- Award ID(s):
- NSF-PAR ID:
- Publisher / Repository:
- Date Published:
- Journal Name:
- The Plant Journal
- Page Range / eLocation ID:
- p. 1348-1368
- Medium: X
- Sponsoring Org:
- National Science Foundation
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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
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 S2‐ SLF1 ( SLF1 with an S2‐haplotype) of Petunia inflatafor co‐immunoprecipitation (Co‐ IP) and mass spectrometry ( MS), and identified Pi CUL1‐P (a pollen‐specific Cullin1), Pi SSK1 (a pollen‐specific Skp1‐like protein) and Pi RBX1 (a conventional Rbx1) as components of the SCFS2– SLF1complex. Using pollen extracts containing Pi SSK1: FLAG: GFPfor Co‐ IP/ MS, we identified two additional SLFs ( SLF4 and SLF13) that were assembled into SCFSLFcomplexes. As 17 genes ( SLF to SLF1 ) have been identified in SLF17 S2and S3pollen, 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/ MSprocedure, including the addition of style extracts from four different S‐genotypes to pollen extracts containing Pi SSK1: 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 Pi SSK1: FLAG: GFP. Moreover, of the 179 other F‐box proteins predicted by S2and S3pollen transcriptomes, only a pair with 94.9% identity and another pair with 99.7% identity co‐immunoprecipitated with Pi SSK1: FLAG: GFP. These results suggest that SCFSLFcomplexes have evolved specifically to function in self‐incompatibility.
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.more » « less
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