1. Volume 12, Issue 9, Pages 1211-1226 (September 2019)
A GmNINa-miR172c-NNC1 Regulatory Network Coordinates the Nodulation and Autoregulation of Nodulation Pathways in Soybean 
Lixiang Wang, Zhengxi Sun, Chao Su, Yongliang Wang, Qiqi Yan, Jiahuan Chen, Thomas Ott, Xia Li 
Molecular Plant 
Volume 12, Issue 9, Pages (September 2019)
DOI: /j.molp
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2. 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).
Molecular Plant  , DOI: ( /j.molp )
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3. 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 <
(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 200× 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.
Molecular Plant  , DOI: ( /j.molp )
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4. 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 μm.
(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 anti-GFP 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.
Molecular Plant  , DOI: ( /j.molp )
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5. 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, NNC1AP2-His, 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.
Molecular Plant  , DOI: ( /j.molp )
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6. 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), STTM in EV3 and EV2, STTM in EV3 and 35S:GmNINa in EV2, STTM 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 × 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.
Molecular Plant  , DOI: ( /j.molp )
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7. 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 <
(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.
Molecular Plant  , DOI: ( /j.molp )
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8. 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.
Molecular Plant  , DOI: ( /j.molp )
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