Critical Review Post-translational Modifications in Regulation of Saikat Bhattacharjee* Jameeta Noor Pathogen Surveillance and Signaling in Plants: Jewel Bornali Gohain Hitika Gulabani The Inside- (and Perturbations from) Outside Ingole Kishor Story Dnyaneshwar Ankit Singla

Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, Haryana 121001, India

Abstract In its lifetime a plant is exposed to pathogens of diverse types. Although methods of surveillance are broadly pathogenindividualized, immune signaling ultimately connect to common core networks maintained by key protein hubs. Defense elicitations modulate these hubs to re-allocate energy from central metabolic pathway into processes that execute immunity. Because unregulated defenses severely decrease growth and productivity of the host, signaling regulators within the networks function to achieve cellular equilibrium once the threat is minimized. Protein modifications by post-translational processes regulate the molecular switches and crosstalks between interconnected pathways spatially and temporally.

Keywords: plant innate translational modifications

immunity;

effectors;

NB-LRR;

post-

Introduction Under constant pathogen threat, plants have evolved multilayered defense strategies. A class of receptor-like kinases (RLKs) or pattern-recognition receptors (PRRs) span the outer membrane of the plant cell and recognize conserved structural determinants (pathogen-associated molecular patterns; PAMPs) of the pathogen (1). Classic examples of these recepC 2015 International Union of Biochemistry and Molecular Biology V

Volume 67, Number 7, July 2015, Pages 524–532 *Address correspondence to: Saikat Bhattacharjee, Regional Centre for Biotechnology, NCR Biotech Science Cluster, Gurgaon-Faridabad Expressway 3rd Milestone towards Gurgaon, Village Bhakri, Faridabad, Haryana, India 121001. Tel: 191-129-2848837. E-mail: [email protected] Received 17 June 2015; Accepted 17 June 2015 DOI 10.1002/iub.1398 Published online 15 July 2015 in Wiley Online Library (wileyonlinelibrary.com)

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Covalent modification of host targets connected to hubs are strategies used by most virulent effectors and result in rerouting signals to suppress host defenses. Resistance is a result of activation of specialized classes of receptors that short-circuit effector activities by co-localizing via posttranslational modifications (PTMs) with effector targets. Despite advancement in proteome methodologies, our understanding of how PTMs regulate plant defenses remains elusive. This review presents protein-modifications as forefront C regulators of plant innate immunity. V 2015 IUBMB Life, 67(7):524–532, 2015

tors include FLAGELLIN-SENSITIVE 2 (FLS2), CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) and EF-TU RECEPTOR (EFR) kinase that sense flagellin, chitin, and EF-tu, respectively. Elicitation as a result of receptor–ligand interaction leads to activation of MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) cascade causing Ca21 spikes, callose deposition, and oxidative burst. Collectively, these responses are termed PAMP-triggered immunity (PTI) (2). Evolutionary arms race for survival, however, has generated adapted pathogens that secrete effectors (termed virulence proteins) into the host cytoplasm and suppress PTI to cause disease. But ongoing arms race is not biased toward the invaders. Plants in turn have coevolved polymorphic family of nucleotide-binding leucine-rich repeats (NB-LRR) resistance proteins as receptors that sense specific effector activities. The output is a qualitatively and quantitatively stronger immune responses than PTI and is experimentally supported by observations that both pathways recruit similar core signal transduction machinery (3). This second layer of defense is called effector-triggered immunity

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(ETI) and the corresponding effector is called avirulence (Avr) factor (reviewed in ref. 4). In some instances, these responses lead to a programmed cell death (PCD) in local tissue termed hypersensitive response (HR). Coined from Flor’s seminal studies on flax rust (5), a genefor-gene resistance model was proposed wherein immunity results from genetic pairs of pathogen Avr and the cognate NB-LRR brought together upon pathogen invasion. In simplest of ETI-trigger direct receptor–ligand type associations were detected between a Avr and the counterpart NB-LRR protein. In other interceptions, termed “guard hypothesis” or variations thereof NB-LRR proteins “guard” alterations caused on targets of Avr (guardees) in the pathogen’s attempt to establish virulence (6). A complicated interplay of signaling networks ensue resulting in massive transcriptional reprogramming aptly termed as ETI. Interestingly, PTI and ETI responses impinge on the signaling networks differently. Katagiri and Tsuda describe intricate web-like architectures consisting of signaling sectors that interconnect to signaling hubs (7). Both feedforward positive and negative regulatory relationships between the sectors balance the demands of the host via sectorswitching to mount defenses and minimize the fitness cost associated with these responses. Interestingly, only some parts of the signaling network is utilized whereas others serve as reservoirs recruited when the operative network is perturbed by the invader. Unlike PTI which consists of sectorcooperativity causing additive integration of signaling, a rapid and robust ETI uses compensatory mechanisms between the signaling sectors. The compensation mode is achieved by functionally redundant sectors operating through mechanistically distinct components. PTI sensors, the PRRs, are mostly connected to stepwise linear arrangement of signal transducers (namely MAPK cascade) whereas ETI receptors (NB-LRRs or the Avr targets) are often associated with key signaling hubs. PTI thus has features which effectors can perturb at any/many step(s) of signal transduction while ETI relation to hubs intercepts any of these PTI perturbations for trigger (8). In this complicated immune network, how is immunity downregulated in unperturbed cells and how upon a effector entry the cognate NB-LRR protein responses are activated? Post-translational modifications (PTMs) offer tantalizing clues to the above mystery. Under homeostasis, cellular PTMs impart characteristic signatures to target proteins (9). Negative defense players are stabilized whereas cellular levels of positive modulators are balanced with energy needs of the cell. Identification of MAPK pathway laid the foundation of importance of PTMs in defense signaling in interacting networks (10). Its linear signal transduction mode by sequential phosphorylation cascades allows rapid reprogramming of the host transcriptome, induction of defense-related genes, and establishment of PTI. Perhaps this simplicity in the first line of defense made a ground for breach by secreted effectors that employ a competing PTM, target MAPK transducers, and dampen immune signaling. Effector-mediated PTM alterations sensed by the NB-LRR proteins in turn formed the fundamen-

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tal basis of the ETI trigger in resistant accessions. In its elegance, thus, simple covalent attachment of chemical groups or small protein moieties to targets has the potential to generate molecular switches for signal transduction. A recent review describes PTM activities of effectors on their targets (11). While Howden and Huitema focus on effector-mediated interference of host immune pathways, PTM involvement in immune regulation remains shadowed. In mankind’s implementation of selective breeding and transgenic approaches for generating pathogen resistant plants, the most common bottleneck encountered is durability and sustainability. It is increasingly evident that merely stacking NB-LRRs or creating hybrids with resistant accessions result in limited success (12). A black box in these efforts is our lack of understanding of molecular deployment of immune sentinels at key locales, interactome necessary for defense functions, and processes that regulate misfiring of immune responses. In this review, we have attempted to present this perspective utilizing recent advances predominantly from the Arabidopsis thaliana–Pseudomonas syringae pathosystem. We maintain our highest regard for findings from other equally important host– pathogen interactions and have included some of these as examples. In instances where a specific PTM and its involvement is not reported from a plant system, we have suggested their existence either through bioinformatic findings or based on specific PTM activities of a targeting effector. Considering most effectors activities originate either by horizontal gene transfer or by convergent evolution the indulgences of these yet to be identified PTMs is likely realistic. Although we sincerely have made efforts to present an updated coverage of most breakthrough literatures, it is possible that we may have missed several interesting studies. We apologize to our readers in advance for this oversight.

Phosphorylation/Dephosphorylation Energy driven cellular processes rely on the abundance of reservoirs of nucleotide polyphosphates. Resident high energy delivering phosphodiester bonds are targets of nucleophiles and result in reversible covalent attachment of phosphates (phosphorylation) to hydroxyl side chain amino acids such as serine, tyrosine, or threonine, a process catalyzed by kinases. The donor in most instances is ATP. In plant defenses, phosphorylation is an integral part of signaling network exemplified by the complex MAPK circuitry and the identification of massive number of phosphopeptides after pathogen elicitor treatment (10). Several high affinity PRR-RLKs localized at plasma membrane undergo auto-phosphorylation essential for trans-phosphorylating downstream substrates upon ligand binding (13). These include the brassinolid-receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1) and tomato kinase Pto. Auto-phosphorylation-deficient mutants of Pto does not bind the cognate effector AvrPto, has abolished kinase activities, and are impaired in eliciting HR. Protein phosphorylation also influence stability of FLS2 and the rice PRR XA21

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(Xanthomonas resistance 21) by competing with proteasomal degradation by ubiquitination (14,15). Phosphorylation in some instances synergizes with ubiquitination and prevents immune misfiring by recycling positive regulators. NON-EXPRESSOR OF PR1 (NPR1), a signaling mediator of phytohormone salicylic acid (SA), undergoes phosphorylation-dependent turnover in the nucleus preventing mis-expression of defense-related genes in the absence of pathogen (16). In specific instances, defense-associated kinase substrates either are maintained in de-phosphorylated states in un-triggered cells or undergo, among other processes, inactivation via dephosphorylation. Interactions between multiple LRR-RLKs and protein phosphatases thus introduce a regulatory layer of surveillance in the assembly and activation of immune players (17). Autophosphorylated and activated rice PRR XA21 bind XA21binding protein 15 (XB15), a typical protein phosphatase suggestive of their role in recycling of activated XA21 (18). Being of the central importance in defense responses, phosphorylation indeed is a mode for virulence by multiple pathogen effectors. At least two P. syringae (Ps) effectors AvrRpm1 and AvrB cause the phosphorylation of the positive immune regulator RPM1-INTERACTING PROTEIN 4 (RIN4). Surprisingly, neither effectors possess kinase activities and instead rely on a host RPM1-INDUCED PROTEIN KINASE (RIPK) held in close association with their target RIN4 (19,20). Another Ps effector HopAO1 is a functional tyrosine phosphatase that targets phosphorylated EFR activated by the bacterial elicitor EF-Tu (21). PAMP-responses similarly are also dampened by phosphothreonine lyase activity on MAPK targets by the bacterial effector HopAI1 (22).

Ubiquitination Ubiquitination involves three-step covalent attachment of ubiquitin (Ub) to a lysine e-amino group of substrates (23). Most poly-ubiquitinated proteins are degraded via the proteasome whereas mono-ubiquitinated proteins are altered in cellular location, activity, and partner interactions. Increasing evidences point to role of ubiquitination in dynamic regulation of PRRs and in defense hormones such as jasmonic acid (JA) and auxin signaling (24). SA induction against biotrophs and hemibiotrophs recruits NPR1 and cause targeted degradation of repressors such as TGA2 to activate PR genes. Upon SA accumulation NPR1 binds an Ub-ligase and is recycled by the proteasomal complex (16). Multiple regulators of the phytohormone ethylene (ET) closely associate with MAPKs and upon PAMP perception are selectively ubiquitinated (24). Thus, ubiquitination maintain hormonal equilibrium and its cross-talks in defense signaling networks. E3 ligases of PUB (plant U-box) types are associated with both positive and negative regulation of defense genes (25). Arabidopsis pub22 pub23 pub24 triple mutant display enhanced oxidative burst to various PAMPs suggesting their close association with multiple PRRs (26). These PUBs interact with and target regulatory subunits of the proteasome com-

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plex. As a consequence of this self-regulation, significant reduction in proteasome occurs perhaps fine tuning the balance in stability of defense regulators. The virulence function of a nematode effector UBCEP12 (ubiquitin carboxyl extension protein 12) presents a unique mode of regulating host ubiquitination. Upon host entry, UBCEP12 is processed to liberate mono-ubiquitin domain and carboxyl extension peptide (CEP12) which interferes with proteasomal processes and PCD, respectively (27). Based on CEP12 activity, the authors speculate the possible existence of small endogenous peptides with roles in suppression of immunity. In a recent finding, an Arabidopsis ubiquitin-conjugating enzymes E2 (AtUBC2) was demonstrated to function as a negative regulator of disease resistance (28). Mutants of UBC2 display enhanced resistance toward virulent Ps and have upregulated levels of defense cochaperones REQUIRED FOR MLA12 RESISTANCE1 (RAR1) and SUPPRESSOR OF G-TWO ALLELE OF SKP1 (SGT1). AtUBC2 interaction with SGT1 directly connects ubiquitination to NBLRR protein stability. Considering their central importance in defenses it is not surprising that several pathogen effectors have evolved to exploit host ubiquitination and perturb defense signaling (see ref. 29 for review). Ps effector AvrPtoB exhibits E3 Ub ligase activity and mediates proteasomal degradation of kinases Fen and FLS2. Arabidopsis MIN7, an ADP ribosylation factorguanyl nucleotide exchange factor (ARF-GEF) involved in vesicular trafficking is targeted for degradation by the Pseudomonas effector HopM1 (30). Avirulent effectors prevent HopM1-dependent degradation of AtMIN7 suggesting that ETI responses likely induce counter-PTMs to protect effector damages. Recent studies have demonstrated that some effector activities cause stability of their targets especially negative regulators of defense. PopP2, the Ralstonia solanacearum effector interacts with and stabilizes both resistant and susceptible alleles of the negative NB-LRR RRS1, an effect that is mimicked by the application of proteasome inhibitor MG132 (31). A RXLR-type effector AvR3a from Phytophthora infestans suppresses cell death by protecting a host U-box E3 ubiquitin ligase (32). A comprehensive elucidation of ubiquitination and ubiquitinated proteins is essential to unravel the magnitude of ubiquitin interplay in plant innate immunity.

SUMOylation and Sumo-Binding PTMs by small Ub-like modifier (SUMO) involve stepwise covalent attachment to a e-amino group of conserved lysine residues in a target proteins. SUMOylation has been implicated in chromatin modeling, nuclear import, and transcription and is upregulated by stress (33). Unlike ubiquitination, SUMOylated proteins are not degraded instead they are altered in stability, localization, and interactions. Arabidopsis null mutants of SIZ1, a SUMO-ligase display cpr (constitutive activation of PR genes) phenotype. Elevated levels of SA and increased expression of defense-related genes such as ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), PHYTOALEXIN-DEFICIENT4 (PAD4),

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and SA-biosynthesis gene ISOCHORISMATE SYNTHASE1 (ICS1) in siz1 plants likely account for their enhanced resistance to Ps (34). Similar upregulation of immune-responsive genes are also noted in SUMO-knockdown or over-expression plants (33). Interestingly, SUMOylation prediction softwares (GPS-SUMO; www.sumosp.biocuckoo.org) identify conserved SUMOylation motifs in EDS1 and PAD4. Whether the steady-state levels of these defense regulators are influenced by SUMO is a subject of future endeavors. Nevertheless, the above reports seem to suggest SUMOylation as a negative regulator of defenses. In a screen for SUMOylated targets from Arabidopsis overrepresentation of transcription factors, chromatin remodeling and DNA repair proteins were noted suggesting SUMOylation predominantly affects transcriptional processes (35). Interestingly, a histone deacetylase HDA19, involved in repression of defense genes, and TOPLESS-RELATED1 (TPR1) a corepressor functioning with NB-LRR SNC1 featured in the identified SUMOylated proteins. SUMOylated lysines gain new interaction platforms for SUMO-interaction motifs (SIMs)-containing proteins. Indeed a bioinformatic software (GPS-SBM; sbm.biocuckoo.org) predicts multiple SIMs in SNC1 and EDS1. The occurrence of EDS1 homo- and EDS1-SNC1 heterodimers raises the possibility of SIM-SUMO mode of interaction between these proteins (36). Proteins with SIM domains therefore present an additional mode of SUMO involvement regulated by steady-state SUMOylation and by free SUMO levels. Several pathogen effectors manipulate host SUMOylation pathways in their virulence roles. AvrBsT, AvrXv4, and XopD from different pathovars of Xanthomonas campestris have SUMO protease activities (9). Equipped with an EAR (EFR-associated amphiphilic repression) DNA-binding motif XopD alters nuclear architecture and inhibits positive transcription factor MYB30-mediated defense activations (37). A recent report identified interaction between XopD and HFR1, a bHLH transcription factor implicated in photomorphogenesis (38). It is proposed that XopD by de-SUMOylating HFR1 cause transcriptional repression of defense-responsive genes. PTM by ubiquitination in some cases competes with SUMOylation for common targets (16). Indeed several WRKYs that are substrates for MAPKs also feature as SUMOylation substrates (35). The nature of interplay between SUMOylation and phosphorylation of these WRKYs however remains unknown. In a yeast two-hybrid screen for SUMO interactors, several ubiquitin E3 ligases were identified (39) suggesting that SUMOylation in some instances may target protein for degradation. Currently possible roles of SUMO in direct assembly of NB-LRRs such as SNC1 or other defenseassociated proteins remains speculative.

Fatty Acyl Modifications: NMyristoylation and S-Acylation The covalent attachment of palmitic or stearic acid is referred to as S-acylation. N-myristoylation involves covalent modification of a glycine residue by attachment of myristate via an

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amide linkage. Both PTMs regulate the substrate protein stability, correct localization, and complex formation characteristics and have been implicated in immune responses (40). Sophisticated proteomic detection techniques demonstrate the extent of S-acylation and identified about 600 targets from root cultures of Arabidopsis (41). These included calcium channels, LRR-RLKs, subunit of heterotrimeric G protein, SNAREs, ATPases among others. The enrichment of a significant number of defense-related proteins strongly suggests the importance of this modification is immunity. S-Acylation deficient fls2 although unaltered in localization at the plasma membrane (PM) are compromised in PTI functions (41). Because residues that undergo N-myristoylation and Sacylation often occur in close proximity, it is likely that for FLS2, N-myristoylation predominantly drives the PM localization. The tomato Pto kinase by interacting with the NB-LRR protein Prf sense effectors AvrPto and AvrPtoB to trigger ETI. Although Pto is myristoylated, neither Pto nor Prf is membrane localized. In contrast, both N-myristoylation and S-acylation is essential for AvrPto activity in suppressing FLS2 and BAK1 (40). Since a myristoylation mutant of AvrPto still retains interaction with Pto, it is likely that ETI trigger occurs en route of AvrPto localization to the PM. Arabidopsis RLK PBS1 is a target of AvrPphB, an effector from Pseudomonas phaseolicola. PBS1 coordinate with downstream PRRs such as FLS2 and EFR in transduction of immune signaling. Cognate NB-LRR recognizing AvrPphB, RPS5 is myristoylated and S-acylated and co-localizes with PBS1 (42). S-acylation-deficient mutants of PBS1 mislocalize and are incapable of activating RPS5. Interestingly, AvrPphB exploit host fatty-acylation pathway for self-modification and accessing its target PBS1. Dynamics of the secondary messenger Ca21 facilitate signal transduction in different stress pathways including pathogen attack. A class of Ca21 responders CBLs (Calcineurin Blike proteins) and CDPKs (calcium-dependent protein kinases) transduce signals into phosphorylation events which ultimately lead to physiological responses such as stomatal closure and ROS production (43). Mutations in N-myristoylated residues of Arabidopsis CPK5, a positive regulator of innate immunity, mislocalize and are impaired in their activities on the ROS producer RBOH (44,45). Transgenic plants with constitutive expression of AtCPK5 are enhanced resistance to DC3000 and have upregulated transcripts for ICS1 and PR1. In contrast to CDPKs, CBLs mediate indirect phosphorylation via their interaction with CIPKs. Multiple members of CBLs undergo lipid modification necessary for their appropriate localization (46). S-Acylated CBLs govern correct localization of interacting CIPKs on functional membrane microdomains. Fatty-acylations drive crucial molecular switches such as the Rho-GTPases of plants (ROPs). These proteins cycle between GDP-bound inactive and GTP-bound active forms and are important regulators of plant immunity (47). ROP interactors include CERK1, SGT1, RAR1, RBOH, several MAPKs, and NB-LRRs. PTM by fattyacylations, therefore, are key mediator of co-localization and communication between defense players of same pathway.

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Glycosylphosphatidylinositol-Anchored Proteins Glycosylphosphatidylinositol (GPI) anchor is a glycolipid structure that is added post-translationally to the C-terminus of many eukaryotic proteins to tether them to the PM (48). This PTM is initiated in the ER and involves stepwise addition of sugar residues to phosphatidylinositol followed by a transamidase reaction to transfer the anchor to targeted proteins. The GPI-anchored proteins (GAPs) are then secreted from Golgi compartment and attach to the PM. The sugar moiety in a GPI-anchor interacts with cell wall components to maintain continuum across these two barriers. Plant GAPs are implicated in diverse roles in immune signaling (reviewed in ref. 48). The Arabidopsis AGP17, a member of arabinogalactan (AGP) family of GAPs downregulates PR1 induction upon agrobacterium inoculation suggesting its role as a negative regulator of defense. Several AGPs have been predicted to associate with RLKs such as Xa21, Wak1, and SERK1 and function in immune-related gene expression during pathogenesis. Arabidopsis GAP PMR6 is involved in defense suppression and pmr6 mutants are resistant to the powdery mildew fungus possibly because of altered cell wall composition. Protein-modifications by GPI anchor therefore confer adaptor functions to substrates linking cell wall components to RLKs and downstream transcriptional changes. NON-RACE SPECIFIC DISEASE RESISTANCE1 (NDR1) constitutes the central node for signaling via CC-NB-LRR class of resistance proteins. Plasma membrane localized NDR1 contains a C-terminal GPI anchor (49). Overexpression of NDR1 results in increased SA levels and PR1 accumulation causing enhanced resistance to virulent Ps suggesting its role as a positive immune regulator. In contrast, ndr1-1 plants have compromised PTI. NDR1 interacts with RIN4, the common guardee of NB-LRRs RPM1 and RPS2, and a PTM target of multiple Pseudomonas effectors (described in earlier and later sections). NDRI possibly sequesters RIN4 preventing misfiring of the associated NB-LRRs. Physical interaction of NDR1 with FLS2 demonstrates cross-talks between PTI and ETI with NDR1 role as an adaptor (50). Arabidopsis GAP COBRA maintains cell wall integrity and involved in oriented root cell expansion and cellulose deposition. A cob-5 mutant has extreme cell wall defects that mimic a pathogen attack causing hyper-accumulation of JA-, SA-responsive, and disease resistance transcripts (51). A significant overlap is noted between the upregulated transcripts in cob-5 to those induced upon virulent Pseudomonas infection. GPI-anchor of proteins are cleaved by specific phospholipases generating intracellular messengers such as phosphatidylinositol, phosphoglycans, or AGPs. These potent signaling mediators may link the warfare occurring at PM to downstream defense-related transcriptions.

S-Nitrosylation Nitrous oxide (NO) a ubiquitous gaseous moiety can covalently attach to side chains of reactive cysteine residues in several

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proteins, a PTM known as S-nitrosylation (52). The resulting S-nitrosothiols (SNO) are sensitive to cellular redox and can be reversed by reducing agents such as glutathione and ascorbate. S-Nitrosylation primarily affects gene transcriptions. Under un-elicited conditions SA master regulator NPR1 hexamers, stabilized by intermolecular disulfide bonds, localizes at cytoplasm. SA-induced redox changes releases monomeric NPR1 that translocate to the nucleus and functions in positive regulation of defense-responsive genes (52). Several proteins including central metabolic regulators such as phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehyrogenase (GAPDH) are S-nitrosylated at the onset of HR. Interestingly GAPDH, in addition to its glycolytic function, transnitrosylates HDA19 causing its release from chromatin structures de-repressing defense-associated gene expressions. Carbonic anhydrase activity and SA-binding propensity of the Arabidopsis SA-binding protein SABP3 is abolished by Snitrosylation and is indicative of a negative feedback loop in plant defenses. S-nitrosylation turnover is mediated by GSNO reductase (GSNOR) and thioredoxins. De-nitrosylation by thioredoxins/ thioredoxin reductase modulates signal transduction and are responsible for generating monomeric NPR1. In contrast, the cellular reducing agent glutathione is S-nitrosylated to generate S-nitrosoglutathione (GSNO), a mobile reservoir and donor for biologically active NO. An Arabidopsis GSNO reductase (GSNOR) protein has been identified the loss of which severely compromises disease resistance against bacterial and oomycete pathogens (53). PR1 transcript accumulation is significantly lower and delayed in atgsnor1 plants suggesting deficiencies in SA-induced defense gene expression. Contradicting results reported by Rusterucci et al (54) demonstrate that antisense mediated downregulation of GSNOR1 enhances basal defense against the virulent strain of the oomycete pathogen Peronospora parasitica. Although the authors ascribe multiple factors that may contribute to the contrasting results, further investigations are necessary to clear the contradictions. Nevertheless, GSNOR functions as defense regulators remain firm.

Cysteine Proteases HR responses in plant–pathogen interactions bear strong resemblance biochemically and physiologically to apoptosis in animals (55). A common mediator of PCD is the type-members of cysteine protease superfamily. Papain-like cysteine proteases such as tomato RCR3 and Arabidopsis RD19 are required for HR in incompatible interactions (55). The subfamily of vacuolar processing enzymes (VPE) particularly play regulatory roles in membrane collapse during plant HR and have been extensively documented (for review see ref. 56). HR mostly considered a secondary response in ETI is often deployed by the host as a last suicidal resort when the pathogen load is immense. Ectopic expression of cystatin, an endogenous cysteine protease inhibitor abolish HR triggered by a avirulent pathogen in soybean suggesting that plant cells

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possess regulatory mechanisms to prevent mis-timed PCD (57). Incidentally, cystatin expression is repressed by SA further supporting the notion that HR is non-essential in defense signaling. The Yersinia effector YopJ is the establishing member of peptidase family of effectors and contains a conserved catalytic triad of His, Glu/Asp, and Cys. Effectors of this superfamily have been associated with diverse roles. Among them AvrBsT, AvrXv4, and XopD target host SUMOylation and have been described earlier (9). Two effectors from Ps AvrPphB and AvrRpt2 resemble cysteine proteases and cause proteolytic cleavage of their respective targets PBS1 and RIN4 (9). Interestingly, RIN4 cleavage products are potent suppressors of PTI (8). HopN1, a functional cysteine protease from tomato pathovar of Ps inhibits ROS production and callose deposition in Arabidopsis (58). A component of photosystem II PbsQ is a target of HopNI indicating the role of photosynthesis-derived oxidative stress in plant defenses.

Acetylation Acetylation of histones influence transcriptomic and epigenetic processes of plant immunity and is not a focus of this review. Among the few characterized acetylated non-nuclear proteins, the cytoskeletal component tubulin is a substrates for lysineacetylation and is intricately linked to ROS and stress signaling (59). Tubulin assemblies form the basis of secretory pathways and likely are targets for the Pseudomonas effector HopZ1a (60). HopZ1a also acetylates and marks JAZ repressors for degradation thereby creating cellular environment of activated JA and suppressed SA pathway favorable to the biotrophic pathogen (61). As expected the secretion of this effector supplements the absence of toxin coronatine during pathogenesis. Of recent interest is the study of maintenance of genetically linked resistance gene-pairs such as RRS1 and RPS4 for recognition of multiple effectors from unrelated pathogens and mount effective ETI. Two breakthrough publications have linked PopP2 effector-dependent acetylation in dissection of communication between these linked resistance proteins (62,63). In virulence, pursuit PopP2 acetylates lysine residues of several WRKY transcription factors abolishing their DNAbinding and defense activation functions. In resistant accessions of Arabidopsis RRS1-R, the resistance allele of RRS1, held in association with RPS4 is also an acetylation target for PopP2. As a consequence, RRS1-R-mediated inhibition of defense gene is removed and bound RPS4 is activated to mount ETI. PopP2 acetyltransferase activity is suggestive of cellular regulation of RRS1-RPS4 pair misfiring possibly by a host deacetylase. However till date best characterized deacetylases function on histones. Competing PTMs of acetylation such as phosphorylation or methylation may likely be involved in inactivation of mis-primed defenses. A variant mode of acetyl modification involves the Nterminal acetylation (Nt-acetylation). Catalyzed by N-acetyltransferases (NATs), these PTMs affect protein stability and

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localization in most eukaryotic systems (64). Contrary to popular belief Nt-acetylation functions instead as a degron for the marked protein. Accumulation of immune receptors SNC1 and RPM1, in a recent report, was suggested to be regulated by specific NATs (65). In this study, an spiced-variant mutant (muse6-1/naa15-1) of a Arabidopsis NAT subunit displays autoimmune growth phenotypes, expresses elevated levels of defense-related PR genes, and is enhanced resistance to virulent oomycete Hyaloperonospora arabidopsidis. Demonstration of direct acetylation of SNC1 by multiple NATs in conjunction with fate of these acetylated SNC1 in planta implicates Ntacetylations as key PTM determinants in regulated expression/ turnover of NB-LRR proteins.

NMPylation Ribonucleotide polyphosphates, in addition to their well characterized roles in protein phosphorylation, also potentiate other PTMs. Nucleotide monophosphates can be covalently linked to threoine, tyrosine, and serine residues of targets to diversify their functions (66). Proteins containing conserved Fic (filamentation induced by cAMP) and adenylyl transferase domains are predicted AMPylators and have been associated with cellular processes such as DNA-binding, cytoskeleton dynamics, and vesicular targeting in animal systems. Fic domains share common ancestral lineage to doc (death on curing) domains and together form the “fido” superfamily of proteins. Although direct NMPylations of host targets are yet to be identified in plant processes, the wide conservation of fido domains across animals and utilization of this PTM by phytopathogenic effectors for virulence strongly suggest of their existence. Pseudomonas Type-III effector HopU1 is a functional ADP-ribosyltransferase and AMPylates the Arabidopsis glycine-rich RNA-binding protein 7 (GRP7) inhibiting association with FLS2 and EFR transcripts. This impedes ROS production and callose deposition allowing bacterial proliferation (67). PTI signaling is also dampened by the mono-ADP ribosylyltransferase activity of HopF2 on several defense signaling transducers (68). A Fic-domain containing effector from X. campestris, AvrAC was recently shown to UMPylylate RLKs BIK1, and RIPK (69). Interestingly, UMP-modified serine and threonine residues of BIK1 and RIPK reside in the kinase activation domain and antagonize auto-phosphorylation essential for kinase activities of these proteins. In an earlier study, a domain in the Pseudomonas effector AvrB although lacking conserved residues was reported to have similar core structural conformation with fido domains (70). Co-crystalization of AvrB with ADP (71) and its recruitment of RIN4phosphorylating RIPK raises the interesting possibility of fidolike proteins in plants and their role in preventing misfiring of immune responses via regulating proteins such as RIPK. Recent developments in detecting protein NMPylation using synthetic adenylylated peptides, NMP-specific antibodies, affinity tag NTP-conjugations, and metal catalyzed reactions that

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FIG 1

Schematic representation of importance of PTMs in innate immunity of plants. A section of a plant cell is highlighted showing strategic localization of positive immune modulators (PRRs, NB-LRRs, and effector targets) utilizing endogenous proteinmodification processes. Direct or indirect PTM activities of several pathogen effectors (numbers or single letter abbreviations) are depicted on their respective host targets. PTM processes indicated are: P, Phosphorylation; S, SUMOylation; L, Fattyacylation; 1, Ubiquitination; 2, Cysteine protease; 3, SUMO protease; 4, Acetylation; 5, NMPylation; and 6, S-Nitrosylation.

identify modified targets will definitely reveal the true extend of this PTM in plants (66,72).

Conclusion Technological advances in protein detection methodologies especially high resolution mass spectroscopy have increased our understanding of qualitative and quantitative differences between proteome capacity and gene annotations. PTMs are major contributors to this complexity. In a highly networked plant cell, calculated and balanced coordination among different pathways via PTMs maintain regular growth and development. In this steady state, a pathogen attack is indeed unwarranted. Phytopathogenic effectors alter host PTMs either directly or indirectly to suppress host signaling at multiple nodes. But rather than being just a victim, evolution has equipped plants with distinct immune mediators that recruit diverse PTMs to assemble with its peers at correct locales, detect a pathogen invasion severity and risk, and simultaneously connect to downstream defense responses. Figure 1 presents a schematic overview of such immune assemblies. Cross-talks among PTMs result in synergistic or antagonistic relations between signaling hubs and function to modulate sig-

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nal transduction during innate immunity. As more and more effector functions are being elucidated, more intricate networking of plant immunity is being revealed. A major hurdle is the development of further advanced proteomic technologies for detection of new and rare PTMs. Once we better our understanding of these processes, our efforts to engineer sustained pathogen resistant crops will bear success.

Acknowledgements This work is supported by grants from Regional Centre for Biotechnology Core Grant and Ramalingaswami Re-entry Fellowship, Department of Biotechnology, India to Dr. Saikat Bhattacharjee. The authors declare no conflict of interest in any matter in the subjects discussed in this review.

References [1] Zipfel, C. (2014) Plant pattern-recognition receptors. Trends Immunol. 7, 345– 351. [2] Dodds, P. N. and Rathjen, J. P. (2010) Plant immunity: towards an integrated view of plant-pathogen interactions. Nat. Rev. Genet. 11, 539–548. [3] Maekawa, T., Kufer, T. A., and Schulze-Lefert, P. (2011) NLR functions in plant and animal immune systems: so far and yet so close. Nat. Immunol. 12, 817– 826.

Regulation of Pathogen Surveillance and Signaling in Plants

[4] Cui, H., Tsuda, K., and Parker, J. E. (2014). Effector-triggered immunity: from pathogen perception to robust defense. Annu. Rev. Plant Biol. 66, 6.1–6.25. [5] Flor, H. H. (1971) Current status of the gene-for-gene concept. Annu. Rev. Phytopathol. 9, 275–296. [6] van der Hoorn, R. A. and Kamoun, S. (2008) From guard to decoy: a new model for perception of plant pathogen effectors. Plant Cell 20, 2009–2017. [7] Katagiri, F. and Tsuda, K. (2010) Understanding the plant immune system. Mol. Plant Microbe Interact. 23, 1531–1536. [8] Gassmann, W. and Bhattacharjee, S. (2012) Effector-triggered immunity signaling: from gene-for-gene pathways to protein–protein interaction networks. Mol. Plant Microbe Interact. 25, 862–868. [9] Stulemeijer, I. J. E. and Joosten, M. H. A. (2008) Post-translational modification of host proteins in pathogen-triggered defense signaling in plants. Mol. Plant Pathol. 9, 545–560. [10] Meng, X. and Zhang, S. (2013) MAPK cascades in plant disease resistance signaling. Annu. Rev. Phytopathol. 51, 245–266. [11] Howden, A. J. M. and Huitema, E. (2012) Effector-triggered posttranslational modifications and their role in suppression of plant immunity. Front. Plant Sci. 3, 1–6. [12] Ellis, J. G., Lagudah, E. S., Spielmeyer, W., and Dodds, P. N. (2014) The past, present and future of breeding rust resistant wheat. Front. Plant Sci. 5, 1–13. [13] Park, C. J., Caddell, D. F., and Ronald, P. C. (2012) Protein phosphorylation in plant immunity: insights into the regulation of pattern recognition receptor-mediated signaling. Front. Plant Sci. 3, 1–9. [14] Xu, W. H., Wang, Y. S., Liu, G. Z., Chen, X., Tinjuangjun, P., et al. (2006) The autophosphorylated Ser686, Thr688, and Ser689 residues in the intracellular juxtamembrane domain of XA21 are implicated instability control of rice receptor-like kinase. Plant J. 45, 740–751.  mez-Go  mez, L. and Boller, T. (2000) FLS2: a LRR receptor like kinase [15] Go involved in recognition of the flagellin elicitor in Arabidopsis. Mol. Cell 5, 20–21. [16] Spoel, S. H., Mou, Z., Tada, Y., Spivey, N. W., Genschik, P., et al. (2009) Proteasome-mediated turnover of the transcription co-activator NPR1 plays dual roles in regulating plant immunity. Cell 137, 860–872. [17] Becraft, P. W. (2002) Receptor kinase signaling in plant development. Annu. Rev. Cell. Dev. Biol. 18, 163–192. [18] Park, C. J., Peng, Y., Chen, X., Dardick, C., Ruan, D., et al. (2008) Rice XB15, a protein phosphatase 2C, negatively regulates cell death and XA21- mediated innate immunity. PLoS Biol. 6, e231. [19] Chung, E. H., da Cunha, L., Wu, A. J., Gao, Z., Cherkis, K., et al. (2011) Specific threonine phosphorylation of a host target by two unrelated type III effectors activates a host innate immune receptor in plants. Cell Host Microbe 9, 125–136. [20] Liu, J., Elmore, J. M., Lin, Z. J., and Coaker, G. (2011) A receptor-like cytoplasmic kinase phosphorylates the host target RIN4, leading to the activation of a plant innate immune receptor. Cell Host Microbe 9, 137–146. [21] Macho, A. P., Schwessinger, B., Ntoukakis, V., Brutus, A., Segonzac, C., et al. (2014) A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation. Science. 343, 1509–1512. [22] Zhang, J., Shao, F., Li, Y., Cui, H., Chen, L., et al. (2007) A Pseudomonas syringae effector inactivates MAPKs to suppress PAMP-induced immunity in plants. Cell Host Microbe 1, 175–185. [23] Komander, D. and Rape, M. (2012) The ubiquitin code. Ann. Rev. Biochem. 81, 203–229. [24] Marino, D., Peeters, N., and Rivas, S. (2012) Ubiquitination during plant immune signaling. Plant Physiol. 160, 15–27. [25] Yee, D. and Goring, D. R. (2009) The diversity of plant U-box E3 ubiquitin ligases: from upstream activators to downstream target substrates. J. Exp. Bot. 60, 1109–1121. [26] Trujillo, M., Ichimura, K., Casais, C., and Shirasu, K. (2008) Negative regulation of PAMP-triggered immunity by an E3 ubiquitin ligase triplet in Arabidopsis. Curr. Biol. 18, 1396–1401. [27] Chronis, D., Chen, S., Lu, S., Hewezi, T., Carpenter, S. C., et al. (2013) A ubiquitin carboxyl extension protein secreted from a plant-parasitic nema-

Bhattacharjee et al.

tode Globodera rostochiensis is cleaved in planta to promote plant parasitism. Plant J. 74, 185–196. [28] Unver, T., T€ urktas¸, M., and Budak, H. (2012) In planta evidence for the involvement of a ubiquitin conjugating enzyme (UBC E2 clade) in negative regulation of disease resistance. Plant Mol. Biol. Rep. 31, 323–334. [29] Banfield, M. (2015) Perturbation of host ubiquitin systems by plant pathogen/pest effector proteins. Cell. Microbiol. 17, 18–25. [30] Nomura, K., Mecey, C., Lee, Y. N., Imboden, L. A., Chang, J. H., et al. (2011) Effector-triggered immunity blocks pathogen degradation of an immunityassociated vesicle traffic regulator in Arabidopsis. Proc. Natl. Acad. Sci. USA 108, 10774–10779. [31] Tasset, C., Bernoux, M., Jauneau, A., Pouzet, C., Brie`re, C., et al. (2010) Autoacetylation of the Ralstonia solanacearum effector PopP2 targets a lysine residue essential for RRS1-R-mediated immunity in Arabidopsis. PLoS Pathog. 6, e1001202. [32] Gilroy, E. M., Taylor, R. M., Hein, I., Boevink, P., Sadanandom, A., et al. (2011) CMPG1-dependent cell death follows perception of diverse pathogen elicitors at the host plasma membrane and is suppressed by Phytophthora infestans RXLR effector AVR3a. New Phytol. 190, 653–666. [33] van den Burg, H. A., Kini, R. K., Schuurink, R. C., and Takken, F. L. (2010) Arabidopsis small ubiquitin-like modifier paralogs have distinct functions in development and defense. Plant Cell 22, 1998–2016. [34] Lee, J., Nam, J., Park, H. C., Na, G., Miura, K., et al. (2007) Salicylic acidmediated innate immunity in Arabidopsis is regulated by SIZ1 SUMO E3 ligase. Plant J. 49, 79–90. [35] Miller, M. J., Barrett-Wilt, G. A., Hua, Z., and Vierstra, R. D. (2010) Proteomic analyses identify a diverse array of nuclear processes affected by small ubiquitin-like modifier conjugation in Arabidopsis. Proc. Natl. Acad. Sci. USA 107, 16512–16517. [36] Bhattacharjee, S., Garner, C. M., and Gassmann, W. (2013) New clues in the nucleus: transcriptional reprogramming in effector triggered immunity. Front. Plant Sci. 13, 364. €l, L. D., Arechaga, I., Pichereaux, C., et al. [37] Canonne, J., Marino, D., Noe (2010) Detection and functional characterization of a 215 amino acid Nterminal extension in the Xanthomonas type III effector XopD. PLoS One 5, e15773. [38] Tan, C. M., Li, M. Y., Yang, P. Y., Chang, S. H., Ho, Y. P., et al. (2015) Arabidopsis HFR1 is a potential nuclear substrate regulated by the Xanthomonas type III effector XopDXcc8004. PLoS One 10, e0117067. [39] Elrouby, N., Bonequi, M. V., Porri, A., and Coupland, G. (2013) Identification of Arabidopsis SUMO-interacting proteins that regulate chromatin activity and developmental transitions. Proc. Natl. Acad. Sci. USA 110, 19956– 19961. [40] Boyle, P. C. and Martin, G. B. (2015) Greasy tactics in the plant-pathogen molecular arms race. J. Exp. Bot. 66, 1607–1616. [41] Hemsley, P. A., Weimar, T., Lilley, K. S., Dupree, P., and Grierson, C. S. (2013) A proteomic approach identifies many novel palmitoylated proteins in Arabidopsis. New Phytologist 197, 805–814. [42] Qi, D., Dubiella, U., Kim, S. H., Sloss, D. I., Dowen, R. H., et al. (2014) Recognition of the protein kinase AVRPPHB SUSCEPTIBLE1 by the disease resistance protein resistance to pseudomonas Syringae5 is dependent on Sacylation and an exposed loop in AVRPPHB SUSCEPTIBLE1. Plant Physiol. 164, 340–351. [43] Romeis, T. and Herde, M. (2014) From local to global: CDPKs in systemic defense signaling upon microbial and herbivore attack. Curr. Opin. Plant Biol. 20, 1–10. [44] Lu, S. X. and Hrabak, E. M. (2013) The myristoylated amino-terminus of an Arabidopsis calcium-dependent protein kinase mediates plasma membrane localization. Plant Mol. Biol. 82, 267–278. [45] Dubiella, U., Seybold, H., Durian, G., Komander, E., Lassig, R., et al. (2013) Calcium-dependent protein kinase/NADPH oxidase activation circuit is required for rapid defense signal propagation. Proc. Natl. Acad. Sci. USA 110, 8744–8749.

531

IUBMB LIFE

[46] Batistic, O., Waadt, R., Steinhorst, L., Held, K., and Kudla, J. (2010) CBLmediated targeting of CIPKs facilitates the decoding of calcium signals emanating from distinct cellular stores. Plant J. 61, 211–222. [47] Kawano, Y., Kaneko-Kawano, T., and Shimamoto, K. (2014) Rho family GTPase dependent immunity in plants and animals. Front. Plant Sci. 5, 1– 12. [48] Paulick, M. G. and Bertozzi, C. R. (2008) The glycosylphosphatidylinositol anchor: a complex membrane-anchoring structure for proteins. Biochemistry 47, 6991–7000. [49] Knepper, C., Savory, E. A., and Day, B. (2011) The role of NDR1 in pathogen perception and plant defense signaling. Plant Signal. Behav. 6, 1114–1116. [50] Qi, Y., Tsuda, K., Glazebrook, J., and Katagiri, F. (2011) Physical association of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) immune receptors in Arabidopsis. Mol. Plant Pathol. 12, 702–708. [51] Ko, J. H., Kim, J. H., Jayanty, S. S., Howe, G. A., and Han, K. H. (2006) Loss of function of COBRA, a determinant of oriented cell expansion, invokes cellular defense responses in Arabidopsis thaliana. J. Exp. Bot. 57, 2923–2936. [52] Mengel, A., Chaki, M., Shekariesfahlan, A., and Lindermayr, C. (2013) Effect of nitric oxide on gene transcription—S-nitrosylation of nuclear proteins. Front. Plant Sci. 4, 1–7. [53] Feechan, A., Kwon, E., Yun, B. W., Wang, Y., Pallas, J. A., et al. (2005) A central role for S-nitrosothiols in plant disease resistance. Proc. Natl. Acad. Sci. USA 102, 8054–8059. rucci, C., Espunya, M. C., Dıaz, M., Chabannes, M., and Martınez, M. C. [54] Ruste (2007) S-nitrosoglutathione reductase affords protection against pathogens in Arabidopsis, both locally and systemically. Plant Physiol. 143, 1282–1292. [55] van Doorn, W. G. and Papini, A. (2013) Ultrastructure of autophagy in plant cells: a review. Autophagy 9, 1922–1936. [56] Hatsugai, N., Yamada, K., Goto-Yamada, S., and Hara-Nishimura, I. (2015) Vacuolar processing enzyme in plant programmed cell death. Front. Plant Sci. 6, 234. [57] Solomon, M., Belenghi, B., Delledonne, M., Menachem, E., and Levine, A. (1999) The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants. Plant Cell 11, 431–444. lez-Melendi, P., Cuartas-Lanza, R., Ant [58] Rodrıguez-Herva, J. J., Gonza unezLamas, M., Rıo-Alvarez, I., et al. (2012) A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses. Cell Microbiol. 14, 669–681.

532

[59] Nick, P. (2013) Microtubules signaling and abiotic stress. Plant J. 75, 309– 323. [60] Lee, A. H.-Y., Hurley, B., Felsensteiner, C., Yea, C., Ckurshumova, W., et al. (2012) A bacterial acetyltranferase destroys plant microtubule networks and blocks secretion. PLoS Pathog. 8, e1002523. [61] Jiang, S., Yao, J., Ma, K.-W., Zhou, H., Song, J., et al. (2013) Bacterial effector activates Jasmonate signaling by directly targeting JAZ transcriptional repressors. PLoS Pathog. 9, e1003715. mousaygue, D., et al. [62] Le Roux, C., Huet, G., Jauneau, A., Camborde, L., Tre (2015) A receptor pair with an integrated decoy converts pathogen disabling of transcription factors to immunity. Cell 161, 1074–1088. [63] Sarris, P. F., Duxbury, Z., Huh, S. U., Ma, Y., Segonzac, C., et al. (2015) A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors. Cell 161, 1089–1100. [64] Starheim, K. K., Gevaert, K., and Arnesen, T. (2012) Protein N-terminal acetyltransferases: when the start matters. Trends Biochem. Sci. 37, 152–161. [65] Xu, F., Huang, Y., Li, L., Gannon, P., Linster, E., et al. (2015) Two N-terminal acetyltransferases antagonistically regulate the stability of a Nod-like receptor in Arabidopsis. Plant Cell, 27:, 1547–1562. [66] Hedberg, C. and Itzen, A. (2015) Molecular perspectives on protein adenylylation. ACS Chem. Biol. 10, 12–21. [67] Nicaise, V., Joe, A., Jeong, B. R., Korneli, C., Boutrot, F., et al. (2013) Pseudomonas HopU1 modulates plant immune receptor levels by blocking the interaction of their mRNAs withGRP7. EMBO J. 32, 701–712. [68] Zhou, J., Wu, S., Chen, X., Liu, C., Sheen, J., et al. (2014) The Pseudomonas syringae effector HopF2 suppresses Arabidopsis immunity by targeting BAK1. Plant J. 77, 235–245. [69] Feng, F., Yang, F., Rong, W., Wu, X., Zhang, J., et al. (2012) A Xanthomonas uridine 50 -monophosphate transferase inhibits plant immune kinases. Nature 485, 114–118. [70] Kinch, L. N., Yarbrough, M. L., Orth, K., and Grishin, N. V. (2009) Fido, a novel AMPylation domain common to fic, doc, and AvrB. PLoS One 4, e5818. [71] Desveaux, D., Singer, A. U., Wu, A.-J., McNulty, B. C., Musselwhite, L., et al. (2007) Type III effector activation via nucleotide binding, phosphorylation and host target interaction. PLoS Pathog. 3, e48. [72] Yu, X. and LaBaer, J. (2015) High-throughput identification of proteins with AMPylation using self-assembled human protein (NAAPA) microarrays. Nat. Protoc. 10, 756–767.

Regulation of Pathogen Surveillance and Signaling in Plants