Identification and characterization of components of a putative Petunia S-locus F-box-containing E3 ligase complex involved in S-RNase-based self-incompatibility

Petunia inflata S- locus F- box ( Pi SLF) is thought to function as a typical F- box protein in ubiquitin- mediated protein degradation and, along with Skp1, Cullin-1, and Rbx1, could compose an SCF complex mediating the degradation of nonself S- RNase but not self S- RNase. We isolated three P. inflata Skp1s ( Pi SK1, - 2, and - 3), two Cullin- 1s ( Pi CUL1- C and - G), and an Rbx1 ( Pi RBX1) cDNAs and found that Pi CUL1- G did not interact with Pi RBX1 and that none of the three Pi SKs interacted with Pi SLF2. We also isolated a RING- HC protein, S- RNase Binding Protein1 ( Pi SBP1), almost identical to Petunia hybrida SBP1, which interacts with Pi SLFs, S- RNases, Pi CUL1- G, and an E2 ubiquitin- conjugating enzyme, suggesting that Pi CUL1- G, SBP1, and SLF may be components of a novel E3 ligase complex, with Pi SBP1 playing the roles of Skp1 and Rbx1. S- RNases interact more with nonself Pi SLFs than with self Pi SLFs, and Pi SLFs also interact more with nonself S- RNases than with self S- RNases. Bacterially expressed S-1-, S-2-, and S-3- RNases are degraded by the 26S proteasomal pathway in a cell-free system, albeit not in an S- allele - specific manner. Native glycosylated S3- RNase is not degraded to any significant extent; however, deglycosylated S-3- RNase is degraded as efficiently as the bacterially expressed S- RNases. Finally, S- RNases are ubiquitinated in pollen tube extracts, but whether this is mediated by the Pi SLF containing E3 complex is unknown.