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Pseudomonas syringae effector HopZ3 suppresses the bacterial AvrPto1–tomato PTO immune complex via acetylation

['Joanna Jeleńska', 'Department Of Molecular Genetics', 'Cell Biology', 'The University Of Chicago', 'Chicago', 'Illinois', 'United States Of America', 'Jiyoung Lee', 'Andrew J. Manning', 'Donald J. Wolfgeher']

Date: 2021-11

The plant pathogen Pseudomonas syringae secretes multiple effectors that modulate plant defenses. Some effectors trigger defenses due to specific recognition by plant immune complexes, whereas others can suppress the resulting immune responses. The HopZ3 effector of P. syringae pv. syringae B728a (PsyB728a) is an acetyltransferase that modifies not only components of plant immune complexes, but also the Psy effectors that activate these complexes. In Arabidopsis, HopZ3 acetylates the host RPM1 complex and the Psy effectors AvrRpm1 and AvrB3. This study focuses on the role of HopZ3 during tomato infection. In Psy-resistant tomato, the main immune complex includes PRF and PTO, a RIPK-family kinase that recognizes the AvrPto effector. HopZ3 acts as a virulence factor on tomato by suppressing AvrPto1 Psy -triggered immunity. HopZ3 acetylates AvrPto1 Psy and the host proteins PTO, SlRIPK and SlRIN4s. Biochemical reconstruction and site-directed mutagenesis experiments suggest that acetylation acts in multiple ways to suppress immune signaling in tomato. First, acetylation disrupts the critical AvrPto1 Psy -PTO interaction needed to initiate the immune response. Unmodified residues at the binding interface of both proteins and at other residues needed for binding are acetylated. Second, acetylation occurs at residues important for AvrPto1 Psy function but not for binding to PTO. Finally, acetylation reduces specific phosphorylations needed for promoting the immune-inducing activity of HopZ3’s targets such as AvrPto1 Psy and PTO. In some cases, acetylation competes with phosphorylation. HopZ3-mediated acetylation suppresses the kinase activity of SlRIPK and the phosphorylation of its SlRIN4 substrate previously implicated in PTO-signaling. Thus, HopZ3 disrupts the functions of multiple immune components and the effectors that trigger them, leading to increased susceptibility to infection. Finally, mass spectrometry used to map specific acetylated residues confirmed HopZ3’s unusual capacity to modify histidine in addition to serine, threonine and lysine residues.

By secreting virulence proteins (effectors) into their hosts, pathogenic bacteria hijack host cellular processes to promote bacterial colonization and disease development. For the plant pathogen Pseudomonas syringae, the coordinated action of effectors often mediates modifications of host defense proteins to inhibit their function. However, plants have evolved the ability to induce innate immunity upon recognition of effector-induced modifications of host proteins. How do pathogens circumvent the immune-inducing activity of certain effectors? They deploy more effectors to suppress these defenses. HopZ3, an acetyltransferase from P. syringae, is unique among plant pathogen effectors characterized so far in its ability to modify not only multiple components of the effector-triggered immune pathway, but also the triggering effector itself. Through the direct acetylation of residues involved in the interaction and activation of the bacterial effector AvrPto1 Psy and tomato kinase PTO, HopZ3 modifications disrupt their binding and block phosphorylations necessary for immune induction. Additionally, HopZ3 acetylates other possible components in the PTO signaling pathway, including activation sites in SlRIPK kinase, leading to suppression of its activity and reduced phosphorylation of SlRIN4s. Our study emphasizes the importance of HopZ3-dependent acetylation of immune complexes and bacterial effectors across plant species in the suppression of effector-induced immunity.

Funding: This work was supported by National Science Foundation ( www.nsf.gov ) grants NSF2010: Functional Genomics of NBS-LRR Mediated Resistance to RWM and JTG (IOS 0822393), Rol:FELS EAGER: Emergent functions of secreted microbial effectors to JTG (NSF MCB 1837824) and NSF: Post-translational Modifications as Modulators of Crop Plant Defense Signaling: a Systems Approach to JTG and SJK (IOS 1238201). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: Yeast two-hybrid screen data are available from https://charge.ucdavis.edu/charge_db/interaction/Y2H/Y2H_interaction.php The MS data have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) with the dataset identifier PXD022953. All other relevant data are within the manuscript and its Supporting Information files.

Copyright: © 2021 Jeleńska et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

We previously found that deletion of HopZ3 decreased the growth of Psy on tomato with functional PTO [ 7 ], raising the possibility that HopZ3 normally suppresses effector-triggered immunity in tomato. In this study, we investigated this hypothesis. Through genetics and biochemical reconstruction, our data point to a mechanism that involves immune suppression via acetylation of AvrPto1 Psy , PTO and other immunity factors.

PsyB728a has AvrPto and AvrPtoB homologues (AvrPto1 Psy and AvrPtoB Psy /HopAB1, hereafter called AvrPtoB Psy ) that induce resistance in tomato. Transfer of a plasmid carrying AvrPto1 Psy to a P. syringae pv. syringae strain that lacks AvrPto and AvrPtoB (Psy61) confers PTO-dependent recognition, whereas plasmid-borne AvrPtoB Psy confers some PTO-independent recognition that involves other members of PTO family [ 26 ]. AvrPto1 Psy is 88% identical at the amino acid level with AvrPto Pto while AvrPtoB alleles share 52% identity. Both AvrPto1 Psy and AvrPtoB Psy can interact with PTO in a yeast two-hybrid assay [ 26 ]. Consistent with these findings, PRF is a major factor that restricts the growth of PsyB728a on tomato [ 10 , 26 ].

Another potential player in PTO/PRF-conferred immunity is SlRIN4-1, one of three RIN4-related proteins in tomato. Infection with P. syringae pv. tomato strain T1 engineered to express AvrPto causes reduction of SlRIN4 protein levels. Downregulation of SlRIN4-1 using RNAi decreases the growth of strain T1 carrying AvrPto but not the growth of strain T1 alone [ 24 ]. Thus, downregulation of SlRIN4-1 seems to specifically enhance PTO-dependent resistance. Moreover, N. benthamina homologue of RIN4 was found in a search for proteins proximal to AvrPto, suggesting their interaction [ 25 ].

Interestingly, in a large screen for interactions between effectors and plant immune signaling proteins ([ 9 ], https://charge.ucdavis.edu/charge_db/interaction/Y2H/Y2H_interaction.php ), we found that HopZ3 interacted with the resistance-inducing effector AvrPto1 Psy and its tomato targets, PTO-like proteins. Moreover, HopZ3 suppressed AvrPto1 Psy -induced cell death in Nicotiana benthamiana [ 8 ]. That suggested that HopZ3 may affect tomato immunity. The interaction between PsyB728a and tomato has not been well characterized; however, resistance to P. syringae pv. tomato has been studied in great detail. Resistant tomato lacks RPM1 but contains PSEUDOMONAS RESISTANCE AND FENTHION SENSITIVITY (PRF), an NB-LRR protein that forms complexes with the kinases PSEUDOMONAS SYRINGAE PV TOMATO RESISTANCE (PTO) and FENTHION SENSITIVITY (FEN) and recognizes effectors AvrPto and AvrPtoB from P. syringae pv. tomato and other pathovars [ 10 ]. PTO, FEN and related cytoplasmic protein kinases in the same family as RIPK show natural variation that affects their functional specificity in promoting immunity in different tomato accessions [ 11 ]. PTO and FEN interact differently with AvrPto and AvrPtoB. Both effectors can bind to PTO and elicit PRF-dependent immune signaling [ 12 – 15 ]. In contrast, FEN can bind and be activated by AvrPto if the key residue N202 (that corresponds to T204 in PTO) is substituted with threonine [ 16 ]. Truncated versions of AvrPtoB (e.g., AvrPtoB 1-387 ) bind to FEN and stimulate immunity; however, due to the C-terminal E3 ubiquitin ligase domain, full-length AvrPtoB causes proteasome-dependent FEN degradation and does not trigger FEN/PRF immunity [ 14 ]. Structure-based biochemical analysis has indicated that AvrPto-PTO binding is a key step that leads to activation of PRF signaling [ 17 ]. The kinase activity of PTO is important for disease resistance triggered by AvrPto [ 18 – 22 ]. PTO acts as a dimer or higher order complex together with PRF [ 17 , 22 , 23 ]. Although AvrPto can inhibit PTO and other kinases [ 17 ], transphosphorylation between unbound PTO molecules and those bound to AvrPto is thought to be needed for downstream signaling [ 17 , 22 , 23 ].

Pseudomonas syringae pv. syringae B728a (PsyB728a) is a bean pathogen that can also grow to moderate levels on Arabidopsis and tomato without causing overt disease symptoms [ 7 , 8 ]. In Arabidopsis, PsyB728a with a deletion of the type III secreted effector HopZ3 (PsyΔHopZ3) causes the activation of RPM1 signaling. This occurs via two interacting effectors with homology to AvrB and AvrRpm1: AvrB3 Psy and AvrRpm1 Psy . In the context of PsyΔHopZ3 infection, both effectors are needed to activate signaling [ 9 ]. HopZ3 belongs to the YopJ acetyltransferase family that comprises several effectors from animal and plant pathogens. The acetyltransferase activity of HopZ3 is necessary for suppression of RPM1 activation in Arabidopsis and several components of the RPM1 immune-effector complex are substrates of HopZ3 [ 9 ]. HopZ3 acetylates the activation loop and active site residues of RIPK, which inhibits its ability to phosphorylate RIN4. Additionally, acetylation of RIN4 prevents its phosphorylation by RIPK. HopZ3 also acetylates residues in AvrB3 that are predicted to disrupt hydrogen bonds at the key interaction sites with RIN4. Thus, HopZ3 suppresses plant immunity through modification of both Arabidopsis and bacterial proteins that act in the same complex.

The plant pathogen Pseudomonas syringae uses type III-secreted proteins to promote its growth during infection of plants. These effector proteins are injected into plant cells, where they often interfere with plant defense signaling either through binding, post-translational modifications (PTMs) and/or destabilization of host factors [ 1 , 2 ]. A major mechanism to suppress P. syringae growth is signaling mediated by plant immune receptors that monitor specific perturbations caused by effectors. A well-studied example of such a receptor is Arabidopsis RESISTANCE TO P. SYRINGAE MACULICOLA 1 (RPM1), a member of the NUCLEOTIDE BINDING-LEUCINE RICH REPEAT (NB-LRR) protein family. Recognition and signaling occur when RPM1 senses a specific phosphorylation (mainly p-T166) of RPM1-INTERACTING PROTEIN 4 (RIN4), an intrinsically disordered hub protein [ 3 ]. Two unrelated effectors, AvrB or AvrRpm1, from different P. syringae strains can strongly trigger RPM1 signaling and are thus considered avirulence factors. These effectors cause the cytoplasmic RIN4-INDUCED PROTEIN KINASE (RIPK and probably additional kinases) to phosphorylate RIN4. RIN4 is also involved in promoting defense signaling in response to conserved microbial patterns. Immune responses are induced by phosphorylations of specific RIN4 residues that are triggered by recognition of effectors or microbial patterns [ 3 – 6 ].

Results

HopZ3 suppresses PTO/PRF defenses triggered by AvrPto1 Psy PsyB728a has a strong epiphytic growth phase modulated by effectors [7]. P. syringae effectors, including AvrPto Pto , are predominantly expressed by bacteria on a leaf surface and delivered to epidermal cells during infection, where they can induce and suppress defenses [7,27]. Deletion of HopZ3 reduced epiphytic growth of PsyB728a in a resistant tomato PtoR (76R), which has a functional PTO [7]. In a transient expression assay in N. benthamiana, HopZ3 suppressed AvrPto1 Psy -induced cell death, a proxy for immune activation [7,8]. Therefore, it seemed plausible that the effect of HopZ3 on the growth of PsyB728a in tomato is dependent on PTO and PRF proteins needed for recognition and resistance triggered by AvrPto1 Psy . Bacterial growth of PsyB728a and PsyΔHopZ3 was indistinguishable in pto11 and prf3 plants lacking functional PTO and PRF, respectively, indicating that the PTO/PRF pathway is needed for the effect of HopZ3 (Fig 1A). As expected, deletion of HopZ3 similarly restricted total (epiphytic + endophytic, Fig 1A and 1C) and epiphytic (Fig 1B and 1D) populations of PsyB728a in PtoR tomato and we tested these populations interchangeably in further experiments. The growth defect of PsyΔHopZ3 was restored only when a plasmid carrying wild-type HopZ3 but not a catalytically inactive version (HopZ3_C300A) was introduced (Fig 1B). HopZ3 and HopZ3_C300A proteins in these strains are produced at the same level in PsyΔHopZ3 [7]. These results suggest that enzymatically active HopZ3 suppresses PTO-mediated plant immunity in tomato. PPT PowerPoint slide

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TIFF original image Download: Fig 1. HopZ3 promotes the growth of PsyB728a on PTO-containing tomato plants (PtoR) and suppresses defenses triggered by AvrPto1 Psy . Plants were spray inoculated with PsyB728a-derived strains at an OD 600 = 0.01 and total (epiphytic + endophytic) or epiphytic bacterial populations were quantified in 8 leaf discs or leaf disc washes, respectively. (A) Total bacterial populations of PsyB728a and PsyΔHopZ3 were different in PtoR but were not statistically different in pto11 and prf-3 plants after 4 days (n = 8, t-test *P<0.05). (B) HopZ3 (Z3), but not the catalytic mutant (Z3_C300A) complements the low growth phenotype of PsyΔHopZ3 in PtoR tomato. (C-D) Deletion of AvrPto1 Psy (ΔA1) from PsyΔHopZ3 (ΔZ3) restores total (C) and epiphytic (D) bacterial growth to WT (PsyB728a or PsyB728a/V) levels in PtoR tomato. (E) Deletion of AvrPto1 Psy from WT PsyB728a does not affect bacterial growth in PtoR tomato. (F) AvrPto1 Psy does not confer resistance in pto11 plants, regardless of the presence of HopZ3. For (B,D,E) epiphytic bacteria were collected by leaf disc washes five (B) or four (D-E) days after inoculation. Different letters indicate significant differences in growth as assessed by ANOVA with Tukey’s test (P<0.0002) or Fisher’s test P<0.0001, n = 8). For C and F, total bacteria were quantified 3 days after spray inoculation. Different letters indicate significant differences in growth (n = 8, ANOVA with Tukey’s test, P<0.05). All experiments were repeated at least twice with similar results. Bars indicate standard errors. https://doi.org/10.1371/journal.ppat.1010017.g001 A possible explanation for why PTO is needed to observe HopZ3’s effect on promoting PsyB728a growth is that HopZ3 suppresses AvrPto1 Psy recognition. If this is true, the effect of deleting HopZ3 should be reversed when AvrPto1 Psy is also deleted. To test this hypothesis, we assessed the growth of a double mutant of PsyB728a that lacks both HopZ3 and AvrPto1 Psy in PtoR tomato. Both total (Fig 1C) and epiphytic (Fig 1D) populations of PsyΔHopZ3ΔAvrPto1 Psy were increased relative to PsyΔHopZ3 to levels similar to WT PsyB728a. The effect of deleting AvrPto1 Psy was complemented when the double mutant was transformed with a plasmid carrying AvrPto1 Psy (Fig 1D). Deletion of AvrPto1 Psy in PsyB728a with intact HopZ3 had no effect on the growth of PsyB728a in PtoR tomato (Fig 1E), as previously reported [28]. AvrPto1 Psy did not confer resistance in pto11 plants due to lack of functional PTO, regardless of the presence of HopZ3 (Fig 1F). Altogether, our genetic analysis indicates that HopZ3 suppresses AvrPto1 Psy -triggered immunity during PsyB728a infections.

HopZ3 acetylates a subset of interacting proteins Since HopZ3 has acetyltransferase activity [9], we tested whether several interacting proteins were its substrates in vitro, in reactions with 14C-acetyl-CoA and the cofactor inositol hexakisphosphate (IP6). Recombinant HopZ3, but not the catalytically inactive variant HopZ3_C300A, acetylated AvrPto1 Psy and its target PTO, SlRIPK, SlRIN4-1, SlRIN4-2 and SlRIN4-3 (Fig 3A and 3B). There was no detectable acetylation of FEN by HopZ3 (Fig 3B). Although AvrPtoB Psy was capable of binding to HopZ3, it was not a good substrate for acetylation (Fig 3C). Despite diversity of substrates, HopZ3 activity is specific, as the enzyme does not acetylate interacting proteins MPK4 [9], FEN and AvrPtoB Psy or non-interacting HopI Psy [9]. PPT PowerPoint slide

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TIFF original image Download: Fig 3. HopZ3 acetylates SlRIN4-1,-2, -3, AvrPto1 Psy , PTO, SlRIPK but not FEN or AvrPtoB Psy . Purified recombinant His-tagged SlRIN4-1, -2, -3, AvrPto1 Psy , AvrPtoB Psy and GST-tagged PTO, FEN and SlRIPK proteins were incubated with His-tagged HopZ3 or HopZ3_C300A mutant (C/A) in the presence of IP 6 and 14C-acetyl-CoA for 2 h at 30°C. Samples were separated by SDS-PAGE and subjected to autoradiography for 14 days. (A) SlRIN4-1, -2, -3 and AvrPto1 Psy were acetylated by HopZ3. (B) PTO and SlRIPK were acetylated by HopZ3; however, FEN acetylation was not detected. (C) AvrPtoB Psy was not significantly acetylated by HopZ3. https://doi.org/10.1371/journal.ppat.1010017.g003

HopZ3 acetylates AvrPto1 Psy residues essential for interaction with PTO and decreases phosphorylation of residues involved in defense activation To gain further insight into molecular mechanisms of immune suppression by HopZ3, we analyzed post-translational modifications of AvrPto1 Psy produced in E. coli and N. benthamiana by LC-MS/MS. By comparing acetylation sites found in E. coli-produced AvrPto1 Psy after in vitro acetylation reactions with 13C-acetyl-CoA, IP6 and HopZ3 or HopZ3_C300A, we found that H125 and H130 were specifically acetylated by HopZ3 (S1 Table). These histidine residues were also specifically acetylated in planta, when AvrPto1 Psy and HopZ3 were co-expressed in N. benthamiana. Several other AvrPto1 Psy residues were acetylated in vitro and in planta to higher levels in the presence of HopZ3 compared to HopZ3_C300A (S1 Table and Figs 4 and S4). T91 and S94 in the AvrPto1 Psy GINP Ω loop that is essential for interaction with PTO [15,17,29,30] were consistently found to be the most highly acetylated in several experiments (S1 Table). S46, which is also important for interaction with PTO [15,29,30] and the virulence function of AvrPto Pto [31], was also acetylated by HopZ3. This residue is not in the binding interface, but likely stabilizes the protein fold [30]. PPT PowerPoint slide

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TIFF original image Download: Fig 4. HopZ3 acetylates multiple sites in AvrPto1 Psy and PTO important for their interaction and signaling. AvrPto1 Psy and PTO co-expressed with HopZ3 or HopZ3_C300A in N. benthamiana were analyzed using mass spectrometry for post translational modifications. (A–B) Models of the AvrPto1 Psy and PTO showing the modifications identified in the in planta experiment that are important for immune signaling. Models were developed using the iTASSER modeling server and algorithm. Major acetylation sites dependent on HopZ3 are shown in red, important phosphorylation sites in blue, sites either acetylated or phosphorylated in purple, known sites of interaction between AvrPto Pto and PTO in yellow, acetylated interaction sites in orange and G2 myristoylation site in green. See also S1 and S2 Tables and S4 and S5 Figs. HopZ3 acetylates sites essential for interaction (orange) and decreases phosphorylation of residue(s) involved in signaling (blue box). (C) Model of HopZ3 acetylation sites in the crystal structure of PTO:AvrPto Pto contact site [17]. AvrPto is shown in green with residues acetylated by HopZ3 in red, and PTO is shown in orange with sites acetylated by HopZ3 in blue. Modifications on either protein are in the known interaction area of the two proteins. https://doi.org/10.1371/journal.ppat.1010017.g004 Many residues in AvrPto1 Psy produced in E. coli or in N. benthamiana were phosphorylated (S1 Table and Figs 4 and S4). Interestingly, S136 was very highly phosphorylated in planta (regardless of the presence of HopZ3), but it was not phosphorylated in the recombinant protein. This plant modification of AvrPto has not been reported previously; its functional significance is unknown and was not further explored. Since HopZ3 also targets serines and threonines, the same residues may also be phosphorylated. S147 and S149 of AvrPto1 Psy were phosphorylated in vitro and in planta, and HopZ3 acetylated a fraction of these residues as well. Importantly, in N. benthamiana expressing HopZ3, phosphorylation of S147 and/or S149 was significantly reduced (S1 Table). These residues were previously shown to be phosphorylated and contribute to the avirulence activity of AvrPto Pto during interactions with resistant tomato [32] and Nicotiana sp. [33], as well as to virulence during susceptible tomato infection [32]. In our LC-MS/MS analysis, we also directly detected myristoylation of G2, a modification that enables membrane localization of AvrPto [32] (S1 Table and Figs 4 and S4). Acetylation of residues in the AvrPto1 Psy Ω loop that interacts with PTO and decreased phosphorylation of residue(s) involved in signaling likely contribute to the mechanism by which HopZ3 reduces the immune response to AvrPto1 Psy .

Residues acetylated by HopZ3 are important for AvrPto1 Psy avirulence during tomato infection Many residues acetylated by HopZ3 are important for the ability of AvrPto1 Psy to trigger a defense response in resistant tomato. For example, S94 and S147/S149 in AvrPto Pto were shown to contribute to triggering PTO-mediated disease resistance and were extensively studied, as discussed above. Although T91 in the GINP Ω loop was not found to affect interaction with PTO in any mutagenesis studies, a T91A variant that we constructed lost the ability to suppress the growth of PsyB728a ΔHopZ3 in PtoR tomato (Fig 5A) and was defective in the induction of cell death in N. benthamiana (S6 Fig). H125/H130 residues are on the opposite side of AvrPto1 Psy molecule from the Ω loop (Fig 4) and their substitutions did not disrupt in vitro binding to PTO (Fig 5B) or cell death induction in N. benthamiana (S6 Fig). Nevertheless, H125A/H130A substitutions reduced the ability of AvrPto1 Psy to suppress bacterial growth in resistant tomato (Fig 5A). Importantly, AvrPto1 Psy variants were expressed in PsyB728a to similar levels as wild-type AvrPto1 Psy (Fig 5C). Therefore, the residues acetylated by HopZ3 are important for the ability of AvrPto1 Psy to trigger a defense response in resistant tomato. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Effect of mutations of AvrPto1 Psy acetylation sites on PsyB728a growth in tomato. (A) AvrPto1 Psy _T91A and H125A/H130A mutants did not reduce PsyB728a growth in PTO-containing tomato in the absence of HopZ3. Plants were spray-inoculated with indicated strains at an OD 600 = 0.01. Epiphytic bacterial populations were quantified in leaf disc washes from eight different plants per strain four days after inoculation. Different letters indicate significant differences in growth as assessed by ANOVA with Tukey’s test (P<0.05). Similar results were found in at least two other experiments (AvrPto1 Psy _H125A/H130A did not reduce the growth of PsyB728a ΔHopZ3 in three out of five experiments). Bars indicate standard errors. V, vector control; A1, AvrPto1 Psy ; Z3, HopZ3; T91, AvrPto1 Psy _T91A; H125/H130 and HH, AvrPto1 Psy _H125A/H130A. (B) H125A/H130A mutation of AvrPto1 Psy did not affect its binding to PTO. AvrPto1 Psy -GST, AvrPto1 Psy _H125A/H130A-GST and PTO-MBP were expressed in E. coli. Purified soluble AvrPto1 Psy or H125A/H130A mutant was pulled down with immobilized PTO-MBP or, alternatively, soluble PTO was pulled down with immobilized AvrPto1 Psy -GST or AvrPto1 Psy _H125A/H130A-GST. Band intensities were quantified from seven experiments. (T-test, P = 0.1). (C) AvrPto1 mutant variants were expressed to similar levels as AvrPto1 Psy in ΔAvrPto1 and ΔhopZ3ΔAvrPto1 PsyB728a grown in type III secretion-inducing conditions. https://doi.org/10.1371/journal.ppat.1010017.g005

HopZ3 acetylates key sites in the activation loop and other residues important for the immune function of PTO and reduces their phosphorylation We used an LC-MS/MS analysis of PTO to gain insight into what specific effect acetylation might have. By comparing acetylation sites found in the presence of HopZ3 and HopZ3_C300A after in vitro acetylation reactions with 13C-acetyl-CoA, we identified T204 in the P+1 activation loop/region of PTO as a specific HopZ3-mediated acetylation site (S2 Table and S5 Fig). T204 is a cognate of T257 in Arabidopsis RIPK, another member of this kinase family that we found to be acetylated by HopZ3 [9]. T204 and T199 were the major acetylation sites in planta in PTO immunoprecipitated from N. benthamiana that also expressed functional HopZ3 (S2 Table and Figs 4 and S5). Both of these residues in the P+1 loop are important for interaction with AvrPto [16,17,20,22]. In addition, the structurally proximal residue K123 was acetylated in PTO co-expressed with HopZ3 in planta. Moreover, phosphorylation of S198/T199 (and T190) was reduced in the presence of HopZ3 compared to HopZ3_C300A (S2 Table and Figs 4 and S5). Since phosphorylation of S198 and T199 is necessary for immune signaling [17,22,23], this may be a part of the mechanism by which HopZ3 reduces the plant defense response to AvrPto1 Psy .

Acetylation of AvrPto1 Psy and PTO affect their binding A key step in the activation of AvrPto Pto -triggered immunity requires its binding to PTO [19]. We hypothesized that modification by HopZ3 may affect the AvrPto1 Psy –PTO interaction because HopZ3 targets several residues in the binding interface (Fig 4 and S1 and S2 Tables). Therefore, we assayed the impact of AvrPto1 Psy or PTO acetylation on their interaction by performing in vitro acetylation reactions with HopZ3 followed by binding experiments. We found that binding was reduced when either AvrPto1 Psy or PTO was acetylated (Fig 6). Thus, part of the HopZ3 mechanism of immune suppression involves inhibition of the formation of the AvrPto1 Psy –PTO complex through their modification. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Effect of acetylation on AvrPto1 Psy -PTO binding. (A) Acetylation of AvrPto1 Psy reduces its interaction with PTO. Beads with immobilized His-AvrPto1 Psy were incubated for 2 h with acetyl-CoA, IP 6 and HopZ3, HopZ3_C300A (C/A), or no HopZ3 (un, untreated). Beads were washed and then incubated with soluble unmodified PTO-GST for 1 h, and after washing and elution, proteins were resolved by SDS-PAGE and stained with Coomassie blue or silver. The last two lanes in gel images are from different gels run at the same time as the other lanes, and interaction was always quantified relative to immobilized protein in the same lane. The mean with the standard error of relative band intensities from at least four experiments is shown, with binding after reaction with HopZ3 set to 1. Different letters indicate significant differences (ANOVA/Fisher’s test P<0.05). Bars indicate standard errors. (B) Acetylation of PTO reduces its binding to AvrPto1 Psy . Experiments with immobilized acetylated PTO-GST and free AvrPto1 Psy -His were done as in (A). https://doi.org/10.1371/journal.ppat.1010017.g006

Amino acid substitutions in PTO and FEN alter their acetylation specificity FEN has an asparagine (N202) at the cognate position to T204 in PTO. Conversion of T204 to N in PTO abolished the acetylation of the protein by HopZ3 in vitro (Fig 7A). Conversely, mutating N202 to T in FEN rendered it susceptible to acetylation by HopZ3 (Fig 7B). The same amino acid substitutions switched the signaling specificity of PTO and FEN in response to AvrPto Pto as assessed by cell death induction in transient expression experiments in N. benthamiana [16]. The loss of in vitro acetylation of PTO_T204N by HopZ3 is consistent with our finding of only one in vitro acetylation site in PTO by LC-MS/MS (S2 Table). PPT PowerPoint slide

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TIFF original image Download: Fig 7. Substitutions in the P+1 activation loop of PTO and FEN affect their acetylation by HopZ3 and their kinase activity. Purified PTO-GST and FEN variants were incubated with HopZ3-His or HopZ3_C300A (C/A) mutant in the presence of IP 6 and 14C-acetyl-CoA (A–B) or γ32P-ATP (C-D). Samples were separated by SDS-PAGE and subjected to autoradiography. (A) PTO but not a T204N P+1 activation loop variant was acetylated by HopZ3. (B) The substitution of Asn202 to Thr in FEN conferred acetylation by HopZ3. (C–D) Kinase activity assay showing PTO and FEN autophosphorylation and transphosphorylation of HopZ3 and HopZ_C300A in vitro. Kinase variants with Thr (wild-type PTO and FEN_N202T) were more active than Arg or Asn variants. https://doi.org/10.1371/journal.ppat.1010017.g007 Amino acid substitutions at position 204/202 greatly affected kinase activities of PTO and FEN, respectively. PTO and FEN variants with the T at 204/202 had higher kinase activity and showed more autophosphorylation than the N or R versions (Fig 7C and 7D; [17]). Together our data suggest that HopZ3 targets an essential residue in PTO that differentiates it from FEN in immune activation ability.

HopZ3 acetylates multiple sites in SlRIN4s and SlRIPK We analyzed modifications of tomato RIN4s and RIPK acetylated in vitro by HopZ3 using 13C-acetyl-CoA and found many residues to be acetylated by HopZ3 (S3 and S4 Tables). We did not observe common modified sites among all three SlRIN4 paralogues and AtRIN4; however, these proteins are not highly conserved ([9], S7 Fig). The lack of conserved acetylations may also result from the intrinsically unstructured nature of RIN4s. We found one residue that is acetylated in tomato and Arabidopsis: S88 in SlRIN4-1/S79 in AtRIN4, respectively. This residue is conserved among RIN4s from many species [9,34]. The main regulatory phosphorylation sites corresponding to AtRIN4, T166 and S141 [6] were not acetylated by HopZ3 in tomato or Arabidopsis. The major acetylation sites in AtRIPK [9] were acetylated by HopZ3 in the tomato orthologue. Similar to Arabidopsis, these sites could often be also phosphorylated (S8 Fig). In particular, SlRIPK K120 (K122 in AtRIPK) in the ATP binding site, S219 (S221 in At) near the ATP binding site, SlRIPK S249/T250 (At S251/T252) in the activation loop and T255/H256 (T257 in At) were specifically acetylated by HopZ3 in both species; in addition, the serines/threonines were phosphorylation sites. K122 and S251/T252 in AtRIPK are necessary for RIPK activity [9] and S251/T252 are uridylated by the Xanthomonas effector AvrAC leading to RIPK inhibition [35]. Moreover, SlRIPK S249/T250 (At S251/T252) correspond to PTO S198/T199, whose phosphorylation is important for immunity [17,22,23] and is decreased by HopZ3 (S2 Table). The highest acetylation by HopZ3 was observed for SlRIPK T255, which corresponds to acetylated T257 in Arabidopsis RIPK and T204 in the PTO activation loop. Therefore, HopZ3 targets important residues conserved in SlRIPK, AtRIPK and PTO and directly acetylates SlRIPK residues necessary for kinase activity, acetylation of which may compete with phosphorylation.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010017

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