Negatively charged phospholipids accelerate the membrane fusion activity of the plant-specific insert domain of an aspartic protease

Abstract

Various plants use antimicrobial proteins/peptides to resist phytopathogens. In the potato, Solanum tuberosum , the plant-specific insert (PSI) domain of an aspartic protease performs this role by disrupting phytopathogen plasma membranes. However, the mechanism by which PSI selects target membranes has not been elucidated. Here, we studied PSI-induced membrane fusion, focusing on the effects of lipid composition on fusion efficiency. Membrane fusion by the PSI involves an intermediate state whereby adjacent liposomes share their bilayers. We found that increasing the concentration of negatively charged phosphatidylserine (PS) phospholipids substantially accelerated PSI-mediated membrane fusion. NMR data demonstrated that PS did not affect the binding between the PSI and liposomes but had seminal effects on the dynamics of PSI interaction with liposomes. In PS-free liposomes, the PSI underwent significant motion, which was suppressed on PS-contained liposomes. Molecular dynamics simulations showed that the PSI binds to PS-containing membranes with a dominant angle ranging from −31° to 30°, with respect to the bilayer, and is closer to the membrane surfaces. In contrast, PSI is mobile and exhibits multiple topological states on the surface of PS-free membranes. Taken together, our data suggested that PS lipids limit the motion of the anchored PSI, bringing it closer to the membrane surface and efficiently bridging different liposomes to accelerate fusion. As most phytopathogens have a higher content of negatively charged lipids as compared with host cells, these results indicate that the PSI selectively targets negatively charged lipids, which likely represents a way of distinguishing the pathogen from the host.

Negatively charged phospholipids doped liposome delivery system for mRNA with high transfection efficiency and low cytotoxicity

“…In previous reports, electrically neutral 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were applied as auxiliary phospholipids to form the skeleton of liposomes (Cheng & Lee, 2016 ; Yanez Arteta et al., 2018 ). However, in this study, phosphatidyl serine (PS) was applied to replace DOPE or DSPC for increasing the safety of the system because the PS as a negatively charged phospholipids can neutralize part of the positive charge of DOTAP (Zhao et al., 2022 ) and reduce the overall charge of the particle ( Figure 1 ). EGFP mRNA was chosen as targeted mRNA loaded by liposome due to the accessibility of the fluorescence of EGFP expressed by the mRNA (Baeken & Yokobayashi, 2022 ; Larson et al., 2022 ).…”