Should I stay or should I go? Traffic control for plant pattern recognition receptors

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ScienceDirect Should I stay or should I go? Traffic control for plant pattern recognition receptors Ma´rcia Frescatada-Rosa, Silke Robatzek and Hannah Kuhn1 Plants employ cell surface-localised receptors to recognise potential invaders via perception of microbe-derived molecules. This is mediated by pattern recognition receptors (PRRs) that bind microbe-associated or damage-associated molecular patterns or perceive apoplastic effector proteins secreted by microorganisms. In either case, effective recognition and initiation of appropriate defence responses rely on a signalling competent pool of receptors at the cell surface. Maintenance of this pool of receptors at the plasma membrane is guaranteed by sorting of properly folded ligand-unbound and ligand-bound receptors via the secretory-endosomal network in an activation-dependent manner. Recent findings highlight that ligand-induced endocytosis is found across members of distinct PRR families suggesting a conserved mechanism by which PRRs and immunity is regulated. Address The Sainsbury Laboratory, Norwich Research Park, Norwich NR4 7UH, United Kingdom Corresponding author: Robatzek, Silke ([email protected]) 1 Permanent address: RWTH Aachen University, Plant Molecular Cell Biology, Worringerweg 1, 52074 Aachen, Germany.

Current Opinion in Plant Biology 2015, 28:23–29 This review comes from a themed issue on Cell biology Edited by Hiroo Fukuda and Zhenbiao Yang

http://dx.doi.org/10.1016/j.pbi.2015.08.007 1369-5266/# 2015 Elsevier Ltd. All rights reserved.

or absence of a kinase domain in the cytoplasmic tail determines their classification as receptor-like kinases (RLKs) or receptor-like proteins (RLPs), respectively (Figure 1) (reviewed in [1,2]). Several PRRs have been identified till date (for detailed review see [1,2]), and some of the best studied examples are bacterial MAMPs perceiving LRR-RLKs such as the flagellin receptor FLAGELLIN SENSING 2 (FLS2), the Arabidopsis (Arabidopsis thaliana) ELONGATION FACTOR THERMO-UNSTABLE RECEPTOR (EFR) and the rice (Oryza sativa) XA21 receptor that recognises sulfated RaxX, a protein conserved in many plant pathogenic Xanthomonas species [3]. Fungal apoplastic proteins such as ethylene-inducing xylanase (EIX) and the Cladosporium fulvum effectors Avr4/Avr9 are recognised by the tomato (Solanum lycopersicum) LRR-RLPs Eix2 and Cf4/Cf-9, respectively [1,2]. To accurately signal during defence, the activity of PRRs needs to be tightly controlled [4]. It is becoming evident that this involves subcellular transport processes to establish and maintain a signalling competent receptor pool at the plasma membrane (PM) (Figure 1). The population of PRRs at the PM results from an interplay between delivery and removal. PRRs are synthesised in the endoplasmic reticulum (ER) where they are processed into functional receptors and are transported to the PM via the secretory pathway [5]. The mechanisms governing PRR secretory traffic have been recently described [6,7]. In this review we focus on recent research advances on regulatory aspects of PRR subcellular transport with an emphasis on endocytosis and sorting mechanisms and highlight future research directions in this field.

Non-activated PRRs undergo constitutive recycling Introduction Plants are constantly challenged by pathogenic and beneficial microbes and the need to trigger the adequate response is a prerequisite for survival. Frontline defences include cell surface-localised pattern recognition receptors (PRRs) that detect non-self epitopes such as microbeassociated molecular patterns (MAMPs) and apoplastic effector proteins or perceive plant-derived damage-associated molecular patterns (DAMPs). The extracellular ligand-binding domains of PRRs consist of leucine-rich repeats (LRRs), epidermal growth factor-like repeats, lysin motifs (LysM) or lectin-domains. A single-pass transmembrane region connects the extracellular N-terminus to the C-terminal intracellular tail. The presence www.sciencedirect.com

Association of proteins with the PM is transient as constitutive recycling of cell surface proteins via the trans-Golgi network/early endosome (TGN/EE) is a feature common to most PM-localised proteins [8]. A prominent pool of ligand-unbound FLS2 is mainly present at the cell surface. Brefeldin A (BFA) treatment additionally revealed an accumulation of FLS2-GFP in cytosolic agglomerations independently of BRASSINOSTEROID INSENSITIVE1 (BRI1)-ASSOCIATED RECEPTOR KINASE1 (BAK1) required for FLS2-mediated immunity [9]. This is indicative of recycling of the FLS2 receptor as BFA inhibits the ARF-GEF (GUANINE NUCLEOTIDE EXCHANGE FACTOR) GNOM, the main player in PM endocytic cycling (Figure 2) [10]. Similar observations Current Opinion in Plant Biology 2015, 28:23–29

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Figure 1

PROPEP1 EF-Tu flagellin Avr4

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Current Opinion in Plant Biology

LRR-RLK and LRR-RLP PRRs undergo ligand-induced endocytosis. Following secretion to the PM (green arrow), distinct LRR PRRs undergo BAK1/SERK3-dependent endocytosis upon activation (red arrow). Binding of the MAMP/epitope (flagellin/flg22, EF-Tu/elf18), processed DAMP peptide (Pep1), fungal apoplastic effector (Avr4) or fungal xylanase (EIX) to the respective receptor induces complex formation with BAK1/SERK3 co-receptors and activation of PRR signalling. Activated receptors are removed from the plasma membrane (PM) and internalised into mobile endosomes destined for vacuolar degradation. PROPEP1, propeptide1.

were made for OsXA21, as treatment of protoplasts or rice root cells with BFA and Cycloheximide, a combination of inhibitors to monitor the traffic of PM-localised proteins without interference of newly synthesised proteins, induced an accumulation of OsXA21-GFP in BFA compartments [11]. Although information regarding other PRRs is lacking, these results suggest that, as with other PM proteins, non-activated PRRs are subjected to BFA-sensitive endocytic recycling at the PM. Vesicle-based delivery of defence compounds to the plant microbe interface is a common theme in plant defence [12] and also FLS2 has been suggested to focally accumulate after pathogen attack and in turn is required for focal deposition of defencerelevant cargoes such as the ABC transporter PEN3 [13,14]. Recycling of PRRs might therefore contribute to focusing of the cellular defence signalling machinery.

Activated PRRs travel along an endosomal route for vacuolar degradation Internalisation of FLS2 from the PM into the early endosomal pathway towards the TGN, is further enforced Current Opinion in Plant Biology 2015, 28:23–29

by flg22 binding and complex formation with the BAK1 co-receptor [15,16]. An activation-dependent uptake of PRRs from the PM into mobile vesicles has also been found for SlEix2, EFR, the DAMP receptor PEPR1 as well as for SlCf-4 and the associated kinase SUPPRESSOR OF BIR1-1 (SOBIR1) required for LRR-RLP function (Figure 1) [17–20] (IMN Mbengue et al., unpublished). Endosomal localisation of SOBIR1 is additionally promoted by the Phytophthora parasitica elicitin ParA1 in tobacco (Nicotiana tabacum) [19]. Furthermore, the finding that the Phytophthora infestans effector Avr3a inhibits elicitin-mediated and flg22-mediated defence as well as FLS2 internalisation provides evidences for endocytic regulation of ELICITIN RESPONSE (ELR), an RLP receptor interacting with SOBIR1 and BAK1/ SERK3 (SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE3) [21,22]. Interestingly, endosomes carrying activated FLS2-GFP are recruited to P. infestans haustoria in Nicotiana benthamiana [23], indicating a similar dynamic localisation pattern of ELR during pathogen infection. The similarities of endocytic traffic www.sciencedirect.com

Traffic control for plant PRRs Frescatada-Rosa, Robatzek and Kuhn 25

Figure 2

FLS2

FLS2

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CHC DRP2b

u u u

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Overview of FLS2 subcellular trafficking along compartments of the secretory-endosomal network. Secretory trafficking of FLS2 to the PM (light green arrow), constitutive recycling (blue arrow) and ligand-induced endocytosis pathways (red arrow) are indicated. Upon ligand binding FLS2 travels via TGN and MVBs for vacuolar degradation. A potential alternative SYP61-independent endocytic pathway is indicated. Discussed regulators of FLS2 subcellular transport are indicated by dark green arrows. Marker proteins of endosomal compartments colocalising with FLS2 are shown by coloured tags. ER, endoplasmic reticulum; PM, plasma membrane; TGN, trans-Golgi network; MVB, multivesicular body; U, ubiquitination; P, phosphorylation.

between RLK and RLP PRRs suggest a conserved mechanism for this process upon activation by their respective ligands. Ligand-induced endocytosis of FLS2 is insensitive to BFA and following internalisation from the PM, the receptor travels along the late endosomal pathway from the TGN/ EE via multivesicular bodies/late endosomes (MVBs/LEs) towards the vacuole [10,24,25] (Figure 2). Targeting of FLS2 for vacuolar degradation is supported by the finding that loss-of-function of VACUOLAR PROTEIN SORTING-ASSOCIATED PROTEIN 37-1 (VPS37-1), a component of the ENDOSOMAL SORTING COMPLEXES REQUIRED FOR TRANSPORT-I (ESCRT-I) involved in sorting of ubiquitinated cargo for degradation, results in failure of FLS2 sorting into the lumen of MVBs [25]. Moreover, Concanamycin A treatment, that interferes with vesicle trafficking from the TGN to MVBs and the vacuole [10], significantly increases flg22-induced FLS2-GFP endosomal numbers, indicating that degradation of the receptor is impaired [10]. As FLS2 protein abundance is closely linked to signalling capability, vacuolar delivery suggests mechanisms for ultimately controlling FLS2 www.sciencedirect.com

activity [10,15,24,26,27]. It has been shown that flg22 perception transiently decreases FLS2 levels and coincides with signal desensitisation [26]. After washout of the elicitor and recovery of FLS2 levels, signalling competence is regained suggesting that ligand-induced endocytosis and degradation of the receptor is required for its inactivation. Trafficking of FLS2-GFP along the late endosomal pathway occurs via ARA7/RAB-F2b-positive and ARA6/RABF1-positive compartments and requires functional Rab (Ras (rat sarcoma)-related in brain) GTPases (Figure 2). Expression of dominant negative Rab5 and Rab11 GTPases ARA7 and RABA6a results in absence of FLS2-GFP endosomes or delayed transition of FLS2 from SYP61 (SYNTAXIN OF PLANTS 61)/ARA7 colabelled compartments, to SYP61 free MVBs, respectively [10,24]. Importantly, other PRRs such as EFR, PEPR1 and Cf-4 colocalise with the same marker proteins upon ligand treatment demonstrating their transport along the same trafficking route [20] (IMN Mbengue et al., unpublished). Existence of an alternative trafficking pathway to MVBs without transit through SYP61-positive Current Opinion in Plant Biology 2015, 28:23–29

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compartments has also been provided as expression of a dominant negative RABA4c reduced the extent of colocalisation between FLS2 and SYP61 but not with ARA7 (Figure 2) [24]. Involvement of TGN subdomains in FLS2 sorting is likewise possible. In N. benthamiana, FLS2 shows localisation at the vicinity or partial overlap with VHAa1 (V-TYPE H+ ATPASE SUBUNIT a1) or SYP61, respectively [24]. This differential colocalisation of FLS2 with these two TGN-localised proteins suggests association of the receptors to specific TGN subcompartments.

Impact of posttranslational modifications on sorting of activated PRRs The fact that PRRs enter two distinct endocytic trafficking routes depending on their activation-status is indicative of strict regulatory mechanisms for proper sorting of the receptors towards the appropriate destination. Hence, the activation status of PRRs is probably detected in a two-step process: first, at the PM to induce endocytosis after activation, and second, at the TGN as a point of divergence of exocytosis and endocytosis (Figure 2). The sorting mechanisms at the TGN seem to be highly effective as association of FLS2-GFP with the TGN marker SYP61 in N. benthamiana is restricted to approximately 30% [24]. This argues for a relatively short halflife of the receptor at this compartment and is notable as FLS2 travels via the TGN during all major trafficking events: exocytosis, recycling as well as ligand-induced endocytosis. Correct sorting of FLS2 towards the late endosomal pathway requires recognition of the activation status of the receptor at the PM and the TGN. As activation of FLS2 coincides with phosphorylation and ubiquitination of its intracellular domain [4,28,29] these posttranslational modifications might be involved in providing the signal for recruitment of trafficking regulators. The requirement of BAK1/SERK3 for endocytosis of activated FLS2 and Cf-4 suggests ligand-induced transphosphorylation for proper sorting [20] (reviewed in [4,30]). Kinase-active BAK1 compensates for the loss of Cf-4 endocytosis after silencing of NbSERK3a/b in N. benthamiana [20]. Involvement of phosphorylation events in FLS2 endocytosis is additionally supported by a requirement for the potential FLS2 T867 phosphorylation site for flg22-induced FLS2 internalisation [15] as well as interference of the phosphatase inhibitor Cantharidin with internalisation of the receptor (Figure 2) [31]. Ubiquitin acts as a sorting signal to trigger internalisation of PM proteins into the endocytic transport route [32,33]. The E3 ubiquitin ligases shown to interact with PRRs are phosphorylated by the receptor itself or the BAK1 co-receptor upon exposure to MAMPs (for review see [32]). This suggests that phosphorylation could serve as a signal for recruitment and Current Opinion in Plant Biology 2015, 28:23–29

activation of E3 ligases. Polyubiquitination of the brassinosteroid receptor BRI1 (BRASSINOSTEROID INSENSITIVE 1) initiates its internalisation from the PM and is essential for vacuolar targeting from the TGN pointing at a similar mechanism for PRR sorting [34]. In line with this, evidence accumulates for a role of differential ubiquitination in targeting FLS2 for degradation: first, FLS2 degradation after flg22 trigger coincides with an enhanced polyubiquitination of the protein [28]; second, BAK1 recruits and phosphorylates the closely related PLANT U-BOX (PUB) E3 ligases PUB12 and PUB13 to the receptor complex in response to flg22 (Figure 2) [29]; third, polyubiquitination of FLS2 by PUB12/13 induces degradation of the activated receptor and downregulation of signalling [28,29]. Accordingly, loss-of-function of pub12 pub13 or dominant negative overexpression of the PUB13 ARMADILLO (ARM) domain required for BAK1 phosphorylation and interaction with the FLS2-BAK1 complex results in impaired degradation of FLS2 and an enhanced immune response [29]. Of note, unchallenged pub13 mutants display spontaneous cell death and hydrogen peroxide accumulation coinciding with increased salicylic acid (SA) levels when grown under long-day and high light conditions [35]. Reduced FLS2 levels upon flg22 stimulus were still observed in pub12 pub13 using a mass spectrometry approach [36]. This hints at additional components in mediation of FLS2 degradation. Although the PM and TGN are expected to be the major sites for activation-dependent sorting of PRRs, it has not been shown at which location polyubiquitination of FLS2 by PUB12/13 occurs. PUB13 exhibits Golgi/TGN-like localisation patterns and was found to interact with the small GTPase RABA4b/ PRA3 [37]. Besides a role in PM trafficking during pollen tube growth, pea RABA4/Pra3 has been suggested to function in endocytic sorting events due to localisation to TGN or MVBs (reviewed in [38]). However, subcellular colocalisation or interaction of PUB12/ 13 with FLS2 as well as a function of RABA4b in FLS2 endocytosis remain to be addressed.

Regulators of ligand-induced PRR endocytic transport MAMP-induced phosphorylation events might be further required for activation of trafficking regulators essential for PRR endocytosis. Pharmacological interference with Dynasore, an inhibitor of dynamins in animals, reduced EIX-induced SlEix2 internalisation [18]. Mass spectrometry revealed an increase in phosphorylated DYNAMINRELATED-PROTEIN2 (DRP2)A and DRP2B after flg22 and fungal xylanase treatment [39,40]. DRP2A/B are large GTPases mediating vesicle fission from donor membranes with a recognised function in clathrin-mediated endocytosis (CME) [41]. Recent studies revealed a role for AtDRP2B and Nb/NtDRP2 in FLS2 liganddependent-internalisation and signalling in Arabidopsis www.sciencedirect.com

Traffic control for plant PRRs Frescatada-Rosa, Robatzek and Kuhn 27

and N. benthamiana [22,42]. Thus, a clathrin-mediated uptake of activated FLS2 can be assumed (Figure 2). Coincidence of NbDRP2 interaction with the P. infestans effector Avr3a and inhibition of FLS2 endocytosis in N. benthamiana indicates that key regulators of PRR endocytic pathways are targeted by pathogenic effectors [22], which highlights the role of endocytic trafficking in immunity. Further evidence for CME in PRR endocytosis is provided by interference of activated SlEix2 uptake as well as SlEix2 and SlCf signalling upon transient overexpression of the accessory adaptor EPSIN15 HOMOLOGY DOMAIN-containing protein AtEHD2 in N. benthamiana. However, this was not observed for flg22-induced responses [17]. Similarly, although SA treatment interferes with clathrin abundance at the PM, it does not affect activated FLS2-GFP endocytosis in Arabidopsis and in agreement, flg22-induced FLS2 degradation seems to be largely SA independent [29,43]. By contrast, Tyrphostin A23, which impedes the interaction of cargo proteins with the ADAPTOR PROTEIN2 (AP2) complex required for clathrin coated vesicle formation, reduced activated FLS2-GFP and GFP-SlEix2 internalisation suggesting that CME is involved in uptake of both RLKs and RLPs [10,44,45]. Silencing of the N. benthamiana clathrin heavy chain genes (NbCHCs) significantly reduced the number of FLS2-GFP endosomes after flg22 treatment, which additionally supports a role for CME in ligand-induced FLS2 internalisation (IMN Mbengue et al., unpublished). Nevertheless, alternative internalisation pathways such as membrane microdomain (MMD)-associated flotillin-dependent endocytosis cannot be excluded. It is becoming apparent that PRRs and other PM-localised receptors are not equally distributed across the PM but rather organised in MMDs that contribute to both CME and flotillin-dependent endocytosis [46–48] (reviewed in [49,50]). A switch towards flotillindependent endocytosis was observed for BRI1 after brassinosteroid treatment [51], indicating that different endocytic pathways might be at work depending on environmental conditions. The association of PRRs with MMDs further suggests that, besides mediating endocytosis, these might influence organisation and regulation of receptor signalling [52]. FLS2 is enriched in MMDs or sterol-rich detergentresistant membranes (DRMs), their biochemical counterpart, in a flg22-dependent manner [53,54]. Accordingly, transiently induced alterations of N. benthamiana sterol composition interferes with SlEix2 internalisation and signalling [55]. The close association of PRRs with MMDs might potentially mediate concerted ligand-induced internalisation of the PRRs. In vivo visualisation of the spatio temporal behaviour of PRRs in MMDs, depending on activation status, will therefore be an exciting aspect of future research. www.sciencedirect.com

Next destinations in research on PRR trafficking Activation-dependent endocytosis is a theme observed for several PRRs contributing to regulation of receptor abundance and localisation at the cell surface. As distinct PRRs travel along the same subcellular route, once activated, common mechanisms of ligand-triggered endocytosis can be anticipated. To what extend this process influences PRR signalling is an aspect of further studies. Also, whether PRR endocytosis could potentially allow signalling of active receptors from endosomal compartments observed for certain animal receptors remains to be answered [56,57]. As discussed, several regulators directing FLS2 towards the late endosomal pathway have been identified. Since activation dependency of FLS2 trafficking presupposes controlled recruitment and/or regulation of these components, elucidation of flg22-induced regulation will contribute to our understanding of receptor sorting. Similarly, important information on trafficking control will be provided by identification of activation-dependent posttranslational modifications of FLS2 that mediate its endocytosis. This future research, in combination with recent advances in proteomics of subcellular trafficking compartments [58,59], has high potential to ultimately determine the interplay between PRR endocytosis and immunity.

Acknowledgements We thank Deirdre McLachlan for critical reading of the manuscript. S.R. laboratory was supported by the Gatsby Charitable Foundation and by a grant of the European Research Council (ERC).

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