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Title: CRISPR/Cas9-mediated knockout of PiSSK1 reveals essential role of S-locus F-box protein-containing SCF complexes in recognition of non-self S-RNases during cross-compatible pollination in self-incompatible Petunia inflata
Self-incompatibility (SI), an inbreeding-preventing mechanism, is regulated in Petunia inflata by the polymorphic S-locus, which houses multiple pollen-specific S-locus F-box (SLF) genes and a single pistil-specific S-RNase gene. S2-haplotype and S3-haplotype possess the same 17 polymorphic SLF genes (named SLF1 to SLF17), and each SLF protein produced in pollen is assembled into an SCF (Skp1–Cullin1– F-box) E3 ubiquitin ligase complex. A complete suite of SLF proteins is thought to collectively interact with all non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome, allowing cross-compatible pollination. For each SCFSLF complex, the Cullin1 subunit (named PiCUL1-P) and Skp1 subunit (named PiSSK1), like the F-box protein subunits (SLFs), are pollen-specific, raising the possibility that they also evolved specifically to function in SI. Here we used CRISPR/Cas9-meditated genome editing to generate frame-shift indel mutations in PiSSK1, and examined the SI behavior of a T0 plant (S2S3) with biallelic mutations in the pollen genome and two progeny plants (S2S2) each homozygous for one of the indel alleles and not carrying the Cas9-containing T-DNA. Their pollen was completely incompatible with pistils of seven otherwise compatible S-genotypes, but fully compatible with pistils of an S3S3 transgenic plant in which production of S3-RNase was completely suppressed by an antisense S3-RNase gene, and with pistils of immature flower buds, which produce little S-RNase. These results suggest that PiSSK1 specifically functions in SI, and support the hypothesis that SLF-containing SCF complexes are essential for compatible pollination.  more » « less
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Plant Reproduction
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National Science Foundation
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  1. Abstract In Petunia (Solanaceae family), self-incompatibility (SI) is regulated by the polymorphic S-locus, which contains the pistil-specific S-RNase and multiple pollen-specific S-Locus F-box (SLF) genes. SLFs assemble into E3 ubiquitin ligase complexes known as Skp1–Cullin1–F-box complexes (SCFSLF). In pollen tubes, these complexes collectively mediate ubiquitination and degradation of all nonself S-RNases, but not self S-RNase, resulting in cross-compatible, but self-incompatible, pollination. Using Petunia inflata, we show that two pollen-expressed Cullin1 (CUL1) proteins, PiCUL1-P and PiCUL1-B, function redundantly in SI. This redundancy is lost in Petunia hybrida, not because of the inability of PhCUL1-B to interact with SSK1, but due to a reduction in the PhCUL1-B transcript level. This is possibly caused by the presence of a DNA transposon in the PhCUL1-B promoter region, which was inherited from Petunia axillaris, one of the parental species of Pe. hybrida. Phylogenetic and syntenic analyses of Cullin genes in various eudicots show that three Solanaceae-specific CUL1 genes share a common origin, with CUL1-P dedicated to S-RNase-related reproductive processes. However, CUL1-B is a dispersed duplicate of CUL1-P present only in Petunia, and not in the other species of the Solanaceae family examined. We suggest that the CUL1s involved (or potentially involved) in the SI response in eudicots share a common origin. 
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  2. Summary

    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 S2SLF1 (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 theSCFS2–SLF1complex. Using pollen extracts containing PiSSK1:FLAG:GFPfor Co‐IP/MS, we identified two additionalSLFs (SLF4 andSLF13) that were assembled intoSCFSLFcomplexes. 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 thatSCFSLFcomplexes have evolved specifically to function in self‐incompatibility.

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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 theS6a‐haplotype ofP. inflataand theSN‐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.

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