Identification and Characterization of Components of a PutativePetunia S-Locus F-Box–Containing E3 Ligase Complex Involved in S-RNase–Based Self-Incompatibility

Abstract

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 SLF 2 . 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 S 3 -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 SLFcontaining E3 complex is unknown.

Pollen Proteins Bind to the C-terminal Domain of Nicotiana alata Pistil Arabinogalactan Proteins

“…SBP1 is implicated in the Petunia S-RNase-based gametophytic SI pollen rejection system (17). This pollen rejection system is controlled by the S-locus; pollen is rejected when its S-haplotype matches either of the S-haplotypes of the diploid pistil (12).…”

“…The S-RNase-binding protein (SBP1) in Petunia pollen was found to interact with a pistil SI protein, S-RNase, in yeast two-hybrid and pull-down assays (16). S-RNase was also found to interact with the SLF (SI-specific pollen S-locus F-Box) protein (17). In tomato pollen tubes, yeast two-hybrid experiments were used to identify the interaction between the extracellular domain of a pollen-specific protein kinase (LePRK) and a small cysteine-rich protein, LeSTIG1, expressed in the pistil (18).…”

NaStEP: A Proteinase Inhibitor Essential to Self-Incompatibility and a Positive Regulator of HT-B Stability inNicotiana alataPollen Tubes

“…In vitro binding assays show that PiSLF in Petunia inflata physically interacts with S-RNases, although this interaction is stronger with nonself SRNases than with self S-RNases (Hua and Kao, 2006). Additional protein-protein interaction assays suggest that SLF/SFB may be a component of an SCF (for Skp1-Cullin1-F-box) or SCF-like complex (Qiao et al, 2004;Hua and Kao, 2006).…”

“…In vitro binding assays show that PiSLF in Petunia inflata physically interacts with S-RNases, although this interaction is stronger with nonself SRNases than with self S-RNases (Hua and Kao, 2006). Additional protein-protein interaction assays suggest that SLF/SFB may be a component of an SCF (for Skp1-Cullin1-F-box) or SCF-like complex (Qiao et al, 2004;Hua and Kao, 2006). Notably, data from Zhao et al (2010) in Petunia hybrida show that reduction of PhSSK1 (for P. hybrida SLF-interacting Skp-like1) and its Antirrhinum hispanicum ortholog, AhSSK1, is also required for cross-pollen compatibility.…”

“…(1) The S-RNase degradation model (Hua and Kao, 2006;Hua et al, 2007Hua et al, , 2008Kubo et al, 2010) focuses on S-RNase-SLF interactions that bring about preferential nonself S-RNase degradation. In this model, strong nonself S-RNase-SLF interactions lead to the degradation of nonself S-RNases by the ubiquitin-26S proteasome system, allowing pollen tubes to escape from its cytotoxic effect.…”