A Flower-Specific Phospholipase D1 is a Stigmatic Compatibility Factor Targeted by the Self-Incompatibility Response in Brassica Napus

Abstract

Self-incompatibility (SI) is a genetic mechanism in hermaphroditic flowers that prevents inbreeding by rejection of self-pollen, while allowing cross or genetically diverse pollen to germinate on the stigma to successfully fertilize the ovules. In Brassica, SI is initiated by the allele-specific recognition of pollen-encoded, secreted ligand (SCR/SP11) by the stigmatic receptor kinase S-locus receptor kinase (SRK), resulting in activation of SRK through phosphorylation. Once activated, this phospho-relay converges on intracellular compatibility factors, which are immediately targeted for degradation by the E3 ligase, ARC1, resulting in the pollen rejection response. Through proteomics approach using proteins from SI activated stigmas of canola (Brassica napus), we identified phospholipase D1 (PLD1) as one of the candidates that is likely targeted for degradation following SI. PLD1 is enriched in the stigmas and functions as a stigmatic compatibility factor as loss of PLD1 compromised compatible pollination, while overexpression of PLD1 in self-incompatible stigmas led to breakdown of SI response. PLD1 overexpression was also associated with enhanced membrane activity following SI pollination, mimicking compatible pollination. PLD1 can be ubiquitinated by ARC1 and accumulate in ARC1- suppressed lines confirming PLD1 as a direct target of ARC1 during SI response. Addition of phosphatidic acid (PA) to PLD1 deficient stigmas was sufficient to rescue compatibility suggesting an essential role for PA generated by PLD1 for compatible interactions. We propose that PA produced by PLD1 activity during compatible pollination promotes vesicle fusion at the membrane to facilitate exocytosis necessary for pollen germination to occur, while SI response could abrogate this process by targeting PLD1 for degradation.