SLFL Genes Participate in the Ubiquitination and Degradation ofS-RNase in Self-Compatible Chinese Peach
AbstractThe 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) isShaplotype-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 mutantPperSFB1m, PperSFB2m, PperSFB4mand normalPperSFB2, and style determinant genesS1-RNase, S2-RNase, S2m-RNaseandS4-RNase.MutantPperSFBswere translated terminated prematurely because of fragment insertion. Yeast two-hybrid showed that mutant PperSFBs and normal PperSFB2 interacted with all S-RNases. NormalPperSFB2was 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 ofPperSFB2HVa-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, namelyPperSLFL1, PperSLFL2andPperSLFL3, 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 threePperSLFLsare 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.