Exploring the role of the RXLR-EER motifs in the secretion and translocation processes of Phytophthora infestans cytoplasmic effectors

Abstract

Phytophthora infestans is an oomycete that causes late blight, a devastating disease of potato and tomato. This disease was responsible for the Irish potato famine in the 1840s and is still threatening crop production today. For successful infection, P. infestans deploys a variety of strategies to breach the host immune system, one of which is to utilize effector proteins that are synthesized inside pathogens and then delivered into the extracellular environment (apoplastic effectors), or into host cells (cytoplasmic effectors). Many P. infestans cytoplasmic effectors are RXLR class proteins, all of which contain the conserved RXLR (arginine (R)-any amino acid (X)-leucine (L)-arginine (R)) motif near the N-terminus and often followed by another motif, EER (glutamic acid (E)-glutamic acid (E)-arginine (R)). In recent years, many host targets of P. infestans cytoplasmic effectors have been identified and how they are exploited by effectors to promote disease has been determined. However, little is known about how RXLR effectors are secreted and translocated and the roles of the RXLR and EER motifs in these processes.

C-terminal mRFP-tagged wild type (WT) and mutant (replacing the RXLR and EER motifs with alanines singly or simultaneously) of Pi04097 (RXLR only) and Pi04314, Pi21388, Pi22926 (RXLR-EER) were overexpressed in P. infestans. When cultured in vitro, WT RXLR effectormRFP fusions are cleaved at the RXLR or EER motifs before secretion, resulting in the presence of two forms of effector fusions with slight size differences in the culture filtrate (CF). Mutating the RXLR or EER motifs disrupts the cleavage at these sites, resulting in the disappearance of the larger or smaller cleaved form of the secreted effector fusion in the CF. Mutating both the RXLR and EER motifs abolishes the cleavage at both sites and effector fusions are therefore cleaved only after the signal peptides. Other RXLR effectors (Pi04145, Pi09160, Pi09216, Pi09224 and Pi22880) undergo similar proteolytic cleavage, indicating that the cleavage at the RXLR and EER motifs is a general processing step for all RXLR effectors during secretion. This is very similar to PEXEL effector processing in Plasmodium falciparum. The position of the RXLR motif is important for the cleavage and the RXLR motif cannot be recognized by plant proteases for cleavage. Moreover, site-1 protease (S1P), which recognizes R-X-L/V/I-X↓motif for proteolytic cleavage, is not involved in the cleavage at the RXLR motif.

In planta, both WT and mutant (AAAA-AAA, RXLR-AAA, and AAAA-EER) effector-mRFP fusions were secreted from haustoria, but only WT and RXLR-AAA effector-mRFP fusions accumulated inside host cell nuclei, suggesting that the RXLR motif, but not the EER motif, is important for effector translocation. In vitro, the secretion of both WT and mutant effector-mRFP fusions was insensitive to brefeldin A (BFA, inhibitor of the conventional ER-GA secretory pathway) treatment but sensitive to endosidin2 (ES2, inhibitor of exocytosis and endosomal recycling) treatment, which indicated that the secretion followed an unconventional secretory pathway. Further, the EER motif of Pi04314 was not involved in the localization and function of this effector within the host cell.

This work demonstrated the independent cleavage at the RXLR and EER motifs of RXLR effectors and proposed a general model of RXLR effector cleavage. The WT and mutant transgenic lines generated in this work can be used for further investigation about the unconventional secretory pathways adopted by RXLR effectors. The silencing-andoverexpressing vector can be used to test other possible proteases that may be responsible for effector cleavage.

Date of Award	2023
Original language	English
Sponsors	China Scholarship Council & The James Hutton Institute
Supervisor	Paul Birch (Supervisor), Stephen Whisson (Supervisor) & Petra C. Boevink (Supervisor)