P-164 - The RXLR-EER motifs determine an unconventional secretion pathway associated with EV production

Phytophthora infestans unconventionally secrets RXLR effectors to suppress host immunity. These effectors are named after a conserved motif Arg-any amino acid-Leu-Arg (RXLR) which is normally followed by another conserved Glu-Glu-Arg (EER) motif. RXLR-EER motifs and their cleavages were shown to be crucial for effector translocation, but little is known about what their exact functions are. In this study, we described a novel method to distinguish conventional and unconventional secretion pathways and demonstrated that the RXLR-EER motif determines the unconventional secretion pathway of RXLR effectors. Confocal microscopy revealed that RXLR-EER effector Pi04314 colocalises with another RXLR effector Pi04097 but not with the apoplastic effector SCR74, suggesting different secretory routes. Introduction of an ER retaining motif KDEL to the C-terminus of tagged effectors significantly reduced the secretion of apoplastic effectors SCR74 and EPIC1 but not the RXLR effector Pi04314. Mutation of RXLR-EER/RXLR motif to Alanines altered the localisation of RXLR effectors Pi04314 and Pi04097 and resulted in significant reductions of their abundance within extracellular vesicle (EV) associated pellets. Moreover, addition of KDEL motifs prevented the secretion of the mutated RXLR effector 04314^AAAA-AAA. Collectively, we conclude that recognition and cleavage of RXLR-EER motifs are important for RXLR effectors to enter a nonconventional secretion pathway.