Figure 4. A truncated S_m-RNase appears to be responsible for the loss of SI in citrus.

a, Cartoon showing the gene structure of the S_x-RNase (top) and its mutant form (middle). S_x-RNase indicates the length and structure of the normal S-RNase with five conserved domains (C1-C5, grey boxes) and five hypervariable domains (H1-H5, blue boxes). S_m-RNase harbors a single nucleotide deletion resulting in a premature stop codon. The ‘recovered’ S_m^R-RNase (bottom) has a single nucleotide inserted (red triangle) that recovered the full length gene. Conserved histidine and cysteine residues are indicated in orange and purple, based on the deduced amino acid sequences. b, Expression of S_m- (S_m) and S₂-RNase (S₂) quantified using RT-PCR of different tissues from a S₂S_m plant (Le: leaf; Ped: pedicel; Pet: petal; Fi: filament; An: anther; Ov: ovary; St: style). The S_m-RNase is expressed at a much lower level than the S₂-RNase, in a tissue-specific manner (only in pistils: ovary and style tissues). c, Absolute copy number of the S₂- and S_m-RNase (extrapolated from the equation in Supplementary Fig. 18). The copy number of S_m-RNase in 50 μg S₂S_m style RNA is much lower than that of S₂-RNase. Error bars are shown for mean copy number ± SEM (n = 3 biological replicates; red dots indicate individual samples). d, IGV tracks displaying sequencing read clusters of S₂- and S_m-RNase gene from S₂S_m style RNA-seq. The green bars depict the number of the reads mapped to the reference. The reads mapped to S₂-RNase are clearly more than the reads mapped to S_m-RNase. Partial alignment of the RNA-mapping is shown below, with pink and blue representing the different read strands. e, SDS-PAGE analysis of recombinant S_m- (S_m) and S_m^R-RNase (S_m^R). The Mr of the fusion protein S_m-RNase-GST (39 kDa) is lower than the “restored” S_m^R-RNase-GST (45 kDa). f, RNase-activity gel of the recombinant S_m- (S_m) and S_m^R-RNase (S_m^R). The mutated S_m-RNase and the “recovered” S_m^R-RNase have similar RNase activities. g, The recombinant S_m- (i) and S_m^R-RNase (ii) both degrade citrus 28S and 18S rRNA (agarose gel assay). Experiments were repeated independently twice times for panel b and three times for panels e and g, with the similar results for each experiments. h, The S_m^R-RNase recombinant protein displays inhibitory activity against pollen. Box plots show the distribution of individual pollen tube lengths in an in vitro bioassay of recombinant S_m-, S_m^R- and S₂ RNase against pollen from a plant with S₂S_m genotype. The S_m-RNase did not significantly (NS) inhibit pollen tubes from a S₂S_m plant when compared against untreated (UT) pollen; the S_m^R-RNase exhibited significant inhibitory activity (**) reducing the length of pollen tubes by ~40% and was not significantly different (NS) from that of the S₂-RNase. The length of >50 pollen tubes was measured for each replicate (n = 3 biologically independent replicates, >150 in total). One-way ANOVA analysis was used to compare the pollen tube lengths (treatment vs untreated control). The elements in box and whisker plots are the same as in Fig 2d.