S-locus F-Box proteins are solely responsible for S-RNase-based self-incompatibility of Petunia Pollen

Self-incompatibility (SI) in Petunia is regulated by a polymorphic S-locus. For each S-haplotype, the S-locus contains a pistilspecific S-RNase gene and multiple pollen-specific S-locus F-box (SLF) genes. Both gain-of-function and loss-of-function experiments have shown that S-RNase alone regulates pistil specificity in SI. Gain-of-function experiments on SLF genes suggest that the entire suite of encoded proteins constitute the pollen specificity determinant. However, clear-cut loss-offunction experiments must be performed to determine if SLF proteins are essential for SI of pollen. Here, we used CRISPR/ Cas9 to generate two frame-shift indel alleles of S ₂ -SLF1 (SLF1 of S ₂ -haplotype) in S ₂ S ₃ plants of P. inflata and examined the effect on the SI behavior of S ₂ pollen. In the absence of a functional S ₂ -SLF ₁ , S ₂ pollen was either rejected by or remained compatible with pistils carrying one of eight normally compatible S-haplotypes. All results are consistent with interaction relationships between the 17 SLF proteins of S ₂ -haplotype and these eight S-RNases that had been determined by gain-offunction experiments performed previously or in this work. Our loss-of-function results provide definitive evidence that SLF proteins are solely responsible for SI of pollen, and they reveal their diverse and complex interaction relationships with S-RNases to maintain SI while ensuring cross-compatibility.