SLFL Genes Participate in the Ubiquitination and Degradation of S-RNase in Self-Compatible Chinese Peach

The gametophytic self-incompatibility (SI) mediated by S-RNase of Rosaceae, Solanaceae and Plantaginaceae, is controlled by two tightly linked genes located at highly polymorphic S-locus: the S-RNase for pistil specificity and the F-box gene (SFB/SLF) for pollen specificity, respectively. The F-box gene of peach (Prunus persica) is S haplotype-specific F-box (SFB). In this study, we selected 37 representative varieties according to the evolution route of peach and identified their S genotypes. We cloned pollen determinant genes mutant PperSFB1m, PperSFB2m, PperSFB4m and normal PperSFB2, and style determinant genes S1-RNase, S2-RNase, S2m-RNase and S4-RNase. Mutant PperSFBs were translated terminated prematurely because of fragment insertion. Yeast two-hybrid showed that mutant PperSFBs and normal PperSFB2 interacted with all S-RNases. Normal PperSFB2 was divided into four parts: box, box-V1, V1-V2 and HVa-HVb. Protein interaction analyses showed that the box portion did not interact with S-RNases, both of the box-V1 and V1-V2 had interactions with S-RNases, while the hypervariable region of PperSFB2 HVa-HVb only interacted with S2-RNase. Bioinformatics analysis of peach genome revealed that there were other F-box genes located at S-locus, and of which three F-box genes were specifically expressed in pollen, namely PperSLFL1, PperSLFL2 and PperSLFL3, respectively. Phylogenetic analysis showed that PperSFBs and PperSLFLs were classified into two different clades. Yeast two-hybrid analysis revealed that as with PperSFBs, the three F-box proteins interacted with PperSSK1. Yeast two-hybrid and BiFC showed that PperSLFLs interacted with S-RNases with no allelic specificity. In vitro ubiquitination assay showed that PperSLFLs could tag ubiquitin molecules to PperS-RNases. In all, the above results suggest that three PperSLFLs are the appropriate candidates for the general inhibitor, which would inactivate the S-RNases in pollen tubes, and the role of three PperSLFL proteins is redundant, as S-RNase repressors involved in the self-incompatibility of peach.