All 17 S-locus F-box proteins of the S2- and S3-haplotypes of Petunia inflata are assembled into similar SCF complexes with a specific function in self-incompatibility

The collaborative non-self-recognition model for S-RNase-based self-incompatibility predicts that multiple S-locus F-box proteins (SLFs) produced by pollen of a given S-haplotype collectively mediate ubiquitination and degradation of all non-self S-RNases, but not self S-RNases, in the pollen tube, thereby resulting in cross-compatible pollination but self-incompatible pollination. We had previously used pollen extracts containing GFP-fused S-2-SLF1 (SLF1 with an S-2-haplotype) of Petunia inflata for co-immunoprecipitation (Co-IP) and mass spectrometry (MS), and identified PiCUL1-P (a pollen-specific Cullin1), PiSSK1 (a pollen-specific Skp1-like protein) and PiRBX1 (a conventional Rbx1) as components of the SCFS2-SLF1 complex. Using pollen extracts containing PiSSK1:FLAG:GFP for Co-IP/MS, we identified two additional SLFs (SLF4 and SLF13) that were assembled into SCFSLF complexes. As 17 SLF genes (SLF1 to SLF17) have been identified in S-2 and S-3 pollen, here we examined whether all 17 SLFs are assembled into similar complexes and, if so, whether these complexes are unique to SLFs. We modified the previous Co-IP/MS procedure, including the addition of style extracts from four different S-genotypes to pollen extracts containing PiSSK1:FLAG:GFP, to perform four separate experiments. The results taken together show that all 17 SLFs and an SLF-like protein, SLFLike1 (encoded by an S-locus-linked gene), co-immunoprecipitated with PiSSK1:FLAG:GFP. Moreover, of the 179 other F-box proteins predicted by S-2 and S-3 pollen transcriptomes, only a pair with 94.9% identity and another pair with 99.7% identity co-immunoprecipitated with PiSSK1:FLAG:GFP. These results suggest that SCFSLF complexes have evolved specifically to function in self-incompatibility. Significance Statement Seventeen types of pollen-specific S-locus F-box proteins (SLFs) of S-2- and S-3-haplotypes collectively mediate ubiquitination and degradation of respective non-self S-RNases to allow cross-compatible pollination. In this work, we report that, all 17 types of SLFs, an SLF-like protein, and only four of the 179 non-SLF F-box proteins predicted to be expressed in pollen, are assembled into similar SCF complexes, suggesting that these complexes specifically evolved to function in self-incompatibility.