Recognition of S-RNases by an S locus F-box like protein and an S haplotype-specific F-box like protein in the Prunus-specific self-incompatibility system.

KEY MESSAGE: S-RNase was demonstrated to be predominantly recognized by an S locus F-box-like protein and an S haplotype-specific F-box-like protein in compatible pollen tubes of sweet cherry. Self-incompatibility (SI) is a reproductive barrier that rejects self-pollen and inhibits self-fertilization to promote outcrossing. In Solanaceae and Rosaceae, S-RNase-based gametophytic SI (GSI) comprises S-RNase and F-box protein(s) as the pistil and pollen S determinants, respectively. Compatible pollen tubes are assumed to detoxify the internalized cytotoxic S-RNases to maintain growth. S-RNase detoxification is conducted by the Skp1-cullin1-F-box protein complex (SCF) formed by pollen S determinants, S locus F-box proteins (SLFs), in Solanaceae. In Prunus, the general inhibitor (GI), but not pollen S determinant S haplotype-specific F-box protein (SFB), is hypothesized to detoxify S-RNases. Recently, SLF-like proteins 1-3 (SLFL1-3) were suggested as GI candidates, although it is still possible that other proteins function predominantly in GI. To identify the other GI candidates, we isolated four other pollen-expressed SLFL and SFB-like (SFBL) proteins PavSLFL6, PavSLFL7A, PavSFBL1, and PavSFBL2 in sweet cherry. Binding assays with four PavS-RNases indicated that PavSFBL2 bound to PavS1, 6-RNase while the others bound to nothing. PavSFBL2 was confirmed to form an SCF complex in vitro. A co-immunoprecipitation assay using the recombinant PavS6-RNase as bait against pollen extracts and a mass spectrometry analysis identified the SCF complex components of PavSLFLs and PavSFBL2, M-locus-encoded glutathione S-transferase (MGST), DnaJ-like protein, and other minor proteins. These results suggest that SLFLs and SFBLs could act as predominant GIs in Prunus-specific S-RNase-based GSI.