Figure 3. MIKKI negatively regulates the level of miR171.

(A) Repression of osa-miR171b ~ f (top) and derepression of its target gene (bottom) in MIKKI overexpression lines. RNA was extracted from panicles, leaves and roots. Error bars represent mean ± sd of three biological replicates performed in technical triplicate. The asterisks indicate statistical differences in comparison to the same tissues of wildtype (wt) determined by Student’s t-test. **p<e-10; *p<e-05; n.s., not significant. Wt Nipponbare was segregated from hemizygous overexpressor lines. (B) Abnormal spikelet development in MIKKI-overexpressing lines. Bar = 1 cm. (C) Percentage of unfertilized spikelets in overexpression lines. Data are mean of 10 panicles for each genotype. The asterisks indicate statistical differences determined by Student’s t-test. **p<e-10. (D) Derepression of osa-miR171b ~ f (top) and repression of the target gene (bottom) in mikki mutant plants. RNA was extracted from panicles, leaves and roots. Error bars represent mean ± sd of three biological replicates performed in technical triplicate. Wt Nipponbare was segregated from heterozygous mutant plants. The asterisks indicate statistical differences in comparison to the same tissues of wt determined by Student’s t-test. **p<e-10; *p<e-05; n.s., not significant. (E) Shoot and root length of wt and the mutants. Data are presented as mean ± sd; n = 15. The asterisks indicate statistical differences in comparison to the same tissues of wt determined by Student’s t-test. **p<e-10; n.s., not significant. (F) Confocal microscopy images of meristematic regions of wt and the mutants. fifth cortical layer was chosen for comparison. 10 consecutive cells below transition point of meristematic to elongation zone are indicated as red dots. Bar indicates 10 µm. (G) Comparison of cell length (left) and width (right) between wt and the mutants. Data are presented as mean ± sd; n = 15. The asterisks indicate significant statistical differences as determined by Student’s t-test. **p<e-10; n.s., not significant.

Figure 3—figure supplement 1. MIKKI overexpression.

(A) The levels of MIKKI mRNA in overexpressing lines of rice determined by RT-qPCR. Error bars are mean ± sd of three biological replicates performed in technical triplicate and the values are log2 converted. The asterisks indicate statistical differences determined by Student’s t-test. *p<e-05. (B) Overexpression of MIKKI in Arabidopsis. From the top panel down: levels of MIKKI, ath-miR171a and AtSCL6 RNA. The levels of MIKKI and AtSCL6 RNA were normalized to UBQ10 and ath-miR171a was related to ath-miR166. Data are presented as mean ± sd of three biological replicates performed in technical triplicate. The asterisks indicate statistical differences to Col-0 wt determined by Student’s t-test. **p<0.005; *p<0.05. (C) Typical phenotype of Arabidopsis flowers from plants overexpressing MIKKI. Bar indicates 1 cm.

Figure 3—figure supplement 2. MIKKI mutation.

(A) The sequence alignment of the wt and two independent mikki mutant alleles around the targeted region. The regions corresponding to different families of retrotransposons are coloured indicated as in Figure 2C. The lines above show the CRISPR-Cas9 targeted region and the miR171-binding region. One premature stop codon generated in the mikki-1 mutant is highlighted as red letters. (B) Sanger sequencing trace images of the regions around targeted site. Sequences were read in reverse orientation. Shaded box indicates the deleted nucleotides in mikki-1 mutant. (C) MIKKI transcript levels in wt and the two mutant lines. Primers used for the analysis were MIKKI_genotype-F and MIKKI_RT-R. Error bars are mean ± sd of three biological replicates performed in technical triplicate and the values are log2 converted. The asterisks indicate statistical differences determined by Student’s t-test. **p<0.005; n.s., not significant. (D) Short root phenotype of mikki and osscl21 mutant plants grown side-by-side. The image was taken at day two after germination. Bar indicates 1 cm. (E) small RNA blot analyses of wt and the mikki mutants. RNA was extracted from roots. Wt Nipponbare was segregated from heterozygous mutant plants.

Figure 3—figure supplement 3. Identification of osscl21 mutant plants.

(A) Schematic diagram of OsSCL21 gene and T-DNA insertion sites. Box represents exon and line is intergenic region. Arrow indicates transcriptional start site and T-DNA insertion sites are shown as triangles. Primers used in Figure 3—figure supplement 3B are indicated as arrowheads. (B) Genotyping of osscl21-1 and osscl21-2 mutants. (C–E) RT-qPCR analyses on wt and osscl21 mutants. Validation of OsSCL21 (C), osa-miR171b ~ f (D) and MIKKI (E) RNA levels. The levels were normalized to eEF1α (C and E) and osa-miR166 (D), respectively. Error bars are mean ± sd of three biological replicates performed in technical triplicate. The asterisks indicate statistical differences determined by Student’s t-test. **p<0.005; n.s., not significant.