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Pseudomonas syringae effector HopZ3 suppresses PTO immune pathway in tomato by acetylation

ABSTRACT: The plant pathogen Pseudomonas syringae secretes multiple effectors to suppress plant defenses. Some effectors are recognized by plants while others can function to suppress the resulting immune responses. HopZ3 of Psy B728a is an aminotransferase targeting multiple components of both plant immune complexes and Psy effectors, leading to the activation of these complexes. In Arabidopsis, HopZ3 acetylates the host RPM1 complex and Psy effectors AvrRpm1 and AvrB3 s. In P. syringae resistant tomato, the main immune complex includes PRF and PTO. We found that HopZ3 acts as a virulence factor on tomato by suppressing defenses triggered by Psy AvrPto1. HopZ3 interacts with members of the PTO complex and acetylates PTO, AvrPto1, SlRIPK and SlRIN4s in vitro and in planta. Mapping HopZ3-dependent acetylation sites in planta revealed that AvrPto1 is modified on histidine, serine and threonine residues. The most highly acetylated AvrPto1 residues are T91 and S94 in a loop essential for interaction with PTO. HopZ3 also acetylates, in planta, T204 and T199 in the PTO activation loop which is important for kinase activity and recognition of AvrPto1. In agreement with these findings, we showed that acetylation of either AvrPto1 or PTO by HopZ3 reduces their binding in vitro. Mutation of acetylation sites T91A or H125A/H135A in AvrPto1 does not affect PTO binding in vitro but decreases AvrPto1 recognition in tomato in the absence of HopZ3 and increases bacterial growth. We also found decreased in planta phosphorylation in the presence of HopZ3 for some residues in AvrPto1 and PTO involved in immune signaling. Moreover, acetylation of SlRIPK by HopZ3 reduces its activity as well as phosphorylation of HopZ3 and SlRIN4s. Psy modulation of both its own avirulence effectors and their plant targets leads to decreased defenses and increased susceptibility to infection.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Pseudomonas Syringae Pv. Syringae

SUBMITTER: Donald Wolfgeher

LAB HEAD: Dr. Jean T. Greenberg

PROVIDER: PXD022953 | Pride | 2021-10-18

REPOSITORIES: Pride

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Project description:In this study we focus on the MYST-type acetyltransferase MOF (males-absent-on-the-first, KAT8), which is the effector enzyme of two transcription regulator complexes in Dro-sophila. As part of the male-specific-lethal dosage compensation complex (MSL-DCC, or DCC for short), the enzyme is targeted to transcribed genes on the X chromosome, where it acetylates H4K16 to unfold the chromatin fiber to boost transcription (15-17). In the context of the NSL complex (16) MOF has been reported to acetylate different combina-tions of histone H4 lysines 5, 8, 12 and 16 at promoters in different cells, illustrating the potential of complex subunits to modulate substrate specificity (18-21). Defining the substrate selectivity of KATs in vivo poses several challenges (1). The intrin-sic substrate selectivity of enzymes may be modulated by its molecular environment, be it the complex assembly or the chromatin context. Redundant activities of different en-zymes, indirect effects and the action of lysine deacetylases (KDACs) may occlude the consequences of loss-of-function manipulation. Furthermore, the specificity of the anti-bodies that are commonly used to detect distinct acetylated lysines in histones may be compromised due to low-level off-target effects, interference of modifications next to acetylated lysines, and the tendency of Kac-antibodies to react with relaxed selectivity with oligo-acetylated histone tails (22-25). A more reliable strategy involves the detection of modified histone peptides by mass spectrometry, which unequivocally detects individ-ual modifications and defined combinations of modifications and has a much better dy-namic range than traditional Western blotting (26-30). In a complementary approach one may determine the intrinsic substrate selectivity of defined recombinant enzymes and KAT complexes in biochemical assays (31). We re-cently reconstituted a recombinant MOF-containing core DCC (here termed ‘4-MSL’, since it contains the subunits MOF, MSL1, MSL2 and MSL3 (32)) and now report on a study of its substrate selectivity on recombinant nucleosome arrays. Pilot experiments using traditional Western blotting of diagnostic antibodies suggested that, in addition to the expected H4K16 acetylation, H4K12 was modified. Since this finding disagrees with the prevailing view that the DCC selectively acetylates H4K16, we applied the targeted mass spectrometry described in (26) to our in vitro reactions, focusing on acetylation of the H4 N-terminus. We compared the acetylation reaction of the 4-MSL complex with another MYST enzyme, dTip60 (KAT5), in the context of a trimeric complex, akin to the yeast piccolo NuA4 complex, which is thought to have a more relaxed lysine selectivity on the H4 N-terminus (33-35). We found that during short reaction times, the 4-MSL complex acetylated H4K16 with excellent selectivity, while the dTip60piccolo complex preferentially modified H4K12. To our surprise, we also observed that during longer incubations the 4-MSL complex progres-sively acetylated K12, K8 and K5, leading to a complex mixture of oligo-acetylated H4. Mathematical modeling suggests that MOF recognizes and acetylates H4K16 with high selectivity, but remains bound to the substrate and continues to acetylate more N-terminal H4 lysines in a processive manner. Because the MSL-DCC in vivo contains long, non-coding roX RNA (15,36-38), we ex-plored whether RNA affected the specificity of the reaction. We observed that the addi-tion of RNA to the HAT reaction improves the specificity of the reaction and suppresses the generation of oligo-acetylated forms, possibly by reducing the dwell time of the en-zyme on the nucleosome substrate. The comparison of MOF and dTip60 activities in the context of recombinant HAT com-plexes revealed significant differences in activity and specificity. Our study describes a powerful experimental approach and conceptual framework for the analysis of physiolog-ically relevant components that modulate intrinsic activities.

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Pseudomonas syringae effector HopZ3 suppresses PTO immune pathway in tomato by acetylation

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