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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 AvrPto1Psy-triggered immunity. HopZ3 acetylates AvrPto1Psy 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 AvrPto1Psy-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 AvrPto1Psy 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 AvrPto1Psy 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., Author summary 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 AvrPto1Psy 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.

RSK1, a downstream kinase of the MAPK pathway, has been shown to regulate multiple cellular processes and is essential for lytic replication of a variety of viruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV). Besides phosphorylation, it is not known whether other post-translational modifications play an important role in regulating RSK1 function. We demonstrate that RSK1 undergoes robust SUMOylation during KSHV lytic replication at lysine residues K110, K335, and K421. SUMO modification does not alter RSK1 activation and kinase activity upon KSHV ORF45 co-expression, but affects RSK1 downstream substrate phosphorylation. Compared to wild-type RSK1, the overall phosphorylation level of RxRxxS*/T* motif is significantly declined in RSK1K110/335/421R expressing cells. Specifically, SUMOylation deficient RSK1 cannot efficiently phosphorylate eIF4B. Sequence analysis showed that eIF4B has one SUMO-interacting motif (SIM) between the amino acid position 166 and 170 (166IRVDV170), which mediates the association between eIF4B and RSK1 through SUMO-SIM interaction. These results indicate that SUMOylation regulates the phosphorylation of RSK1 downstream substrates, which is required for efficient KSHV lytic replication., Author summary RSK1 is an essential celluar kinase for lytic replication of Kaposi’s sarcoma-associated herpesvirus (KSHV). Besides phosphorylation, it is not known whether other post-translational modifications play an important role in regulating RSK1 function. Here, we found that RSK1 is SUMOylated at lysine residues K110, K335, and K421, which is enhanced by KSHV lytic replication. RSK1 SUMOylation is required for effient KSHV lytic replication and mutations on its SUMOylation sites leads to reduced lytic gene expressions and impaired progeny virus production. Interestingly, SUMO modification does not alter RSK1 activation and kinase activity upon KSHV ORF45 co-expression, but affects RSK1 downstream substrate phosphorylation. Specifically, SUMOylation deficient RSK1 cannot efficiently phosphorylate eIF4B since SUMO-SIM interaction mediates the association between eIF4B and RSK1. These results indicate that SUMOylation regulates the phosphorylation of RSK1 downstream substrates, which is required for efficient KSHV lytic replication.