SBT3.3 and pro-PME17 are secreted through distinct protein secretion pathways in the apoplast

Pectin Methylesterases (PMEs) are frequently organized as ProPMEs whose activity can be finely tuned at post-transcriptional level in plant development by a class of proteases named Subtilases (SBTs) (Del Corpo et al. 2020). PMEs are critical factors in plant-fungus interaction. SBT3.3 was previously found in the extracellular compartment, but its secretion pathway was never explored. Pro-PME17 was predicted to be an apoplastic protein, but its localization was never demonstrated. With the aim of deepening our knowledge on the secretion patterns and on the final subcellular destination of the two proteins, a fluorescent variant of SBT3.3 and Pro-PME17 was constructed and used to explore SBT3.3 and Pro-PME17 targeting and subcellular localization. The SBT3.3-YFP and Pro-PME17-GFP constructs were transiently expressed in the epidermal cells of tobacco leaves and observed by laser scanning confocal microscopy. Both proteins accumulated in the apoplast. While Pro-PME17 follows a conventional secretion pathway, SBT3.3 undergoes an unconventional route colocalizing with Exo70E2, a subunit of the exocyst complex. Our results indicate that SBT3.3 and Pro-PME17 are secreted using different secretion pathways and in a temporally staggered manner to colocalize in the cell wall.