Pseudomonas syringae HopN1 Binds Plant VAP12 and a Rho-GTPase,Suggesting a Role in Membrane-Associated Processes

Abstract

Many Gram-negative bacterial pathogens deploy type III effector proteins (T3Es) to manipulate host cellular processes and suppress immune responses. Increasing evidence suggests that certain T3Es mimic eukaryotic FFAT (two phenylalanines in an acidic tract) motifs, enabling interaction with vesicle-associated membrane protein (VAMP)-associated proteins (VAPs). These interactions likely help pathogens target and exploit host membrane contact sites. However, the significance and distribution of FFAT mimicry across different bacterial pathogens remain poorly understood, which is crucial to uncovering its role in pathogenic strategies. In this study, we analyzed the T3E repertoire of the model plant pathogenic bacterium Pseudomonas syringae pv. tomato (Pst) DC3000 to identify potential FFAT motifs. Our preliminary data reveal that HopN1, a Pst T3E belonging to the YopT/AvrPphB family of cysteine proteases, contains at least one functional FFAT motif. Yeast two-hybrid and in planta co-immunoprecipitation assays confirmed that HopN1 interacts with plant VAP proteins. This interaction suggests that VAP binding may facilitate its localization to specific membrane compartments. Furthermore, HopN1 was shown to interact with a plant RHO-GTPase, hinting at a functional parallel to YopT in mammals. Our findings demonstrate that HopN1 interacts with VAP12 and a plant RHO-GTPase, suggesting a potential role in membrane-associated processes. However, whether HopN1 actively exploits VAP proteins for subcellular localization remains to be determined. While FFAT motif mimicry may contribute to effector targeting in plant-pathogenic bacteria, further studies are required to establish its functional significance in HopN1 virulence.

Introduction

The pathogenicity of many Gram-negative plant pathogenic bacteria relies on effector proteins that are translocated from the bacterium into the host cell via a type-III secretion system (Büttner, 2016). These type-III effector proteins (T3Es) target various host cell compartments to suppress immune responses and create a favorable niche for bacterial infection. One remarkable feature of bacterial T3Es is their evolution within prokaryotic cells to function effectively in eukaryotic cells. This adaptation requires T3Es to be accurately localized within the host cell, interact with substrates distinct from endogenous bacterial proteins, and exhibit enzymatic activity suited to the eukaryotic intracellular environment.

The Gram-negative bacterium Pseudomonas syringae pv. tomato (Pst) DC3000 is one of the most extensively studied plant pathogens and serves as a model organism for investigating host-microbe interactions, bacterial virulence mechanisms, pathogen adaptation, and microbial evolution, ecology, and epidemiology (Xin et al., 2018). Pst translocates more than 30 effector proteins into a host cell, with the majority of host effector targets being membrane-localized (Wei et al., 2015; Khan et al., 2018). These targets primarily include membrane-associated kinases that are critical in plant pattern-triggered immunity as well as membrane-localized components of effector-triggered immunity (Khan et al., 2018). This is largely due to the central role that secretory and endocytic membrane trafficking pathways play in plant immune responses (Gu et al., 2017; Yun and Kwon, 2017; Jeon and Segonzac, 2023).

To achieve proper localization, some effectors possess organelle-targeting signals (Li et al., 2014), while others are modified by the attachment of lipid groups after translocation into the cytoplasm of eukaryotic cells. For instance, lipidation of effector proteins promotes membrane association and is required for their function. This process involves modifications such as S-palmitoylation, N-myristoylation, and prenylation, all carried out by the host cell enzymatic machinery (reviewed in Schreiber et al., 2021). However, there are also effectors that associate with host membranes through mechanisms that remain poorly understood (Dowen et al., 2009).

Effectors typically function by binding to or enzymatically modifying host molecules, which fall into two primary functional categories: (1) “targets” that are directly altered by effectors to influence host processes, and (2) “helpers” or “facilitators” that are co-opted by effectors to carry out their functions, such as trafficking within host cells or aiding in interactions with target proteins (Win et al., 2012). It has recently been shown that the T3E XopM from the bacterium Xanthomonas campestris pv. vesicatoria (Xcv) binds to host vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) by virtue of two so called FFAT (two phenylalanines in an acidic tract) motifs within the effector polypeptide sequence (Brinkmann et al., 2024). VAPs are type II ER – integral proteins which act as common major players for generating tethers between the ER and other compartments such as the plasma membrane (PM) at ER – PM contact sites (EPCS) (Stefan et al., 2011; Murphy and Levine, 2016; Wang et al., 2016). In plants, EPCSs function as vital centers for maintaining phospholipid homeostasis and cell integrity (Schapire et al., 2008; Ruiz-Lopez et al., 2021). They also play roles in endocytosis (Stefano et al., 2018), autophagy (Wang et al., 2019a), and the regulation of cell-to-cell transport at plasmodesmata (Levy et al., 2015; Ishikawa et al., 2020).

Available evidence suggests that VAP binding of XopM requires at least one functional FFAT motif; however, the ability of XopM to bind VAP via its FFAT motifs appears independent of XopM's ability to suppress plant immune responses (Brinkmann et al., 2024). This led to the hypothesis that VAPs do not represent the direct virulence targets of the effector. Instead, XopM may use VAP binding via FFAT motifs as a targeting mechanism to access specific membrane subcompartments, such as EPCSs (Brinkmann et al., 2024). Such a mechanism potentially allows fine-tuning of effector virulence by focusing its activity on a subpopulation of target proteins.

In addition to XopM from Xanthomonas, the T3E IncV from the human intracellular pathogen Chlamydia trachomatis has also been shown to interact with VAPs via two FFAT motifs (Stanhope et al., 2017). Binding of IncV to VAP tethers the membrane of the bacteria-containing vacuole inside the host cell to the host ER membrane, providing a structural requirement for bacterial infection (Stanhope et al., 2017).

Given that XopM from Xanthomonas and IncV from Chlamydia appear to have independently acquired eukaryotic FFAT motifs for their respective virulence functions, it is tempting to speculate that other type-III effectors may employ similar strategies to promote their functions.

HopN1 is a T3E produced by Pst and belongs to the YopT/AvrPphB effector family of cysteine proteases (Shao et al., 2003; Dowen et al., 2009). The prototype effector from this family, YopT from Yersinia, disrupts the actin cytoskeleton of the host cell by inhibiting membrane-tethered Rho proteins through proteolytic cleavage. This cleavage results in rounding of the host cell and contributes to the antiphagocytic effect of Yersinia (Iriarte and Cornelis, 1998; Aepfelbacher et al., 2003). The P. syringae pv. phaseolicola effector AvrPphB specifically cleaves the Arabidopsis protein kinase PBS1 to initiate an RPS5-dependent hypersensitive response (HR) (Shao et al., 2003). Upon delivery into the host cell, AvrPphB autoproteolytically processes to reveal a novel amino terminus containing sites for both N-myristoylation and S-palmitoylation (Nimchuk et al., 2000). Host-dependent lipidation directs plasma membrane localization and is required for the avirulence activity of AvrPphB (Dowen et al., 2009).

Although its virulence mechanism is not completely understood, HopN1 was shown to be one of eight T3Es sufficient to rescue much of the virulence of an “effector-less” Pst mutant strain in the plant Nicotiana benthamiana (Cunnac et al., 2011). In a previous study, HopN1 has been suggested to localize to chloroplasts where it interacts with the photosynthesis protein PsbQ to promote its degradation (Rodriguez-Herva et al., 2012). This has been tied to HopN1's ability to suppress PTI responses such as ROS production and callose deposition (Rodriguez-Herva et al., 2012). However, in a recent report Gonzales-Fuentes et al. (2025) show that HopN1 induces the formation of processing bodies (P-bodies) in plant cells and partially co-localizes with the P-body component DCP5. The authors suggest that by manipulating P-body function HopN1 reprograms host translation in favor of the pathogen (Gonzales-Fuentes et al., 2025). In addition, biochemical data suggest that HopN1 utilizes an unknown acylation-independent mechanism to localize to the plasma membrane (Dowen et al., 2009).

In this study, we provide preliminary insight that HopN1 contains at least one functional FFAT motif and that the effector can bind VAP proteins. Further interaction studies demonstrate that HopN1 also interacts with a plant RHO-GTPase, suggesting it might have a similar virulence function in plants as YopT from Yersinia in mammals.

Results

Pst Effector Proteins Feature Predicted FFAT Motifs

Based on previous findings with XopM from Xanthomonas (Brinkmann et al., 2024) and other data from the literature, we hypothesized that FFAT motifs might represent a common strategy to target pathogen-secreted effector proteins to VAP-containing membrane compartments. To test this hypothesis, we screened the well-catalogued T3E effector repertoire of Pst DC3000 for the presence of predicted FFAT motifs using the algorithm developed by Murphy and Levine (2016). From the 29 known active Pst DC3000 effector proteins (Wei et al., 2015), two were identified as containing a putative FFAT motif within their polypeptide sequences (Table 1).

Table 1.

List of Pst DC3000 Effector Proteins That Contain a Putative FFAT Motif Within Their Polypeptide Sequence.

ANALYSED SEQUENCE / PROTEIN	MOTIF SEQUENCE	MOTIF SCORE
PERFECT FFAT+		0
HOPN1		2.0
AVRPTOB		2.5

+ = Murphy and Levine (2016).

FFAT = two phenylalanines [FF] in an acidic tract (black letters) whereas the F residue at the second position is highly conserved (underlined), core motif marked in yellow.

Motif score: 0 = perfect, 2.5 OK, 4 weak, 7 poor.

AvrPtoB is a conserved Pst effector protein with E3-ubiquitin ligase activity that mediates the degradation of multiple plant components, including the leucine-rich repeat pattern recognition receptor kinase FLAGELLIN SENSITIVE 2 (FLS2) and the salicylic acid (SA) defense pathway regulator NON-EXPRESSER OF PR GENES 1 (NPR1) (Rosebrock et al., 2007; Chen et al., 2017). HopN1 is a cysteine protease belonging to the YopT effector family, whose virulence mechanism is not completely understood (Cunnac et al., 2011; Dowen et al., 2009).

Both candidates display the highly conserved phenylalanine (F) residue at the second position of the core motif, where any residue other than F or tyrosine (Y) is heavily penalized by the scoring system. While many proteins with one conserved FFAT motif also possess a second or even third, less conserved FFAT-related motif (Murphy and Levine, 2016), no additional FFAT motifs with a relevant motif score (<5.5) were identified in these effector proteins (Table 1).

The Pst Effector Protein HopN1 Interacts with NtVAP12 in Yeast and in Planta

A direct interaction test in yeast between the candidate Pst T3Es and VAP12 from tobacco confirmed the ability of HopN1 to interact with VAP, while AvrPtoB showed no interaction with VAP12 (Figure 1A). Auto-activation of yeast reporter gene expression was excluded (Supplementary Figure S1). The VAP protein family consists of different isoforms (Wang et al., 2016); however; a test for interaction of AvrPtoB with other isoforms or VAPs from other plant species also showed no interaction (Supplementary Figure S2). Thus, further experiments were carried out with HopN1 only.

Figure 1.

Direct nInteraction Test in Yeast Between the Candidate Pst T3Es and VAP12 from Tobacco Revealed the Ability of HopN1 to Interact with NtVAP12. (A) Pst Effectors with Putative FFAT Motifs HopN1 and AvrPtoB Were Fused to the GAL4 Activation Domain (AD) and co-Expressed with NtVAP12 Fused to GAL4 Binding Domain (BD). –LT, Yeast Growth on medium Without Leu and Trp. –HTL, Yeast Growth on medium Lacking His, Leu, and Trp, Indicating Expression of the HIS3 Reporter Gene. LacZ, Activity of the LacZ Reporter Gene. (B) GFP Pull-Down Assay Confirmed NtVAP12-myc as in Planta HopN1-GFP, but not Free GFP, Interaction Partner. Co-Immunoprecipitation (co-IP) of the HopN1-NtVAP12 Interaction. NtVAP12-myc was co-Expressed with HopN1-GFP or Free GFP via Agrobacterium-Mediated Transient Expression in N. benthamiana Leaves. 24 h After Infiltration the Total Protein Content was Extracted (Input) and a Pull-Down Assay Using GFP-Trap^® was Performed (IP). Samples Were Detected by Western Blot Analysis Using Anti-GFP and Anti-myc Antibodies. – LT = Yeast Grown on Selective medium Lacking Leu and Trp. - LTH = Yeast Grown on Selective medium Lacking Leu Trp and His. LacZ, Activity of the LacZ Reporter Gene. Nt = N. tabacum.

To determine if HopN1 interacts with NtVAP12 in planta, a GFP pull-down assay was performed. To this end, we transiently expressed either HopN1-GFP or free GFP each combined with or without NtVAP12-myc in N. benthamiana. One day after infiltration with Agrobacteria, a pull-down of XopM-GFP using GFP-Trap beads was performed and the eluates were analyzed by immunoblotting with anti-GFP and anti-myc antibodies. As shown in Figure 1B, HopN1-GFP, but not free GFP, was able to pull down NtVAP12-myc, verifying the specific interaction of both proteins in planta.

Structure Function Analyses Suggest a Contribution of FFAT Motifs to the HopN1/VAP12 Interaction

Structural predictions and mutation experiments have demonstrated that most VAP interaction partners bind through conserved residues within the MSP domain, particularly those possessing a FFAT motif (Loewen and Levine, 2005; Slee and Levine, 2019). Previous data suggest that T46 and T47 of NtVAP12 are critical for binding protein partners via their FFAT motif (Brinkmann et al., 2024). In yeast, alanine substitution of neither T46 nor T47 abolished the interaction of NtVAP12 with HopN1 (Figure 2A and B), although the T47A substitution appeared to weaken NtVAP12 binding to HopN1 as indicated by reduced yeast growth on selective media (Figure 2B). A double substitution of T46 and T47 did not appear to affect interaction strength any further (Figure 2B).

Figure 2.

Structure-function Analysis of the HopN1-NtVAP12 Interaction. (A) HopN1 Fused to GAL4 Binding Domain (BD) was Co-expressed with Wildtype or VAP12 T to A Substitutions Fused to the GAL4 Activation Domain (AD). (B) HopN1 – BD was Co-expressed as in (A), the Optical Density at 600 nm (OD₆₀₀) was Adjusted to 1 and a Dilution Series was Plated on Selective Medium. (C) The Third Residue of the HopN1 FFAT Motif was Substituted. BD-HopN1_F57A was Co-expressed with AD-NtVAP12 and the pAD empty vector control. (D) The FFAT mutant BD-HopN1_F57A and AD-NtVAP12 mutants were Co-expressed. – LT = yeast Grown on Selective Medium Lacking Leu and Trp. - LTH = yeast grown on Selective Medium Lacking Leu Trp and His. LacZ, Activity of the lacZ Reporter Gene. Nt = N. tabacum. (E) Structural Modelling of XopN Interacting with NtVAP12 using AlphaFold and Highlighting the Predicted site of Interaction. HopN1 is Displayed in blue, VAP in green. The Position of the Two Residues (F56 of HopN1 and T47 of VAP) Implicated in the Interaction are Highlighted. Images were Prepared using EzMol.

These observations suggest that similar structural motifs within the NtVAP12 protein sequence as required for FFAT motif interaction contribute to binding to HopN1. However, additional factors also appear to mediate the interaction between the two proteins.

Alignment of the FFAT motif containing region of HopN1 proteins from different plant pathogenic Pseudomas species and pathovars, indicates a high degree of sequence conservation of critical amino acid residues (Supplementary Figure S3), further supporting the functional significance of the motif. To further explore the structural requirements for the XopM/NtVAP12 interaction, the third residue of the FFAT motif of HopN1 (F57) was substituted by alanine and a direct interaction test was carried out in yeast. Although this residue is adjacent to the critical F residue at position 2 of the FFAT motif (Murphy and Levine, 2016), the resulting construct allowed us to assess whether residues flanking the motif contribute to VAP binding. Especially since the F at position 3 in predicted HopN1 FFAT motifs from different Pseudomonads is also highly conserved (Supplementary Figure S3). The HopN1 variant HopN1_F57A still interacted with wildtype NtVAP12 (Figure 2C), showing thatthe interaction of these two proteins depends on additional structural motifs than just the suggested FFAT motif of HopN1. However, when tested against NtVAP_T47A or NtVAP12_T46A/T47A, HopN1_F57A did not show VAP12 binding in yeast (Figure 2D), suggesting structural motifs of both binding partners make a quantitative contribution to the interaction and a mutation of F57 to A can affect binding under certain conditions. A structural prediction of the interaction between HopN1 and VAP using AlphaFold (Fowler and Williamson, 2022) suggests that F56 at position 2 of HopN1 and T47 of VAP are arranged in close spatial proximity to each other (Figure 2E) which is in line with a predicted critical role of this residue for FFAT motif function. Previous data suggest that lysine residues within the MSP of VAP create a positively charged surface that attracts the acidic flanking regions of FFAT motifs (Loewen and Levine, 2005). According to the structural model, K45 and K122 of NtVAP12 could potential play a similar role; however, this requires further experimental validation (Figure 2E).

HopN1 Does not Inhibit flg22 – Induced ROS Production in N. Benthmiana

Previous studies demonstrated that the VAP-binding Xanthomonas T3E XopM interferes with PAMP-induced ROS production through an unknown mechanism (Brinkmann et al., 2024). The specific plant defense responses targeted by HopN1 remain unclear. To investigate whether HopN1 inhibits flg22-induced ROS production, HopN1 fused to GFP, along with appropriate controls, was transiently expressed in N. benthamiana leaves using Agrobacterium-mediated transformation. In contrast to XopM and the Pst T3E AvrPto, HopN1 did not significantly suppress the ROS burst triggered by flg22 treatment (Figure 3A). These findings suggest that, although HopN1 and XopM both contain putative FFAT motifs and bind to VAPs, they likely have different virulence functions in plant cells. Proper expression of all proteins was verified by immunoblotting (Figure 3B).

Figure 3.

HopN1 did not Significantly Suppress a ROS Burst Triggered by flg22 Treatment. (A) the flg22-Induced Production of Reactive Oxygen species (ROS) was Measured in Leaf Discs Transiently Expressing Effector Proteins Using a Luminol Based Assay. Leaf Discs Were Treated with flg22 and ROS Production Measured as Relative Light Units (RLU) Over an Hour. Leaves Infiltrated with MgCl₂ Were not Treated with flg22 as a Negative Control (- flg22). At Least ten Samples Were Measured (n = ≥ 10), Except for the Negative Control Where Only Four Samples Were Measured (n = 4). Individual Values are Plotted with Bars Representing the Mean and Error Bars Give the Standard Deviation (± SD). Statistical Significance was Calculated by a one-way ANOVA with a Dunnett´s Multiple Comparison Test. the Different Asterisks Indicating Significant Differences (* = p-Value 0.05, *** = p-Value 0.001). (B) Immunoblot Verification of the Expression of GFP-Tagged Proteins Used in ROS Assay. Leaf Tissue was Harvested 24 h After Infiltration and Visualized Using Western Blot Analysis with an Anti-GFP Antibody. Amidoblack Staining of RuBisCo is Used as Loading Control.

HopN1 Interacts with a RHO-GTPase in Planta and in Yeast

It has previously been hypothesized that VAP proteins are not the primary virulence targets of XopM in plants. Instead, the effector may use VAP binding to fine-tune its function by aiding localization to specific membrane compartments (Brinkmann et al., 2024). Thus, proteins other than VAPs might be required for the effector's virulence function. To investigate whether HopN1 could use a similar mechanism, we attempted to identify additional HopN1-interacting proteins in plants. A pull-down assay using transient expression of either GFP-tagged HopN1 or free GFP (negative control) was conducted in N. benthamiana leaves. The interactome of HopN1-GFP-tagged proteins was queried by co-immunoprecipitation (co-IP) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Among the proteins exclusively interacting with HopN1, but not with the GFP control, were several VAP12 isoforms, including NbVAP1-2, NbVAP1-3, NbVAP2-1, and NbVAP2-2, further supporting the notion that HopN1 interacts with VAPs in plants (Figure 4 and Supplementary Table 1). Additionally, a number of proteins functionally related to membrane trafficking processes were associated with HopN1. These include subunits of the coatomer complex, involved in membrane trafficking through the Golgi, different syntaxin isoforms likely involved in membrane fusion, and Sec61 complex subunits related to protein insertion into the ER membrane, among others (Figure 4 and Supplementary Table 1). Surprisingly, the HopN1 interactome also included a range of plastidic proteins, possibly explained by VAPs’ roles in organizing ER-chloroplast contact sites (Renna et al., 2024), which might target HopN1 to protein complexes bridging organellar membranes. Another large group of HopN1 interactors included ribosomal proteins, potentially hinting at an association of the effector with host protein translation (Supplementary Table 1).

Figure 4.

Identification of Additional HopN1 Interacting Proteins in planta. Heat Map of Proteins Identified as Potential HopN1 Binding Partners by Affinity MS. Interaction Partners Pulled down by GFP EV or HopN-GFP, respectively. Heat Map is Illustrated as Gradient Colormap from Largest Value Dark Blue (1 × 10⁷) to 0 (white). Each Cell is Labeled with its Grouped Abundance from Three Technical Replicates.

HopN1 is a member of the YopT family of T3Es, and YopT itself has been shown to function as a cysteine protease that cleaves Rho family GTPases (Shao and Dixon, 2003). The list of potential HopN1-associated proteins includes several RAB GTPases and a protein annotated as a rac-like GTP-binding protein, RHO1 (Figure 4). To investigate whether HopN1 interacts directly with RHO1, a direct interaction assay was conducted in yeast. As shown in Figure 5, HopN1 interacted with NtRHO1 in yeast, while XopM and NtVAP12 did not. The NtRHO1-HopN1 interaction was further confirmed in planta via GFP pull-down, as described above (Figure 5). These results suggest that while XopM and HopN1 both bind to VAP12, they also target different other host proteins, potentially contributing to their virulence functions. Furthermore, HopN1 appears to directly interact with NtRHO1 without requiring VAP12.

Figure 5.

HopN1 Interacts with NtRHO1 in Yeast and in Planta. (A) NtRHO1 was Identified as a Potential HopN1 Interaction Partner and Fused to GAL4 Activation Domain (AD). It was co-Expressed with the Effectors HopN1 and XopM as Well as NtVAP12 Fused to GAL4 Binding Domain (BD). – LT = Yeast Grown on Selective medium Lacking Leu and Trp. - LTH = Yeast Grown on Selective medium Lacking Leu Trp and His. LacZ, Activity of the LacZ Reporter Gene. (B) Co-Immunoprecipitation (co-IP) of NtRHO1-myc with XopM-GFP and HopN1-GFP in Planta. NtRHO1 Fused to 3xmyc tag was co-Expressed via Agrobacterium-Mediated Transient Expression in N. benthamiana Leaves with GFP-Tagged XopM and HopN1. 24 h After Infiltration Total Protein was Extracted (Input) and a Pull-Down Assay Using GFP-Trap^® was Performed (IP). Samples Were Detected by Western Blot Analysis Using Anti-GFP and Anti-myc Antibodies. Nt = N. tabacum.

Discussion

Members of the VAP protein family reside on the cytoplasmic face of the ER and are common components of membrane contact sites that mediate rapid signal transduction and the exchange of biomaterial between different membrane compartments. These proteins are the sole receptors for cytoplasmic proteins on this large organelle (Murphy and Levine, 2016). Many cytoplasmic proteins interacting with VAP contain FFAT motifs, which bind to VAP through specific amino acid residues in the MSP domain (Murphy and Levine, 2016). Increasing evidence suggests that bacterial pathogens exploit FFAT motifs to hijack VAP, enabling the manipulation of ER-related processes during host infection (Stanhope and Derre, 2018; Vormittag et al., 2023; Angara et al., 2024). Examples include the Chlamydia effector protein IncV, which uses two FFAT motifs to tether the pathogen inclusion to the host ER (Stanhope et al., 2017), and the Coxiella effector protein CbEPF1, which establishes inter-organelle contact sites between host lipid droplets and the ER (Angara et al., 2024).

The occurrence of FFAT motifs in effector proteins from extracellular bacterial pathogens, such as many plant pathogenic bacteria, has been less explored. Recently, Brinkmann et al. (2024) showed that the effector protein XopM translocated by the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv), binds to VAP via mimicry of two FFAT motifs. XopM is widely distributed across Xanthomonads, and the strong conservation of these motifs in sequenced Xanthomonas genomes highlights the functional importance of VAP binding (Brinkmann et al., 2024).

Given the presence of FFAT motif-bearing effector proteins in intracellular animal pathogens and the plant pathogen Xcv, we hypothesized that FFAT motif mimicry might be more widespread and relevant for diverse virulence activities. To test this hypothesis, we analyzed the type-III effector (T3E) repertoire of the well-characterized plant-pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pst) for the presence of FFAT motifs.

In this study, we identified that the Pst effector protein HopN1 contains a putative FFAT motif and interacts with VAP12 in plant cells. However, the role of VAP binding in HopN1 function remains unclear. Unlike XopM, which suppresses a flg22 induced ROS burst, transient HopN1 expression in leaves does not affect ROS production, suggesting functional divergence between these effectors. Additionally, HopN1 interacts with a plant RHO-GTPase, which is reminiscent of the activity of YopT in mammals, yet direct cleavage of RHO1 by HopN1 has not been demonstrated. The discovery of plastid-associated interactors in the HopN1 pull-down experiment suggests a potential functional link to chloroplast-related processes. This observation aligns with previous findings by Rodriguez-Herva et al. (2012), which indicate that HopN1 may be imported into chloroplasts. However, a role of VAP in in organizing ER-chloroplast contact sites has recently been described (Renna et al., 2024), which could also explain a co-purification of HopN1 with plastidic proteins mediated by VAP binding. Furthermore, a recent preprint (Gonzalez-Fuente et al., 2025) proposes that HopN1 contributes to P-body formation, which could reflect a broader role in host cell manipulation independent of chloroplasts.

Given that FFAT motif-containing effectors from diverse pathogens, including Xanthomonas and Chlamydia, target VAP proteins for distinct purposes, it remains an open question whether HopN1 utilizes a similar strategy for membrane localization or whether VAP binding serves a different function. Future studies should address whether HopN1-mediated targeting is functionally linked to its interaction with RHO1 and whether this interaction influences host signaling pathways.

Another Pst T3E, AvrPtoB, was also predicted to contain a FFAT motif with a significant cut-off value of 2.5. However, initial yeast two-hybrid assays did not confirm AvrPtoB binding to VAP. This could indicate a false-positive prediction, technical limitations of the assay, or the absence of the relevant VAP isoform. Despite this, VAP binding by AvrPtoB remains plausible, given that many of its target proteins are located at the plasma membrane (Göhre et al., 2008; Xiang et al., 2008; Gimenez-Ibanez et al., 2009; Wang et al., 2019b). To date, no direct evidence suggests modifications enhancing AvrPtoB's membrane binding beyond interactions with membrane-localized target proteins. Further experimentation is required to resolve this ambiguity.

For HopN1, we identified a single FFAT motif with a cut-off value of 2.0. Interaction assays in yeast and in planta confirmed its binding to VAP12. However, mutating T47 within VAP's MSP domain, a residue critical for FFAT binding, weakened but did not abolish the interaction in yeast, as evidenced by reduced growth on selective media. This is in contrast to previous findings obtained with the Xanthomonas T3E XopM, which lost binding to VAP upon a T47 to A substitution within the MSP (Brinkmann et al., 2024). Residual binding could either be explained by structural differences in XopM/VAP vs. HopN1/VAP binding or by technical limitations. The yeast two-hybrid system is very sensitive as it can detect very weak or only transient interactions that escape detection by other methods (Brückner et al., 2009). Thus, to clarify the effect of the substitution of individual T residues of VAP on HopN1 binding requires more experimentation under physiological conditions. Additionally, alanine substitution of the conserved F residue (F57) at position 3 within HopN1's FFAT motif did not significantly affect VAP binding. Only the simultaneous mutation of both protein partners disrupted their interaction, supporting the functional involvement of the FFAT motif in HopN1's VAP binding while suggesting additional contributing factors. In future studies, a more comprehensive mutagenesis of the motif and the use of complementary experimental methods to assess the effect on protein-protein interactions would help clarify individual residue contributions. This also needs to include the conserved F residue at position 2 of the FFAT motif.

Strikingly, all other secreted effector proteins known to bind VAP via FFAT motifs, such as IncV from Chlamydia, CbEPF1 from Coxiella, and XopM from Xanthomonas, contain two FFAT motifs (Stanhope et al., 2017; Angara et al., 2024; Brinkmann et al., 2024). In these cases, simultaneous mutation of both motifs was required to abolish interaction with VAP, suggesting that at least one functional FFAT motif is sufficient for binding. While our prediction did not identify a second FFAT motif in HopN1, our findings and comparisons with other effectors suggest the presence of an additional, unrecognized motif. Functional FFAT motifs deviating from the classical sequence may evade our detection methods. For example, phospho-FFAT motifs, which require phosphorylation to activate VAP binding, and FFNT motifs, which replace acidic residues with neutral ones, represent potential alternatives (Cabukusta et al., 2020; Di Mattia et al., 2020). Future research is needed to investigate whether HopN1 contains such FFAT motif variants.

Previously, it was demonstrated that XopM binds VAP via FFAT motifs and suppresses flg22-triggered ROS production, a likely virulence mechanism to inhibit host immunity (Brinkmann et al., 2024). Interestingly, XopM's VAP binding and ROS suppression are structurally independent and functionally separable. This led to the hypothesis that XopM uses VAP to reach specific membrane compartments, such as ER-PM contact sites, but exerts its virulence function on other target proteins (Brinkmann et al., 2024). Similarly, the finding that HopN1 binds VAP but does not affect flg22-induced ROS production suggests it might use VAP as a facilitator for interactions with target proteins at the membrane. This aligns with previous reports that HopN1 employs an unknown acylation-independent mechanism for PM localization (Dowen et al., 2009).

HopN1 belongs to the YopT family of cysteine protease effector proteins (Shao et al., 2003). The archetype, YopT from Yersinia, specifically cleaves Rho proteins, such as RhoA, near their isoprenylation sites, releasing them from membrane anchors and inactivating them. This disrupts the actin cytoskeleton and inhibits phagocytosis (Iriarte and Cornelis, 1998; Aepfelbacher et al., 2003). Our data suggest that, like YopT, HopN1 specifically interacts with the rac-like GTP-binding protein RHO1. RHO1 is critical for maintaining cell polarity during pollen tube growth and mediating exocytosis (Denninger, 2024). Thus, it is tempting to speculate that RHO1 might also play a role in exocytotic vesicle trafficking during immunity.

Future studies should address whether RHO1 is a proteolytic substrate for HopN1 and clarify how this activity may contribute to the effector's ability to utilize VAP-decorated membrane compartments. It is important to note that the limited data set presented here only allows for preliminary conclusions regarding HopN1 binding to VAP and its potential virulence function. While all interaction studies were performed in N. benthamiana, we acknowledge that future validation in natural hosts such as tomato or Arabidopsis would further strengthen the physiological relevance of these findings. A further understanding of HopN1 binding to VAP could significantly enhance our knowledge of virulence strategies employed by pathogens.

Methods

Plant Material and Growth Conditions

Nicotiana benthamiana and N. benthamiana roq1 (Gantner et al., 2019) plants were grown in soil in a growth chamber with daily watering, and subjected to a 16 h light : 8 h dark cycle (25°C : 20°C) at 240-300 µmol m^–2 s^–1 light and 75% relative humidity.

Plasmid Construction

To generate plasmids containing the corresponding gene of interest, the entire open-reading frame either with or without the stop codon was amplified from cDNA using the primers listet in Supplementary Table 2. The resulting fragments were inserted into the pENTR-D/TOPO vector according to the manufacturer's instructions (ThermoFisher, Waltham, MA, USA) and verified by sequencing. For Y2H experiments, fragments containing a stop codon were recombined into Gateway-compatible versions of the GAL4 DNA BD vector pGBT-9 and the activation domain (AD) vector pGAD424 (Clontech, Mountain View, CA, USA) using L/R-clonase (ThermoFisher). To generate translational fusions between the protein of interest and GFP, CDSs without stop codon were recombined as described above into the binary vector pK7FWG2 (Karimi et al., 2002). To tag proteins with myc, fragments were inserted into pGWB620 vector (Nakamura et al., 2010).

Transient Expression of Proteins in N. benthamiana

For infiltration of N. benthamiana leaves, A. tumefaciens (Agrobacterium) strain C58C1 was infiltrated into the abaxial air space of 4-week-old plants, using a 1 mL sterile needleless syringe. Agrobacteria were cultivated overnight in YEB medium (5 g/L Difco bovine extract, 1 g/L yeast extract, 1 g/L bacto peptone, 5 g/L saccharose, 2 mM MgSO₄) containing appropriate antibiotics at 28 °C with shaking. The cultures were harvested by centrifugation and the pellet was resuspended in infiltration buffer (10 mM MgCl₂, 10 mM MES pH 5.7, 200 mM acetosyringone; Sigma–Aldrich) and incubated for 2 h in the dark with shaking prior to infiltration. Cultures were infiltrated at an OD₆₀₀ = 0.5. Infiltrated plants were kept in the growth chamber under 16-h light:8-h dark cycle (25 °C:20 °C) at 240–300 mmol m^–2 s^–1 light and 75% relative humidity.

Measurement of ROS Production and POX Activity

ROS production upon flg22 treatment was monitored using the luminol-based quantification (Liang et al., 2013). To this end, leaf disks (0.5 cm²) were excised from N. benthamiana leaves transiently expressing the respective construct and incubated in a 48-well-plate with water overnight. After incubation, water was removed from each well and exchanged with 100 μl of reaction solution (34 mg/μl Luminol; 20 mg/μl HRP in ddH₂O) with or without 100 nM flg22 was added. The solution was added in the same intervals and order as the reader measured the samples. Measurement started immediately after adding the reaction solution and lasted for 90 min in Tecan (Infinite^® 200 PRO, Tecan). Measurement of plant peroxidase (POX) was carried out as described (Mott et al., 2018). To this ent, leaf discs with a diameter of 0.5 cm were left to recover in 100 μl of ½ MS for at least 1 h in a clear 96 well plate. Four wells were left without a leaf disc for a blank measurement. The media was carefully removed from the wells and 60 μl of ½ MS containing 100 nM flg22 was added. The plate was sealed using parafilm and left for 20 h at 22 °C with gentle shaking (30 rpm, New Brunswick Innova^® 42). After a brief centrifugation (1 min, 15 rcf), 50 μl of solution from each well was transferred to a new 96 well plate. 50 μl assay substrate solution (1 mg/ml 5-aminosalicylic acid solution (pH 6.0); 0.01% hydrogen peroxide) was added to each well. The reaction was stopped after 2 min 30 s by the addition of 20 μl of 2 N NaOH. The assay was quantified by measuring the optical density at 600 nm (OD₆₀₀).

Yeast Two-Hybrid Assays

Direct interaction of two proteins was investigated by co-transformation of the respective plasmids in the yeast strain Y190 using the LiAc method (Gietz et al., 1995), followed by selection of transformants on medium lacking Leu and Trp at 30°C for 3 days and subsequent transfer to medium lacking Leu, Trp, and His for growth selection and lacZ activity testing of interacting clones. All manipulations of yeast cells were performed according to the yeast protocols handbook (Clontech).

In Planta GFP Pull-Down Assay

Approximately 1 g of frozen N. benthamiana leaf material was ground in liquid nitrogen and thawed in 4 ml extraction buffer (100 mM Tris-HCl, pH 8.0; 100 mM NaCl; 5 mM EDTA; 5 mM EGTA; 20 mM DTT; 10 mM NaF; 10 mM Na₃VO₄; 2 µl/ml plant protease inhibitor cocktail [Sigma P9599]; 0.5% [v/v] Triton X-100). After centrifugation, the supernatant was incubated with 50 μL of GFP-Trap^® coupled to magnetic beads (50% [v/v] slurry, Chromotek) and incubated for 60 min at 4°C. After five washing steps (100 mM Tris-HCl, pH 8.0; 100 mM NaCl; 0.5 mM EDTA; 1 mM DTT) the purified GFP-tagged protein was either used for immunoblot or for LC-MS/MS analysis.

Immunoblotting

Transiently expressed proteins were extracted from N. benthamiana leaf material using protein extraction buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM NaF, 10 mM DTT, 0.1% (v/v) Triton X-100 and 1% (v/v) protease inhibitor cocktail; Sigma–Aldrich). Protein extracts were boiled at 95°C for 10 min in 1× sodium-dodecyl sulfate (SDS) loading buffer (4×; 200 mM Tris–HCl pH 6.8, 0.4 M DTT, 40% (v/v) glycerol, 6 mM bromophenol blue and 8% (w/v) SDS) and then subjected to SDS-polyacrylamide gel electrophoresis. Separated proteins were transferred onto nitrocellulose membrane (Amersham biosciences, Amersham, UK), blocked with 5% (w/v) skimmed milk in TBS-T and incubated with respective antibodies in 1% (w/v) skimmed milk in TBS-T. Immunoblotting was carried out with anti-myc antibody (1:2.500, Abcam), followed by Goat Anti-Rabbit IgA alpha chain (HRP) (1:5.000) secondary antibody (Abcam). GFP was detected using a horseradish peroxidase-conjugated anti-GFP antibody (1:1.000; Santa Cruz Biotechnology Inc., cat. no. sc-9996 HRP). Proteins were detected using the Clarity Western ECL substrate (BioRad, Hercules, CA, USA). Signals were visualized using chemiluminescence (Thermo Fisher Scientific) with a ChemiDoc Imaging system (Biorad).

Large-Scale GFP Pull-Down and LC-MS/MS Analysis

Pull-down followed by LC-MS/MS analysis was carried out as previously described (Bortlik et al., 2023), proceeding from GFP-tagged protein purified as described above. To this end, on bead trypsin digest of GFP-tagged proteins were treated with 100 µl of 0.1% RapiGest SF (Waters, Eschborn) followed by reduction and alkylation. Trypsin (1:50 (w/w) trypsin/protein) was added and incubated at 37 °C overnight. Subsequently, desalting of peptides was carried out according to Witzel et al. (2019). Digested proteins were analysed using the Thermo Fisher Q Exactive high field mass spectrometer by reverse-phase HPLC-ESI-MS/MS using the Dionex UltiMate 3000 RSLC nano System coupled to the Q Exactive High Field (HF) Hybrid Quadrupole Orbitrap MS (Thermo Fisher Scientific) and a Nano- electrospray Flex ion source (Thermo Fisher Scientific) as described by Bortlik et al. (2023) with slight modifications. In short, samples were separated on a 25 cm Acclaim PepMap 100 C18 column (0.075 × 150 mM, 3 µm, Thermo Scientific at a flow rate of 300 nl min⁻¹. The mobile phases consisted of 0.1% formic acid (solvent A) and 0.1% formic acid in 80% ACN (solvent B). Peptides were separated chromatographically by a linear 100 min gradient from 5% to 44% solvent B, with the column temperature set at 40°C. Spray voltage was set at 1.80 kV, capillary temperature at 275°C, and S-lens RF level at 60. Mass spectra were acquired in positive ion and data-dependent mode. Full-scan spectra (375 to 1,500 m/z) were acquired at 140,000 resolution. Automated gain control (AGC) target was 3 × 10⁶ with a max. injection time of 65 ms. Triplicates of each sample were measured. Proteome discoverer software (PD2.4) was used to analyse and align the LC-MS raw data files, with its built-in MS Amanda, MS Mascot and Sequest HT search engine (Thermo Scientific) (Witzel et al., 2019). The MS data was searched against common contaminants, separated FASTA files of the tagged bait proteins as well as a N. benthamiana database based on the Niben1.0.1 draft genome (Kourelis et al., 2019). The target false discovery rate (FDR) for proteins and peptides with medium confidence (relaxed) was set to 0.05 and for proteins and peptides with high confidence FDR was set to 0.01. Further parameters for database search were used: 0.02 Da fragment ion mass tolerance, 10 ppm parent peptide ion tolerance, full tryptic digestion with maximum two missed cleavage. Carbamidomethylation of cysteine as fixed modification and oxidation of methionine as variable modification was set. The result lists were filtered for high confident peptides and their signals were mapped across all LC-MS experiments. Only unique peptides were selected for quantification and abundances. Grouped abundances were calculated by the mean of three technical replicates (PD2.4).

Supplemental Material

sj-docx-1-ctc-10.1177_25152564251376890 - Supplemental material for Pseudomonas syringae HopN1 Binds Plant VAP12 and a Rho-GTPase, Suggesting a Role in Membrane-Associated Processes

Supplemental material, sj-docx-1-ctc-10.1177_25152564251376890 for Pseudomonas syringae HopN1 Binds Plant VAP12 and a Rho-GTPase, Suggesting a Role in Membrane-Associated Processes by Charlotte Brinkmann, Jennifer Bortlik and Frederik Börnke in Contact

Supplemental Material

sj-docx-2-ctc-10.1177_25152564251376890 - Supplemental material for Pseudomonas syringae HopN1 Binds Plant VAP12 and a Rho-GTPase, Suggesting a Role in Membrane-Associated Processes

Supplemental material, sj-docx-2-ctc-10.1177_25152564251376890 for Pseudomonas syringae HopN1 Binds Plant VAP12 and a Rho-GTPase, Suggesting a Role in Membrane-Associated Processes by Charlotte Brinkmann, Jennifer Bortlik and Frederik Börnke in Contact