In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA

Abstract

We show, with miR171, that plant miRNA genes are modular independent transcription units in which the fold-back pre-miRNA is sufficient for miRNA processing, and that the upstream region contains highly specific promoter elements. Processing depends on flanking sequences within the miRNA stem-loop precursor rather than the miRNA sequence itself, and mutations affecting target pairing at the center and 5' but not 3' region of the miRNA compromise its function in vivo. Inactivation of the SDE1 RNA-dependent-RNA-polymerase was mandatory for accurate representation of miRNA activity by sensor constructs in Arabidopsis. Work in sde1 background revealed a near-perfect spatial overlap between the patterns of miR171 transcription and activity, supporting the idea that plant miRNAs enable cell differentiation.

Figure 1.

Processing depends on structure rather than sequence of miR171 precursor. (A) Schematic of the miR171 locus (IGR171) and miR171 precursor (miR171prec). Sizes of the IGR and putative promoter-containing fragments are indicated. miR171 is boxed in red. (B) Cultures of Agrobacterium strains containing IGR171 and miR171prec constructs were injected into N. benthamiana leaves. Total RNA was extracted at 4 dpi and miR171 levels analyzed by Northern blot using 15 μg of RNA. (rRNA) Sample loading control by ethidium bromide staining; (n.i.) noninfiltrated tissue. (C) Predicted secondary structure of miR171prec (left) and miRGFPprec (right). miRNAs are boxed in red and green, respectively. (D, top) Predicted pairing between miRGFP and its target sequence in transcript GFPa. (Bottom) Gel blot analysis of 10 μg total RNA confirms synthesis of miRGFP in N. benthamiana leaves at 4 dpi. (E) Visual inspection (top), protein blot analysis (middle), and Northern analysis (10 μg total RNA, bottom) of samples cotreated with GFPa and miRGFPprec (lane 3) or miR171prec (lane 2), at 4 dpi. (F) Construct GFPb contains five successive synonymous mutations (indicated in red) at the site of miRGFP-target pairing. The experiments in E were repeated with GFPb. Total prot, sample loading controls by Coomassie staining.

Figure 2.

Target pairing requirements for miR171 function in vivo. (A) Schematic of the GFP-miR171 target sequence fusions. The GFP stop codon is underlined. The three nucleotides in red were used as linkers. Nucleotides in green located 3′ of the miR171 target sequence (blue) define a BamHI restriction site for generic cloning. Nucleotide substitutions in miR171 targets are in bold and underlined. The predicted ΔG values for the miR171-target duplex are on the right. Arrow indicates prominent cleavage site for the SCL6-IV mRNA, as mapped by Llave et al. (2002). The pictures of N. benthamiana leaves were taken at 4 dpi. (B) Gel blot analysis of 5 μg total RNA (top) and total protein (bottom) extracted from the tissues in A. Plus sign (+) indicates cotreatment with miR171prec. The antibody was GFP-specific. (C) ΔG values of the pairing of miR171 to target sequences in GFP-171.4 and GFP-171.5. The experiment was as described in A and B. Total prot and rRNA, sample loading controls, as in Figure 1.

Figure 3.

miR171-triggered transitive RNA silencing of GFP-target fusion constructs. (A) GFP-171.1 plants with an SDE1 genetic background are uniformly silenced for GFP. Pictures were taken under UV illumination without a band-pass filter so that tissues were visible due to chlorophyll red fluorescence. (B) GFP transformants with the SDE1 background are uniformly green. (C) Gel blot analysis of 8 μg of total RNA for detection of secondary GFP siRNAs. RNA was extracted from leaves of the progeny of two independent lines as depicted in A (lanes 4,5) or E and F (lanes 2,3). (C, lane 1) RNA from GFP silenced Arabidopsis provided a positive control. (D) Closer view of a leaf from a GFP transformant with the sde1 background. (E,F) Vein-specific GFP phenotype of the GFP-171.1 plants with the sde1 background. (G-I) Tranverse section of the leaf in D observed under CLSM. (vI) Primary vein; (vII) secondary vein; (ep) epidermis; (par) parenchyma. (Inset) Closer view of the tissue near the primary vein. The green channel was cut off in H to monitor chlorophyll fluorescence only. The panel in I is an overlay of G and H revealing the epidermal layer. (J-L) Same as G-I but with a leaf from a GFP-171.1 plant, as depicted in E,F. (M) Gel blot analysis of two independent GFP-171.1 lines in the SDE1 (lanes 1-4) or sde1 (lanes 5-8) background. Total RNA was extracted from leaves (L) or inflorescences (F), and 5 μg was subjected to gel blot analysis. Leaf RNA from a GFP line with SDE1 (lane 9) or sde1 (lane 10) background provided positive controls, and leaf RNA from nontransformed plants (SDE1 or sde1 background) provided negative controls (lanes 11,12). (N) miR171 levels in leaves (L) and inflorescences (F) of nontransgenic sde1 plants, as assessed by gel blot analysis of 10 μg total RNA. (O) Same as in M but with tissues from representative GFP-171.2 lines in SDE1 or sde1 background. (P) Sensor construct for miR164. The target sequence is as found in the NAC1 transcript (At1g56010). (Q) Transversal leaf sections of GFP-164.1 transformants with a wild-type (WT, top) or sde1 background (bottom).

Figure 4.

Comparison of the miR171 transcription and activity patterns in transgenic Arabidopsis. (A) Transversal section of a leaf from a GFP-171.2 representative T2 line with the sde1 background and the corresponding overlays, as in Figure 3. (B) Longitudinal views of the leaf in A. The second panel is an enlargement. (C) Transversal section of the central stem of the plant in A. (D-G) Same as A-C but for a pmiR171-GFP transformant (sde1 background). (H-J) Same as A-C but for a GFP-171.1 transformant (sde1 background). (K) Typical flower of a GFP-171.2 line with the sde1 genetic background. GFP expression is high in all organs (petal [K1], sepal [K2], stamen [K3], and carpel [K4]). (L) Transversal section of the carpel in K4 and the corresponding overlays, observed by CLSM. (M) Roots from a GFP-171.2 representative line (sde1 background). (N-P) Same as K-M, but with tissues from pmiR171-GFP lines (sde1 background). The inlay in P shows the root imaged under transmitted light. (Q-S) Same as K-M, but with tissues from GFP-171.1 plants (sde1 background). (g) Guard cells; (ep) epidermis; (c) cortex; (sta) stamen; (carp) carpel; (pet) petal; (sep) sepal; (stig) stigma; (sty) style; (v) valve; (rep) replum; (mes) mesocarp; (ex) exocarp; (ov) ovule.