A pathogen effector co-opts a host RabGAP protein to remodel pathogen interface and subvert defense-related secretion

Abstract

Pathogens have evolved sophisticated mechanisms to manipulate host cell membrane dynamics, a crucial adaptation to survive in hostile environments shaped by innate immune responses. Plant-derived membrane interfaces, engulfing invasive hyphal projections of fungal and oomycete pathogens, are prominent junctures dictating infection outcomes. Understanding how pathogens transform these host-pathogen interfaces to their advantage remains a key biological question. Here, we identified a conserved effector, secreted by plant pathogenic oomycetes, that co-opts a host Rab GTPase-activating protein (RabGAP), TOPGAP, to remodel the host-pathogen interface. The effector, PiE354, hijacks TOPGAP as a susceptibility factor to usurp its GAP activity on Rab8a, a key Rab GTPase crucial for defense-related secretion. By hijacking TOPGAP, PiE354 purges Rab8a from the plasma membrane, diverting Rab8a-mediated immune trafficking away from the pathogen interface. This mechanism signifies an uncanny evolutionary adaptation of a pathogen effector in co-opting a host regulatory component to subvert defense-related secretion, thereby providing unprecedented mechanistic insights into the reprogramming of host membrane dynamics by pathogens.

Fig. 1.. Conserved effectors from Phytophthora species target TOPGAP.

(A) TIKI interacts with TOPGAP in planta. RFP:TIKI or RFP:EV was transiently co-expressed with GFP:TOPGAP or GFP:EV. IPs were obtained with anti-GFP antibody. Total protein extracts were immunoblotted. Red asterisks indicate expected band sizes. Numbers on the right indicate kilodalton values. (B) Structural alignment of the effectors TIKI (blue) from P. palmivora and PiE354 (orange) and PiE355 (brown) from P. infestans. Structural predictions were obtained via AF2. The model shows overall structural conservation of the effectors, without their secretion signals and RXLR motifs, as these regions are cleaved off and are not part of the mature effector protein and are not required for their virulence function (53). (C) PiE355 and PiE354 interact with TOPGAP in planta. GFP:TOPGAP was transiently co-expressed with RFP:PiE355, RFP:PiE354, or RFP:EV. IPs were obtained with anti-GFP antibody. Total protein extracts were immunoblotted. Red asterisks indicate expected band sizes. Numbers on the right indicate kilodalton values. (D) TOPGAP colocalizes with TIKI, PiE354, and PiE355 in puncta in planta. Confocal micrographs of N. benthamiana leaf epidermal cells transiently expressing either RFP:TIKI, RFP:PiE354, RFP:PiE355, or RFP:EV, with GFP:TOPGAP. Presented images are single-plane images. Overlay panel transects correspond to line intensity plots showing relative fluorescence across the marked distance. Scale bars, 5 μm. A.U., arbitrary units.

Fig. 2.. PiE354 targets the N-terminal RBD fragment of TOPGAP.

(A) AF2-M–predicted model of PiE354 targeting TOPGAP. PiE354, RBD of TOPGAP, and TBCD of TOPGAP are depicted in yellow, pink, and black, respectively. The key residues responsible for the GAP activity of TOPGAP, R466 and Q483, are highlighted in cyan. The putative bonds between PiE354 and TOPGAP are predicted by ChimeraX with a distance of 5 Å. The colors of the bonds are based on the AF2-calculated predicted aligned error (PAE) score. A low PAE score, indicating high-confidence contacts, is shown by blue lines. A high PAE score, indicating low-confidence contacts, is shown by red lines. The curved gray dashed lines indicate the predicted disordered regions in TOPGAP. aa, amino acids. (B) PiE354 interacts with full-length TOPGAP and RBDF of TOPGAP but not with TBCDF of TOPGAP and EV. GFP:PiE354 was transiently co-expressed with RFP:TOPGAP, RFP:RBDF, RFP:TBCDF, or RFP:EV. (C) AF2-M–predicted model of PiE354 (yellow) targeting the RBD of TOPGAP (pink), depicting their interacting residues. The bonds between PiE354 and TOPGAP are predicted by PyMOL with a distance of 3 Å. The PiE354-RBD interaction interface consists of seven key residues on both proteins, colored as blue on PiE354 and as magenta on RBD. (D) PiE354 targets TOPGAP through six key residues on PiE354. GFP:TOPGAP was transiently co-expressed with either RFP:PiE354, RFP:PiE354^6A, or RFP:EV. (E) TOPGAP interacts with PiE354 through seven key residues on TOPGAP. RFP:PiE354 was transiently co-expressed with either GFP:TOPGAP, GFP:TOPGAP^7M, or GFP:EV. For all co-IP assays, IPs were obtained with anti-GFP antibody. Total protein extracts were immunoblotted. Red asterisks indicate expected band sizes. Numbers on the right indicate kilodalton values.

Fig. 3.. TOPGAP negatively regulates plant immunity through its GAP function.

(A) Structural alignment of TOPGAP and its GAP mutant TOPGAP^GAP using AF2 predictions. (B) N. benthamiana leaves expressing TOPGAP, TOPGAP^GAP, or EV control were infected with P. infestans, and pathogen growth was calculated by measuring infection lesion size at 8 days postinoculation (dpi). (C) N. benthamiana leaves expressing TOPGAP, TOPGAP^GAP, or EV control were infected with tdTomato-expressing P. infestans, and pathogen growth was calculated by measuring hyphal growth using fluorescence stereomicroscope at 5 dpi. (D) N. benthamiana leaves expressing RNAi:TOPGAP or RNAi:GUS control were infected with P. infestans, and pathogen growth was calculated by measuring infection lesion size at 8 dpi. (E) N. benthamiana leaves expressing RNAi:TOPGAP or RNAi:GUS control were infected with tdTomato-expressing P. infestans, and pathogen growth was calculated by measuring hyphal growth using fluorescence stereomicroscope at 5 dpi. [(B) to (E)] Each color represents an independent biological replicate. Each dot represents the average of three infection spots on the same leaf. Statistical differences were analyzed by Student’s t test or Mann-Whitney U test in R. Scale bars, 10 μm. Measurements were highly significant when ***P < 0.001. Detailed statistical analyses can be found in table S9. (F) N. benthamiana leaves were agroinfiltrated to express GFP:TOPGAP, GFP:TOPGAP^GAP, or GFP:EV. The infiltrated leaves were challenged with P. infestans extract at 3 dpi, and proteins were extracted from the apoplast and leaf tissue at 4 dpi and immunoblotted. Red asterisks show expected band sizes. Numbers on the right indicate kilodalton values. CBB, Coomassie Brilliant Blue staining.

Fig. 4.. PiE354 co-opts TOPGAP to subvert plant immunity and defense-related secretion.

(A) N. benthamiana leaves expressing PiE354, PiE354^6A, or EV control were infected with WT P. infestans, and pathogen growth was calculated by measuring infection lesion size at 8 dpi. Each color represents an independent biological replicate. Each dot represents an infection spot. Statistical differences were analyzed by Mann-Whitney U test in R. Measurements were highly significant when ***P < 0.001. Detailed statistical analyses can be found in table S9. n.s., not significant. (B and C) For PR1 secretion assays, the infiltrated leaves were challenged with P. infestans extract at 3 dpi, and proteins were extracted from the apoplast and leaf tissue at 4 dpi and immunoblotted. (B) Western blot shows PiE354 suppresses antimicrobial PR1 secretion into the apoplast, dependent on the interaction with its host target TOPGAP. N. benthamiana leaves were infiltrated to express GFP:PiE354, GFP:PiE354^6A (mutant that lacks the capability to interact with TOPGAP), or GFP:EV. (C) Western blot shows that the ability of PiE354 to inhibit PR1 secretion is less pronounced when TOPGAP is silenced, compared to that under GUS-silenced control condition. N. benthamiana leaves were infiltrated to express RNAi:TOPGAP or RNAi:GUS control, with either PiE354 or EV control. Red asterisks show expected band sizes. Numbers on the right indicate kilodalton values.

Fig. 5.. Rab8a is a GAP substrate of TOPGAP.

(A) TOPGAP interacts with Rab8a in planta. RFP:TOPGAP was transiently co-expressed with GFP:Rab8a or the GFP:EV control. RFP:EV served as a control for RFP:TOPGAP. IPs were obtained with anti-GFP antibody. For Western blotting results, total protein extracts were immunoblotted, red asterisks indicate expected band sizes, and numbers on the right indicate kilodalton values. (B) Rab8a colocalizes with TOPGAP in puncta in planta. Confocal micrographs of N. benthamiana leaf epidermal cells transiently expressing RFP:TOPGAP or RFP:EV, with GFP:Rab8a. Presented images are single-plane images. Overlay panel transects correspond to line intensity plots showing relative fluorescence across the marked distance. Scale bars, 5 μm. (C) AF2-M modeling of Rab8a with individual RBD and TBCD of TOPGAP in complex. (Left) Rab8a interacts with both the RBD and TBCD of TOPGAP. (Right) The AF2-M model colors are based on the AF2-calculated prediction confidence score [predicted local distance difference test (pLDDT)] shown in the rectangular box. (D) Rab8a interacts with full-length TOPGAP, RBDF of TOPGAP, and TBCDF of TOPGAP. GFP:Rab8a was transiently co-expressed with either RFP:TOPGAP, RFP:RBDF, RFP:TBCDF, or RFP:EV control. IPs were obtained with anti-GFP antibody. (E) Predicted AF2-M model of Rab8a in complex with full-length TOPGAP, focusing on TBCDF of TOPGAP. The catalytic dual fingers of the TBCD, R446 and Q483 (cyan), are positioned across the GTP-binding pocket of Rab8a, flanked by switch-1 (pink) and switch-2 (orange) regions, and making contacts with the GTP molecule within the pocket. (F) TOPGAP stimulates the GTPase activity of Rab8a dependent on its GAP activity. A luciferase-based GTPase assay was used to quantify the amount of GTP levels. The bar graph illustrates the effect of TOPGAP, TOPGAP^GAP, or GFP control on the GTPase activity of Rab8a across three technical repeats. No enzyme control does not contain Rab8a. “No substrate control” does not contain added GTP. RLU, relative light unit.

Fig. 6.. TOPGAP negatively regulates immunity by restricting Rab8a-mediated subcellular trafficking toward the cell surface.

(A and B) TOPGAP diverts Rab8a localization from the plasma membrane to the tonoplast dependent on its GAP activity. (A) Confocal micrographs of N. benthamiana leaf epidermal cells transiently co-expressing either RFP:TOPGAP, RFP:TOPGAP^GAP, or RFP:EV, with GFP:Rab8a. Presented images are single-plane images. Scale bars, 5 μm. (B) Box plot illustrates that TOPGAP expression significantly reduces the relative intensity ratio of Rab8a in plasma membrane to tonoplast compared to that in the EV control, while TOPGAP^GAP expression has no significant effect. Black dots denote outliers as indicated by ggplot2 in R. (C) The significant increase in P. infestans lesion size caused by TOPGAP expression is negated when Rab8a is silenced. RNAi:Rab8a or RNAi:GUS control was co-expressed with either TOPGAP or EV control in WT leaves. The agroinfiltrated leaves were infected with WT P. infestans, and pathogen growth was calculated by measuring infection lesion size at 7 dpi. (D) The significant increase in P. infestans hyphal growth caused by TOPGAP expression is negated when Rab8a is silenced. RNAi:Rab8a or RNAi:GUS control was co-expressed with either TOPGAP or EV control in WT leaves. The agroinfiltrated leaves were infected with tdTomato-expressing P. infestans, and pathogen growth was calculated by measuring hyphal growth using fluorescence stereomicroscope at 5 dpi. Scale bars, 10 μm. [(C) and (D)] Each color represents an independent biological replicate. Each dot represents the average of three infection spots on the same leaf. Statistical differences were analyzed by Student’s t test, or Mann-Whitney U test in R. Measurements were highly significant when ***P < 0.001. Detailed statistical analyses can be found in table S9.

Fig. 7.. PiE354 targets the TOPGAP-Rab8a complex and re-routes Rab8a trafficking toward the vacuole.

(A) AF2-M–predicted model of PiE354 in complex with the TOPGAP-Rab8a pair. The model indicates that in the presence of PiE354, Rab8a and the RBD of TOPGAP no longer interact. Bond colors reflect the AF2-calculated predicted aligned error (PAE) score. (B) Confocal micrographs of N. benthamiana leaf epidermal cells transiently co-expressing RFP:PiE354, RFP:PiE54^6A, or RFP:EV, with BFP:TOPGAP and GFP:Rab8a. Overlay panel transects correspond to line intensity plots showing relative fluorescence across the marked distance. (C) PiE354 binds to the TOPGAP-Rab8a pair dependent on its interaction with TOPGAP. GFP:Rab8a and 3xHA:TOPGAP were transiently co-expressed with RFP:PiE354, RFP:PiE354^6A, or RFP:EV. IPs were obtained with anti-GFP antibody. Red asterisks indicate expected band sizes. Numbers on the right indicate kilodalton values. (D and E) PiE354 diverts Rab8a localization from the plasma membrane to the tonoplast dependent on its interaction with TOPGAP. (D) Confocal micrographs of N. benthamiana cells transiently expressing RFP:PiE354, RFP:PiE354^6A, or RFP:EV, with GFP:Rab8a. (E) Box plot illustrates that expression of EV control or PiE354^6A predominantly localizes Rab8a to the plasma membrane, while PiE354 redirects Rab8a primarily to the tonoplast. (F and G) Silencing TOPGAP nullifies the ability of PiE354 diverting Rab8a localization from plasma membrane to the tonoplast. (F) Confocal micrographs of N. benthamiana cells transiently expressing RNAi:TOPGAP, or RNAi:GUS control, with RFP:PiE354 and GFP:Rab8a. (G) Box plot illustrates that, under PiE354 expression, silencing TOPGAP leads to Rab8a predominantly localizing to the plasma membrane, while, in the GUS-silenced control condition, Rab8a primarily localizes to the tonoplast. All presented images are single-plane images. Scale bars, 5 μm. Black dots in box plots denote outliers indicated by ggplot2 in R. Statistical differences were analyzed by Mann-Whitney U test in R. Measurements were significant when **P < 0.01. Detailed statistical analyses can be found in table S9.

Fig. 8.. Summary models of the effector PiE354 co-opting the RabGAP TOPGAP and suppressing plant immunity by redirecting defense-related secretion.

(A) Following penetration by P. infestans into the host plant cell, the effector PiE354 is secreted into the cytoplasm through its haustoria. Subsequently, PiE354 targets the host RabGAP protein TOPGAP, facilitating TOPGAP to deactivate its cognate RabGAP Rab8a. This leads to the redirection of Rab8a trafficking toward the vacuole instead of the plasma membrane, resulting in the suppression of plant immunity by inhibiting defense cargo secretion toward the pathogen interface. (B) In the absence of pathogen effectors, Rab8a interacts with both the RBD and TBCD of TOPGAP. Introduction of the effector PiE354, which binds to the RBD, causes a shift in the positioning of Rab8a toward the TBCD, where the GAP function is housed. Consequently, this facilitates the deactivation of Rab8a through increased GTP hydrolysis.