Figure 1.
Expression of candidate NIN-target genes in roots that were ectopically expressing the NIN protein.
RT-PCR was used to analyze gene expression in nin-2 roots that were transformed with Pro35S-NIN-GR. Roots were treated as indicated in the figure. For CHX plus DEX, the DEX was added after pre-incubation with CHX for 30 min. chr4.CM0179.190.r2.m encodes a plastocyanin-like domain-containing protein (PLDP) and chr6.CM1757.140.r2.m encodes an AP2/ERF family protein. These genes are listed in Figure S2. The promoter of the former gene possesses NIN-binding nucleotide sequences (see Figure 2). The latter gene was used as a negative control for DEX treatment. The means and SDs from 3 biological repeats are shown.
Figure 2.
NIN directly targets LjNF-YA1 and LjNF-YB1.
(A) A diagram of the LjNF-YA1 and LjNF-YB1 promoter regions. These regions were used for the promoter-GUS reporters (see Figure 3). Gray lines indicate 5′-UTRs. Regions analyzed by RT-PCR for the ChIP assays in (B) are shown as blue lines and probes used for EMSA in (C) and (D) are shown as green lines. The red numbers indicate regions and probes that gave positive results in the ChIP assays and EMSAs. Arrowheads indicate positions of the NIN-binding sites. (B) ChIP assays using either ProLjUb-NIN-myc roots or control (empty vector) roots. The means and SDs from 3 biological repeats are shown. (C,D) EMSAs for analyzing NIN binding to LjNF-YA1 (C) and LjNF-YB1 (D) promoter regions. NIN-myc (Full), NIN(520–878)-myc (C-ter), and in vitro translation products without templates (control) were incubated with 32P-labeled probes shown in (A). Arrowheads indicate mobility-shifted bands specifically detected when incubated with NIN proteins. (E) An alignment of the partial nucleotide sequences of probes that were bound by NIN. yB1a, yB1b, yA1, E16a, are E16b correspond to NBSs found in the promoters of LjNF-YB1, LjNF-YA1, and the PLDP-encoding gene. Red and blue lines indicate nucleotides that are required for NIN-binding to NBS-yB1a, or that influence binding, respectively (see Figure S5). NBS-yB1m is an NBS-yB1a derivative with nucleotide substitutions. Comparison of NIN-binding sequences illustrated by logo is shown at the bottom. (F) GUS expression in tobacco leaf disks transformed with the indicated constructs. Bar: 1 mm. (G) EMSA for analyzing NIN binding to NBS-yB1a and the NBS-yB1a-like sequences. Arrowheads indicate probes that were bound by NIN proteins.
Figure 3.
Spatial expression patterns of LjNF-YA1 and LjNF-YB1 during root nodule organogenesis and their protein interactions in planta.
(A–F) GUS expression in roots that were transformed with either ProLjNF-YA1-GUS (A–C) or ProLjNF-YB1-GUS (D–F). GUS staining was detected in root nodule primordia (A, D), developing root nodules (B, E), and in spontaneous root nodules caused by Pro35S-gof-LHK1 (C, F). (G–L) BiFC analysis of interactions between LjNF-YA and LjNF-YB subunits in N. benthamiana leaves. Confocal images of eYFP fluorescence (green) from nuclei and chloroplast autofluorescence (red) are shown. ProLjUb-eYFPC-LjNF-YA1 was co-introduced with either ProLjUb-eYFPN-LjNF-YB1 (G) or ProLjUb-eYFPN-LjNF-YB2 (H). ProLjUb-eYFPC-LjNF-YA2 was co-introduced with either ProLjUb-eYFPN-LjNF-YB1 (I), ProLjUb-eYFPN-LjNF-YB2 (J), or ProLjUb-eYFPN (K). ProLjUb-eYFPN-LjNF-YB1 was co-introduced with ProLjUb-eYFPC (L). Bars: 0.2 mm in (A–F); 50 µm in (G–L).
Figure 4.
Inhibition of root nodule development by the knockdown of LjNF-YA1.
Roots that were transformed with either ProLjUb-RNAi-LjNF-YA1 (A,C) or an empty vector (B,D) were inoculated with DsRed-labeled M. loti for 14 days. (A,B) Root nodule formation in transformed roots. Fluorescence is visible from GFP (the root transformation marker) and from DsRed expressed in M. loti. Arrowheads indicate root nodules. The fractions of plants that formed root nodules on GFP-positive roots are shown in parentheses. (C,D) Infection thread formation in transformed roots. Fluorescence from GFP and DsRed is shown in the upper panels and bright field images are shown in the bottom panels of the same roots. Arrowheads indicate infection threads visualized by DsRed-labeled M. loti. Bars: 5 mm in (A,B), 0.2 mm in (C,D). (E) RT-PCR analyses of gene expression in roots that were transformed with either the empty vector, ProLjUb-RNAi-LjNF-YA1, or ProLjUb-RNAi-LjNF-YB1. Roots were inoculated with (2 dai; days after inoculation) or without (−) M. loti. Expression was analyzed for LjNF-YB1 and three L. japonicus NF-Y subunit A genes (LjNF-YA1, LjNF-YA2, and CBF-A22 [57]). The means and SDs from 3 biological repeats are shown.
Figure 5.
NIN overexpression induces cortical cell division.
(A) A Gifu (wild-type) root was transformed with ProLjUb-NIN and then cultured for 6 weeks in the absence of M. loti. Bumps (arrowheads) and malformed lateral roots (arrows) are indicated. (B,C) Cleared roots that were transformed with either an empty vector (B) or ProLjUb-NIN (C). The fractions of plants with bumps are shown in parentheses. (D) A transverse section of a root nodule primordium (10 dai) formed on a MG-20 (wild-type) root that was transformed with the empty vector. (E,F) Transverse sections of bumps formed on uninoculated MG-20 roots that were transformed with ProLjUb-NIN. Blue and red lines in (F) represent the outer edges of the endodermis and the boundary of the region with dividing cortical cells, respectively. (G–I) in situ RNA hybridization of ENOD40-1 in transverse sections of bumps caused by NIN overexpression, using either antisense (G,H) or sense probes (I). Asterisks indicate the central xylem. Arrows indicate the pericycle with in situ signals. Bars: 5 mm in (A); 0.2 mm in (B,C); 0.1 mm in (D–F); 50 µm in (G–I).
Figure 6.
NIN and NF-Y subunits regulate cell division.
(A–C) Lateral roots formed on MG-20 roots that were transformed with either an empty vector (A), ProLjUb-LjNF-YA1 (B), or ProLjUb-LjNF-YA1 Pro35S-LjNF-YB1 (C). Roots were cultured in the absence of M. loti. (D–G) Longitudinal sections of lateral roots formed on MG-20 roots that were transformed with either the empty vector (D), ProLjUb-LjNF-YA1 Pro35S-LjNF-YB1 (E,F), or ProLjUb-NIN (G). An asterisk in (F) indicates an additional lateral root meristem-like structure. (H,I) Magnified images of the boxed regions in (D) and (E), respectively. (J) A longitudinal section of the root that was transformed with ProLjUb-LjNF-YA1 and Pro35S-LjNF-YB1. Note the presence of small cells that were generated by cortical cell division. Bars: 1 mm in (A–C); 0.1 mm in (D–G,J); 0.2 mm in (H,I).
Figure 7.
RT–PCR analysis of the expression of early nodulin genes and LjNF-Y subunit genes.
Expression was analyzed for ENOD40-1 (A), ENOD40-2 (B), ENOD2 (C), LjNF-YA1 (D), LjNF-YB1 (E), and NIN (F). Plants were cultured for 3 weeks without M. loti inoculation. Total RNA was isolated from roots transformed with either an empty vector, ProLjUb-LjNF-YA1 plus Pro35S-LjNF-YB1 (NF-YA1 NF-YB1 OE), or ProLjUb-NIN (NIN OE). ProLjUb-NIN roots that exhibited altered structures (+) were harvested separately from roots with no morphological alterations (−). The means and SDs from 3 biological repeats are shown.