Figure 3. The MpFRH1 locus produces a miRNA that targets the MpRSL1 transcript.

(A) RNA folding prediction of the 150 bp sequence sufficient to reproduce the few rhizoids phenotype when over-expressed in the wild type carried out using RNAfold (Gruber et al., 2008). The colours represent base-pairing probabilities. The small RNA sequences corresponding to MpFRH1 miRNA (mpo-MIR11861) and the complementary *miRNA are indicated. (B) MpRSL1 gene model, the MpFRH1 miRNA target site is indicated in red. (C) MpFRH1 negatively regulates MpRSL1 transcript level. qRT-PCR quantification of steady state MpRSL1 transcript levels in 15 day old gemmae of wild type, the four MpFRH1^GOF mutant lines and four proACT:FRH1 lines with a strong few rhizoid phenotype. MpRSL1 transcript levels were normalised against MpAPT1 and MpCUL3..

Figure 3—figure supplement 1. Stem-loop PCR detection of the MpFRH1 miRNA.

RNA extracted from 15 day old wild type and MpFRH1^GOF1-3 gemmae was used as a template for the stem-loop PCR amplification. Amplification products after 28 PCR cycles were visualised on a EtBr stained 2% agarose gel. The tRNA band was used as a loading control.

Figure 3—figure supplement 2. Predicted targets of MpFRH1 miRNA.

Lower scores indicate a more likely target based on sequence similarity between the miRNA and the target transcript. Transcript number corresponds to the M. polymorpha gametophyte transcriptome (Honkanen et al., 2016). Mapoly gene ID correspond to the M. polymorpha genome sequence published in Bowman et al. (2017).

Figure 3—figure supplement 3. Transcripts of one of the four predicted MpFRH1 miRNA targets are less abundant in MpFRH1^GOF2 compared to wild type.

(A) RT-PCR quantification of transcript levels of the four predicted FRH1 miRNA target mRNAs in WT and MpFRH1^GOF2 plants. (B) qRT-PCR quantification of transcript levels of MpRSL1. MpRSL1 transcript levels were normalised against MpAPT1 and MpCUL3.