The plasma membrane proton (H +)-ATPases of plants generate steep electrochemical gradients and activate osmotic solute uptake. H +-ATPase-mediated proton pumping orchestrates cellular homeostasis and is a prerequisite for plastic cell expansion and plant growth. All evidence suggests that the population of H +-ATPase proteins at the plasma membrane reflects a balance of their roles in exocytosis, endocytosis, and recycling. Auxin governs both traffic and activation of the plasma membrane H +-ATPase proteins already present at the membrane. As in other eukaryotes, in plants, SNARE-mediated membrane traffic influences the density of several proteins at the plasma membrane. Even so, H +-ATPase traffic, its relationship with SNAREs, and its regulation by auxin have remained enigmatic. Here, we identify the Arabidopsis (Arabidopsis thaliana) Qa-SNARE SYP132 (Syntaxin of Plants132) as a key factor in H +-ATPase traffic and demonstrate its association with endocytosis. SYP132 is a low-abundant, secretory SNARE that primarily localizes to the plasma membrane. We find that SYP132 expression is tightly regulated by auxin and that augmented SYP132 expression reduces the amount of H +-ATPase proteins at the plasma membrane. The physiological consequences of SYP132 overexpression include reduced apoplast acidification and suppressed vegetative growth. Thus, SYP132 plays unexpected and vital roles in auxin-regulated H +-ATPase traffic and associated functions at the plasma membrane.
The vesicle trafficking SYNTAXIN OF PLANTS132 (SYP132) drives hormone-regulated endocytic traffic to suppress the density and function of plasma membrane H+-ATPases. In response to bacterial pathogens, it also promotes secretory traffic of antimicrobial pathogenesis-related (PR) proteins. These seemingly opposite actions of SYP132 raise questions about the mechanistic connections between the two, likely independent, membrane trafficking pathways intersecting plant growth and immunity. To study SYP132 and associated trafficking of plasma membrane H+-ATPase 1 (AHA1) and PATHOGENESIS-RELATED PROTEIN1 (PR1) during pathogenesis, we used the virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) bacteria for infection of Arabidopsis (Arabidopsis thaliana) plants. SYP132 overexpression suppressed bacterial infection in plants through the stomatal route. However, bacterial infection was enhanced when bacteria were infiltrated into leaf tissue to bypass stomatal defences. Tracking time-dependent changes in native AHA1 and SYP132 abundance, cellular distribution, and function, we discovered that bacterial pathogen infection triggers AHA1 and SYP132 internalisation from the plasma membrane. AHA1 bound to SYP132 through its regulatory SNARE Habc domain, and these interactions affected plasma membrane H+-ATPase traffic. Remarkably, using the Arabidopsis aha1 mutant, we discovered that AHA1 is essential for moderating SYP132 abundance and associated secretion of PR1 at the plasma membrane for pathogen defence. Thus, we show that, during pathogenesis, SYP132 coordinates AHA1 with opposing effects on the traffic of AHA1 and PR1.
The yeast Tri-SUS system can be used to study tripartite protein–protein interactions between bait and prey protein pairs by modulation of expression of their binding partner
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