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 responsemore »
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 more »
- Award ID(s):
- 1645557
- Publication Date:
- NSF-PAR ID:
- 10047759
- Journal Name:
- Plant Reproduction
- ISSN:
- 2194-7953
- Sponsoring Org:
- National Science Foundation
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Self‐incompatibility in Petunia is controlled by the polymorphic S‐locus, which contains S‐RNase encoding 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 inflata using bacterial artificial chromosome clones collectively containing all 17 SLF genes, SLFLike1, and S‐RNase. Two SLF pseudogenes 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. inflata and 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 SLF genes, resulting in highly similar flanking regions between different types of SLF and between alleles of the same type of SLF of 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 ofmore »
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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.
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Heterostyly is a breeding system that promotes outbreeding through a combination of morphological and physiological floral traits. In Turnera these traits are governed by a single, hemizygous S-locus containing just three genes. We report that the S-locus gene, BAHD, is mutated and encodes a severely truncated protein in a self-compatible long homostyle species. Further, a self-compatible long homostyle mutant possesses a T. krapovickasii BAHD allele with a point mutation in a highly conserved domain of BAHD acyl transferases. Wild type and mutant TkBAHD alleles were expressed in Arabidopsis to assay for brassinosteroid (BR) inactivating activity. The wild type but not mutant allele caused dwarfism, consistent with the wild type possessing, but the mutant allele having lost, BR inactivating activity. To investigate whether BRs act directly in self-incompatibility, BRs were added to in vitro pollen cultures of the two mating types. A small morph specific stimulatory effect on pollen tube growth was found with 5 µM brassinolide, but no genotype specific inhibition was observed. These results suggest that BAHD acts pleiotropically to mediate pistil length and physiological mating type through BR inactivation, and that in regard to self-incompatibility, BR acts by differentially regulating gene expression in pistils, rather than directly onmore »
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Accepted Manuscript1.0
- Publisher's Version of Record
- https://doi.org/10.1007/s00497-017-0314-1
