Primary restriction of S-RNase cytotoxicity by a stepwise ubiquitination and degradation pathway in Petunia hybrida.

In self-incompatible Petunia species, the pistil S-RNase acts as cytotoxin to inhibit self-pollination but is polyubiquitinated by the pollen-specific non-self S-locus F-box (SLF) proteins and subsequently degraded by the ubiquitin-proteasome system (UPS), allowing cross-pollination. However, it remains unclear how S-RNase is restricted by the UPS. Using biochemical analyses, we first show that Petunia hybrida S3 -RNase is largely ubiquitinated by K48-linked polyubiquitin chains at three regions, R I, II and III. R I is ubiquitinated in unpollinated, self- and cross-pollinated pistils, indicating its occurrence before PhS3 -RNase uptake into pollen tubes, whereas R II and III are exclusively ubiquitinated in cross-pollinated pistils. Transgenic analyses showed that removal of R II ubiquitination resulted in significantly reduced seed sets from cross-pollination and that of R I and III in less extent, indicating their increased cytotoxicity. Consistent with this, the mutated R II of PhS3 -RNase resulted in a marked reduction of its degradation, whereas that of R I and III in less reduction. Taken together, we demonstrate that PhS3 -RNase R II functions as a major ubiquitination region for its destruction and R I and III as minor ones, revealing that its cytotoxicity is primarily restricted by a stepwise UPS mechanism for cross-pollination in P. hybrida.