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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

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Function of Petunia PiSSK1.

Abstract

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. S 2-haplotype and S 3-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 T 0 plant (S 2 S 3) with biallelic mutations in the pollen genome and two progeny plants (S 2 S 2) 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 S 3 S 3 transgenic plant in which production of S3-RNase was completely suppressed by an antisense S 3-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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Acknowledgements

We thank Yinong Yang and Bastian Minkenberg for providing pGTR plasmid and technical advice on CRISPR/Cas9-mediated genome editing; Ning Zheng for discussion about PiSSK1 sequence features; Shawn Burghard for greenhouse management; Hongli Hao for general laboratory help. This work was supported by Grant IOS-1645557 from the National Science Foundation to Teh-hui Kao.

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Authors and Affiliations

  1. Intercollege Graduate Degree Program in Plant Biology, The Pennsylvania State University, University Park, PA, 16802, USA

    Linhan Sun & Teh-hui Kao

  2. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA, 16802, USA

    Teh-hui Kao

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  1. Linhan Sun
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  2. Teh-hui Kao
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Correspondence to Teh-hui Kao.

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Communicated by Noni Franklin-Tong.

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Sun, L., Kao, Th. 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 . Plant Reprod 31, 129–143 (2018). https://doi.org/10.1007/s00497-017-0314-1

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