Identification of INOSITOL PHOSPHORYLCERAMIDE SYNTHASE 2 (IPCS2) as a new rate-limiting component in Arabidopsis pathogen entry control

SIGNIFICANCE STATEMENTPolarized transport of executive defense gene products to sites of attempted microbial invasion is important for plant pathogen entry control and disease resistance. Here, we provide evidence that INOSITOL PHOSPHORYLCERAMIDE SYNTHASE 2 (IPCS2)-dependent sphingolipid production contributes to the role of the trans-Golgi network as a multi-domain sorting compartment, and mediates proper delivery of an ATP-binding cassette transporter to polarized plasma membrane domains at plant-microbe interaction sites. INOSITOL PHOSPHORYLCERAMIDE SYNTHASE 2 (IPCS2) is involved in the biosynthesis of complex sphingolipids at the trans-Golgi network (TGN). Here, we demonstrate a role of IPCS2 in penetration resistance against non-adapted powdery mildew fungi. A novel ipcs2W205* mutant was recovered from a forward genetic screen for Arabidopsis plants with enhanced epidermal cell entry success of the non-adapted barley fungus Blumeria graminis f. sp. hordei (Bgh). A yeast complementation assay and a sphingolipidomic approach revealed that the ipcs2W205* mutant represents a knock-out and lacks IPCS2-specific enzymatic activity. Further mutant analyses suggested that IPCS2-derived glycosyl inositol phosphorylceramides (GIPCs) are required for cell entry control of non-adapted fungal intruders. Confocal laser scanning microscopy (CLSM) studies indicated that upon pathogen attack, IPCS2 remains at the TGN to produce GIPCs, while focal accumulation of the defense cargo PENETRATION 3 (PEN3) at Bgh penetration sites was reduced in the ipcs2W205* mutant background. Thus, we propose a model in which sorting events at the TGN are facilitated by complex sphingolipids, regulating polar secretion of PEN3 to host-pathogen contact sites to terminate fungal ingress.