Signal Peptide Peptidase and PI4Kß1/2 play opposite roles in plant ER stress response and immunity.

The Arabidopsis pi4kß1,2 mutant is mutated in the phosphatidylinositol 4-kinase (PI4K) ß1 and PI4Kß2 enzymes which are involved in the biosynthesis of phosphatidylinositol 4-phosphate (PI4P), a minor membrane lipid with important signaling roles. pi4kß1,2 plants display autoimmunity and shorter roots. Though the pi4kß1,2 mutant has been extensively characterized, the source of its autoimmunity remains largely unknown. In this study, through a genetic suppressor screen, we identified multiple partial loss-of-function alleles of signal peptide peptidase (spp) that can suppress all the defects of pi4kß1,2. SPP is an intramembrane cleaving aspartic protease. Interestingly, pi4kß1,2 plants display enhanced ER stress response and mutations in SPP can suppress such phenotype. Furthermore, reduced ER stress responses were observed in the spp single mutants. Overall, our study reveals a previously unknown function of PI4Kß and SPP in ER stress and plant immunity.

A novel fluorescent protein pair facilitates FLIM-FRET analysis of plant immune receptor interaction under native conditions.

Elucidating protein-protein interactions is crucial for our understanding of molecular processes within living organisms. Microscopy-based techniques can detect protein-protein interactions in vivo at the single-cell level and provide information on their subcellular location. Fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) is one of the most robust imaging approaches, but it is still very challenging to apply this method to proteins which are expressed under native conditions. Here we describe a novel combination of fluorescence proteins (FPs), mCitrine and mScarlet-I, which is ideally suited for FLIM-FRET studies of low abundance proteins expressed from their native promoters in stably transformed plants. The donor mCitrine displays excellent brightness in planta, near-mono-exponential fluorescence decay, and a comparatively long fluorescence lifetime. Moreover, the FRET pair has a good spectral overlap and a large Förster radius. This allowed us to detect constitutive as well as ligand-induced interaction of the Arabidopsis chitin receptor components CERK1 and LYK5 in a set of proof-of-principle experiments. Due to the good brightness of the acceptor mScarlet-I, the FP combination can be readily utilized for co-localization studies. The FP pair is also suitable for co-immunoprecipitation experiments and western blotting, facilitating a multi-method approach for studying and confirming protein-protein interactions.