SDJ, a pollen-expressed type III J-protein in Prunus, directly binds S-RNase and promotes recruitment of SLFL6 to an S-RNase-associated complex

Abstract

In Prunus, self-incompatibility (SI) is controlled by S-RNases and pollen-expressed F-box proteins, whereas the molecular processes governing S-RNase regulation in pollen remain incompletely understood. Here, we characterized PavSDJ, a novel pollen protein from sweet cherry (Prunus avium), as a candidate factor involved in pollen-side S-RNase-associated processes. Sequence and structural analyses identified PavSDJ as a type III J-protein. Phylogenetic analyses placed PavSDJ within a distinct SDJ-like sublineage of the type III J-protein group, separate from a closely related sister lineage. Consistent with this divergence, PavSDJ was strongly expressed in anthers and pollen, whereas its sister gene was broadly expressed across organs. Transient expression assays showed that PavSDJ–GFP exhibited a predominantly cell-peripheral fluorescence pattern consistent with intracellular localization. Biochemical analyses showed that PavSDJ associated with recombinant PavS-RNases in pollen extracts and in reconstituted pull-down assays, without obvious allele preference. Proteomic analysis of PavSDJ co-immunoprecipitants from pollen extracts identified a complex including PavSLFL6 and PavSSK1. Reconstitution assays further showed that PavSDJ promoted the co-precipitation of PavSLFL6 with S-RNase. These findings identify PavSDJ as a candidate pollen-side factor in the Prunus SI pathway and provide evidence that a specialized J-protein may contribute to SI-related protein complex assembly. PavSDJ is a pollen-expressed type III J-protein that binds S-RNase and promotes recruitment of SLFL6 to an S-RNase-associated complex in Prunus. It may function as a general modifier involved in the GSI system of Prunus.