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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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.
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- Award ID(s):
- 1645557
- NSF-PAR ID:
- 10047759
- Date Published:
- Journal Name:
- Plant Reproduction
- ISSN:
- 2194-7953
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Summary The collaborative non‐self‐recognition model for S‐
RN ase‐based self‐incompatibility predicts that multiple S‐locus F‐box proteins (SLF s) produced by pollen of a givenS ‐haplotype collectively mediate ubiquitination and degradation of all non‐self S‐RN ases, but not self S‐RN ases, in the pollen tube, thereby resulting in cross‐compatible pollination but self‐incompatible pollination. We had previously used pollen extracts containingGFP ‐fused S2‐SLF 1 (SLF 1 with anS 2‐haplotype) ofPetunia inflata for co‐immunoprecipitation (Co‐IP ) and mass spectrometry (MS ), and identified PiCUL 1‐P (a pollen‐specific Cullin1), PiSSK 1 (a pollen‐specific Skp1‐like protein) and PiRBX 1 (a conventional Rbx1) as components of theSCFS 2–SLF 1complex. Using pollen extracts containing PiSSK 1:FLAG :GFP for Co‐IP /MS , we identified two additionalSLF s (SLF 4 andSLF 13) that were assembled intoSCFSLF complexes. As 17SLF SLF 1SLF 17S 2andS 3pollen, here we examined whether all 17SLF s are assembled into similar complexes and, if so, whether these complexes are unique toSLF s. We modified the previous Co‐IP /MS procedure, including the addition of style extracts from four differentS ‐genotypes to pollen extracts containing PiSSK 1:FLAG :GFP , to perform four separate experiments. The results taken together show that all 17SLF s and anSLF ‐like protein,SLFL ike1 (encoded by anS ‐locus‐linked gene), co‐immunoprecipitated with PiSSK 1:FLAG :GFP . Moreover, of the 179 other F‐box proteins predicted byS 2andS 3pollen transcriptomes, only a pair with 94.9% identity and another pair with 99.7% identity co‐immunoprecipitated with PiSSK 1:FLAG :GFP . These results suggest thatSCFSLF complexes have evolved specifically to function in self‐incompatibility. -
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SUMMARY Self‐incompatibility in
Petunia is controlled by the polymorphicS ‐locus, which containsS‐RNase encoding 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 inflata using bacterial artificial chromosome clones collectively containing all 17SLF genes,SLFLike1 , andS‐RNase . TwoSLF pseudogenes and 28 potential protein‐coding genes were identified, 20 of which were also found at theS ‐loci of both theS6a ‐haplotype ofP. inflata and 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 ofSLF genes, resulting in highly similar flanking regions between different types ofSLF and between alleles of the same type ofSLF of 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 withSLF pseudogenes. Based on the hypothesis thatSLF genes were derived by retrotransposition, we identified 10F‐box genes as putativeSLF parent 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 ofSLF genes. -
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Summary Many eukaryotic intracellular processes employ protein ubiquitylation by ubiquitin E3 ligases for functional regulation or protein quality control. In plants, the multi‐subunit Skp1–Cullin1–F‐box (SCF) complexes compose the largest group of E3 ligases whose specificity is determined by a diverse array of F‐box proteins. Although both sequence divergence and polymorphism of
F‐box genes well support a broad spectrum of SCF functions, experimental evidence is scarce due to the low number of identified SCF substrates. Taking advantage of the bridge role of Skp1 between F‐box and Cullin1 in the complex, we systematically analyzed the functional influence of a well‐characterizedArabidopsis Skp1‐Like1 (ASK1 )Ds insertion allele,ask1 , in different Arabidopsis accessions. Through 10 generations of backcrossing with Columbia‐0 (Col‐0), we partially rescued the fertility of this otherwise sterileask1 allele in Landsbergerecta , thus providing experimental evidence showing the polymorphic roles of SCF complexes. Thisask1 mutant produces twisted rosette leaves, a reduced number of petals, fewer viable pollen grains, and larger embryos and seeds compared to Col‐0. RNA‐Seq‐based transcriptome analysis ofask1 uncovered a large spectrum of SCF functions, which is greater than a 10‐fold increase compared with previous studies. We also identified its hyposensitive responses to auxin and abscisic acid treatments and enhanced far‐red light/phyA‐mediated photomorphogenesis. Such diverse roles are consistent with the 20–30% reduction of ubiquitylation events inask1 estimated by immunoblotting analysis in this work. Collectively, we conclude that ASK1 is a predominant Skp1 protein in Arabidopsis and that the fertileask1 mutant allowed us to uncover a comprehensive set of SCF functions. -
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Auxin phytohormones control most aspects of plant development through a complex and interconnected signaling network. In the presence of auxin, AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors are targeted for degradation by the SKP1-CULLIN1-F-BOX (SCF) ubiquitin-protein ligases containing TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB). CULLIN1-neddylation is required for SCFTIR1/AFBfunctionality, as exemplified by mutants deficient in the NEDD8-activating enzyme subunit AUXIN-RESISTANT 1 (AXR1). Here, we report a chemical biology screen that identifies small molecules requiring AXR1 to modulate plant development. We selected four molecules of interest, RubNeddin 1 to 4 (RN1 to -4), among which RN3 and RN4 trigger selective auxin responses at transcriptional, biochemical, and morphological levels. This selective activity is explained by their ability to consistently promote the interaction between TIR1 and a specific subset of AUX/IAA proteins, stimulating the degradation of particular AUX/IAA combinations. Finally, we performed a genetic screen using RN4, the RN with the greatest potential for dissecting auxin perception, which revealed that the chromatin remodeling ATPase BRAHMA is implicated in auxin-mediated apical hook development. These results demonstrate the power of selective auxin agonists to dissect auxin perception for plant developmental functions, as well as offering opportunities to discover new molecular players involved in auxin responses.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1007/s00497-017-0314-1
