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1. All 17 S‐locus F‐box proteins of the S 2 ‐ and S 3 ‐haplotypes of Petunia inflata are assembled into similar SCF complexes with a specific function in self‐incompatibility

   https://doi.org/10.1111/tpj.13222  

   Li, Shu ; Williams, Justin S. ; Sun, Penglin ; Kao, Teh‐hui ( August 2016 , The Plant Journal)

   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 S₂‐SLF1 (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 theSCF^S2–SLF1complex. Using pollen extracts containing PiSSK1:FLAG:GFPfor Co‐IP/MS, we identified two additionalSLFs (SLF4 andSLF13) that were assembled intoSCF^SLFcomplexes. 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 thatSCF^SLFcomplexes have evolved specifically to function in self‐incompatibility.

    

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2. 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

   https://doi.org/10.1007/s00497-017-0314-1  

   Sun, Linhan ; Kao, Teh-hui ( November 2017 , Plant Reproduction)

   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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3. Analyses of Cullin1 homologs reveal functional redundancy in S-RNase-based self-incompatibility and evolutionary relationships in eudicots

   https://doi.org/10.1093/plcell/koac357  

   Sun, Linhan ; Cao, Shiyun ; Zheng, Ning ; Kao, Teh-hui ( December 2022 , The Plant Cell)

   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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4. The long and short of the S ‐locus in Turnera (Passifloraceae)

   https://doi.org/10.1111/nph.15970  

   Shore, Joel S. ; Hamam, Hasan J. ; Chafe, Paul D. J. ; Labonne, Jonathan D. J. ; Henning, Paige M. ; McCubbin, Andrew G. ( July 2019 , New Phytologist)

   Distyly is an intriguing floral adaptation that increases pollen transfer precision and restricts inbreeding. It has been a model system in evolutionary biology since Darwin. Although theS‐locus determines the long‐ and short‐styled morphs, the genes were unknown inTurnera. We have now identified these genes.

   We used deletion mapping to identify, and then sequence,BACclones and genome scaffolds to constructS/shaplotypes. We investigated candidate gene expression, hemizygosity, and used mutants, to explore gene function.

   Thes‐haplotype possessed 21 genes collinear with a region of chromosome 7 of grape. TheS‐haplotype possessed three additional genes and two inversions.TsSPH1was expressed in filaments and anthers,TsYUC6in anthers andTsBAHDin pistils. Long‐homostyle mutants did not possessTsBAHDand a short‐homostyle mutant did not expressTsSPH1.

   Three hemizygous genes appear to determine S‐morph characteristics inT. subulata. Hemizygosity is common to all distylous species investigated, yet the genes differ. The pistil candidate gene,TsBAHD, differs from that ofPrimula, but both may inactivate brassinosteroids causing short styles.TsYUC6is involved in auxin synthesis and likely determines pollen characteristics.TsSPH1is likely involved in filament elongation. We propose an incompatibility mechanism involvingTsYUC6andTsBAHD.

    

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5. The rhg1‐a ( Rhg1 low‐copy) nematode resistance source harbors a copia‐family retrotransposon within the Rhg1‐ encoded α‐SNAP gene

   https://doi.org/10.1002/pld3.164  

   Bayless, Adam M. ; Zapotocny, Ryan W. ; Han, Shaojie ; Grunwald, Derrick J. ; Amundson, Kaela K. ; Bent, Andrew F. ( August 2019 , Plant Direct)

   Soybean growers widely use theResistance toHeteroderaglycines1 (Rhg1) locus to reduce yield losses caused by soybean cyst nematode (SCN).Rhg1is a tandemly repeated four gene block. Two classes of SCN resistance‐conferringRhg1haplotypes are recognized:rhg1‐a(“Peking‐type,” low‐copy number, three or fewerRhg1repeats) andrhg1‐b(“PI 88788‐type,” high‐copy number, four or moreRhg1repeats). Therhg1‐aandrhg1‐bhaplotypes encode α‐SNAP (alpha‐SolubleNSFAttachmentProtein) variants α‐SNAP_Rhg1LC and α‐SNAP_Rhg1HC, respectively, with differing atypical C‐terminal domains, that contribute to SCN resistance. Here we report thatrhg1‐asoybean accessions harbor a copia retrotransposon within theirRhg1 Glyma.18G022500(α‐SNAP‐encoding) gene. We termed this retrotransposon “RAC,” forRhg1alpha‐SNAPcopia. Soybean carries multipleRAC‐like retrotransposon sequences. TheRhg1 RACinsertion is in theGlyma.18G022500genes of all truerhg1‐ahaplotypes we tested and was not detected in any examinedrhg1‐borRhg1_WT(single‐copy) soybeans.RACis an intact element residing within intron 1, anti‐sense to therhg1‐a α‐SNAPopen reading frame.RAChas intrinsic promoter activities, but overt impacts ofRACon transgenic α‐SNAP_Rhg1LC mRNA and protein abundance were not detected. From the nativerhg1‐a RAC⁺genomic context, elevated α‐SNAP_Rhg1LC protein abundance was observed in syncytium cells, as was previously observed for α‐SNAP_Rhg1HC (whoserhg1‐bdoes not carryRAC). Using a SoySNP50K SNP corresponding withRACpresence, just ~42% of USDA accessions bearing previously identifiedrhg1‐aSoySNP50K SNP signatures harbor theRACinsertion. Subsequent analysis of several of these putativerhg1‐aaccessions lackingRACrevealed that none encodedα‐SNAP_Rhg1LC, and thus, they are notrhg1‐a.rhg1‐ahaplotypes are of rising interest, withRhg4, for combating SCN populations that exhibit increased virulence against the widely usedrhg1‐bresistance. The present study reveals another unexpected structural feature of manyRhg1loci, and a selectable feature that is predictive ofrhg1‐ahaplotypes.

    

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