S-RNase Internalisation and Vacuolar Targeting via Clathrin-Dependent Endocytosis Mediate Interspecific Pollen Rejection in Nicotiana.

In Nicotiana, the multiallelic S-locus controls self-incompatibility (SI), which encodes S-RNases in the pollen's pistil and S-locus F-box (SLF) proteins. Their interaction mediates S allele-specific pollen rejection, preventing self-fertilisation. Beyond its role in SI, the S-locus also contributes to unilateral interspecific (UI) incompatibility, where self-incompatible pistils reject pollen from self-compatible species. Experimental evidence indicates that S-RNases alone can mediate UI. In transgenic Nicotiana tabacum-a naturally self-compatible species-expression of the S-RNase is sufficient to trigger self-pollen rejection, demonstrating that S-RNases can function independently of a complete SI system. However, the molecular mechanisms underlying S-RNase-mediated factor-independent UI remain unclear. To explore this, we analysed S-RNase trafficking in N. tabacum, which lacks endogenous S-RNases and SLFs. Pollen tube assays and confocal microscopy showed that S-RNases are internalised via clathrin-mediated endocytosis and transported in vesicles to vacuoles. Disruption of vacuoles releases S-RNases into the cytoplasm, coinciding with RNA degradation. These findings reveal an SLF-independent pathway for S-RNase internalisation, sequestration, and release, supporting that S-RNase compartmentalisation underlies pollen rejection between self-compatible and self-incompatible Nicotiana. This mechanism may be central in evolving interspecific reproductive barriers within the genus.