Genome-wide identification and functional analysis of S-RNase involved in the self-incompatibility of citrus

S-RNase-based self-incompatibility is found in Solanaceae, Rosaceae, and Scrophulariaceae, and is the most widespread mechanism that prevents self-fertilization in plants. Although 'Shatian' pummelo (Citrus grandis), a traditional cultivated variety, possesses the self-incompatible trait, the role of S-RNases in the self-incompatibility of 'Shatian' pummelo is poorly understood. To identify genes associated with self-incompatibility in citrus, we identified 16 genes encoding homologs of ribonucleases in the genomes of sweet orange (Citrus sinensis) and clementine mandarin (Citrus clementine). We preliminarily distinguished S-RNases from S-like RNases with a phylogenetic analysis that classified these homologs into three groups, which is consistent with the previous reports. Expression analysis provided evidence that CsRNS1 and CsRNS6 are S-like RNase genes. The expression level of CsRNS1 was increased during fruit development. The expression of CsRNS6 was increased during the formation of embryogenic callus. In contrast, we found that CsRNS3 possessed several common characteristics of the pistil determinant of self-incompatibility: it has an alkaline isoelectric point (pI), harbors only one intron, and is specifically expressed in style. We obtained a cDNA encoding CgRNS3 from 'Shatian' pummelo and found that it is homolog to CsRNS3 and that CgRNS3 exhibited the same expression pattern as CsRNS3. In an in vitro culture system, the CgRNS3 protein significantly inhibited the growth of self-pollen tubes from 'Shatian' pummelo, but after a heat treatment, this protein did not significantly inhibit the elongation of self- or non-self-pollen tubes. In conclusion, an S-RNase gene, CgRNS3, was obtained by searching the genomes of sweet orange and clementine for genes exhibiting sequence similarity to ribonucleases followed by expression analyses. Using this approach, we identified a protein that significantly inhibited the growth of self-pollen tubes, which is the defining property of an S-RNase.