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Self‐incompatibility inPetuniais controlled by the polymorphicS‐locus, which containsS‐RNaseencoding the pistil determinant and 16–20S‐locus F‐box(SLF) genes collectively encoding the pollen determinant. Here we sequenced and assembled approximately 3.1 Mb of theS₂‐haplotype of theS‐locus inPetunia inflatausing bacterial artificial chromosome clones collectively containing all 17SLFgenes,SLFLike1, andS‐RNase. TwoSLFpseudogenes and 28 potential protein‐coding genes were identified, 20 of which were also found at theS‐loci of both theS_6a‐haplotype ofP. inflataand theS_N‐haplotype of self‐compatiblePetunia axillaris, but not in theS‐locus remnants of self‐compatible potato (Solanum tuberosum) and tomato (Solanum lycopersicum). Comparative analyses ofS‐locus sequences of these threeS‐haplotypes revealed potential genetic exchange in the flanking regions ofSLFgenes, resulting in highly similar flanking regions between different types ofSLFand between alleles of the same type ofSLFof differentS‐haplotypes. The high degree of sequence similarity in the flanking regions could often be explained by the presence of similar long terminal repeat retroelements, which were enriched at theS‐loci of all threeS‐haplotypes and in the flanking regions of allS‐locus genes examined. We also found evidence of the association of transposable elements withSLFpseudogenes. Based on the hypothesis thatSLFgenes were derived by retrotransposition, we identified 10F‐boxgenes as putativeSLFparent genes. Our results shed light on the importance of non‐coding sequences in the evolution of theS‐locus, and on possible evolutionary mechanisms of generation, proliferation, and deletion ofSLFgenes.

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 givenS‐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 containingGFP‐fused S₂‐SLF1 (SLF1 with anS₂‐haplotype) ofPetunia inflatafor 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 theSCF^S2–SLF1complex. Using pollen extracts containing PiSSK1:FLAG:GFPfor Co‐IP/MS, we identified two additionalSLFs (SLF4 andSLF13) that were assembled intoSCF^SLFcomplexes. As 17SLFgenes (SLF1toSLF17) have been identified inS₂andS₃pollen, here we examined whether all 17SLFs are assembled into similar complexes and, if so, whether these complexes are unique toSLFs. We modified the previous Co‐IP/MSprocedure, including the addition of style extracts from four differentS‐genotypes to pollen extracts containing PiSSK1:FLAG:GFP, to perform four separate experiments. The results taken together show that all 17SLFs and anSLF‐like protein,SLFLike1 (encoded by anS‐locus‐linked gene), co‐immunoprecipitated with PiSSK1:FLAG:GFP. Moreover, of the 179 other F‐box proteins predicted byS₂andS₃pollen transcriptomes, only a pair with 94.9% identity and another pair with 99.7% identity co‐immunoprecipitated with PiSSK1:FLAG:GFP. These results suggest thatSCF^SLFcomplexes have evolved specifically to function in self‐incompatibility.