Of guards, decoys, baits and traps: pathogen perception in plants by type III effector sensors

Available online at www.sciencedirect.com

ScienceDirect Of guards, decoys, baits and traps: pathogen perception in plants by type III effector sensors Madiha Khan1, Rajagopal Subramaniam1,2 and Darrell Desveaux1,3 Effector-triggered immunity (ETI) is conferred by dominant plant resistance (R) genes, which encode predominantly nucleotide-binding and leucine-rich repeat domain proteins (NLRs), against cognate microbial avirulence (Avr) genes, which include bacterial type III secreted effectors (T3Es). The ‘guard model’ describes the mechanism of T3E perception by plants, whereby NLRs monitor host proteins (‘sensors’) for T3Einduced perturbations. This model has provided a molecular framework to understand T3E perception and has rationalized how plants can use a limited number of NLRs (160 in Arabidopsis) to contend with a potentially limitless number of evolving effectors. In this review we provide a characteristic overview of plant T3E sensors and discuss how these sensors convey the presence of T3Es to NLR proteins to activate ETI. Addresses 1 Department of Cell and Systems Biology, University of Toronto, 25 Willcocks Street, Toronto, Ontario M5S 3B2, Canada 2 Agriculture and Agri-Food Canada/Agriculture et Agroalimentaire Canada, KW Neatby Building, 960 Carling Avenue, Ottawa, Ontario K1A 0C6, Canada 3 Centre for the Analysis of Genome Function and Evolution, University of Toronto, 25 Willcocks Street, Toronto, Ontario M5S 3B2, Canada Corresponding authors: Subramaniam, Rajagopal ([email protected]) and Desveaux, Darrell ([email protected])

Current Opinion in Microbiology 2016, 29:49–55 This review comes from a themed issue on Host-microbe interactions: bacteria Edited by Elizabeth Hartland and Anthony Richardson

http://dx.doi.org/10.1016/j.mib.2015.10.006 1369-5274/# 2015 Elsevier Ltd. All rights reserved.

Plants possess two main classes of proteins to detect microbial molecules and induce innate immunity. Cell surface pattern recognition receptors (PRRs) bind extracellular microbe-associated molecular patterns (MAMPs) and activate PRR-triggered immunity (PTI). Intracellular NLR proteins recognize pathogen-specific effector molecules and activate ETI. T3Es delivered into plant cells by bacterial type III secretion systems represent an important class of effectors recognized by NLR proteins. As www.sciencedirect.com

proposed by the guard hypothesis of effector perception by plant NLRs, it is T3E-induced modifications of host proteins (hereafter T3E sensors) that are detected by NLRs to activate ETI. T3E sensors include NLR-associated proteins (‘autonomous sensors’) as well as sensor domains [1] of NLR proteins (‘integrated sensors’), which provide direct and indirect perception, respectively (Figure 1a). In addition, recognition of transcription activator-like effectors (TALEs) can involve integrating DNA T3E sensors known as effector-binding elements (EBEs) in the promoters of executor R genes (nonNLR) whose transcriptional activation by TALEs is sufficient to promote ETI (Figure 1b) [2]. T3E sensors include bona fide T3E virulence targets as well as proteins that do not appear to contribute to pathogenesis. In scenarios where the T3E sensors represent proteins that are targeted by T3Es to promote pathogen virulence (aka effector-triggered susceptibility; ETS), the sensors are referred to as ‘guardees’ since the NLR is ‘guarding’ a T3E virulence target [3,4]. In scenarios where the T3E sensors do not represent T3E virulence targets, they are referred to as ‘decoys’ or ‘baits’ and likely represent mimics of operative T3E virulence targets [5,6]. In both cases, the sensor traps the T3E in an NLR resistance complex resulting in ETI (Figure 1c). Since a major virulence function of T3Es is to suppress PTI, it is perhaps not surprising that T3E sensors represent crucial components of the PTI-signaling cascade including MAMP perception, signal transduction and transcriptional reprogramming [7,8].

Protecting the RIN4 hub of plant immunity: on guard for thee RIN4 is one of the best-characterized T3E sensors and serves as a scaffold for numerous post-translational modifications that contribute to both ETI and PTI (Figure 2a,b). It is a small, unstructured protein that is membrane-associated with PRR complexes and is targeted by at least four unrelated T3Es, AvrRpm1, AvrB, AvrRpt2 and HopF2, and monitored by two Arabidopsis NLR proteins, RPM1 and RPS2 as well as the soybean NLR RPG1-B [9–14]. Cleavage of RIN4 by the T3E protease AvrRpt2 leads to RIN4 disappearance and activation of ETI by RPS2 (Figure 2a) [15,16]. As such, rin4 loss-of-function mutants display RPS2-dependent lethality, presumably due to Current Opinion in Microbiology 2016, 29:49–55

50 Host-microbe interactions: bacteria

Figure 1

(a)

Autonomous Sensor

T3E

NLR

Integrated Sensor

NLR

(c)

Guardee Scenario

NLR

-NLR

+NLR

T3E

T3E

Sensor

NLR Sensor

Sensor Sensor T3E

Effector-Triggered Susceptibility

Effector-Triggered Immunity

Effector-Triggered Immunity

Decoy Sensor Scenario -like

(b) Effector-Triggered Susceptibility Susceptibility (S)

Effector-Triggered Immunity

TALE

EBE

EBE

Sensor T3E

Sensor -like

Sensor

NLR

T3E

Resistance (R) Current Opinion in Microbiology

Features of the plant T3E sensors. (a) An NLR-associated T3E sensor or ‘Autonomous Sensor’ and T3E sensor incorporated as a domain of an NLR protein or ‘Integrated Sensor’. In both cases the T3E alters the T3E sensor leading to activation of an NLR protein which induces effectortriggered immunity (ETI). (b) TALEs manipulate host gene expression by binding effector-binding elements (EBEs) in the promoters of plant genes. Expression of Susceptibility (S) genes promotes effector-triggered susceptibility (i.e. disease). On the other hand, plants have evolved EBEs as T3E sensors by incorporating them into the promoters of executor Resistance (R) genes whose expression promotes ETI. (c) T3E sensors can be categorized as ‘guardees’ or ‘decoys’ depending on their role in effector-triggered susceptibility. In the absence of NLR proteins, ‘guardee’ sensors represent virulence targets of T3Es and T3E-induced modifications promote effector-triggered susceptibility (top left). ‘Decoy’ sensors are mimics of the T3E virulence target (sensor-like), but Decoy modification does not promote effector-triggered susceptibility (bottom left). In the presence of NLR proteins, modification of either ‘guardee’ or ‘decoy’ sensors by the T3E leads to activation of NLR-mediated ETI (right).

constitutive ETI activation [11,14]. In the absence of RPS2, AvrRpt2 cleavage products of RIN4 can suppress PTI and promote bacterial growth [17]. On the other hand, the T3Es AvrB and AvrRpm1 enhance the expression of the Arabidopsis kinase RIPK, which phosphorylates RIN4 at an evolutionarily conserved threonine residue (T166; Figure 2b) [18,19]. Phosphorylation of T166 is sufficient and required for RPM1 activation, since the phosphomimetic mutants T166D/E result in effector-independent RPM1 activation, whereas the phosphorylation mutant T166A displays compromised RPM1 activation [18,19]. RIN4 also interacts with the prolyl-peptidyl isomerase (PPIase) ROC1 (also activates the T3E AvrRpt2), which mediates the cis/trans isomerization of RIN4 proline 149 (P149) and influences RPM1mediated ETI (Figure 2b) [20]. Mutation and deletion of P149 can suppress and activate RPM1-mediated ETI, respectively. Phosphorylation of T166 reduces the ROC1/RIN4 interaction supporting a model whereby RPM1 detects ROC1-mediated conformational changes of RIN4 (Figure 2b) [20]. In addition to acting as a T3E sensor during ETI, RIN4 also acts as a PTI sensor. Phosphorylation of RIN4 serine 141 (S141), following activation of the PRRs FLS2 or EFR, enhances PTI responses [21]. Interestingly, RIPKmediated T166 phosphorylation of RIN4 during ETI Current Opinion in Microbiology 2016, 29:49–55

suppresses the PTI-induced phosphorylation of S141, supporting a model whereby the T3Es AvrB and AvrRpm1 enhance RIPK activity to promote T166 phosphorylation and suppression of PTI [21]. In response, RPM1 has evolved to detect RIN4 phosphorylated T166 in order to detect AvrB and AvrRpm1 activity and induce ETI. Thus, RIN4 represents an excellent example of a molecular hub of the plant pathogen arms race. Multiple T3Es target RIN4, presumably to dampen the PTI response, and multiple NLRs guard RIN4 to detect T3E-induced perturbations and trigger an ETI response. As a continuation of the arms race, T3Es can also target RIN4 and RIN4associated proteins in order to interfere with ETI. Proteolytic cleavage of RIN4 by AvrRpt2 disrupts AvrB and AvrRpm1 mediated activation of RPM1 [16]. The cysteine protease HopAR1 (AvrPphB) blocks the recognition of AvrB by cleaving RIPK to prevent RIN4 phosphorylation [22]. The ADP-ribosyltransferase HopF2 interferes with AvrRpt2-induced RIN4 cleavage and blocks RPS2 activation [13]. HopF2 can ribosylate RIN4 and promote pathogen virulence without detectable NLR activation; presumably by inhibiting accumulation of RIN4 phosphorylated S141 (PTI-promoting) without increasing phosphorylated T166 (ETI-promoting) [13,21]. Overall, the role of RIN4 in both PTI and ETI makes it an ideal T3E target for immunosuppression. www.sciencedirect.com

Plant type III effector sensors Khan, Subramaniam and Desveaux 51

Figure 2

RIN4

cRIN4

Pto

Prf

ETI

Prf

Prf

nRIN4

Prf

AvrRpt2

RPS2

(c) RPS2

(a)

ETI

Pto Pto (helper) (sensor)

Pto

P+1 loop disruption

AvrPto/ AvrPtoB

AvrRpt2

AvrPto/ AvrPtoB

(b)

(d)

RI

RPS4

RPS4 RIN4

PK

RIN4

C1

ETI

RRS1

RRS1

WRKY

AvrRps4 WRKY

?

ETI

RI

C1

RO

RPM1

RO

PK

RPM1

cis/trans isomerization

AvrRpm1/ AvrB AvrRpm1/ AvrB

AvrRps4

PopP2

PopP2

Current Opinion in Microbiology

T3E perception by RIN4, Pto and RRS1. (a) RIN4, an autonomous T3E sensor, is targeted for cleavage by the T3E protease AvrRpt2 leading to disappearance of RIN4 and activation of ETI by the associated NLR protein RPS2. (b) Binding of T3Es AvrB and AvrRpm1 promotes RIN4 phosphorylation by RIPK at T166. This phosphorylation disrupts interaction between RIN4 and ROC1 which mediates cis/trans isomerization of RIN4 presumably leading to a conformational change in RIN4 resulting in RPM1 activation. (c) Oligomerization of NLR protein Prf brings two Pto kinases into close proximity. Binding of the T3Es AvrPto or AvrPtoB to one Pto kinase previously autophosphorylated on S198 leads to diruption of the P+1 loop and activation of a second Pto which transphosphorylates the initial Pto kinase at T199. Together these events activate Prfmediated ETI. As such, one Pto molecule acts as the bona fide T3E sensor whereas the other acts as a helper. (d) The T3Es PopP2 and AvrRps4 are recognized by the paired NLR proteins RPS4 and RRS1. Both T3Es target the integrated WRKY domain at the C-terminus of RRS1 to activate ETI. PopP2 acetylates the RRS1 WRKY domain at K1221 whereas the AvrRps4-induced modification is unknown.

Protecting kinases: deploy the decoys Kinases play crucial roles in PTI and represent the largest class of sensors identified to date. Kinase domains are present on PRRs as well as PRR-associated co-receptors that activate MAP kinase cascades to transduce PTI signals [8]. PTI-associated kinases represent important virulence targets of T3Es, which employ a myriad of enzymatic activities to suppress kinase targets and consequently PTI signaling [8,23]. In response, plants have evolved NLRs to guard against perturbations to PTI kinases, mainly by monitoring decoy-kinases that trap T3Es into NLR complexes. The tomato Pto gene encodes a serine/threonine protein kinase that interacts directly with two unrelated T3E proteins AvrPto and AvrPtoB as well as the NLR Prf (Figure 2c) (reviewed in [24]). Oligomerization of Prf brings at least two associated Pto kinases into close proximity, which then function cooperatively to detect T3Es [25–27]. Binding of AvrPto or AvrPtoB to one Pto kinase activates a second Pto molecule, which then trans-phosphorylates the T3E-bound Pto molecule [25,26,28]. Pto phosphorylation events, as well as www.sciencedirect.com

conformational changes in Pto due to T3E binding, result in activation of Prf-mediated ETI (Figure 2c) [25,27,29,30]. Hence, the Pto-Prf complex acts as a trap for T3Es targeting kinases, where one Pto protein acts as the T3E sensor, while a second serves as a helper required to activate Prf [28]. Both AvrPto and AvrPtoB represent kinase inhibitors. AvrPto binds to the catalytic cleft of Pto and PRRs thereby inhibiting kinase activity, whereas AvrPtoB is a relatively weak kinase inhibitor, but possesses an active E3 ligase domain that can ubiquitinate PRR kinase domains leading to proteasomal degradation [24]. Interestingly, Pto kinase is able to avoid AvrPtoB-mediated degradation by phosphorylating and inactivating its E3 ligase domain [31]. In contrast, kinases with low intrinsic activity, such as the PRR FLS2 that detects bacterial flagellin, cannot efficiently inhibit AvrPtoB E3 ligase activity and are susceptible to degradation [31–34]. It is likely that these PTI-associated kinases are the ‘bona fide’ virulence targets of AvrPto and AvrPtoB and that Pto has evolved as a decoy of these kinases to trap the T3Es into the Prf immune complex. Current Opinion in Microbiology 2016, 29:49–55

52 Host-microbe interactions: bacteria

PBS1 is a membrane-associated serine/threonine kinase that is targeted by the T3E AvrPphB (aka HopAR1) and interacts with the NLR RPS5 [35,36]. AvrPphB is a cysteine protease that cleaves PBS1 and other related kinases [37]. Cleavage of PBS1 is necessary and sufficient for the activation of the NLR RPS5 [35–38]. RPS5 distinguishes PBS1 from other related kinases by binding to a polymorphic loop (SEMPH), located on the opposite side of PBS1 from the AvrPphB cleavage site [39]. Cleavage of PBS1 by AvrPphB is proposed to lead to a conformational change of the SEMPH loop, which is detected by RPS5 resulting in ETI activation [38,39]. Like RIN4, PBS1 can be immunoprecipitated with the PRR FLS2 suggesting that it is involved in detecting perturbations of early PTI signaling events [37]. PBLs (PBS1-like kinases), BIK1 and PBL1, also associate with FLS2 and play crucial roles in PTI signaling [37,40]. AvrPphB can also cleave PBL proteins to suppress PTI signaling and promote pathogen virulence. In the decoy model, PBS1, which contributes only minimally to PTI signaling, has evolved to trap AvrPphB into the RPS5 resistance complex and promote ETI signaling [37,40]. Arabidopsis ZED1 is a pseudokinase and lacks the necessary aspartate residue in the conserved ‘HRD’ catalytic kinase motif, present in most active kinases [41]. ZED1 interacts directly with the acetyltransferase HopZ1a as well as the NLR ZAR1, and is required for ZAR1-mediated recognition of HopZ1a [41,42]. ZED1 is acetylated by HopZ1a and it is hypothesized that acetylation of ZED1 by HopZ1a is recognized by ZAR1 leading to activation of ZAR1-mediated ETI [41]. Since ZED1 is not required for HopZ1a virulence activity and does not significantly contribute to PTI, it is more than likely that the virulence targets of HopZ1a include PTI-associated kinases and that ZED1 as a pseudokinase has evolved as a decoy to trap HopZ1a in the ZAR1 resistance complex. Interestingly, it was recently shown that ZAR1 forms a preactivation complex with ZED1-related kinase (RKS1/ZRK1) to indirectly recognize the uridylylation of PBL2 at Serine 253 and Threonine 254 by Xanthomonas campestris effector AvrAC. Since it is not an AvrAC virulence target, it is likely that PBL2 has also evolved as a decoy to trap AvrAC in the ZAR1-RKS1 complex [43]. Thus, the ability of ZAR1 to monitor multiple sensors expands the scope of ETI surveillance.

Protecting transcription: rosetta stone sensors In Arabidopsis, alleles of the genomically linked NLR proteins RPS4 and RRS1 recognize two unrelated T3Es, PopP2 and AvrRps4 (Figure 2d) [44–46]. RPS4 and RRS1 heterodimerize to form an inactive NLR complex [47]. An interesting feature of these paired NLRs is the presence of an integrated sensor consisting of a WRKY transcription factor DNA-binding domain at the C-terminus of RRS1. Current Opinion in Microbiology 2016, 29:49–55

WRKY transcription factors bind DNA using a conserved WRKYGQK motif and play crucial roles in plant defense (reviewed in [48]). PopP2 acetylates the lysine residues of the RRS1 WRKY motif, leading to activation of RPS4-dependent ETI, possibly through reorientation of RRS1/RPS4 complex and homodimerization of RPS4 [47,49,50]. The mechanism of action of AvrRps4 on RRS1 is not known, but, acetylation of RRS1 by PopP2 eliminates its ability to recognize AvrRps4 [50]. The C-terminal WRKY domain of RRS1 likely represents a decoy, since PopP2 acetylates immunity-associated WRKY transcription factors and inhibits their DNA-binding and transcriptional activation properties [49,50]. This, example demonstrates that paired NLRs use integrated sensors as decoys to directly trap T3Es into the NLR resistance complex. Phytopathogens have also evolved to bypass host defense signaling and directly manipulate host gene expression by deploying TALEs (reviewed in [2]). TALEs act as transcription factors that can upregulate S (susceptibility) and executor R (resistance) genes to promote disease or resistance, respectively. Executor R genes include Bs3 which encodes a flavin-dependent monooxygenase and a second group consisting of short proteins predicted to localize to host cellular membranes, as represented by Bs4C-R, Xa27, Xa10 and Xa23 (reviewed in [51]). AvrBs3 from X. campestris pv. vesicatoria contains nuclear localization signals, an acidic transcriptional activation domain and a DNA-binding domain represented by a series 34 amino acid repeats which pair with specific nucleotides using two critical amino acids [52,53]. The transcriptional targets of AvrBs3 (upa genes) are upregulated by the binding of AvrBs3 to a conserved upa-box EBE (TATATAAACCN2-3CC) in the promoters of the upa genes, including susceptibility (S) genes that promote disease [54]. Coincidently, the upa-box is also present in the promoter of a host executor R gene Bs3 whose expression is sufficient to activate ETI [54,55]. The upa-box of Bs3 is an example of integrated sensor (DNA, rather than protein) found within the promoter of the Bs3 R gene. The upa box of Bs3 has evolved to mimic the promoters of AvrBs3 virulence targets, to trap AvrBs3 and promote ETI-activation [54,55]. These examples demonstrate that plants have integrated T3E sensors into ETI-inducing R genes to monitor for pathogenic threats to transcription. Genome analyses of additional plant species, such as wheat and rice, have revealed additional NLR proteins with putative ‘integrated sensors’ [56]. In addition to WRKY transcription factor domain described above, plants encode NLRs that have integrated other protein domains such as kinases and phosphatases suggesting that these rosetta stone sensors www.sciencedirect.com

Plant type III effector sensors Khan, Subramaniam and Desveaux 53

Table 1 Plant T3E sensors Type

Autonomous/ integrated

Guardee/decoya (operative T3E target[s])

Associated NLR

RIN4

Intrinsically disordered protein

Autonomous

Guardee

RPS2 RPM1

EDS1

Lipase-like

Autonomous

Guardee

Pto

Kinase

Autonomous

Decoy (PRRs)

RPS4 RPS6 Prf

PBS1 PBL2 ZED1 MPK4 RRS1 WRKY domain Bs3 promoter

Kinase Kinase f Pseudokinase Kinase Transcription factor

Autonomous Autonomous Autonomous Autonomous Integrated

Decoye (PBLs) Decoy (BIK1) Decoy (ZRKs?) Guardee Decoy (WRKY TFs)

RPS5 ZAR1 g ZAR1 SUMM2? RRS1 i

Gene promoter

Integrated

Decoy (upa genes; upa20)

Bs3 j

Sensor

T3E(s) sensed AvrRpt2 AvrRpm1 and AvrB AvrRps4 HopA1 AvrPto AvrPtoB AvrPphB AvrAC HopZ1a HopAI1 PopP2 AvrRps4 AvrBs3

T3E modification sensed Proteolysis Phosphorylation b NLR complex disruption?c NLR complex disruption? c Perturbation d Proteolysis Uridylylation h Acetylation? Phosphothreonine lyase? Acetylation Unknown Transcriptional activation

a

According to current pathosystems tested. Indirectly by the kinase RIPK [18,19]. c AvrRps4 and HopA1 disrupt RPS4/EDS1 and RPS6/EDS1 interactions [57,58]. d T3E binding, Pto conformational change and subsequent phosphorylation events [24]. e PBS1 contributes to PTI responses but much less than related kinases and pbs1 plants do not display altered susceptibility to P. syringae [12,40]. f Kinase activity not required for recognition of AvrAC [43]. g Recruited to ZAR1 complex following uridylylation by AvrAC [43]. h Recognition requires ZRK1 pseudokinase [43]. i Paired NLR with RPS4. j Encodes a flavin dependent monooxygenases (FMO), not an NLR [55]. b

have evolved to protect more than transcription (reviewed in [56]).

Conclusions Plants have evolved a plethora of sensors to detect T3Es and activate ETI (Table 1). Some of the features highlighted from the examples outlined above are: 1) sensors can be targeted by multiple T3Es; 2) sensors can be monitored by multiple NLR proteins; 3) one NLR can monitor multiple sensors; 4) sensors include T3Es virulence targets (‘guardees’) as well as mimics of these virulence targets (‘decoys’); 5) sensors can be autonomous from the NLR or integrated as a domain of the NLR; 6) TALEs can be detected using DNA sensors that activate ETI-promoting genes (i.e. executor R genes). Of the examples described in this review, RIN4 represents the most convincing example of a ‘guardee’, whereas the other T3E sensors discussed are likely decoys of T3E virulence targets. However, the immune regulator EDS1 and the MAP kinase MPK4 may also represent NLR guardees. EDS1 is a critical susceptibility node of plant immunity that is required for numerous ETI responses and optimal resistance to various pathogens. EDS1 associates with the NLR proteins RPS4 and RPS6 and is required for their ability to activate ETI in response to the T3Es AvrRps4 and HopA1, respectively [57,58]. Both AvrRps4 and HopA1 interact with EDS1 and can disrupt its interaction with RPS4 and RPS6 [57,58]. It is www.sciencedirect.com

speculated that these T3E-induced disruptions of EDS1 involving both cytoplasmic and nuclear NLR complexes activate ETI. An intriguing question is whether RPS4 uses both EDS1 and RRS1 as sensors to detect T3Es [49,50,57,58]. MPK4 is as another likely guardee that is targeted by phosphothreonine lyase HopAI1 and negatively regulates the NLR SUMM2. It is hypothesized that inhibition of MPK4 by HopAI1 activates SUMM2-mediated ETI [59]. It is tempting to speculate that decoys evolved from T3E virulence targets allowing them the flexibility to optimize T3E detection without compromising any PTI-associated functions. As such T3E sensors would be evolving from dual functions in PTI and ETI (e.g. RIN4; ‘guardees’) to ETI-specific roles (e.g. ‘decoys’). This specialization would also increase the burden on phytopathogens trying to suppress both PTI and ETI, requiring that two different proteins be targeted rather than one. Both Pto and ZED1 are part of a genomic cluster of related kinases that may include the ancestral virulence targets of their cognate T3Es. Interestingly, RKS1 in the ZED1 genomic cluster has been identified to be an important quantitative trait locus required for optimal resistance to multiple plant pathogens, and could represent an operative T3E virulence target [60]. Overall T3E sensors represent crucial molecular hubs of the plant-pathogen interface that integrate numerous Current Opinion in Microbiology 2016, 29:49–55

54 Host-microbe interactions: bacteria

host and pathogen derived post-translational modifications (PTMs) to activate plant immunity. An important avenue of future research will be to determine the molecular mechanisms by which these PTMs are perceived by NLRs to activate ETI.

Acknowledgements GS is supported by the Agriculture and Agri-Food Canada Crop Genomics Initiative. DD is supported by an Natural Sciences and Engineering Research Council of Canada Discovery Award D.D.; a Canada Research Chair in Plant-Microbe Systems Biology and the Centre for the Analysis of Genome Evolution and Function.

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