- Award ID(s):
- NSF-PAR ID:
- Date Published:
- Journal Name:
- The Plant Cell
- Page Range / eLocation ID:
- 673 to 699
- 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.
Petuniais controlled by the polymorphic S‐locus, which contains S‐RNaseencoding the pistil determinant and 16–20 S‐locus F‐box( SLF) genes collectively encoding the pollen determinant. Here we sequenced and assembled approximately 3.1 Mb of the S2‐haplotype of the S‐locus in Petunia inflatausing bacterial artificial chromosome clones collectively containing all 17 SLFgenes, SLFLike1, and S‐RNase. Two SLFpseudogenes and 28 potential protein‐coding genes were identified, 20 of which were also found at the S‐loci of both the S6a‐haplotype of P. inflataand the SN‐haplotype of self‐compatible Petunia axillaris, but not in the S‐locus remnants of self‐compatible potato ( Solanum tuberosum) and tomato ( Solanum lycopersicum). Comparative analyses of S‐locus sequences of these three S‐haplotypes revealed potential genetic exchange in the flanking regions of SLFgenes, resulting in highly similar flanking regions between different types of SLFand between alleles of the same type of SLFof different S‐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 the S‐loci of all three S‐haplotypes and in the flanking regions of all S‐locus genes examined. We also found evidence of the association of transposable elements with SLFpseudogenes. Based on the hypothesis that SLFgenes were derived by retrotransposition, we identified 10 F‐boxgenes as putative SLFparent genes. Our results shed light on the importance of non‐coding sequences in the evolution of the S‐locus, and on possible evolutionary mechanisms of generation, proliferation, and deletion of SLFgenes.
In self‐incompatible Solanaceae, the pistil protein S‐RNase contributes to
S‐specific pollen rejection in conspecific crosses, as well as to rejecting pollen from foreign species or whole clades. However, S‐RNase alone is not sufficient for either type of pollen rejection. We describe a thioredoxin (Trx) type h from Nicotiana alata, NaTrxh, which interacts with and reduces S‐RNase in vitro. Here, we show that expressing a redox‐inactive mutant, NaTrxhSS, suppresses both S‐specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia. Biochemical experiments provide evidence that NaTrxh specifically reduces the Cys155‐Cys185disulphide bond of SC10‐Rnase, resulting in a significant increase of its ribonuclease activity. This reduction and increase in S‐RNase activity by NaTrxh helps to explain why S‐RNase alone could be insufficient for pollen rejection.
Genome sequencing has uncovered tremendous sequence variation within and between species. In plants, in addition to large variations in genome size, a great deal of sequence polymorphism is also evident in several large multi-gene families, including those involved in the ubiquitin-26S proteasome protein degradation system. However, the biological function of this sequence variation is yet not clear. In this work, we explicitly demonstrated a single origin of retroposed Arabidopsis Skp1-Like ( ASK ) genes using an improved phylogenetic analysis. Taking advantage of the 1,001 genomes project, we here provide several lines of polymorphism evidence showing both adaptive and degenerative evolutionary processes in ASK genes. Yeast two-hybrid quantitative interaction assays further suggested that recent neutral changes in the ASK2 coding sequence weakened its interactions with some F-box proteins. The trend that highly polymorphic upstream regions of ASK1 yield high levels of expression implied negative expression regulation of ASK1 by an as-yet-unknown transcriptional suppression mechanism, which may contribute to the polymorphic roles of Skp1-CUL1-F-box complexes. Taken together, this study provides new evolutionary evidence to guide future functional genomic studies of SCF-mediated protein ubiquitylation.more » « less