription for miR172c and miR1520d was performed as described previously (Chen et al., 2005). qRT–PCR was performed using SuperReal PreMix Plus (SYBR Green; Tiangen Biotech) with the gene-specific primers listed in Supplemental Table 1.

ChIP–qPCR Assay One gram of transgenic roots (10 DAI) expressing 35S:NNC1m6-GFP, 35S:GmNINa-FLAG, or 35S:GFP were used for ChIP assays. Roots were crosslinked with 1% formaldehyde at room temperature for 30 min and neutralized with 0.125 M glycine. Roots were ground to fine power in liquid nitrogen and the nuclei isolated. Immunoprecipitation was done using anti-GFP (ab290, Abcam) or anti-FLAG (A2220, ANTI-FLAG M2 Affinity Gel, Sigma) antibodies. Chromatin precipitated without the use of antibodies was used as a negative control, while chromatin isolated before precipitation was used as an input control. qPCR analysis was performed, and soybean ELF1b was used as an internal control. The specific primers used in this experiment are listed in Supplemental Table 1. Three independent biological repeats were performed for each analysis.

EMSA EMSA was performed as described previously (Wang et al., 2014). MBPtagged proteins were expressed in E. coli BL21 cells. An EMSA was performed using a Light Shift Chemiluminescent EMSA Kit (Pierce, Rockford,

1222 Molecular Plant 12, 1211–1226, September 2019 ª The Author 2019.

Molecular Plant

A GmNIN-miR172c Network in Nodulation IL) according to the manufacturer’s protocol. The binding activities of proteins were analyzed using an oligonucleotide labeled with biotin at the 50 end (Invitrogen, Carlsbad, CA). For unlabeled probe competition, 200fold excess unlabeled probes were added to the reactions. For GmNNC1, the mutated probe sequence was cgcttcg; For GmNINa, the mutated derivatives were modified from Soyano T’s protocol (Soyano et al., 2013).

To detect GFP/FLAG or GFP/FLAG fusion proteins, we used anti-GFP/ FLAG antibodies (Clontech, Mountain View, CA). Immunoreactive proteins were detected using an ECL Plus Chemiluminescence Kit (GE Healthcare, Chicago, IL). Ponceau S-stained polyvinylidene fluoride membranes or anti-tubulin immunoblotting were used to ensure equal loading.

Bioinformatics Analysis Transcriptional Activity Analysis in N. benthamiana Leaves Agrobacterium tumefaciens strain GV3101 was used in the experiments. A. tumefaciens GV3101 containing binary vectors was cultured overnight, then the OD600 of the culture was adjusted until the final OD600 was 0.3 using 10 mM MgCl2. For transiently expressing one gene or co-expressing two genes, each culture alone or a mixed culture with an equal volume (1:1) of each culture were used for injection (the final OD600 was 0.3 for each). At 2 days after injection, GFP fluorescence in the transformed N. benthamiana leaf cells was detected with a Leica SP8 confocal microscope and the leaf materials were collected for immunoblot analysis to confirm the observation.

Transcriptional Activity Analysis in Soybean Hairy Roots

Analyses of the promoters of the GmRIC1, GmRIC2, and miR172c genes, and the functional RIC orthologous genes in other legumes were performed using the online toolkit PLACE (http://www.dna.affrc.go.jp/ PLACE). The 2000-bp regions located upstream of the start codons (ATG) of GmRIC1, GmRIC2, and miR172c (http://www.phytozome.net) were used as promoter sequences for analyzing cis elements that can be bound by GmNINa and NNC1. The amino acid sequences of GmNINa, LjNIN, and MtNIN were aligned using the software MEGA5. The conserved domains PB1_NLP and RWP-RK of GmNINa and the orthologs were analyzed using the NCBI Conserved Domains software (http://www. ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).

Statistical Analysis

A. rhizogenes K599 strains containing binary vectors EV1, 35S:NNC1m6, and miR172cpro:GUS (2000 bp) were used for hairy root transformation of germinating W82 soybean seedlings. The construct combinations EV1 + miR172cpro:GUS and 35S:NNC1m6 + miR172cpro:GUS were co-transformed in hairy roots, For the histochemical analysis of GUS (b-glucuronidase) expression in roots and nodules, multiple roots or nodules at different developmental stages from at least 10 independent lines were stained for each construct. GUS activity was analyzed histochemically as described previously (Hu et al., 2013).

The gene expression and phenotypic analysis data in this study were analyzed using SigmaPlot 10.0 (Systat Software) and GraphPad Prism 5 (GraphPad Software). The averages and standard deviations (SD) of all results were calculated, and one-way ANOVA and multi-paired Student’s t-test were performed to generate P values. When there were statistically significant differences, Student–Newman–Keuls tests were conducted.

SUPPLEMENTAL INFORMATION Supplemental Information is available at Molecular Plant Online.

Y2H Assays The full-length coding sequence of GmNINa was amplified with the listed primers (Supplemental Table 1). Gateway PCR products were cloned into the pGBKT7/pGADT7 vectors by BP and LR reactions. We performed Y2H assays following the Matchmaker GAL4 two-hybrid system (Clontech). Yeast transformants were exhaustively selected on SD/-Ade/-His/-Leu/Trp (SD/-4) medium. For verification of the interactions between GmNINa/its domains and NNC1/its domains, GmNINa/NNC1 and their related domains were fused with the BD domain in the pGBKT7 vector individually. Primers used for the constructs are listed in Supplemental Table 1. For testing interactions the above constructs were cotransformed into the yeast strain Saccharomyces cerevisiae AH109. Transformation was confirmed by growth on SD/-Leu/-Trp medium. Interactions were assayed by spreading 5 ml of suspended transformed yeast on plates containing SD/-Ade/-His/-Leu/-Trp medium. The interactions were observed after 2–3 days of incubation at 30 C.

BiFC Assays NNC1 and GmNINa coding sequences were cloned in frame with the N-terminal end of YFP and the C-terminal end of YFP in vectors through a Gateway reaction with the pDONR vector system (Invitrogen). For verification of the interactions between GmNINa/its domains and NNC1/its domains, GmNINa/NNC1 and their related domains were fused with the pEARLYGATE201/202 vector individually. The primers used for vector construction are listed in Supplemental Table 1. The resulting constructs were transformed in A. tumefaciens strain GV3101, and successfully transformed clones were injected into. N. benthamiana leaf epidermal cells. Plants were then cultured for at least another 36 h before observation. YFP fluorescence was detected using a Leica confocal laser scanning microscope (Leica Microsystems). For visualizing nuclei, leaves were stained with 2 mg/ml 40 ,6-diamidino-2phenylindole (DAPI) for 2 h before observation.

Immunoblotting Assays Total proteins were extracted from N. benthamiana leaves transformed with the target gene(s) and the immunoblotting assays were performed.

FUNDING This research was supported by the National Key Research and Development Program of China (2016YFA0500503), the National Natural Science Foundation of China (31730066 and 31230050), the Ministry of Agriculture of the People’s Public of China (2018ZX0800919B and 2014ZX0800929B), and Huazhong Agricultural University Scientific & Technological Selfinnovation Foundation (2015RC014).

AUTHOR CONTRIBUTIONS X.L. and L.W. designed research; L.W., Z.S., Y.W., Q.Y., and J.C performed research; L.W., Z.S., and C.S. analyzed data; L.W., X.L., and T.O. wrote the paper.

ACKNOWLEDGMENTS We thank Prof. Peter Gresshoff (University of Queensland, Australia) for kindly providing the Bragg cultivar and nts1007 mutant. We thank Drs. Youning Wang, Zhijuan Wang, and Hongtao Ji at Huazhong Agricultural University (China) for their help in techniques and manuscript preparation. We thank Ms. Xingke Zhang for her technical support. No conflict of interest declared. Received: October 14, 2018 Revised: June 2, 2019 Accepted: June 4, 2019 Published: June 12, 2019

REFERENCES Abdelaty, S., and Montserrat, P. (2003). Plant AP2/ERF transcription factors. Genetika 35:37–50. Arrighi, J.F., Barre, A., Amor, B.B., Bersoult, A., Soriano, L.C., Mirabella, R., Carvalho-Niebel, F., Journet, E.P., Ghe´rardi, M., Huguet, T., et al. (2006). The Medicago truncatula LysM motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes. Plant Physiol. 142:265–279.

Molecular Plant 12, 1211–1226, September 2019 ª The Author 2019. 1223

Molecular Plant Azarakhsh, M., Lebedeva, M.A., and Lutova, L.A. (2018). Identification and expression analysis of Medicago truncatula isopentenyl transferase genes (IPTs) involved in local and systemic control of nodulation. Front. Plant Sci. 9:304. Bartel, D.P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297. Caetano-Anolle´s, G., and Gresshoff, P.M. (1990). Early induction of feedback regulatory responses governing nodulation in soybean. Plant Sci. 71:69–81.

A GmNIN-miR172c Network in Nodulation Indrasumunar, A., Kereszt, A., Searle, I., Miyagi, M., Li, D., Nguyen, C.D.T., Men, A., Carroll, B.J., and Gresshoff, P.M. (2010). Inactivation of duplicated Nod Factor Receptor 5 (NFR5) genes in recessive loss-of-function non-nodulation mutants of allotetraploid soybean (Glycine max L. Merr.). Plant Cell Physiol. 51:201–214. Indrasumunar, A., Searle, I., Lin, M., Kereszt, A., Men, A., Carroll, B.J., and Gresshoff, P.M. (2011). Nodulation Factor Receptor kinase 1 alpha controls nodule organ number in soybean (Glycine max L. Merr). Plant J. 65:39–50.

Cai, Z., Wang, Y., Zhu, L., Tian, Y., Chen, L., Sun, Z., Ullah, I., and Li, X. (2017). GmTIR1/GmAFB3-based auxin perception regulated by miR393 modulates soybean nodulation. New Phytol. 215:672–686.

Jose´, M.F., Irene, L.V., Jose´, L.C., Marta, G., Pablo, V., and Roberto, S. (2014). DNA-binding specificities of plant transcription factors and their potential to define target genes. Proc. Natl. Acad. Sci. U S A 111:2367– 2372.

Chardin, C., Girin, T., Roudier, F., Meyer, C., and Krapp, A. (2014). The plant RWP-RK transcription factors: key regulators of nitrogen responses and of gametophyte development. J. Exp. Bot. 65:5577– 5587.

Konishi, M., and Yanagisawa, S. (2013). Arabidopsis NIN-like transcription.factors.have a central role in nitrate signalling. Nat. Commun. 4:1617.

Chen, C., Ridzon, D.A., Broomer, A.J., Zhou, Z., Lee, D.H., Nguyen, J.T., Barbisin, M., Xu, N.L., Mahuvakar, V.R., Andersen, M.R., et al. (2005). Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 33:e179. Combier, J.P., Frugier, F., de Billy, F., Boualem, A., El-Yahyaoui, F., Moreau, S., Vernie´, T., Ott, T., Gamas, P., Crespi, M., and Niebel, A. (2006). MtHAP2-1 is a key transcriptional regulator of symbiotic nodule development regulated by microRNA169 in Medicago truncatula. Genes Dev. 20:3084–3088. Desbrosses, G.J., and Stougaard, J. (2011). Root nodulation: a paradigm for how plant-microbe symbiosis influences host developmental pathways. Cell Host Microbe 10:348–358. Fang, Y., and Hirsch, A.M. (1998). Studying early nodulin gene ENOD40 expression and induction by nodulation factor and cytokinin in transgenic Alfalfa. Plant Physiol. 116:53–68. Ferguson, B.J., and Mathesius, U. (2003). Signaling interactions during nodule development. J. Plant Growth Regul. 22:47–72. Guan, D., Stacey, N., Liu, C., Wen, J., Mysore, K.S., Torres-Jerez, I., Vernie´, T., Tadege, M., Zhou, C., Wang, Z., et al. (2013). Rhizobial infection is associated with the development of peripheral vasculature in nodules of Medicago truncatula. Plant Physiol. 162:107–115. Heckmann, A., Lombardo, F., Miwa, H., Perry, J., Bunnewell, S., Parniske, M., Wang, T., and Downie, J. (2006). Lotus japonicus nodulation requires two GRAS domain regulators, one of which is functionally conserved in a non-legume. Plant Physiol. 142:1739– 1750. Heckmann, A.B., Sandal, N., Bek, A.S., Madsen, L.H., Jurkiewicz, A., Nielsen, M.W., Tirichine, L., and Stougaard, J. (2011). Cytokinin induction of root nodule primordia in Lotus japonicus is regulated by a mechanism operating in the root cortex. Mol. Plant Microbe Interact. 24:1385–1395. Hiratsu, K., Matsui, K., Koyama, T., and Ohme-Takagi, M. (2003). Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabidopsis. Plant J. 34:733–739. Holt, D.B., Gupta, V., Meyer, D., Abel, N.B., Andersen, S.U., Stougaard, J., and Katharina, M. (2015). MicroRNA 172 (mir172) signals epidermal infection and is expressed in cells primed for bacterial invasion in Lotus japonicus roots and nodules. New Phytol. 208:241–256. Hu, Y., Chen, L., Wang, H., Zhang, L., Wang, F., and Yu, D. (2013). Arabidopsis transcription factor WRKY8 functions antagonistically with its interacting partner VQ9 to modulate salinity stress tolerance. Plant J. 74:730–745.

Konishi, M., and Yanagisawa, S. (2019). The role of protein-protein interactions mediated by the PB1 domain of NLP transcription factors in nitrate-inducible gene expression. BMC Plant Biol. 19:90. Kosslak, R.M., and Bohlool, B.B. (1984). Suppression of nodule development of one side of a split-root system of soybeans caused by prior inoculation of the other side. Plant Physiol. 75:125–130. Kouchi, H., Imaizumi-Anraku, H., Hayashi, M., Hakoyama, T., Nakagawa, T., Umehara, Y., Suganuma, N., and Kawaguchi, M. (2010). How many peas in a pod? Legume genes responsible for mutualistic symbioses underground. Plant Cell Physiol. 51:1381– 1397. Krusell, L., Madsen, L.H., Sato, S., Aubert, G., Genua, A., Szczyglowski, K., Duc, G., Kaneko, T., Tabata, S., Bruijn, F., et al. (2002). Shoot control of root development and nodulation is mediated by a receptor-like kinase. Nature 420:422–425. Lelandais-Brie`re, C., Moreau, J., Hartmann, C., and Crespi, M. (2016). Noncoding RNAs, emerging regulators in root endosymbioses. Mol. Plant Microbe Interact 29:170–180. Libault, M., Farmer, A., Brechenmacher, L., Drnevich, J., Langley, R.J., Bilgin, D.D., Radwan, O., Neece, D.J., Clough, S.J., May, G.D., et al. (2010). Complete transcriptome of the soybean root hair cell, a single-cell model, and its alteration in response to Bradyrhizobium japonicum infection. Plant Physiol. 152:541–552. Licausi, F., Ohme-Takagi, M., and Perata, P. (2013). Apetala2/ethylene responsive factor (AP2/ERF) transcription factors: mediators of stress responses and developmental programs. New Phytol. 199: 639–649. Li, D., Kinkema, M., and Gresshoff, P.M. (2009). Autoregulation of nodulation (AON) in Pisum sativum (Pea) involves signalling events associated with both nodule primordia development and nitrogen fixation. J. Plant Physiol. 166:955–967. Ligero, F., Caba, J.M., Lluch, C., and Olivares, J. (1991). Nitrate inhibition of nodulation can be overcome by the ethylene inhibitor aminoethoxyvinylglycine. Plant Physiol. 97:1221–1225. Lim, C.W., Lee, Y., and Hwan, C. (2011). Soybean nodule-enhanced CLE peptides in roots act as signals in GmNARK-mediated nodulation suppression. Plant Cell Physiol. 52:1613–1627. Luis, A.D., Markmann, K., Cognat, V., Holt, D.B., Charpentier, M., Parniske, M., Stougaard, J., and Voinnet, O. (2012). Two microRNAs linked to nodule infection and nitrogen-fixing ability in the legume Lotus japonicus. Plant Physiol. 160:2137–2154. Madsen, E.B., Madsen, L.H., Radutoiu, S., Olbryt, M., Rakwalska, M., Szczyglowski, K., Sato, S., Kaneko, T., Tabata, S., Sandal, N., et al. (2003). A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals. Nature 425:637–640.

1224 Molecular Plant 12, 1211–1226, September 2019 ª The Author 2019.

Molecular Plant

A GmNIN-miR172c Network in Nodulation Marsh, J.F., Rakocevic, A., Mitra, R.M., Brocard, L., Sun, J., Eschstruth, A., Long, S., Schultze, M., Ratet, P., and Oldroyd, G.E. (2007). Medicago truncatula NIN is essential for rhizobialindependent nodule organogenesis induced by autoactive calcium/ calmodulin-dependent protein kinase. Plant Physiol. 144:324–335. Mathews, A., Carroll, B.J., and Gresshoff, P.M. (1989). Development of bradyrhizobium, infections in supernodulating and non-nodulating mutants of soybean (Glycine max, [l.] Merrill). Protoplasma 150:40–47. Matsubayashi, Y. (2011). Small post-translationally modified peptide signals in Arabidopsis. Arabidopsis Book 9:e0150. Matsunami, T., Kaihatsu, A., Maekawa, T., Takahashi, M., and Kokubun, M. (2004). Characterization of vegetative growth of a supernodulating soybean genotype. Plant Prod. Sci. 7:165–171. Mortier, V., Den Herder, G., Whitford, R., Van de Velde, W., Rombauts, S., D’Haeseleer, K., Holsters, M., and Goormachtig, S. (2010). CLE peptides control Medicago truncatula nodulation locally and systemically. Plant Physiol. 153:222–237. Mortier, V., Holsters, M., and Goormachtig, S. (2012). Never too many? How legumes control nodule numbers. Plant Cell Environ. 35:245–258. Nishida, H., Handa, Y., Tanaka, S., Suzaki, T., and Kawaguchi, M. (2016). Expression of the CLE-RS3 gene suppresses root nodulation in Lotus japonicus. J. Plant Res. 129:909–919. Nishida, H., Tanaka, S., Handa, Y., Ito, M., Sakamoto, Y., Matsunaga, S., Betsuyaku, S., Miura, K., Soyano, T., Kawaguchi, M., et al. (2018). A NIN-LIKE PROTEIN mediates nitrate-induced control of root nodule symbiosis in Lotus japonicus. Nat. Commun. 9:499. Nishimura, R., Hayashi, M., Wu, G.J., Kouchi, H., Imaizumi-Anraku, H., Murakami, Y., Kawasaki, S., Akao, S., Ohmori, M., Nagasawa, M., et al. (2002). HAR1 mediates systemic regulation of symbiotic organ development. Nature 420:426–429. Nova-Franco, B., I´n˜iguez, L.P., Valde´s-Lo´pez, O., AlvaradoAffantranger, X., Leija, A., Fuentes, S.I., Ramı´rez, M., Paul, S., Reyes, J.L., Girard, L., et al. (2015). The microRNA172cAPETALA22-1 node as a key regulator of the common beanrhizobium etli nitrogen fixation symbiosis. Plant Physiol. 168:273–291. Okamoto, S., Ohnishi, E., Sato, S., Takahashi, H., Nakazono, M., Tabata, S., and Kawaguchi, M. (2009). Nod factor/nitrate-induced CLE genes that drive HAR1-mediated systemic regulation of nodulation. Plant Cell Physiol. 50:67–77. Okamoto, S., Shinohara, H., Mori, T., Matsubayashi, Y., and Kawaguchi, M. (2013). Root-derived CLE glycopeptides control nodulation by direct binding to HAR1 receptor kinase. Nat. Commun. 4:2191. Oldroyd, G.D., and Downie, J.M. (2008). Coordinating nodule morphogenesis with rhizobial infection in legumes. Ann. Rev. Plant Biol. 59:519–546. Oldroyd, G.E., Murray, J.D., Poole, P.S., and Downie, J.A. (2011). The rules of engagement in the legume-rhizobial symbiosis. Annu. Rev. Genet. 45:119–144. Papadopoulou, K., Roussis, A., and Katinakis, P. (1996). Phaseolus ENOD40 is involved in symbiotic and non-symbiotic organogenetic processes: expression during nodule and lateral root development. Plant Mol. Biol. 30:403–417.

Reid, D.E., Ferguson, B.J., and Gresshoff, P.M. (2011). Inoculation- and nitrate-induced CLE peptides of soybean control NARK-dependent nodule formation. Mol. Plant Microbe Interact. 24:606–618. Sakuma, Y., Liu, Q., Dubouzet, J.G., Abe, H., Shinozaki, K., and Yamaguchi- Shinozaki, K. (2002). DNA-binding specificity of the ERF/AP2 domain of arabidopsis drebs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochem. Biophys. Res. Commun. 290:998–1009. Sasaki, T., Suzaki, T., Soyano, T., Kojima, M., Sakakibara, H., and Kawaguchi, M. (2014). Shoot-derived cytokinins systemically regulate root nodulation. Nat. Commun. 5:4983. Santi, C., Bogusz, D., and Franche, C. (2013). Biological nitrogen fixation in non-legume plants. Ann. Bot. 111:743–767. Schauser, L., Roussis, A., Stiller, J., and Stougaard, J. (1999). A plant regulator controlling development of symbiotic root nodules. Nature 402:191–195. Schnabel, E., Journet, E.P., De Carvalho-Niebel, F., Duc, G., and Frugoli, J. (2005). The Medicago truncatula SUNN gene encodes a CLV1-like leucine-rich repeat receptor kinase that regulates nodule number and root length. Plant Mol. Biol. 58:809–822. Searle, I.R., Men, A.E., Laniya, T.S., Buzas, D.M., Iturbe-Ormaextxe, M., Carroll, B.J., and Gresshoff, P.M. (2003). Long-distance signalling in nodulation directed by a clavata1-like receptor kinase. Science 299:109–112. Soyano, T., and Kawaguchi, M. (2014). Systemic regulation of root nodule formation. In Advances in Biology and Ecology of Nitrogen Fixation, T. Ohyama, ed. (Croatia: INTECH), pp. 89–109. Soyano, T., Kouchi, H., Hirota, A., and Hayashi, M. (2013). Nodule inception directly targets NF-Y subunit genes to regulate essential processes of root nodule development in Lotus japonicus. PLoS Genet. 9:e1003352. Soyano, T., Hirakawa, H., Sato, S., Hayashi, M., and Kawaguchi, M. (2014). Nodule Inception creates a long-distance negative feedback loop involved in homeostatic regulation of nodule organ production. Proc. Natl. Acad. Sci. U S A 111:14607–14612. Streeter, J.G. (1980). Carbohydrates in soybean nodules. Plant Physiol. 66:471–476. Suzuki, A., Hara, H., Kinoue, T., Abe, M., Uchiumi, T., Kucho, K., Higashi, S., Hirsch, A.M., and Arima, S. (2008). Split-root study of autoregulation of nodulation in the model legume Lotus japonicus. Plant Res. 121:245–249. Takahara, M., Magori, S., Soyano, T., Okamoto, S., Yoshida, C., Yano, K., Sato, S., Tabata, S., Yamaguchi, K., Shigenobu, S., et al. (2013). Too much love, a novel kelch repeat-containing f-box protein, functions in the long-distance regulation of the legume-rhizobium symbiosis. Plant Cell Physiol. 54:433–447. Tsikou, D., Yan, Z., Holt, D.B., Abel, N.B., and Markmann, K. (2018). Systemic control of legume susceptibility to rhizobial infection by a mobile microrna. Science 362:233–236. Vernie´, T., Moreau, S., de Billy, F., Plet, J., Combier, J.P., Rogers, C., Oldroyd, G., Frugier, F., Niebel, A., and Gamas, P. (2008). EFD is an ERF transcription factor involved in the control of nodule number and differentiation in Medicago truncatula. Plant Cell 20:2696–2713.

Porebski, S., Bailey, L.G., and Baum, B.R. (1997). Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol. Biol. Rep. 15:8–15.

Vernie´, T., Kim, J., Frances, L., Ding, Y., Sun, J., Guan, D., Niebel, A., Gifford, M.L., de Carvalho-Niebel, F., and Oldroyd, G.E. (2015). The NIN transcription factor coordinates diverse nodulation programs in different tissues of the Medicago truncatula root. Plant Cell 27:3410–3424.

Radutoiu, S., Madsen, L.H., Madsen, E.B., Felle, H.H., Umehara, Y., Grønlund, M., Sato, S., Nakamura, Y., Tabata, S., Sandal, N., et al. (2003). Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature 425:585–592.

Wang, Y., Wang, L., Zou, Y., Chen, L., Cai, Z., Zhang, S., Zhao, F., Tian, Y., Jiang, Q., Ferguson, B.J., et al. (2014). Soybean miR172c targets the repressive AP2 transcription factor NNC1 to activate ENOD40 expression and regulate nodule initiation. Plant Cell 26:4782–4801.

Molecular Plant 12, 1211–1226, September 2019 ª The Author 2019. 1225

Molecular Plant Wang, Y., Li, K., Chen, L., Zou, Y., Liu, H., Tian, Y., Li, D., Wang, R., Zhao, F., Ferguson, B.J., et al. (2015). MicroRNA167-directed regulation of the auxin response factors, GmARF8a and GmARF8b, is required for soybean (Glycine max L.) nodulation and lateral root development. Plant Physiol. 168:984–999. Yan, J., Gu, Y., Jia, X., Kang, W., Pan, S., Tang, X., Chen, X., and Tang, G. (2012). Effective small RNA destruction by the expression of a short tandem target mimic in arabidopsis. Plant Cell 24:415–427.

A GmNIN-miR172c Network in Nodulation Yan, Z., Hossain, M.S., Wang, J., Valde´s-Lo´pez, O., Liang, Y., Libault, M., Qiu, L., and Stacey, G. (2013). miR172 regulates soybean nodulation. Mol. Plant Microbe Interact. 26:1371–1377. Yang, W., de Blank, C., Meskiene, I., Hirt, H., Bakker, J., van Kammen, A., Franssen, H., and Bisseling, T. (1994). Rhizobium nod factors reactivate the cell cycle during infection and nodule primordium formation, but the cycle is only completed in primordium formation. Plant Cell 6:1415–1426.

1226 Molecular Plant 12, 1211–1226, September 2019 ª The Author 2019.