S-Locus F-Box Proteins Are Solely Responsible for S-RNase-Based Self-Incompatibility of <i>Petunia</i> Pollen

Abstract

Self-incompatibility (SI) in <i>Petunia</i> is regulated by a polymorphic S-locus. For each S-haplotype, the S-locus contains a pistil-specific <i>S-RNase</i> gene and multiple pollen-specific <i>S-locus F-box</i> (<i>SLF</i>) genes. Both gain-of-function and loss-of-function experiments have shown that <i>S-RNase</i> alone regulates pistil specificity in SI. Gain-of-function experiments on <i>SLF</i> genes suggest that the entire suite of encoded proteins constitute the pollen specificity determinant. However, clear-cut loss-of-function 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 <i>S₂</i> -<i>SLF1</i> (<i>SLF1</i> of <i>S₂</i> -haplotype) in <i>S₂S₃</i> plants of <i>P. inflata</i> and examined the effect on the SI behavior of <i>S₂</i> pollen. In the absence of a functional S₂-SLF1, <i>S₂</i> pollen was either rejected by or remained compatible with pistils carrying one of eight normally compatible <i>S</i>-haplotypes. All results are consistent with interaction relationships between the 17 SLF proteins of <i>S₂</i> -haplotype and these eight S-RNases that had been determined by gain-of-function 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.