- Email: [email protected]
Emerging Insights into the Functions of Pathogenesis-Related Protein 1
Review
Emerging Insights into the Functions of PathogenesisRelated Protein 1 Susan Breen,1,3 Simon J. Williams,1,3 Megan Outram,2 Bostjan Kobe,2 and Peter S. Solomon1,* The members of the pathogenesis-related protein 1 (PR-1) family are among the most abundantly produced proteins in plants on pathogen attack, and PR-1 gene expression has long been used as a marker for salicylic acid-mediated disease resistance. However, despite considerable interest over several decades, their requirement and role in plant defence remains poorly understood. Recent reports have emerged demonstrating that PR-1 proteins possess sterol-binding activity, harbour an embedded defence signalling peptide, and are targeted by plant pathogens during host infection. These studies have reenergised the field and provided long-awaited insights into a possible PR-1 function. Here we review the current status of PR-1 proteins and discuss how these recent advances shed light on putative roles for these enigmatic proteins. The PR-1 Family PR-1 family members were first identified in the 1970s from Nicotiana tabacum infected with tobacco mosaic virus (TMV) [1]. Using this pathosystem it was shown that some, but not all, tobacco PR-1 proteins were upregulated during TMV infection [2–5]. We now know that PR-1 proteins are ubiquitous across plant species. They occur in multigene families within plant genomes and can be broadly characterised into classes (acidic or basic) depending on their theoretical isoelectric point. They are members of a broader protein family known as the cysteine-rich secretory protein, antigen 5, and pathogenesis-related-1 (CAP) protein superfamily [6]. The CAP domain comprises approximately 150 amino acids and CAP-containing proteins are represented in more than 2500 species including bacteria, fungi, plants, and animals, with diverse roles in reproduction, cancer, immune defence, sterol binding and export, and ion binding (http://pfam.xfam.org/family/PF00188) [7]. In plants PR-1 proteins are among the most abundantly produced proteins during defence responses and have been reported to constitute 2% of the total leaf protein in pathogeninfected tobacco [8]. Most members of the PR-1 family are thought to be secreted and accumulate in the extracellular/apoplastic space, which is facilitated by their N-terminal secretion peptide. However, research in tobacco has also shown that some PR-1 proteins accumulate in the vacuoles of protoplasts [9] and the vacuoles of specialised cells known as crystal idioblasts [10]. PR-1 proteins are not exclusively associated with host defence. Numerous reports have shown that PR-1 genes are responsive to abiotic stimuli, suggesting important roles in abiotic stress responses [11–17]. There is also evidence to suggest that PR-1 proteins have a role in plant growth or development that is independent of stress responses [18]. For example, PR-1 proteins were shown to accumulate strongly in the senescing leaves of adult flowering plants [19]. A subsequent study by Lotan et al. showed that PR-1 protein
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Trends Recent studies have shown that plant pathogenesis-related protein 1 (PR-1) family members bind sterols. This function is responsible for antimicrobial activity towards sterol auxotrophs such as Phytophthora species. However, the link between sterol binding and the proposed broader antimicrobial function of PR-1 remains unclear. PR-1 proteins harbour an embedded C-terminal peptide (CAPE) involved in plant immune signalling. Evidence suggests that CAPE has a signalling role that facilitates defence responses against microbial pathogens and also herbivores. The CAPE response is independent of other defence signalling pathways such as those elicited by recognised pathogen-associated molecular patterns. The significance of PR-1 proteins during plant–microbe interactions is now realised, with a growing list of identified pathogen effector proteins that directly interact with PR-1 during infection.
1 Plant Sciences Division, Research School of Biology, The Australian National University, Canberra 2601, Australia 2 School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, Australia 3 These authors contributed equally to the manuscript.
*Correspondence: [email protected] (P.S. Solomon).
http://dx.doi.org/10.1016/j.tplants.2017.06.013 © 2017 Elsevier Ltd. All rights reserved.
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accumulated in the sepals of developing flowers, further alluding to a potential role in plant flowering [20]. Yet, their function in plant growth or development is unknown. However, it is their upregulation in salicylic acid-dependent defence responses and their subsequent use as a defence marker that are their most distinguishable features in plants. The regulation of PR-1 gene expression has been well described and the pathways that lead to PR-1 induction during host defence have been identified [21]. Functional studies over the past 30 years have indicated a potential role for PR-1 proteins in host defence, but it is reasonable to suggest that much of this evidence is circumstantial. Recent reports, however, have provided some tantalising evidence of possible roles, and this review focusses on these studies and how they contribute to our understanding of the function of these ubiquitous proteins.
Are PR-1 Proteins Antimicrobial? The upregulation and abundance of PR-1 proteins during infection, combined with their localisation to the apoplast (an important interface for plant–microbe interactions) lend themselves to a potential antimicrobial function. Such functions have been linked to other PR proteins that are also upregulated by salicylic acid such as PR-2 (an acidic form of beta-1,3glucanase) and PR-5 (a thaumatin-like protein), which possess recognised enzymatic activities known to inhibit microbial growth [22,23]. Several groups have demonstrated that overexpression of PR-1 in transgenic plants results in increased resistance to fungi [24] oomycetes [8,25], and bacteria [25,26] but not viruses [27]. Consequently, it was considered that the observed increased resistance was a result of broad antimicrobial activity by PR-1. Niderman and colleagues first presented evidence of anti-oomycete activity by incubating PR-1 proteins isolated from tomato (Solanum lycopersicum) and tobacco (Nicotiana tabacum) with Phytophthora infestans zoospores and assaying for inhibition of germination. They demonstrated that PR-1 protein concentrations of 20–200 mg/ml (depending on the PR-1 protein assayed) were sufficient to inhibit 90% of zoospore germination. Furthermore, exogenous application of these proteins also inhibited P. infestans colonisation of tomato leaf discs [28]. The yields the authors obtained from infected plants (0.6–4 mg/g tissue fresh weight) were lower than the concentrations required for in vitro anti-oomycete activity; however, the authors argue that the local concentration within extracellular spaces would likely be significantly higher (10) in vivo (based on PR-1 localisation towards infection sites) and suggest a direct PR-1 protein anti-oomycete function. The application of PR-1 proteins produced in Escherichia coli also restricted P. infestans zoospore germination [29] and the growth of some fungal plant pathogens, albeit at high PR-1 protein concentrations (200–600 mg/ml). A more recent study by Gamir and colleagues demonstrated that the heterologously expressed PR-1 proteins P14c (from tomato) and PR-1a (from tobacco) inhibited the growth of the oomycete pathogen Phytophthora brassicae at concentrations of 20 mg/ml (1.1 mM) [30]. These data added to a host of evidence that PR-1 proteins have anti-oomycete properties. However, using the same experimental procedure there was no observed effect on the growth of Aspergillus niger or Botrytis cinerea, questioning the antifungal activity of PR1 proteins. Importantly, this work described the first biochemical function for PR-1 proteins. Armed with the knowledge that the CAP domain from the yeast proteins PRY1 and PRY2 have a sterol-binding function [31] (discussed further below), Gamir and colleagues demonstrated that P14c and PR-1a can genetically complement yeast sterol-transport-deficient mutants. Purified P14c and PR-1a proteins were shown to bind cholesterol with modest affinity (Kd 30 mM) and P14c had indistinguishable binding preferences for the fungusspecific sterol ergosterol, the plant-derived sterol stigmasterol, and cholesterol. The inhibition of P. brassicae growth by P14c was prevented when cholesterol was added to the medium and when a mutant unable to bind sterols (P14cC146S) was substituted for P14c, providing a
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critical link between sterol binding and anti-oomycete activity [30]. The authors suggested that the antimicrobial activity of PR-1 towards P. brassicae is due to the oomycete’s inability to produce its own sterols (sterol auxotrophy). They subsequently showed that the addition of non-lethal doses of fungicide that block sterol biosynthesis in A. niger and B. cinerea made the fungi susceptible to P14c. From their data Gamir and colleagues present a model suggesting that PR-1 proteins are antimicrobial by sequestering sterols from the membranes of microbes and are more effective against sterol auxotrophs, which must obtain sterols from the environment. However, if PR-1 proteins are not effective in vitro against sterol prototrophs such as fungi, how could antifungal activity be achieved in vivo? Gamir and colleagues suggest that the vacuole-targeted PR-1 proteins may provide the answer. They speculate that the delivery of a large PR-1 payload (from within the vacuole) to an invading fungus may provide the concentration of PR-1 protein that is required to sequester more sterols than can be replaced by the sterol biosynthesis machinery, achieving in vivo antifungal properties [30]. While this is an interesting idea, their work clearly shows that further experimental validation is required before a true, physiological antifungal function can be attributed to PR-1 proteins. This also highlights that maybe we should not be thinking of PR-1 proteins as purely pathogen killers. After all, how could this single function explain the role of PR-1 proteins in host responses independent of pathogens? An explanation of the expanded functions of PR-1 in pathogen inhibition and regulation under abiotic stress conditions may be found in the recent discovery that PR-1 proteins carry an embedded stressresponse peptide [32].
CAP-Derived Peptide 1 (CAPE1) Stress-Signalling Peptides Are Embedded within PR-1 Proteins Recently, Chen and colleagues identified a peptide that was strongly induced in response to wounding and methyl jasmonate treatment in tomato [32]. Subsequent analysis of the peptide revealed that it comprised the last 11 amino acids from the C terminus of the tomato PR-1 protein and thus it was named CAPE1. Growth experiments with Spodoptera litura larvae fed on tomato leaves pretreated with synthetic CAPE1 demonstrated suppressed growth and a 20% reduction in larval weight. In addition, plants pretreated with CAPE1 showed no significant symptoms of infection when inoculated with Pseudomonas syringae pv tomato (Pst) strain DC3000 and reduced bacterial populations compared with controls [32]. Gene expression studies of CAPE1-treated leaves demonstrated that application of the peptide induced multiple defence-related genes, including proteinase inhibitor 1 (PI-1), PI-2, PR-2, PR7, ethylene response factor 5 (ERF5), and the precursor protein PR-1b [32]. The induction of these genes in the absence of biotic stress strongly suggests that CAPE1 has a role as a defence-signalling molecule as opposed to a direct antimicrobe/herbivore function. Interestingly, CAPE1 did not upregulate WRKY TRANSCRIPTION FACTOR53 (WRKY53), which is highly upregulated in response to the application of the pathogen-associated molecular pattern (PAMP) peptide flg22 [32]. These data suggest that the CAPE1 peptide does not induce the canonical PAMP-triggered immunity (PTI) signalling pathway described in other peptide-mediated responses and implies that the CAPE1 peptide primes defence through an independent signalling pathway. Chen and colleagues compared PR-1 sequences from various monocot and dicot plants and identified the conserved consensus motif PxGNxxxxxPY in the CAPE1 peptide [32]. This consensus sequence was experimentally validated by assessing the efficacy of the Arabidopsis thaliana AtCAPE-PR-1 peptide on Pst DC3000 infection in tomato. As observed with CAPE1, the application of AtCAPE1-PR-1 on tomato resulted in increased immunity. This is significant as AtCAPE-PR-1 shares only the amino acids within the consensus motif with CAPE1, proving the importance of the conserved residues for the CAPE-induced defence response. A highly
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conserved CNYx motif positioned N terminal to the CAPE peptide was also predicted to represent the CAPE cleavage motif (Figure 1). A subsequent study confirmed the requirement for the CNYx motif for CAPE1 cleavage through a site-directed mutation approach; however, the mechanism of cleavage is unknown [33].
Figure 1. Putative and Established Features of the Enigmatic Plant Pathogenesis-Related Protein 1 (PR-1). (A) The NMR structure of tomato cysteine-rich secretory protein, antigen 5, and pathogenesis-related (CAP) protein (Sl) pathogenesis-related protein 14a (SlP14a) solved by NMR spectroscopy [43], shown in cartoon representation. Residues shown in stick representation are labelled. Disulfide bonds are shown in yellow. Additional highlighted features of the structure identify functional and putative functional regions. Magenta, CAP-derived peptide (CAPE) with conserved residues within the consensus sequence labelled [32]. Pink, CAPE cleavage motif as described in [33]. Light blue, caveolin-binding motif (CBM) representing the putative sterol-binding motif from pathogen-related yeast 1 (PRY1) proteins [46]. Orange, conserved residues representing the CAP tetrad and coordinate metals in the PRY1 CAP domain structure [47]. Blue, residues involved in fatty acid binding in tablysin-15, conserved in PRY1 and PR-1 proteins. (B) ClustalW sequence alignment of plant PR-1 domains and the CAP domain from the yeast protein PRY1 prepared using ESPript 3.0 [55]. Secondary-structure elements above and below the alignment are derived from the structure of SlP14a (PDB ID: 1cfe) and PRY1 (PDB ID: 5jys). Features highlighted in (A) are also highlighted on the alignment with the same colour scheme. CBM and CAPE are labelled, while orange and blue stars represent the CAP tetrad and conserved tablysin-15 fatty acid-binding residues. SlP14a and SlP14c are from tomato [32,43], NtPR-1a is from tobacco [30], TaPR-1-1 and TaPR-1-7 are from wheat [41], TaPR-1_RLK_1_1 and _2 represent the first and second PR-1 domain in TaPR-1-receptor-like kinase (RLK1) from wheat [54], TcPR-1g_RLR is the PR-1 domain from PR-1-RLK1 from cocoa [53], and PRY1 is from yeast [31].
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Interestingly, the CAPE peptide is not restricted to PR-1 proteins implicated in pathogen defence. For example, of the 22 CAP proteins in A. thaliana nine were identified to contain the consensus sequence defined by Chen and colleagues [32,33] despite only AtPR-1 being upregulated in infected tissue [34]. The CAP gene AT4G33730 (designated PROAtCAPE1) from A. thaliana is a salt-responsive gene, whereby expression is reduced on salt treatment [33]. Silencing of PROAtCAPE1 resulted in transgenic plants with increased survival on highsalt media, while exogenous application of AtCAPE1 to the same transgenic plants resulted in reduced growth and salt-sensitive phenotypes. AtCAPE1 was subsequently shown to downregulate salt tolerance by suppressing genes that are associated with salt tolerance [33]. The authors subsequently suggested that CAPE peptides could function in a tradeoff between pathogen defence and salt tolerance [33]. The identification of CAPE peptides and their observed roles in stress (biotic and abiotic) responses represents a significant advancement in our understanding of PR-1 protein function. Undoubtedly, significant research challenges remain. How are CAPE peptides perceived? Is there a CAPE receptor? How are CAPE peptides cleaved from PR-1 proteins? What is clear is that both PR-1 proteins and CAPE peptides are important host defence molecules, making these conceivable targets for pathogens to overcome host defence responses.
PR-1 Proteins: A Pathogen Effector Hub? Despite the absence of an in vivo function, PR-1 proteins are clearly an integral component of host defence that need to be countered by colonising pathogens. Accordingly, recent studies have identified three pathogen effectors that interact with PR-1 proteins [35–37]. The best characterised of these effectors are the ToxA and Tox3 proteins. The ToxA effector has been identified in the wheat pathogens Parastagonospora nodorum, Pyrenophora tritici-repentis, and Bipolaris sorokiniana, while Tox3 is unique to P. nodorum [38–40]. Independent yeast-twohybrid (Y2H) library screens showed that ToxA and Tox3 both interact with wheat PR-1 proteins [36,37]. Despite being effectors from the same pathogen, their interactions with PR-1 appear to be fundamentally different. The ToxA interaction is highly specific, occurring only with the basic protein PR-1-5, whereas Tox3 is far more promiscuous, interacting with six of the eight tested wheat PR-1 proteins, including acidic and basic proteins but not basic PR-1 proteins with a Cterminal extension (CTE) [36,37,41]. In both studies the effectors appeared to interact with amino acids towards the C terminus of PR-1. In the case of ToxA, the mutation of a relatively well-conserved asparagine (PR-1-5 N141A) resulted in loss of interaction with ToxA [36]. Interestingly, this residue is conserved in a non-interacting wheat PR-1 protein, PR-1-1, which shares 90% sequence identity with PR-1-5, suggesting that other residues in PR-1-5 must provide specificity for the interaction. Mutation of the same asparagine in PR-1-1 did not disrupt the Tox3–PR-1-1 interaction and suggests that the dynamics of the ToxA/Tox3–PR-1 interactions differ [37]. The two PR-1 proteins (PR-1-18 and -21) that did not interact with Tox3 belong to the basic PR-1 proteins with CTEs. Interaction between Tox3 and the C-terminal section of PR-1-18 could be restored by substituting the predicted surface-exposed amino acids that differed between PR-1-18 and PR-1-1 [37]. However, these amino acid substitutions were not enough to induce an interaction with the full-length PR-1-18 protein and it appears that amino acids at the N terminus of these PR-1 proteins also control Tox3 specificity [37]. The interaction between these effectors and the PR-1 proteins also appears to influence the susceptibility of the host. The Tox effectors are major virulence determinants in the P. nodorum– wheat interaction; however, their effectiveness relies on the presence of host-susceptibility genes [42]. The wheat gene Tsn1 is required for ToxA-induced necrosis, while Tox3-induced necrosis relies on the presence of Snn3. Wheat lines lacking these genes, Tsn1 and Snn3, are resistant to isolates of P. nodorum expressing ToxA or Tox3, respectively. This gene-for-gene interaction has an inverse effect on the plant compared with the classically described gene-for-
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gene interaction in biotrophic pathogens. In the case of ToxA, Lu and colleagues showed that co-infiltration of ToxA and PR-1-5 into sensitive wheat lines enhanced the necrosis phenotype induced by ToxA alone. By contrast, the non-interacting PR-1-5N141A mutant did not, suggesting that the interaction between ToxA and PR-1-5 plays a role in promoting necrosis in ToxAsensitive wheat (Tsn1-containing wheat) [36]. Tox3 wheat leaves pretreated with synthetic TaCAPE1 (sequence from the wheat PR-1-1 protein) before infection with P. nodorum exhibited increased disease symptoms [37]. This phenotype was observed only in wheat cultivars that were susceptible to Tox3 (Snn3-containing wheat) [37]. Therefore, the interaction of the ToxA and Tox3 effectors with the PR-1 proteins appears to facilitate infection of the host in a genotype-specific manner. It remains to be seen how Tox3 utilises TaCAPE1 for this function and what role Snn3 (gene currently unknown) plays in the interaction. Could Tox3 induce the cleavage of the TaCAPE1 peptide, triggering the activation of host-defence responses that the necrotrophic pathogen P. nodorum is able to hijack and induce increased programmed cell death? Intriguingly, while CAPE had a protective function against P. syringae [32] the observation that a wheat cultivar lacking Snn3 showed no inhibition of P. nodorum disease when treated with TaCAPE1 suggests that CAPE is ineffective against some pathogens or is primarily effective against biotrophic pathogens. The third effector identified to interact with PR-1 is CSEP0055 from powdery mildew fungus [Blumeria graminis f.sp. hordei (Bgh)] [35]. This effector–host protein interaction was identified using a Y2H assay on a library generated from Bgh-infected barley leaves. The authors identified PR-17a, PR-17c, PR-1a, and PR-1b as host proteins interacting with CSEP0055 [31]. PR-17c was found in multiple prey clones and downstream work focused on the CSEP0055–PR-17c interaction over the PR-1 interaction [35]. Unfortunately, no further information has been reported describing the biological effect of the CSEP0055–PR-1 interaction. To date, three effectors from different fungal plant pathogens have been found to interact with host PR-1 proteins. Given the importance of PR-1 proteins in plant defence, it will be interesting to see whether more effectors from diverse plant pathogens target these proteins and associated pathways in the future.
What Can We Learn from Structural and Biochemical Studies of CAP Domains? The first CAP domain structure to be determined corresponds to the NMR structure of the tomato P14a [43] (Figure 1). To date this is the only published structure of a plant PR-1 protein; however, since then the structures of 21 CAP-containing proteins have been solved. The CAP domain folds into a unique a-b-a sandwich, generally comprising four a helices and four b strands, and is stabilised by conserved disulfide bonds (Figure 1). While many CAP domaincontaining proteins contain additional functional domains [44], PR-1 proteins contain only the CAP domain with relatively short C- and N-terminal extensions, suggesting that it is the function of the CAP domain that determines their role in plants. Therefore, structural and biochemical studies of CAP domains from other proteins can assist our understanding of PR-1 function. The sterol-binding ability of the CAP domain was first reported in the yeast proteins PRY1 and PRY2 and the human protein CRISP2 [31]. The same function was described in the Schistosoma mansoni venom allergen-like protein SmVAL4 [45] and subsequently in the PR-1 proteins P14c and PR-1a (discussed above) [30]. To date, sterol binding has not been captured in a 3D structure. Despite this, computational modelling of the yeast PRY1 identified a flexible loop with sequence similarity to the caveolin-binding motif (CBM), which suggested that this region is involved in sterol binding [46] (Figure 1). Point mutations in the CBM motif of PRY1 (F239L, F244L, P242C) resulted in loss of cholesteryl acetate binding in PRY1 [46].
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A recent crystallographic study of the CAP domain from PRY1 demonstrated that magnesium binding to distal regions of the CAP domain is required for sterol binding [47]. In this structure magnesium is coordinated by the so-called CAP tetrad, encompassing direct binding between two well-conserved histidine residues that are positioned relative to two conserved glutamate residues (Figure 1). These conserved residues have also been shown to bind Zn2+ in other CAP domains, including the human glioma PR-1 protein and a snake-venom cysteine-rich secretory protein, where it was shown to be functionally important [48,49]. PRY1 is known to dimerise in solution and it is suggested that the Mg2+-binding site and sterol-binding regions would be connected within a PRY1CAP dimer [47]. Despite this, neither dimerisation nor Mg2+ binding appears to be required for sterol binding by the SmVAL4 CAP domain, which binds sterols, lacks the CAP tetrad, and is a monomer in solution [45]. For plant PR-1 proteins, it remains unclear whether the designated CBM motif and CAP tetrad influence the sterol-binding and ionbinding functions, respectively. While the CAP tetrad is conserved (Figure 1), there are divergent features in the CBM motif of PR-1 proteins, including two additional cysteine residues that form an additional disulfide bond. This would be likely to affect the flexibility of this region, which could influence sterol binding. Despite this, the aromatic residues that are important for the sterol-binding function of PRY1 are generally conserved (Figure 1), supporting a shared CBM function. A striking feature of the CBM region with reference to the NMR structure of P14a is that this region accommodates (at least in part) the embedded C-terminal CAPE peptide (Figure 1). If the CBM region is involved in sterol binding in PR-1 proteins, it is difficult to explain how a PR-1 protein could accommodate both sterol and CAPE simultaneously. This observation provides a tantalising possibility that sterol binding may be associated with the repositioning of the embedded C-terminal CAPE amino acids. Perhaps this conformation change is involved in CAPE cleavage; alternatively, CAPE may help to regulate the sterol-binding activity of PR-1 proteins. Interestingly, the CAP domain of other proteins has also been shown to bind other lipids. The crystal structure of the horsefly (Tabanus yao) CAP domain-containing protein tablysin-15 revealed that the E. coli-produced protein was bound to palmitic acid [50]. Xu and colleagues subsequently demonstrated that tablysin-15 could bind the proinflammatory fatty acid leukotriene, implicating the CAP domain in the protein’s anti-inflammatory function [50]. Recently, the yeast protein PRY1 has also been shown to bind fatty acids, with micromolar affinity [51]. This function appears to be independent of PRY1’s sterol-binding activity, suggesting that a single CAP domain can accommodate the binding of multiple, structurally diverse lipids. While fatty acid binding has, to date, not been reported for PR-1 proteins, a number of the hydrophobic residues involved in fatty acid binding in tablysin-15 are conserved in PR-1 proteins (Figure 1). The literature reviewed above suggests that the CAP domain of PR-1 proteins might have multiple functions (i.e., sterol, ion, and lipid binding and peptide signalling); however, to date there are no publications that describe a mechanistic relationship between these functions. In light of insights provided by research on other CAP family members, it seems possible, even likely, that the multiple functions associated with the CAP domain are dependent on each other. For example, we could speculate based on the CBM accommodation of the CAPE peptide that PR-1 proteins act as sterol sensors, utilising CAPE for signalling. Interestingly, in line with these hypothesised multiple functions two recent reports [53,54] have shown that the PR-1 domain is associated with a diverse class of plant receptors that are implicated in sensing molecules in the extracellular environment.
PR-1 Receptor-Like Kinases (RLKs): Part of the PR-1 Puzzle? RLKs are one of the most abundant classes of plant proteins and are implicated in perceiving signals from the extracellular environment, enabling plant cells to react to these accordingly. The majority of RLKs possess a single transmembrane domain, an intracellular kinase domain,
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and an extracellular ligand-binding domain (ectodomain). RLKs are generally classified according to the ectodomain, which can comprise many different functional domains. In plant immunity, RLKs play important roles in the perception of both PAMPs and damage-associated molecular patterns (DAMPs), which are associated with pathogen invasion [52]. Recently, genes encoding extracellular PR-1 domains fused to transmembrane and kinase domains have been found in cocoa [53], wheat, rice, and several other plant species [54]. In cocoa, PR-1RLKs are upregulated on challenge with the biotrophic pathogen Moniliopthora perniciosa, the causal agent of cacao’s witch’s broom disease [53]. Similarly, the expression of two PR-1-RLK genes identified in wheat was induced in response to biotic (including barley stripe mosaic virus) and abiotic (including high salt) stress. In wheat, dual PR-1 ectodomains are observed in PR-1RLK, while a single PR-1 ectodomain is present in the proteins encoded in cocoa [53,54]. The integrated PR-1 domains from the wheat PR-1-RLK proteins share 50% identity with the PR1 proteins shown to be involved in sterol binding and contain some of the conserved aromatic residues in the CBM (Figure 1). While their functions in stress responses remain unclear, in light of the general role that RLKs play in the recognition and induction of defence responses, it is tempting to speculate the PR-1-RLKs may play a role in sterol sensing. Alternatively, given that effectors are known to target PR-1 proteins, perhaps PR-1-RLKs function in effector recognition.
Outstanding Questions Is there a link between sterol binding and CAPE signalling? Does sterol binding promote CAPE release? Do PR-1 proteins bind other lipids, such as fatty acids? How is CAPE cleaved from PR-1 proteins? Are peptidases involved or can PR-1 cleave itself in a caspase-like process? What is the identity of the CAPE peptide receptor? What pathways act downstream of CAPE detection and/or accumulation? Does the peptide itself possess functional properties beyond signalling (i.e., cell lysis or antimicrobial properties)? Do PR-1 domains in the PR-1-RLKs sense sterols or are they an integrated effector-recognition domain? Do PR1-RLKs signal using mechanisms similar to those of known RLK pathways?
Concluding Remarks and Future Perspectives In recent years, clear progress has been made towards understanding the role of PR-1 proteins in plant defence. The emergence of a biochemical function implicating PR-1 proteins in sterol binding and the identification of the CAPE peptide and PR-1-RLKs suggest multiple roles, from antimicrobial function and defence signal amplification to potential sterol or effector recognition. These PR-1 functions all signify challenges for plant pathogens and it remains to be seen whether more plant pathogens target PR-1 proteins during defence. Finally, the knowledge gleaned from other CAP domain proteins has guided and will continue to guide PR-1 functional studies to help ascertain their role in plants. Here we highlight the present research regarding the role of PR-1, but many more questions remain that will drive future research on this topic in the coming years (see Outstanding Questions). References 1. van Loon, L.C. and van Kammen, A. (1970) Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacum var. ‘Samsun’ and ‘Samsun NN’. Virology 40, 199–211
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2. van Loon, L.C. (1975) Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacum var. ‘Samsun’ and ‘Samsun NN’. Physiol. Plant Pathol. 6, 289–300
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16. Liu, W.-X. et al. (2013) Arabidopsis Di19 functions as a transcription factor and modulates PR1, PR2, and PR5 expression in response to drought stress. Mol. Plant 6, 1487–1502
8. Alexander, D. et al. (1993) Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesisrelated protein-1a. Proc. Natl. Acad. Sci. U. S. A. 90, 7327–7331
17. Kothari, K.S. et al. (2016) Rice stress associated protein 1 (OsSAP1) interacts with aminotransferase (OsAMTR1) and pathogenesis-related 1a protein (OsSCP) and regulates abiotic stress responses. Front. Plant Sci. 7, 1057
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How does the relatively modest affinity for sterol binding affect the growth of oomycetes? Is the affinity for sterols altered by other functions (i.e., CAPE release) or putative functions such as fatty acid binding? Could this subsequently result in broader antimicrobial effectiveness, particularly towards sterol prototrophs like fungi, in an in vivo context? Do other pathogens contain effector proteins that target PR-1 proteins?
19. Fraser, R.S.S. (1981) Evidence for the occurrence of the “pathogenesis-related” proteins in leaves of healthy tobacco plants during flowering. Physiol. Plant Pathol. 19, 69–76 20. Lotan, T. et al. (1989) Pathogenesis-related proteins are developmentally regulated in tobacco flowers. Plant Cell 1, 881–887 21. Durrant, W.E. and Dong, X. (2004) Systemic acquired resistance. Annu. Rev. Phytopathol. 42, 185–209 22. Leah, R. et al. (1991) Biochemical and molecular characterization of three barley seed proteins with antifungal properties. J. Biol. Chem. 266, 1564–1573 23. Selitrennikoff, C.P. (2001) Antifungal proteins. Appl. Environ. Microbiol. 67, 2883–2894 24. Kiba, A. et al. (2007) Pathogenesis-related protein 1 homologue is an antifungal protein in Wasabia japonica leaves and confers resistance to Botrytis cinerea in transgenic tobacco. Plant Biotechnol. 24, 247–253 25. Sarowar, S. et al. (2005) Overexpression of a pepper basic pathogenesis-related protein 1 gene in tobacco plants enhances resistance to heavy metal and pathogen stresses. Plant Cell Rep. 24, 216–224 26. Shin, S.H. et al. (2014) An acidic pathogenesis-related 1 gene of Oryza grandiglumis is involved in disease resistance response against bacterial infection. Plant Pathol. J. 30, 208–214 27. Cutt, J.R. et al. (1989) Disease response to tobacco mosaic virus in transgenic tobacco plants that constitutively express the pathogenesis-related PR1b gene. Virology 173, 89–97 28. Niderman, T. et al. (1995) Pathogenesis-related PR-1 proteins are antifungal. Isolation and characterization of three 14-kilodalton proteins of tomato and of a basic PR-1 of tobacco with inhibitory activity against Phytophthora infestans. Plant Physiol. 108, 17–27 29. Li, J. et al. (2009) Expression and purification of tobacco PR-1a protein for function analysis. Asian J. Chem. 21, 3697–3707 30. Gamir, J. et al. (2016) The sterol-binding activity of pathogenesisrelated protein 1 reveals the mode of action of an antimicrobial protein. Plant J. 89, 502–509 31. Choudhary, V. and Schneiter, R. (2012) Pathogen-related yeast (PRY) proteins and members of the CAP superfamily are secreted sterol-binding proteins. Proc. Natl. Acad. Sci. U. S. A. 109, 16882–16887 32. Chen, Y.-L. et al. (2014) Quantitative peptidomics study reveals that a wound-induced peptide from PR-1 regulates immune signalling in tomato. Plant Cell 26, 4135–4148 33. Chien, P.S. et al. (2015) A salt-regulated peptide derived from the CAP superfamily protein negatively regulates salt-stress tolerance in Arabidopsis. J. Exp. Bot. 66, 5301–5313 34. van Loon, L.C. et al. (2006) Significance of inducible defenserelated proteins in infected plants. Annu. Rev. Phytopathol. 44, 135–162 35. Zhang, W.-J. et al. (2012) Interaction of barley powdery mildew effector candidate CSEP0055 with the defence protein PR17c. Mol. Plant Pathol. 13, 1110–1119 36. Lu, S. et al. (2014) A dimeric PR-1-type pathogenesis-related protein interacts with ToxA and potentially mediates ToxAinduced necrosis in sensitive wheat. Mol. Plant Pathol. 15, 650–663 37. Breen, S. et al. (2016) Wheat PR-1 proteins are targeted by necrotrophic pathogen effector proteins. Plant J. 88, 13–25
38. Friesen, T.L. et al. (2006) Emergence of a new disease as a result of interspecific virulence gene transfer. Nat. Genet. 38, 953–956 39. Oliver, R.P. et al. (2012) Stagonospora nodorum: from pathology to genomics and host resistance. Annu. Rev. Phytopathol. 50, 23–43 40. McDonald, M.C. et al. (2017) The discovery of the virulence gene ToxA in the wheat and barley pathogen Bipolaris sorokiniana. Mol. Plant Pathol. Published online January 17, 2017. http://dx. doi.org/10.1111/mpp.12535 41. Lu, S. et al. (2011) Molecular characterization and genomic mapping of the pathogenesis-related protein 1 (PR-1) gene family in hexaploid wheat (Triticum aestivum L.). Mol. Genet. Genomics 285, 485–503 42. Oliver, R.P. and Solomon, P.S. (2010) New developments in pathogenicity and virulence of necrotrophs. Curr. Opin. Plant Biol. 13, 415–419 43. Fernández, C. et al. (1997) NMR solution structure of the pathogenesis-related protein P14a. J. Mol. Biol. 266, 576–593 44. Gibbs, G.M. et al. (2006) The cysteine-rich secretory protein domain of Tpx-1 is related to ion channel toxins and regulates ryanodine receptor Ca2+ signaling. J. Biol. Chem. 281, 4156–4163 45. Kelleher, A. et al. (2014) Schistosoma mansoni venom allergenlike protein 4 (SmVAL4) is a novel lipid-binding SCP/TAPS protein that lacks the prototypical CAP motifs. Acta Crystallogr. D Biol. Crystallogr. 70, 2186–2196 46. Choudhary, V. et al. (2014) The caveolin-binding motif of the pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is required for in vivo export of cholesteryl acetate. J. Lipid Res. 55, 883–894 47. Darwiche, R. et al. (2016) Structural and functional characterization of the CAP domain of pathogen-related yeast 1 (Pry1) protein. Sci. Rep. 6, 28838 48. Asojo, O.A. et al. (2011) Structural studies of human glioma pathogenesis-related protein 1. Acta Crystallogr. D Biol. Crystallogr. 67, 847–855 49. Wang, Y.-L. et al. (2010) Cobra CRISP functions as an inflammatory modulator via a novel Zn2+- and heparan sulfate-dependent transcriptional regulation of endothelial cell adhesion molecules. J. Biol. Chem. 285, 37872–37883 50. Xu, X. et al. (2012) Structure of protein having inhibitory disintegrin and leukotriene scavenging functions contained in single domain. J. Biol. Chem. 287, 10967–10976 51. Darwiche, R. et al. (2017) The pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is a fatty acidbinding protein. J. Biol. Chem. 292, 8304–8314 52. Zipfel, C. (2014) Plant pattern-recognition receptors. Trends Immunol. 35, 345–351 53. Teixeira, P.J.P.L. et al. (2013) Novel receptor-like kinases in cacao contain PR-1 extracellular domains. Mol. Plant Pathol. 14, 602– 609 54. Lu, S. et al. (2017) Molecular cloning and characterization of two novel genes from hexaploid wheat that encode double PR-1 domains coupled with a receptor-like protein kinase. Mol. Genet. Genomics 292, 435–452 55. Robert, X. and Gouet, P. (2014) Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324
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Emerging Insights into the Functions of PathogenesisRelated Protein 1 Susan Breen,1,3 Simon J. Williams,1,3 Megan Outram,2 Bostjan Kobe,2 and Peter S. Solomon1,* The members of the pathogenesis-related protein 1 (PR-1) family are among the most abundantly produced proteins in plants on pathogen attack, and PR-1 gene expression has long been used as a marker for salicylic acid-mediated disease resistance. However, despite considerable interest over several decades, their requirement and role in plant defence remains poorly understood. Recent reports have emerged demonstrating that PR-1 proteins possess sterol-binding activity, harbour an embedded defence signalling peptide, and are targeted by plant pathogens during host infection. These studies have reenergised the field and provided long-awaited insights into a possible PR-1 function. Here we review the current status of PR-1 proteins and discuss how these recent advances shed light on putative roles for these enigmatic proteins. The PR-1 Family PR-1 family members were first identified in the 1970s from Nicotiana tabacum infected with tobacco mosaic virus (TMV) [1]. Using this pathosystem it was shown that some, but not all, tobacco PR-1 proteins were upregulated during TMV infection [2–5]. We now know that PR-1 proteins are ubiquitous across plant species. They occur in multigene families within plant genomes and can be broadly characterised into classes (acidic or basic) depending on their theoretical isoelectric point. They are members of a broader protein family known as the cysteine-rich secretory protein, antigen 5, and pathogenesis-related-1 (CAP) protein superfamily [6]. The CAP domain comprises approximately 150 amino acids and CAP-containing proteins are represented in more than 2500 species including bacteria, fungi, plants, and animals, with diverse roles in reproduction, cancer, immune defence, sterol binding and export, and ion binding (http://pfam.xfam.org/family/PF00188) [7]. In plants PR-1 proteins are among the most abundantly produced proteins during defence responses and have been reported to constitute 2% of the total leaf protein in pathogeninfected tobacco [8]. Most members of the PR-1 family are thought to be secreted and accumulate in the extracellular/apoplastic space, which is facilitated by their N-terminal secretion peptide. However, research in tobacco has also shown that some PR-1 proteins accumulate in the vacuoles of protoplasts [9] and the vacuoles of specialised cells known as crystal idioblasts [10]. PR-1 proteins are not exclusively associated with host defence. Numerous reports have shown that PR-1 genes are responsive to abiotic stimuli, suggesting important roles in abiotic stress responses [11–17]. There is also evidence to suggest that PR-1 proteins have a role in plant growth or development that is independent of stress responses [18]. For example, PR-1 proteins were shown to accumulate strongly in the senescing leaves of adult flowering plants [19]. A subsequent study by Lotan et al. showed that PR-1 protein
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Trends Recent studies have shown that plant pathogenesis-related protein 1 (PR-1) family members bind sterols. This function is responsible for antimicrobial activity towards sterol auxotrophs such as Phytophthora species. However, the link between sterol binding and the proposed broader antimicrobial function of PR-1 remains unclear. PR-1 proteins harbour an embedded C-terminal peptide (CAPE) involved in plant immune signalling. Evidence suggests that CAPE has a signalling role that facilitates defence responses against microbial pathogens and also herbivores. The CAPE response is independent of other defence signalling pathways such as those elicited by recognised pathogen-associated molecular patterns. The significance of PR-1 proteins during plant–microbe interactions is now realised, with a growing list of identified pathogen effector proteins that directly interact with PR-1 during infection.
1 Plant Sciences Division, Research School of Biology, The Australian National University, Canberra 2601, Australia 2 School of Chemistry and Molecular Biosciences, Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre, University of Queensland, Brisbane, Australia 3 These authors contributed equally to the manuscript.
*Correspondence: [email protected] (P.S. Solomon).
http://dx.doi.org/10.1016/j.tplants.2017.06.013 © 2017 Elsevier Ltd. All rights reserved.
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accumulated in the sepals of developing flowers, further alluding to a potential role in plant flowering [20]. Yet, their function in plant growth or development is unknown. However, it is their upregulation in salicylic acid-dependent defence responses and their subsequent use as a defence marker that are their most distinguishable features in plants. The regulation of PR-1 gene expression has been well described and the pathways that lead to PR-1 induction during host defence have been identified [21]. Functional studies over the past 30 years have indicated a potential role for PR-1 proteins in host defence, but it is reasonable to suggest that much of this evidence is circumstantial. Recent reports, however, have provided some tantalising evidence of possible roles, and this review focusses on these studies and how they contribute to our understanding of the function of these ubiquitous proteins.
Are PR-1 Proteins Antimicrobial? The upregulation and abundance of PR-1 proteins during infection, combined with their localisation to the apoplast (an important interface for plant–microbe interactions) lend themselves to a potential antimicrobial function. Such functions have been linked to other PR proteins that are also upregulated by salicylic acid such as PR-2 (an acidic form of beta-1,3glucanase) and PR-5 (a thaumatin-like protein), which possess recognised enzymatic activities known to inhibit microbial growth [22,23]. Several groups have demonstrated that overexpression of PR-1 in transgenic plants results in increased resistance to fungi [24] oomycetes [8,25], and bacteria [25,26] but not viruses [27]. Consequently, it was considered that the observed increased resistance was a result of broad antimicrobial activity by PR-1. Niderman and colleagues first presented evidence of anti-oomycete activity by incubating PR-1 proteins isolated from tomato (Solanum lycopersicum) and tobacco (Nicotiana tabacum) with Phytophthora infestans zoospores and assaying for inhibition of germination. They demonstrated that PR-1 protein concentrations of 20–200 mg/ml (depending on the PR-1 protein assayed) were sufficient to inhibit 90% of zoospore germination. Furthermore, exogenous application of these proteins also inhibited P. infestans colonisation of tomato leaf discs [28]. The yields the authors obtained from infected plants (0.6–4 mg/g tissue fresh weight) were lower than the concentrations required for in vitro anti-oomycete activity; however, the authors argue that the local concentration within extracellular spaces would likely be significantly higher (10) in vivo (based on PR-1 localisation towards infection sites) and suggest a direct PR-1 protein anti-oomycete function. The application of PR-1 proteins produced in Escherichia coli also restricted P. infestans zoospore germination [29] and the growth of some fungal plant pathogens, albeit at high PR-1 protein concentrations (200–600 mg/ml). A more recent study by Gamir and colleagues demonstrated that the heterologously expressed PR-1 proteins P14c (from tomato) and PR-1a (from tobacco) inhibited the growth of the oomycete pathogen Phytophthora brassicae at concentrations of 20 mg/ml (1.1 mM) [30]. These data added to a host of evidence that PR-1 proteins have anti-oomycete properties. However, using the same experimental procedure there was no observed effect on the growth of Aspergillus niger or Botrytis cinerea, questioning the antifungal activity of PR1 proteins. Importantly, this work described the first biochemical function for PR-1 proteins. Armed with the knowledge that the CAP domain from the yeast proteins PRY1 and PRY2 have a sterol-binding function [31] (discussed further below), Gamir and colleagues demonstrated that P14c and PR-1a can genetically complement yeast sterol-transport-deficient mutants. Purified P14c and PR-1a proteins were shown to bind cholesterol with modest affinity (Kd 30 mM) and P14c had indistinguishable binding preferences for the fungusspecific sterol ergosterol, the plant-derived sterol stigmasterol, and cholesterol. The inhibition of P. brassicae growth by P14c was prevented when cholesterol was added to the medium and when a mutant unable to bind sterols (P14cC146S) was substituted for P14c, providing a
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critical link between sterol binding and anti-oomycete activity [30]. The authors suggested that the antimicrobial activity of PR-1 towards P. brassicae is due to the oomycete’s inability to produce its own sterols (sterol auxotrophy). They subsequently showed that the addition of non-lethal doses of fungicide that block sterol biosynthesis in A. niger and B. cinerea made the fungi susceptible to P14c. From their data Gamir and colleagues present a model suggesting that PR-1 proteins are antimicrobial by sequestering sterols from the membranes of microbes and are more effective against sterol auxotrophs, which must obtain sterols from the environment. However, if PR-1 proteins are not effective in vitro against sterol prototrophs such as fungi, how could antifungal activity be achieved in vivo? Gamir and colleagues suggest that the vacuole-targeted PR-1 proteins may provide the answer. They speculate that the delivery of a large PR-1 payload (from within the vacuole) to an invading fungus may provide the concentration of PR-1 protein that is required to sequester more sterols than can be replaced by the sterol biosynthesis machinery, achieving in vivo antifungal properties [30]. While this is an interesting idea, their work clearly shows that further experimental validation is required before a true, physiological antifungal function can be attributed to PR-1 proteins. This also highlights that maybe we should not be thinking of PR-1 proteins as purely pathogen killers. After all, how could this single function explain the role of PR-1 proteins in host responses independent of pathogens? An explanation of the expanded functions of PR-1 in pathogen inhibition and regulation under abiotic stress conditions may be found in the recent discovery that PR-1 proteins carry an embedded stressresponse peptide [32].
CAP-Derived Peptide 1 (CAPE1) Stress-Signalling Peptides Are Embedded within PR-1 Proteins Recently, Chen and colleagues identified a peptide that was strongly induced in response to wounding and methyl jasmonate treatment in tomato [32]. Subsequent analysis of the peptide revealed that it comprised the last 11 amino acids from the C terminus of the tomato PR-1 protein and thus it was named CAPE1. Growth experiments with Spodoptera litura larvae fed on tomato leaves pretreated with synthetic CAPE1 demonstrated suppressed growth and a 20% reduction in larval weight. In addition, plants pretreated with CAPE1 showed no significant symptoms of infection when inoculated with Pseudomonas syringae pv tomato (Pst) strain DC3000 and reduced bacterial populations compared with controls [32]. Gene expression studies of CAPE1-treated leaves demonstrated that application of the peptide induced multiple defence-related genes, including proteinase inhibitor 1 (PI-1), PI-2, PR-2, PR7, ethylene response factor 5 (ERF5), and the precursor protein PR-1b [32]. The induction of these genes in the absence of biotic stress strongly suggests that CAPE1 has a role as a defence-signalling molecule as opposed to a direct antimicrobe/herbivore function. Interestingly, CAPE1 did not upregulate WRKY TRANSCRIPTION FACTOR53 (WRKY53), which is highly upregulated in response to the application of the pathogen-associated molecular pattern (PAMP) peptide flg22 [32]. These data suggest that the CAPE1 peptide does not induce the canonical PAMP-triggered immunity (PTI) signalling pathway described in other peptide-mediated responses and implies that the CAPE1 peptide primes defence through an independent signalling pathway. Chen and colleagues compared PR-1 sequences from various monocot and dicot plants and identified the conserved consensus motif PxGNxxxxxPY in the CAPE1 peptide [32]. This consensus sequence was experimentally validated by assessing the efficacy of the Arabidopsis thaliana AtCAPE-PR-1 peptide on Pst DC3000 infection in tomato. As observed with CAPE1, the application of AtCAPE1-PR-1 on tomato resulted in increased immunity. This is significant as AtCAPE-PR-1 shares only the amino acids within the consensus motif with CAPE1, proving the importance of the conserved residues for the CAPE-induced defence response. A highly
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conserved CNYx motif positioned N terminal to the CAPE peptide was also predicted to represent the CAPE cleavage motif (Figure 1). A subsequent study confirmed the requirement for the CNYx motif for CAPE1 cleavage through a site-directed mutation approach; however, the mechanism of cleavage is unknown [33].
Figure 1. Putative and Established Features of the Enigmatic Plant Pathogenesis-Related Protein 1 (PR-1). (A) The NMR structure of tomato cysteine-rich secretory protein, antigen 5, and pathogenesis-related (CAP) protein (Sl) pathogenesis-related protein 14a (SlP14a) solved by NMR spectroscopy [43], shown in cartoon representation. Residues shown in stick representation are labelled. Disulfide bonds are shown in yellow. Additional highlighted features of the structure identify functional and putative functional regions. Magenta, CAP-derived peptide (CAPE) with conserved residues within the consensus sequence labelled [32]. Pink, CAPE cleavage motif as described in [33]. Light blue, caveolin-binding motif (CBM) representing the putative sterol-binding motif from pathogen-related yeast 1 (PRY1) proteins [46]. Orange, conserved residues representing the CAP tetrad and coordinate metals in the PRY1 CAP domain structure [47]. Blue, residues involved in fatty acid binding in tablysin-15, conserved in PRY1 and PR-1 proteins. (B) ClustalW sequence alignment of plant PR-1 domains and the CAP domain from the yeast protein PRY1 prepared using ESPript 3.0 [55]. Secondary-structure elements above and below the alignment are derived from the structure of SlP14a (PDB ID: 1cfe) and PRY1 (PDB ID: 5jys). Features highlighted in (A) are also highlighted on the alignment with the same colour scheme. CBM and CAPE are labelled, while orange and blue stars represent the CAP tetrad and conserved tablysin-15 fatty acid-binding residues. SlP14a and SlP14c are from tomato [32,43], NtPR-1a is from tobacco [30], TaPR-1-1 and TaPR-1-7 are from wheat [41], TaPR-1_RLK_1_1 and _2 represent the first and second PR-1 domain in TaPR-1-receptor-like kinase (RLK1) from wheat [54], TcPR-1g_RLR is the PR-1 domain from PR-1-RLK1 from cocoa [53], and PRY1 is from yeast [31].
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Interestingly, the CAPE peptide is not restricted to PR-1 proteins implicated in pathogen defence. For example, of the 22 CAP proteins in A. thaliana nine were identified to contain the consensus sequence defined by Chen and colleagues [32,33] despite only AtPR-1 being upregulated in infected tissue [34]. The CAP gene AT4G33730 (designated PROAtCAPE1) from A. thaliana is a salt-responsive gene, whereby expression is reduced on salt treatment [33]. Silencing of PROAtCAPE1 resulted in transgenic plants with increased survival on highsalt media, while exogenous application of AtCAPE1 to the same transgenic plants resulted in reduced growth and salt-sensitive phenotypes. AtCAPE1 was subsequently shown to downregulate salt tolerance by suppressing genes that are associated with salt tolerance [33]. The authors subsequently suggested that CAPE peptides could function in a tradeoff between pathogen defence and salt tolerance [33]. The identification of CAPE peptides and their observed roles in stress (biotic and abiotic) responses represents a significant advancement in our understanding of PR-1 protein function. Undoubtedly, significant research challenges remain. How are CAPE peptides perceived? Is there a CAPE receptor? How are CAPE peptides cleaved from PR-1 proteins? What is clear is that both PR-1 proteins and CAPE peptides are important host defence molecules, making these conceivable targets for pathogens to overcome host defence responses.
PR-1 Proteins: A Pathogen Effector Hub? Despite the absence of an in vivo function, PR-1 proteins are clearly an integral component of host defence that need to be countered by colonising pathogens. Accordingly, recent studies have identified three pathogen effectors that interact with PR-1 proteins [35–37]. The best characterised of these effectors are the ToxA and Tox3 proteins. The ToxA effector has been identified in the wheat pathogens Parastagonospora nodorum, Pyrenophora tritici-repentis, and Bipolaris sorokiniana, while Tox3 is unique to P. nodorum [38–40]. Independent yeast-twohybrid (Y2H) library screens showed that ToxA and Tox3 both interact with wheat PR-1 proteins [36,37]. Despite being effectors from the same pathogen, their interactions with PR-1 appear to be fundamentally different. The ToxA interaction is highly specific, occurring only with the basic protein PR-1-5, whereas Tox3 is far more promiscuous, interacting with six of the eight tested wheat PR-1 proteins, including acidic and basic proteins but not basic PR-1 proteins with a Cterminal extension (CTE) [36,37,41]. In both studies the effectors appeared to interact with amino acids towards the C terminus of PR-1. In the case of ToxA, the mutation of a relatively well-conserved asparagine (PR-1-5 N141A) resulted in loss of interaction with ToxA [36]. Interestingly, this residue is conserved in a non-interacting wheat PR-1 protein, PR-1-1, which shares 90% sequence identity with PR-1-5, suggesting that other residues in PR-1-5 must provide specificity for the interaction. Mutation of the same asparagine in PR-1-1 did not disrupt the Tox3–PR-1-1 interaction and suggests that the dynamics of the ToxA/Tox3–PR-1 interactions differ [37]. The two PR-1 proteins (PR-1-18 and -21) that did not interact with Tox3 belong to the basic PR-1 proteins with CTEs. Interaction between Tox3 and the C-terminal section of PR-1-18 could be restored by substituting the predicted surface-exposed amino acids that differed between PR-1-18 and PR-1-1 [37]. However, these amino acid substitutions were not enough to induce an interaction with the full-length PR-1-18 protein and it appears that amino acids at the N terminus of these PR-1 proteins also control Tox3 specificity [37]. The interaction between these effectors and the PR-1 proteins also appears to influence the susceptibility of the host. The Tox effectors are major virulence determinants in the P. nodorum– wheat interaction; however, their effectiveness relies on the presence of host-susceptibility genes [42]. The wheat gene Tsn1 is required for ToxA-induced necrosis, while Tox3-induced necrosis relies on the presence of Snn3. Wheat lines lacking these genes, Tsn1 and Snn3, are resistant to isolates of P. nodorum expressing ToxA or Tox3, respectively. This gene-for-gene interaction has an inverse effect on the plant compared with the classically described gene-for-
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gene interaction in biotrophic pathogens. In the case of ToxA, Lu and colleagues showed that co-infiltration of ToxA and PR-1-5 into sensitive wheat lines enhanced the necrosis phenotype induced by ToxA alone. By contrast, the non-interacting PR-1-5N141A mutant did not, suggesting that the interaction between ToxA and PR-1-5 plays a role in promoting necrosis in ToxAsensitive wheat (Tsn1-containing wheat) [36]. Tox3 wheat leaves pretreated with synthetic TaCAPE1 (sequence from the wheat PR-1-1 protein) before infection with P. nodorum exhibited increased disease symptoms [37]. This phenotype was observed only in wheat cultivars that were susceptible to Tox3 (Snn3-containing wheat) [37]. Therefore, the interaction of the ToxA and Tox3 effectors with the PR-1 proteins appears to facilitate infection of the host in a genotype-specific manner. It remains to be seen how Tox3 utilises TaCAPE1 for this function and what role Snn3 (gene currently unknown) plays in the interaction. Could Tox3 induce the cleavage of the TaCAPE1 peptide, triggering the activation of host-defence responses that the necrotrophic pathogen P. nodorum is able to hijack and induce increased programmed cell death? Intriguingly, while CAPE had a protective function against P. syringae [32] the observation that a wheat cultivar lacking Snn3 showed no inhibition of P. nodorum disease when treated with TaCAPE1 suggests that CAPE is ineffective against some pathogens or is primarily effective against biotrophic pathogens. The third effector identified to interact with PR-1 is CSEP0055 from powdery mildew fungus [Blumeria graminis f.sp. hordei (Bgh)] [35]. This effector–host protein interaction was identified using a Y2H assay on a library generated from Bgh-infected barley leaves. The authors identified PR-17a, PR-17c, PR-1a, and PR-1b as host proteins interacting with CSEP0055 [31]. PR-17c was found in multiple prey clones and downstream work focused on the CSEP0055–PR-17c interaction over the PR-1 interaction [35]. Unfortunately, no further information has been reported describing the biological effect of the CSEP0055–PR-1 interaction. To date, three effectors from different fungal plant pathogens have been found to interact with host PR-1 proteins. Given the importance of PR-1 proteins in plant defence, it will be interesting to see whether more effectors from diverse plant pathogens target these proteins and associated pathways in the future.
What Can We Learn from Structural and Biochemical Studies of CAP Domains? The first CAP domain structure to be determined corresponds to the NMR structure of the tomato P14a [43] (Figure 1). To date this is the only published structure of a plant PR-1 protein; however, since then the structures of 21 CAP-containing proteins have been solved. The CAP domain folds into a unique a-b-a sandwich, generally comprising four a helices and four b strands, and is stabilised by conserved disulfide bonds (Figure 1). While many CAP domaincontaining proteins contain additional functional domains [44], PR-1 proteins contain only the CAP domain with relatively short C- and N-terminal extensions, suggesting that it is the function of the CAP domain that determines their role in plants. Therefore, structural and biochemical studies of CAP domains from other proteins can assist our understanding of PR-1 function. The sterol-binding ability of the CAP domain was first reported in the yeast proteins PRY1 and PRY2 and the human protein CRISP2 [31]. The same function was described in the Schistosoma mansoni venom allergen-like protein SmVAL4 [45] and subsequently in the PR-1 proteins P14c and PR-1a (discussed above) [30]. To date, sterol binding has not been captured in a 3D structure. Despite this, computational modelling of the yeast PRY1 identified a flexible loop with sequence similarity to the caveolin-binding motif (CBM), which suggested that this region is involved in sterol binding [46] (Figure 1). Point mutations in the CBM motif of PRY1 (F239L, F244L, P242C) resulted in loss of cholesteryl acetate binding in PRY1 [46].
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A recent crystallographic study of the CAP domain from PRY1 demonstrated that magnesium binding to distal regions of the CAP domain is required for sterol binding [47]. In this structure magnesium is coordinated by the so-called CAP tetrad, encompassing direct binding between two well-conserved histidine residues that are positioned relative to two conserved glutamate residues (Figure 1). These conserved residues have also been shown to bind Zn2+ in other CAP domains, including the human glioma PR-1 protein and a snake-venom cysteine-rich secretory protein, where it was shown to be functionally important [48,49]. PRY1 is known to dimerise in solution and it is suggested that the Mg2+-binding site and sterol-binding regions would be connected within a PRY1CAP dimer [47]. Despite this, neither dimerisation nor Mg2+ binding appears to be required for sterol binding by the SmVAL4 CAP domain, which binds sterols, lacks the CAP tetrad, and is a monomer in solution [45]. For plant PR-1 proteins, it remains unclear whether the designated CBM motif and CAP tetrad influence the sterol-binding and ionbinding functions, respectively. While the CAP tetrad is conserved (Figure 1), there are divergent features in the CBM motif of PR-1 proteins, including two additional cysteine residues that form an additional disulfide bond. This would be likely to affect the flexibility of this region, which could influence sterol binding. Despite this, the aromatic residues that are important for the sterol-binding function of PRY1 are generally conserved (Figure 1), supporting a shared CBM function. A striking feature of the CBM region with reference to the NMR structure of P14a is that this region accommodates (at least in part) the embedded C-terminal CAPE peptide (Figure 1). If the CBM region is involved in sterol binding in PR-1 proteins, it is difficult to explain how a PR-1 protein could accommodate both sterol and CAPE simultaneously. This observation provides a tantalising possibility that sterol binding may be associated with the repositioning of the embedded C-terminal CAPE amino acids. Perhaps this conformation change is involved in CAPE cleavage; alternatively, CAPE may help to regulate the sterol-binding activity of PR-1 proteins. Interestingly, the CAP domain of other proteins has also been shown to bind other lipids. The crystal structure of the horsefly (Tabanus yao) CAP domain-containing protein tablysin-15 revealed that the E. coli-produced protein was bound to palmitic acid [50]. Xu and colleagues subsequently demonstrated that tablysin-15 could bind the proinflammatory fatty acid leukotriene, implicating the CAP domain in the protein’s anti-inflammatory function [50]. Recently, the yeast protein PRY1 has also been shown to bind fatty acids, with micromolar affinity [51]. This function appears to be independent of PRY1’s sterol-binding activity, suggesting that a single CAP domain can accommodate the binding of multiple, structurally diverse lipids. While fatty acid binding has, to date, not been reported for PR-1 proteins, a number of the hydrophobic residues involved in fatty acid binding in tablysin-15 are conserved in PR-1 proteins (Figure 1). The literature reviewed above suggests that the CAP domain of PR-1 proteins might have multiple functions (i.e., sterol, ion, and lipid binding and peptide signalling); however, to date there are no publications that describe a mechanistic relationship between these functions. In light of insights provided by research on other CAP family members, it seems possible, even likely, that the multiple functions associated with the CAP domain are dependent on each other. For example, we could speculate based on the CBM accommodation of the CAPE peptide that PR-1 proteins act as sterol sensors, utilising CAPE for signalling. Interestingly, in line with these hypothesised multiple functions two recent reports [53,54] have shown that the PR-1 domain is associated with a diverse class of plant receptors that are implicated in sensing molecules in the extracellular environment.
PR-1 Receptor-Like Kinases (RLKs): Part of the PR-1 Puzzle? RLKs are one of the most abundant classes of plant proteins and are implicated in perceiving signals from the extracellular environment, enabling plant cells to react to these accordingly. The majority of RLKs possess a single transmembrane domain, an intracellular kinase domain,
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and an extracellular ligand-binding domain (ectodomain). RLKs are generally classified according to the ectodomain, which can comprise many different functional domains. In plant immunity, RLKs play important roles in the perception of both PAMPs and damage-associated molecular patterns (DAMPs), which are associated with pathogen invasion [52]. Recently, genes encoding extracellular PR-1 domains fused to transmembrane and kinase domains have been found in cocoa [53], wheat, rice, and several other plant species [54]. In cocoa, PR-1RLKs are upregulated on challenge with the biotrophic pathogen Moniliopthora perniciosa, the causal agent of cacao’s witch’s broom disease [53]. Similarly, the expression of two PR-1-RLK genes identified in wheat was induced in response to biotic (including barley stripe mosaic virus) and abiotic (including high salt) stress. In wheat, dual PR-1 ectodomains are observed in PR-1RLK, while a single PR-1 ectodomain is present in the proteins encoded in cocoa [53,54]. The integrated PR-1 domains from the wheat PR-1-RLK proteins share 50% identity with the PR1 proteins shown to be involved in sterol binding and contain some of the conserved aromatic residues in the CBM (Figure 1). While their functions in stress responses remain unclear, in light of the general role that RLKs play in the recognition and induction of defence responses, it is tempting to speculate the PR-1-RLKs may play a role in sterol sensing. Alternatively, given that effectors are known to target PR-1 proteins, perhaps PR-1-RLKs function in effector recognition.
Outstanding Questions Is there a link between sterol binding and CAPE signalling? Does sterol binding promote CAPE release? Do PR-1 proteins bind other lipids, such as fatty acids? How is CAPE cleaved from PR-1 proteins? Are peptidases involved or can PR-1 cleave itself in a caspase-like process? What is the identity of the CAPE peptide receptor? What pathways act downstream of CAPE detection and/or accumulation? Does the peptide itself possess functional properties beyond signalling (i.e., cell lysis or antimicrobial properties)? Do PR-1 domains in the PR-1-RLKs sense sterols or are they an integrated effector-recognition domain? Do PR1-RLKs signal using mechanisms similar to those of known RLK pathways?
Concluding Remarks and Future Perspectives In recent years, clear progress has been made towards understanding the role of PR-1 proteins in plant defence. The emergence of a biochemical function implicating PR-1 proteins in sterol binding and the identification of the CAPE peptide and PR-1-RLKs suggest multiple roles, from antimicrobial function and defence signal amplification to potential sterol or effector recognition. These PR-1 functions all signify challenges for plant pathogens and it remains to be seen whether more plant pathogens target PR-1 proteins during defence. Finally, the knowledge gleaned from other CAP domain proteins has guided and will continue to guide PR-1 functional studies to help ascertain their role in plants. Here we highlight the present research regarding the role of PR-1, but many more questions remain that will drive future research on this topic in the coming years (see Outstanding Questions). References 1. van Loon, L.C. and van Kammen, A. (1970) Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacum var. ‘Samsun’ and ‘Samsun NN’. Virology 40, 199–211
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