投稿日: 投稿者: OHTAAtsushi

STT3A Is Required for Recognition of Pathogen-Derived Sphingolipids in Arabidopsis

Abstract

Plants recognize pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors, leading to the activation of pattern-triggered immunity in response to pathogen attack. Phytophthora infestans ceramide D (Pi-Cer D) is a sphingolipid from the oomycete pathogen P. infestans. Pi-Cer D is cleaved by the plant extracellular ceramidase NEUTRAL CERAMIDASE 2 (NCER2), and the resulting 9-methyl-branched sphingoid base is recognized by the plant receptor RESISTANT TO DFPM-INHIBITION OF ABSCISIC ACID SIGNALING 2 (RDA2) at the plasma membrane to transduce a defense signal. However, additional components are likely involved in sphingolipid recognition, which remain to be identified. Here, we employed a screen based on Lumi-Map technology to look for Arabidopsis (Arabidopsis thaliana) mutants with altered defense responses to Pi-Cer D. We identified three mutants showing diminished responses to Pi-Cer D and elf18, each carrying mutations in STAUROSPORIN AND TEMPERATURE SENSITIVE 3-LIKE A (STT3A), which encodes an oligosaccharyltransferase. The stt3a mutants exhibited higher susceptibility to the pathogen Colletotrichum higginsianum than the wild type. In stt3a mutants, the molecular mass of NCER2 and RDA2 proteins appeared smaller, indicating STT3A is involved in post-translational modification of the proteins. Enzymatic deglycosylation assay revealed that NCER2 and RDA2 are N-glycosylated. These findings suggest that STT3A contributes to plant immunity via post-translational modification of proteins including NCER2 and RDA2.

STT3A is involved in the recognition of Pi-Cer D in plants. (A) STT3A gene structure and thelocations of the causative mutations in the three mutants, stt3a-3 (L-08), stt3a-4 (L-13), and stt3a-5 (L-51). Black rectangles and white rectangles indicate exons and UTRs respectively; lines represent introns.The locations of causative mutations are represented by white triangles, with their mutation types indicated.The red arrows indicate the primer positions for RT-qPCR with STT3A. (B) Genetic complementation of thethree mutants with a wild-type of the STT3A gene. The wild-type STT3A gene was introduced into eachmutant background (10.7-kb genomic fragment with its native promotor). The pWRKY33-LUC reporter line(WT), the L-08, L-13, L-51 mutants, and complementation lines for each mutant were treated with 0.17 µMPi-Cer D (means ± SE); bioluminescence was monitored using a continuous bioluminescence monitoringsystem. (C) Relative WRKY33 expression levels in Col-0 and the T-DNA insertion lines stt3a-2 and rda2-3, asdetermined by RT-qPCR. Seven-day-old seedlings were used for the analysis. The seedlings were sampled at0, and 1 h of treatment with 0.17 µM Pi-Cer D. *, p < 0.01 in two-tailed t-tests compared with thecorresponding values for Col-0 at each time point.