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

Abstract

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.

Isolation and Characterization of <i>S-RNase</i>-homologous Genes Expressed in Styles in ‘Hyuganatsu’ (<i>Citrus tamurana</i> hort. ex Tanaka)

“…Some T2 RNase genes in citrus are reportedly involved in fruit development (Liang et al, 2017). Liang et al (2017) found that one gene (CsRNS1) out of nine T2 RNase genes was continuously up-regulated in sweet orange fruit from flowering to 240 d after flowering, suggesting that the S-like RNase genes obtained here may be involved in fruit development. However, none of the T2 RNase genes isolated in this study were expressed in fruit tissues, indicating that their function is not associated with fruit development.…”

“…Six and seven genes annotated as ribonuclease T2 genes were identified from the sweet orange and clementine genome databases, respectively, using database searches (Table 3). Liang et al (2017) used a similar approach to obtain T2 RNase genes by surveying different databases from those used in this study; they obtained seven and nine T2 RNase genes from the clementine v.1.0 and sweet orange v.2.0 databases, respectively. In this study, obtained amino acid sequences were queried using a tblastx search of the EST database that resulted in five hits.…”

“…Their noninvolvement in fruit development is also supported by the distant phylogenetic position of CtRNS1-CtRNS3 relative to the Class II clade. Most Class II T2 RNase genes are constitutively expressed, suggesting that they may have housekeeping roles (MacIntosh, 2011); the citrus T2 RNase genes related to fruit development and ovary senescence also belong to this clade (Chai et al, 2011;Liang et al, 2017).…”

“…'Hyuganatsu' is an SI cultivar with a very low level of parthenocarpy (Miwa, 1951;Yamashita, 1978), resulting in seedy fruit that are contrary to consumer preference. Although similar attempts have previously been made to isolate genes homologous to Rosaceae, Solanaceae, and Plantaginaceae S-RNases from some Citrus species (Chai et al, 2011;Liang et al, 2017;Miao et al, 2011), no confirmed S-RNases have been identified in citrus to date (Zhang et al, 2018). However, recent technological advances in DNA sequencing and successful completion of citrus genome sequencing projects have resulted in the public availability of annotated genome sequences of several citrus species (Shimizu et al, 2017;Wu et al, 2014;Xu et al, 2013) that will facilitate the identification of homologous genes.…”

Temperature-Dependent Compatible and Incompatible Pollen-Style Interactions in Citrus clementina Hort. ex Tan. Show Different Transglutaminase Features and Polyamine Pattern

“…An increasing number of studies have recently focused on the SI mechanism in Citrus . Comparative transcriptome approaches have been applied in order to identify proteins putatively involved in the SI response; however, only in C. grandis L. Osbeck (pummelo), an S-RNase gene (homolog to those of Rosaceae and Solanaceae) has been identified, whose product was able to inhibit pollen tube growth ( Liang et al., 2017 ). Recently, the S-RNase-based SI system was verified in pummelo, and S-RNases were confirmed to be the pistil S determinants ( Liang et al., 2020 ).…”