SNARE SYP132 mediates divergent traffic of plasma membrane H+-ATPase AHA1 and antimicrobial PR1 during bacterial pathogenesis

Abstract

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.

Small secreted proteins and exocytosis regulators: do they go along?

“…We were especially interested in the co-expression context of the defense-related PR1 At2g14610, therefore we further investigated the PR1-positive RLP23/WAK1 cluster as obtained by STRING, connected with SNARE/EXO70 cluster, and also expanded by SNAREs and Rabs previously reported to be related with PR1 localization and secretion 26 , 28 , 29 in Genevestigator co-expression analysis. We applied the hierarchical clustering tool with Pearson correlation applied as a distance measure.…”

“… 27 . The AtPR1 trafficking to the extracellular space has been found to be dependent on the presence of RING E3 ligase KEEP-ON-GOING, and vesicular pathway regulators SNARE (BET12, MEMB12, or SYP132) or exocyst complex [ 28 , 29 , 30 , 31–33 ] . PR1-GFP expressed transiently in N. benthamiana leaves exhibits a complex dual trafficking mode, as it is localized in the extracellular space and intracellularly localized in the multivesicular bodies and vacuolar lumen.…”

Secretory membrane traffic in plant–microbe interactions

“…2). The reduced resistance in SYP132 ‐downregulated Nicotiana benthamiana to Pseudomonas syringae pv tabaci (Kalde et al ., 2007) but elevated defense in SYP132 ‐upregulated Arabidopsis against flood‐inoculated P. syringae DC3000 (Baena et al ., 2022), and the necessity of VAMP721/722 for Arabidopsis defense against P. syringae DC3000 (Kim et al ., 2021) suggest that the SYP132–VAMP721/722 interaction is important for plant immunity against bacterial pathogens. Furthermore, this implicates that plants may use SYP121 for fungal pathogens but SYP132 for bacterial ones as VAMP721/722‐interacting Qa‐SNAREs in immune exocytosis.…”

“…Severe growth defects in the vamp721 vamp722 double mutant (Kwon et al ., 2008) make it difficult to precisely understand what is transported by these VAMP721/722 vesicles. Secretion of PR1 into the apoplast was diminished in N. benthamiana in which SYP132 is silenced (Kalde et al ., 2007) or the SYP132 trafficking was compromised by expressing a dominant‐negative SYP132 variant (Baena et al ., 2022). This indirectly suggests that PR1 is transported via VAMP721/722 vesicles, because SYP132 and VAMP721/722 are important for plant defense against bacterial pathogens.…”

“…This indirectly suggests that PR1 is transported via VAMP721/722 vesicles, because SYP132 and VAMP721/722 are important for plant defense against bacterial pathogens. A study using transgenic Arabidopsis overexpressing SYP132 or a dominant‐negative SYP132 variant revealed that the abundance of PM H + ‐ATPase AHA1, which is bound to and endocytosed with SYP132, is important for regulating SYP132 levels at the PM and SYP132‐mediated PR1 secretion (Baena et al ., 2022). The reduced observation of the flagellin‐recognizing FLAGELLIN‐SENSING 2 (FLS2) receptor at the PM in syp121 plants (Wang et al ., 2020) and the atypical RESISTANCE TO POWDERY MILDEW 8 (RPW8) resistance (R) protein at the fungus‐invaginated PM in VAMP721/722‐depeleted plants (Kim et al ., 2014) provide further evidence that some PM‐targeted immune proteins are also transported via VAMP721/722 vesicles (Fig.…”