Targeting of plant pattern recognition receptor-triggered immunity by bacterial type-III secretion system effectors

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ScienceDirect Targeting of plant pattern recognition receptortriggered immunity by bacterial type-III secretion system effectors Alberto P Macho and Cyril Zipfel During infection, microbes are detected by surface-localized pattern recognition receptors (PRRs), leading to an innate immune response that prevents microbial ingress. Therefore, successful pathogens must evade or inhibit PRR-triggered immunity to cause disease. In the past decade, a number of type-III secretion system effector (T3Es) proteins from plant pathogenic bacteria have been shown to suppress this layer of innate immunity. More recently, the detailed mechanisms of action have been defined for several of these effectors. Interestingly, effectors display a wide array of virulence targets, being able to prevent activation of immune receptors and to hijack immune signaling pathways. Besides being a fascinating example of pathogen-host co-evolution, effectors have also emerged as valuable tools to dissect important biological processes in host cells. Addresses The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, United Kingdom Corresponding author: Zipfel, Cyril ([email protected])

Current Opinion in Microbiology 2015, 23:14–22 This review comes from a themed issue on Host–microbe interactions Edited by David Holden and Dana Philpott

http://dx.doi.org/10.1016/j.mib.2014.10.009 1369-5274/# 2014 Elsevier Ltd. All right reserved.

Introduction Major bacterial pathogens of plants include members of the Pseudomonas, Xanthomonas, Ralstonia, Agrobacterium and Pectobacterium genera, amongst others. As all would-be pathogens, they face efficient innate immune responses when first coming into contact with plant cells. Surface-localized pattern recognition receptors (PRRs) ensure initial perception of this infectious non-self [1,2]. Plant PRRs can perceive conserved bacterial molecules, usually termed pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs), such as bacterial flagellin, elongation factor Tu (EF-Tu), peptidoglycan (PGN) or lipopolysaccharides [3,4]. Current Opinion in Microbiology 2015, 23:14–22

Plant PRRs are either plasma membrane-resident receptor kinases (RKs) or receptor-like proteins (RLPs) [3,4]. Plant PRRs exist within dynamic protein complexes composed of PRRs themselves, co-receptors, regulatory proteins, and components directly involved in intracellular signaling [3,5]. Upon PAMP perception, the activation of different PRR complexes leads to the phosphorylation and activation of cytoplasmic receptor-like cytoplasmic kinases (RLCKs) [5,6]. Downstream immune responses include the activation of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), the rapid generation of reactive oxygen species (ROS), the expression of immune-related genes, and the deposition of callose at the cell wall [7,8]. Collectively, these responses culminate in PRR- or PAMP-triggered immunity (PTI), which is sufficient to halt the ingress or growth of most microbes. Therefore, successful pathogens must either evade or actively suppress this important layer of plant innate immunity in order to cause disease. Several Gramnegative bacterial pathogens do so by using the type III-secretion system (T3SS), which resembles a molecular syringe that injects proteins directly inside the host cell [9]. These proteins are usually referred to as type III effectors (T3Es), and promote bacterial infection by manipulating host cell functions, including immunity. The relevance of this secretion system in the overall virulence of plant pathogenic bacteria is best proven by the near-complete loss of infectivity of strains unable to secrete T3Es [10]. However, resistant plants employ intracellular immune receptors, most often of the nucleotide-binding domain-LRR receptor (NLR) type, which directly or indirectly recognize T3Es, thereby inducing effector-triggered immunity (ETI; [2]. Plant pathogenic bacteria also employ additional virulence strategies, such as evasion of PAMP detection or production of toxins, among others [7,11]. Here, we will review the different strategies evolved by T3Es from plant pathogenic bacteria to impede PTI by directly targeting PRR themselves, their biogenesis, or key signaling components acting downstream of PRR activation. We will mainly focus on T3Es for which molecular plant targets are known. Therefore, we apologize to colleagues whose work on other effectors is not covered. www.sciencedirect.com

Inhibition of plant PTI by bacterial type-III effectors Macho and Zipfel 15

Effectors that target de novo PRR biogenesis In response to PAMP perception, transcripts of a large number of PRRs are rapidly up-regulated [12–14]. This may represent an amplification mechanism, but also could serve to replenish the plasma membrane with ligand-free PRRs for subsequent waves of PAMP recognition and PRR activation. The targeting of this de novo PRR biogenesis could therefore represent a virulence strategy to block the establishment of sustained PTI during infection (Figure 1). Interestingly, the T3E HopU1 from Pseudomonas syringae pv. tomato (Pto) DC3000 is an mono-ADP-ribosyltransferase that specifically modifies several plant RNA binding proteins, such as GRP7 [15,16]. The action of HopU1 impedes their RNA-binding function and inhibits PTI [15,16]. Notably, GRP7 can bind mRNAs encoding the leucine-rich repeat (LRR)-RKs FLS2 and EFR (which

are the PRRs for bacterial flagellin and EF-Tu, respectively), potentially contributing to the pathogen-induced translation of these mRNAs. HopU1 affects this binding, causing reduced PRR protein levels in planta upon infection with Pto DC3000 [17] (Figure 1). Notably, the T3E HopQ1 from Pto DC3000 activates the production of the plant hormone cytokinin, which results in reduced FLS2 expression [18] (Figure 1). HopQ1 exhibits similarities with nucleoside hydrolases and might contribute to enhanced cytokinin production by catalysing the conversion of its precursor N6-(2-isopentenyl)adenine-9-riboside-5’-monophosphate (iPRMP) [18]. It is worth mentioning that other studies on HopQ1 from Pto DC3000 or P. syringae pv. phaseolicola (Pph) 1448A identified plant 14-3-3 proteins as interactors for this effector, but have failed to observe alterations in early

Figure 1

(a)

(c)

(b) Time upon contact with bacteria

T3SS T3E T3E T3E

HopU1

Immune signalling

T3Es

T3E T3E

T3E

mRNAbinding proteins

PRR mRNAs

Immune signalling HopQ1

High cytokinin levels

Current Opinion in Microbiology

Targeting of de novo PRR biogenesis during bacterial infection. (A) Basal levels of PRRs perceive the initial bacterial PAMPs present in the apoplast and trigger immune signaling in the plant cell. (B) Immune signaling leads to a first wave of defense-related responses, which include de novo synthesis of immune components. (C) As a consequence, PRRs and other signaling components accumulate at higher levels in order to build up defenses against a potential subsequent bacterial invasion. Pseudomonas syringae T3Es HopU1 and HopG1 are able to suppress this mechanism of plant reinforcement by targeting de novo PRR biosynthesis (B). www.sciencedirect.com

Current Opinion in Microbiology 2015, 23:14–22

16 Host–microbe interactions

PTI outputs triggered by the flagellin-derived immunogenic epitope flg22 upon HopQ1 expression in planta [19– 21]. In addition, HopQ1 orthologs from Pto DC3000, Pph 1448A and Xanthomonas oryzae pv. oryzae (Xoo) KACC10331 do not exhibit nucleoside hydrolase activity [19–21], and a recent structural study on XopQ1Xoo revealed that it can bind adenosine diphosphate ribose (ADPR) [22]. It is therefore possible that HopQ1 has different substrates to modulate PTI via distinct mechanisms.

Effectors that target directly PRRs and their co-receptors Several T3Es have been found to target PRRs directly, causing their degradation and/or the inhibition of their PAMP-induced activation (Figure 2). An example of T3E that exerts both functions is AvrPtoB, which is a multi-domain protein with orthologs in several P. syringae pathovars. AvrPtoB uses a C-terminal E3 ligase

domain to ubiquitinate and induce the proteasomemediated degradation of PRRs, such as FLS2, and the LysM-RK CERK1, which is involved in PGN perception [23–25]. Moreover, AvrPtoB possesses both an N-terminal and a central domain that can act as kinase inhibitors to target important kinases involved in PTI. The Nterminal domain binds the LysM-RK Bti9, which is the tomato ortholog of CERK1 [26]. In contrast, the central domain can interact with FLS2 and BAK1 [27,28], an LRR-RK that acts as an important co-receptor for several LRR-type PRRs [3,5]. Similarly, another P. syringae effector, AvrPto, targets FLS2 and EFR to inhibit their kinase activity and block PTI signaling [29,30]. AvrPto may target additional PRRs, as expression of AvrPto in plants leads to the inhibition of immune responses triggered by multiple PAMPs [28]. Notably, AvrPto can also interact with BAK1, thereby inhibiting its interaction with FLS2 [28]. Despite the fact that AvrPto is a potent inhibitor

Figure 2

PRRs Co-receptors PAMP

AvrPtoB

AvrPto

? AvrPto

AvrPtoB HopF2 HopF2 HopAO1

AvrRpt2

Xoo2875

AvrB AvrPphB

HopF2

MEKK MKK MPK

RLCKs

RIN4

XopAC

Xoo1488 HopAI1

?

AvrRpm1

IMMUNE RESPONSES

Current Opinion in Microbiology

Multiple targeting of early PTI components by T3Es. Simplified diagram of early PTI components targeted by T3Es from Pseudomonas and Xanthomonas strains. Current Opinion in Microbiology 2015, 23:14–22

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Inhibition of plant PTI by bacterial type-III effectors Macho and Zipfel 17

of PRR-BAK1 complex activation, whether PRRs and/or BAK1 represent(s) the true physiological target(s) during infection remains a matter of debate [29,31].

recently revealed for the recognition of HopZ1a (an acetyltransferase from P. syringae pv syringae A2) action on the RLCK ZED1 by the NLR ZAR1 [46].

HopAO1 (formerly HopPtoD2) from Pto DC3000 exhibits tyrosine phosphatase activity that is important for suppression of PAMP-induced responses [32–34]. A recent study revealed that HopAO1 interacts directly with FLS2 and EFR [35]. Interestingly, HopAO1 dephosphorylates tyrosine residues on EFR to inhibit the activation of the EFR-BAK1 complex and therefore subsequent initiation of immune signalling [35].

The T3E Xoo1488 from Xoo MAFF311018 targets several rice RLCKs, including OsRLCK185. These RLCKs act downstream of the rice LysM-RK CERK1 [47], which is involved in the perception of bacterial PGN and fungal chitin [48,49]. The mechanisms by which Xoo1488 impedes OsRLCK185 activation remain to be characterized.

Effectors that target MAP kinases The Pto DC3000 T3E HopF2 is an mono-ADP-ribosyltransferase that also suppresses early PTI outputs [36– 38]. HopF2 contains a potential myristoylation site required for its plasma membrane localization and virulence functions [39]. Recently, it was found that BAK1 is a major virulence target of HopF2 [31], although the underlying mechanisms remain unknown. BAK1 seems to be a conserved target for T3Es from different bacterial species that infect different plant hosts. The T3E Xoo2875 from Xoo KACC10331 interacts with the rice ortholog of BAK1 and increases rice susceptibility to Xoo, although the mechanism of PTI suppression remains unknown [40].

Effectors that target PRR-associated cytoplasmic kinases The T3E AvrPphB from Pph 1448A is a cysteine protease related to Yersinia pestis YopT [41]. It can inhibit PTI signaling by cleaving multiple members of the BIK1 family of RLCKs that are direct substrates of multiple PRR complexes [5,42] (Figure 2). Interestingly, plants harboring the intracellular immune receptor RPS5, an NLR protein, are able to detect AvrPphB-mediated cleavage of one of those RLCKs, PBS1, to initiate ETI [43]. This is good illustration that, while T3Es are key virulence factors, they can also betray the pathogen by revealing their presence to plants that have evolved direct or indirect surveillance mechanisms for these effectors or their action. An additional interesting mechanism to target RLCKs is displayed by the T3E AvrAC/XopAC from X. campestris pv. campestris (Xcc) 8004. This effector displays a previously uncharacterized uridine 5’-monophosphate transferase enzymatic activity to promote bacterial virulence [44]. AvrAC transfers uridine 5’-monophosphate to important phosphorylation sites in the activation loop of several RLCKs, including BIK1, inhibiting its kinase activity, thereby blocking the activation of PTI responses [44]. Notably, AvrAC triggers ETI in specific Arabidopsis accessions in a manner dependent on the RLCKs PBL2 and RIPK [45]. This suggests that their modifications may be sensed by an intracellular NLR, as www.sciencedirect.com

HopAI1 from Pto DC3000 inactivates MPK3, MPK4 and MPK6 in an irreversible manner [50,51]. This is achieved through a phosphothreonine lyase activity, similar to what is observed in the Shigella flexneri T3E OspF, which targets animal MAPKs [50,52]. However, in plants carrying the NLR SUMM2, inactivation of MPK4 by HopAI1 results in ETI [51]. The fact that both RLCKs and MAPKs are guarded by resistance proteins support the notion that they constitute key components of plant immune signaling targeted by pathogen virulence factors. In addition to targeting BAK1 (see above), HopF2 also targets MKK5 through ADP-ribosylation to block its phosphorylation and consequent activation of downstream MAPKs [53]. It is thus apparent that HopF2 has multiple host targets. It also seems to regulate post-transcriptional gene silencing, via yet unknown mechanisms [54]. Furthermore, HopF2, as well as the orthologous HopF1 from Pph 1449b, also directly interact with RIN4, an important regulator of plant immunity [11,55,56]. Notably, several other T3Es from P. syringae (e.g. AvrRpm1, AvrRpt2 and AvrB) target RIN4 and suppress flg22-induced responses [57,58]. Interestingly, RIN4 cleavage by AvrRpt2 leads to ETI via activation of the NLR RPS2, while RIN4 phosphorylation promoted by AvrRmp1 or AvrB activities leads to ETI via the NLR RPM1 [59–61]. It is however still unclear how perturbation of RIN4 by AvrRpm1, AvrRpt2 or AvrB regulates PTI.

Effectors that target vesicle trafficking to suppress PTI Vesicle trafficking is an important cellular function. Regarding plant immune functions, vesicle trafficking is necessary for the transport of immune receptors and associated proteins, and for the secretion of immune related molecules and antimicrobial compounds upon pathogen detection [62]. HopM1 from Pto DC3000 interacts with and induces the degradation of the ADP ribosylation factor (ARF) guanine nucleotide exchange factor (GEF) protein AtMIN7 in a Current Opinion in Microbiology 2015, 23:14–22

18 Host–microbe interactions

proteasome-dependent manner [63]. AtMIN7 localizes to the trans-Golgi/early endosome network and is important for PTI, potentially mediating immune-related vesicle trafficking [63–65]. Several T3Es from X. campestris pv. vesicatoria (Xcv) target plant protein secretion pathways to suppress PTI. As an example, XopJ, a member of the YopJ family of SUMO

peptidases and acetyltransferases, interferes with protein secretion to inhibit immune-associated callose deposition at the cell wall [66,67]. Additionally, XopB and XopS suppress PAMP-triggered gene expression [68]. XopB localizes to the Golgi apparatus and cytoplasm and interferes with eukaryotic vesicle trafficking [68]. However, specific PTI-related targets are still unknown for these Xcv effectors.

Table 1 Examples of bacterial type III effectors that suppress PTI. Effector name

Bacterial species

HopU1

Pseudomonas syringae pv. tomato

HopQ1

Pseudomonas syringae pathovars

AvrPtoB

Pseudomonas syringae pathovars

AvrPto HopAO1 HopF1/F2

Pseudomonas syringae pv. tomato Pseudomonas syringae pv. tomato Pseudomonas syringae pathovars

Xoo2875 AvrPphB

Xanthomonas oryzae pv. oryzae Pseudomonas syringae pv. phaseolicola Xanthomonas campestris pv. campestris

AvrAC/XopAC

Activity

PTI-related plant target

Plant species

Mono-ADPribosyltransferase Putative nucleoside hydrolase

GRP7, other RNAbinding proteins Cytokinin biosynthesis/ 14-3-3 proteins

Arabidopsis

[15,16,17] [18,19,21]

E3-ubiquitin ligase/Kinase inhibitor Kinase inhibitor Tyrosine phosphatase ADP-ribosyltransferase

PRRs

Arabidopsis Tomato Bean Arabidopsis

[28–30,31] [32–34,35] [31,36–39,53]

Unknown Cysteine protease

OsSERK2 RLCKs

Arabidopsis Arabidopsis Nicotiana benthamiana Arabidopsis Bean Rice Arabidopsis

Uridine 5’-monophosphate transferase Unknown Phosphothreonine lyase Unknown

RLCKs

Arabidopsis

[44]

RLCKs MAPKs

Rice Arabidopsis

[47] [36,50,51]

Vesicle trafficking/ 14-3-3 proteins

Arabidopsis Nicotiana benthamiana Arabidopsis

[37,63,64,73]

Arabidopsis Tomato Arabidopsis Arabidopsis Arabidopsis

[68] [74,75] [83] [84] [85]

Arabidopsis Arabidopsis Arabidopsis Arabidopsis Nicotiana benthamiana Arabidopsis Nicotiana benthamiana Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis Nicotiana benthamiana

[86] [57] [57] [58] [37]

PRRs EFR and FLS2 BAK1/MAPKs

Xoo1488 HopAI1

Xanthomonas oryzae pv. oryzae Pseudomonas syringae pv. tomato

HopM1

Pseudomonas syringae pv. tomato

XopJ

X. campestris pv. vesicatoria

XopB/XopS XopN XopL XopR AvrBsT

X. campestris X. campestris X. campestris Xanthomonas X. campestris

pv. vesicatoria pv. vesicatoria pv. vesicatoria oryzae pv. oryzae pv. vesicatoria

Putative SUMO peptidase Unknown Unknown E3 Ligase Unknown Acetyltransferase

HopK1 AvrRpt2 AvrRpm1 AvrB AvrE

Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas

syringae syringae syringae syringae syringae

Unknown Cysteine protease Unknown Unknown Unknown

Protein secretion/ callose deposition Vesicle trafficking TFT1 (14-3-3 protein) Unknown Unknown Microtubuleassociated proteins Chroloplasts RIN4 RIN4 RAR1/RIN4 Unknown

HopG1

Pseudomonas syringae pv. tomato

Unknown

Mitochondria

HopS1 HopAF1 HopT1-1 HopT1-2 HopAA1-1 HopC1 NopM

Pseudomonas syringae Pseudomonas syringae Pseudomonas syringae Pseudomonas syringae Pseudomonas syringae Pseudomonas syringae Rhizobium sp.

Unknown Unknown Unknown Unknown Unknown Unknown E3 Ubiquitin ligase

Unknown Unknown Unknown Unknown Unknown Unknown Unknown

pv. tomato pv. tomato pv. maculicola pv. tomato

pv. pv. pv. pv. pv. pv.

tomato tomato tomato tomato tomato tomato

Current Opinion in Microbiology 2015, 23:14–22

Reference(s)

[23,24,26–28]

[40] [42]

[66]

[37,87]

[36] [36] [36] [36] [36] [36] [88]

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Inhibition of plant PTI by bacterial type-III effectors Macho and Zipfel 19

Effectors that target 14-3-3 proteins to suppress PTI Consistent with the importance of protein phosphorylation for immune signaling [5], 14-3-3 proteins are emerging as important players in plant immunity [69–71]. 14-33 proteins bind to specific motifs containing phosphorylated serine or threonine residues, regulating the function of target proteins by several different mechanisms [72]. Several T3Es interact with 14-3-3 proteins, which could serve as a mechanism to disrupt immunity-associated 143-3 functions, or a way to exploit host 14-3-3 proteins to facilitate other functions of the T3E inside the plant cell. The T3E HopM1 from Pto DC3000 targets 14-3-3 proteins and disrupts several PAMP-triggered responses independently of the previously identified target AtMIN7 [73]. Interestingly, chemical disruption of the 14-3-3 interactions with their client proteins similarly disrupts several PAMP-triggered responses and restores virulence of a HopM1-deficient Pto DC3000 mutant [73]. The Xcv effector XopN interacts with the tomato RK TARK1 and several 14-3-3 isoforms [74]. One of these isoforms, TFT1, is a positive regulator of PTI, and its targeting by XopN induces the formation of a TFT1XopN-TARK1 complex which results in the suppression of PTI in tomato [74,75]. Other T3Es from Xcv (AvrRxc and XopQ), and the XopQ1 homologs in Pto and Pph (HopQ1) interact with 14-3-3 in different plant species to contribute to bacterial virulence [19,21,76]. However, it is unclear whether this virulence activity involves suppression of PTI.

Concluding remarks The detailed study of T3Es, mainly from Pseudomonas and Xanthomonas species, has revealed that one major function of these effectors is to actively suppress PTI by directly targeting key components of this important layer of immune recognition. As such, T3Es (and virulence effectors in general) can be considered as very useful biological tools to dissect key biological processes and may also reveal novel activities evolved to achieve their virulence functions [77,78].

components. In addition to the example listed in this review, these two points are also supported by recent large-scale interactomics studies [79]. Particularly, it is becoming obvious that early PTI components are cellular hubs for suppression by effectors from a single bacterial species or from different bacterial genera or species (Figure 2). Of note, even during a successful infection by a bacterial pathogen, PTI suppression is not completely achieved, as exemplified by the observation that alterations of plant immune components (e.g. PRRs) can render the plant hyper- or hypo-susceptible to pathogens [14,80]. Therefore, redundant targeting of a pathway (e.g. PTI signaling) by multiple effectors from a given pathogen may be important to ensure optimal immune suppression over the course of the infection. Our knowledge on the hierarchy and dynamics of single effector delivery within host cells is still limited [81,82]. Furthermore, most studies on T3Es and their function are based on over-expression in host cells. While this approach is essential to understand what a single effector can do, it will be important to determine the precise amount of effector proteins delivered within single cells during infection and what is their sub-cellular localization at these physiological concentrations. This could be particularly important for effectors that do not exhibit enzymatic activities, as their ability to suppress host proteins may depend on the amount of their respective target proteins. The development of more sensitive cell biological, biochemical and proteomics approaches will be required to address this important question. Importantly, not all T3Es are necessarily involved in the suppression of host immunity. They are likely to have additional roles in nutrient acquisition and in the establishment of a compatible environment for the bacterial replication. Targets for many T3Es are still unknown, and T3Es display a wide range of sub-cellular localization within plant cells, targeting diverse organelles such as chloroplasts or mitochondria (Table 1; [11]), which promises further exciting discoveries in the future about how these fascinating proteins remodel the host’s physiology to the benefit of the pathogen.

Acknowledgements A number of general observations can be drawn based on our current knowledge. Namely, a single effector can have multiple targets. While some effectors may carry different activities (e.g. AvrPtoB), other effectors have a unique activity (e.g. kinase inhibition) that can target host proteins without the need for specificity (e.g. AvrPto is a kinase inhibitor that can globally target receptor kinases, whether they are involved in immunity or not). In this sense, effectors can be seen as weapons of mass destruction delivered inside host cells, being evolutionarily selected based on their final contribution to the infection process. Also, different effectors can target the same www.sciencedirect.com

Research in the Zipfel laboratory is funded by the Gatsby Foundation and the European Research Council. We thank all members of the Zipfel laboratory for helpful discussions. We apologize to colleagues whose work could not be cited due to space limits.

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18. Hann DR, Dominguez-Ferreras A, Motyka V, Dobrev PI,  Schornack S, Jehle A, Felix G, Chinchilla D, Rathjen JP, Boller T: The Pseudomonas type III effector HopQ1 activates cytokinin signaling and interferes with plant innate immunity. New Phytol 2013, 201:585-598. This report provides a novel virulence strategy of a T3E, HopQ1, which induces plant cytokinin production. Increased cytokinin levels correlates with a reduction on FLS2 gene expression and protein accumulation.

34. Underwood W, Zhang S, He SY: The Pseudomonas syringae type III effector tyrosine phosphatase HopAO1 suppresses innate immunity in Arabidopsis thaliana. Plant J 2007, 52:658672.

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Inhibition of plant PTI by bacterial type-III effectors Macho and Zipfel 21

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66. Bartetzko V, Sonnewald S, Vogel F, Hartner K, Stadler R, Hammes UZ, Bornke F: The Xanthomonas campestris pv. vesicatoria type III effector protein XopJ inhibits protein secretion: evidence for interference with cell wall-associated defense responses. Mol Plant-Microbe Interact: MPMI 2009, 22:655-664.

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