S-RNase is the female determinant of self-incompatibility (SI) in pear (Pyrus bretschneideri). After translocation to the pollen tube, S-RNase degrades rRNA and induces pollen tube death in an S-haplotype-specific manner. In this study, we found that the actin cytoskeleton is a target of P. bretschneideri S-RNase (PbrS-RNase) and uncovered a mechanism that involves phosphatidic acid (PA) and protects the pollen tube from PbrS-RNase cytotoxicity. PbrS-RNase interacts directly with PbrActin1 in an S-haplotype-independent manner, causing the actin cytoskeleton to depolymerize and promoting programmed cell death in the self-incompatible pollen tube. Proline 156 of PbrS-RNase is essential for the PbrS-RNase-PbrActin1 interaction, and the actin cytoskeleton-depolymerizing function of PbrS-RNase does not require its RNase activity. PbrS-RNase cytotoxicity enhances the expression of phospholipase D (PbrPLDδ1), resulting in increased PA levels in the incompatible pollen tube. PbrPLDδ1-derived PA initially prevents depolymerization of the actin cytoskeleton elicited by PbrS-RNase and delays the SI signaling that leads to pollen tube death. This work provides insights into the orchestration of the S-RNase-based SI response, in which increased PA levels initially play a protective role in incompatible pollen, until sustained PbrS-RNase activity reaches the point of no return and pollen tube growth ceases.

The hydroxyproline-rich glycoprotein (HRGP) superfamily, comprising three families (arabinogalactan-proteins, AGPs; extensins, EXTs; proline-rich proteins, PRPs), is a class of proline-rich proteins that exhibit high diversity and are involved in many aspects of plant biology. In this study, 838 HRGPs were identified from Chinese white pear (Pyrus bretschneideri) by searching for biased amino acid composition and conserved motifs. 405 HRGPs were derived from whole genome duplication (WGD) events which is suggested to be the major force of driving HRGPs expansion and the recent WGD event shared by apple and pear generated most duplicated HRGPs in pear. This duplication event drived the structural variation of the HRGPs encoding hydroxyproline (Hyp)-rich motifs. The rate of HRGPs evolution mainly impacted the Hyp-rich motifs even in chimeric HRGPs. During the evolution of 53 PRPs that are also typified by 7-deoxyloganetin glucosyltransferase-like genes, the duplication from PRP to non-PRP was indirectly modified by positive selection. These results suggested that the rate of HRGP evolution mainly influenced the Hyp-rich motifs even in chimeric HRGPs. The expression divergence of HRGPs was higher than that of other commonly duplicated genes. In pear pistil, 601 HRGPs exhibited expression, while in pear pollen, 285 HRGPs were expressed. The qPCR results revealed that Pbr036330.1 and Pbr010506.1 showed different expression profile in self-incompatibility of pear pistil. The researches indicated that WGD events was the main duplication type during the evolution of HRGPs, and the highly variable Hyp-motifs might be accountable for the expansion, evolution and expression divergence of HRGPs and that this divergence may be responsible for the gain of new functions in plants.