Corruption of host seven-transmembrane proteins by pathogenic microbes: a common theme in animals and plants?

Microbes and Infection 5 (2003) 429–437 www.elsevier.com/locate/micinf

Review

Corruption of host seven-transmembrane proteins by pathogenic microbes: a common theme in animals and plants? Ralph Panstruga *, Paul Schulze-Lefert Department of Plant-Microbe Interactions, Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, 50829 Cologne, Germany

Abstract Human diseases like AIDS, malaria, and pneumonia are caused by pathogens that corrupt host chemokine G-protein coupled receptors for molecular docking. Comparatively, little is known about plant host factors that are required for pathogenesis and that may serve as receptors for the entry of pathogenic microbes. Here, we review potential analogies between human chemokine receptors and the plant seventransmembrane MLO protein, a candidate serving a dual role as docking molecule and defence modulator for the phytopathogenic powdery mildew fungus. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Chemokine receptors; Defence suppression; Molecular docking; Molecular pathogenesis; Plant-microbe interactions

1. Introduction In recent years, it has become evident that innate immunity in plants and animals shares many structural and functional similarities, suggesting a common evolutionary origin of pathogen perception and respective signal transduction in higher eukaryotes [1,2]. Extracellular pathogen-derived molecules that are conserved between different taxa of microbial organisms (also designated pathogen-associated molecular patterns) are recognized by membrane-anchored Toll-like receptors (TLRs) in animals and plants. Drosophila Toll was first shown to function in pattern formation and, several years later, its dual role in insect innate immunity emerged (reviewed in [3]). TLRs consist of an extracellular leucine-rich repeat (LRR) part, a single membrane spanning helix, and either a cytoplasmic domain with significant similarity to the intracellular region of the interleukin-1 receptor (animals) or a serine/threonine kinase domain (plants). Examples of membrane-anchored innate immunity TLRs are the flagellin receptors TLR5 from mouse and FLS2 from Arabidopsis thaliana [1]. Cytosolic counterparts of the membraneanchored TLRs are also present in animals and plants. These may survey the intracellular space for pathogen-associated molecular patterns and/or isolate-specific pathogen effector * Corresponding author. Tel.: +49-221-5062-316; fax: +49-221-5062-353. E-mail address: [email protected] (R. Panstruga). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 1 2 8 6 - 4 5 7 9 ( 0 3 ) 0 0 0 5 3 - 4

components. Examples are vertebrate Nod proteins, typically consisting of a caspase recruitment domain, a nucleotidebinding oligomerization domain (NOD), and LRRs, or variations thereof [4]. The cytoplasmic plant equivalents are represented by the largest subgroup of disease resistance proteins. They are composed of either an N-terminal coiledcoil (CC) domain or a domain with homology to the Toll/interleukin-1 receptor of animals, a central part sharing consensus nucleotide binding and other motifs with NOD, and C-terminal LRRs (reviewed in [5]). Taken together, recognition processes in innate immunity in plants and animals involve proteins that are composed of a limited number of similar building blocks and share a common architecture. Apart from the structural similarities of molecules mediating pathogen recognition in both phyla, a common architecture appears to also exist in downstream signalling pathways. This is well documented in the case of the transmembrane receptors. Signalling includes protein phosphorylation via a serine/threonine kinase, which is in the case of the plant FLS2 an integral module of the receptor, stimulation of a mitogen-activated protein kinase cascade, and subsequent activation of transcriptional regulators like NF-jB (e.g. insects and vertebrates) or WRKY factors (plants; reviewed in [1]). Given the striking similarity between the molecular architecture of innate immunity across kingdoms, one should not be surprised to find further molecular commonalities in pathogenesis-related processes. Here, we discuss whether fundamental pathogenesis pro-

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cesses like molecular docking to target cells and suppression of the host’s defence machinery in animals may have parallels in plant-microbe interactions. 2. Chemokine receptor piracy by pathogens causing AIDS, malaria, and pneumonia Pathogenic microbes use diverse strategies for host cell admission, including entry via lipid rafts [6] and induced membrane ruffling [7]. Attachment of the pathogen to the host membrane might involve binding of microbial proteins to specific host surface proteins. Examples are the attachment of Escherichia coli adhesin FimH to the urothelial receptor uroplakin [8] or binding of internalin to E-cadherin during infection of epithelial cells by Listeria monocytogenes [9]. Likewise, several taxonomically unrelated human pathogens exploit the presence of host seven-transmembrane (7-TM) G-protein coupled receptors (GPCRs) to gain access to their target cells. Known examples include the human immunodeficiency virus type 1 (HIV-1), a protozoan (Plasmodium vivax), and the Gram-positive bacterium Streptococcus pneumoniae (reviewed in [10]). HIV-1 is the main source of the acquired immunodeficiency syndrome (AIDS), whereas P. vivax and S. pneumoniae are major causes of human malaria and pneumonia, respectively. HIV-1 utilizes chemokine GPCRs of cells harbouring the CD4 antigen (CD4+ cells; mainly T lymphocytes) as target, whereas P. vivax enters red blood cells (erythrocytes) via the Duffy chemokine receptor. Similarly, S. pneumoniae gains access to endothelial cells by binding to receptors for plateletactivating factor, a non-chemokine leukocyte chemoattractant lipid [11]. Recently, it was found that myxoma virus, a member of the poxvirus family and the cause of myxomatosis in rodents, also exploits chemokine receptors to invade its host cells [12]. Interestingly and perversely, in all these cases the pathogens exploit components of the host’s immune system. Chemokines comprise a family of small (8–10 kDa) polypeptides that are structurally related and activate mammalian leucocytes for pathogen defence. They are secreted and serve as ligands by binding to the extracellular domains of specific plasma membrane-localized GPCRs, the chemokine receptors. The chemokine system is thought to stimulate leukocytes via binding of chemokines to cognate chemokine receptors and subsequent activation of intracellular downstream events by a signal transduction cascade initiated by heterotrimeric G-proteins [10]. The corruption of chemokine receptors by HIV-1 is the best-studied case of the examples outlined above. The gp120 envelope glycoprotein of HIV-1 binds to the CD4 antigen that acts as the main receptor for docking of the virus to its target cells. It was found that presence of CD4 is necessary but not sufficient to allow effective entry of the virus. Presence of a co-receptor of the chemokine class of GPCRs greatly promotes efficient fusion of viral and host membranes. A number of chemokine receptors of the CC- and

CXC-class, including CCR2, CCR3, CCR4, CCR5, CCR8, CXCR4, CXCR6, and GPR15 were shown to act as HIV-1 co-receptors in vitro. However, it was found that CCR5 and CXCR4 serve as the main co-receptors in vivo. Site-directed mutagenesis of single amino acids revealed the significance of extracellular domains of CCR5 and CXCR4 for coreceptor function [13,14]. The amino terminus of both CCR5 and CXCR4 seems to be of particular importance, since replacements of several amino acids in this region impaired co-receptor activity (reviewed in [14]). In contrast, a carboxyl-terminal truncated variant of CCR5 (D45 amino acids) displayed loss of G-protein-mediated signal transduction but no reduction in gp120-mediated cell fusion or HIV-1 infection, suggesting that intracellular signalling via heterotrimeric G-proteins is not required for co-receptor function [15]. Human individuals homozygous for a naturally occurring defective allele of CCR5 were found to have greatly enhanced resistance to HIV-1 infection. The defect is caused by a deletion of 32 nucleotides (D-32) in the central part of the CCR5 gene, resulting in a CCR5 null allele. Likewise, a mutation in the promoter of the Duffy chemokine receptor gene leads to the absence of the Duffy receptor protein in the erythrocytes of most black Africans. Since Duffy is the receptor used as docking molecule by the protozoan P. vivax, the causal agent of vivax malaria, these individuals are highly resistant to this subtype of the global plague [10]. Interestingly, loss of either CCR5 or Duffy is not associated with a known pathologic phenotype, suggesting that these chemokine receptors are dispensable for life under present conditions. Moreover, the mutation in the Duffy promoter seems to mediate a selective advantage for inhabitants of Africa, since the mutation is fixed at high levels in black African populations (allele frequency of 0.8) but rare among whites [16]. The CCR5 D-32 allele arose apparently in Northern Europeans and its prevalence is low, about 1% homozygous and 15–20% heterozygous carriers in North American Caucasians, and has not yet been identified in individuals of purebred African or Asian origin [17]. 3. Plant candidate genes for pathogen “docking” Comparatively little is known about plant host factors that are required for pathogenesis and that may serve as receptors for the entry of pathogenic microbes. Conceptually, recessive inheritance of resistance to single or closely related pathogen species in plants may be an indicator for the existence of such “compatibility factors” or “docking molecules”. Indeed, genetic analysis of resistant lines in natural plant populations and of induced mutations revealed single recessive resistance loci in a range of plant species. These confer resistance to fungal (mlo in barley, [18], er-1 in pea [19], ol-2 in tomato [20], pmr6 in Arabidopsis [21]), bacterial (xa13 in rice [22], RRS1-R in Arabidopsis [23]), viral (bc-1(2) in bean [24]), and nematode (rk3 in cowpea, [25]) pathogens. Unlike these pathogen species-specific examples of recessive resistance, induced Arabidopsis edr1 mutants confer resistance to both

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Fig. 1. Powdery mildew infection phenotypes of barley Mlo wild-type and mlo mutant plants. Leaves of either genotype (Mlo, left; mlo, right) were inoculated with a high density of Bgh conidiospores. While Mlo wild-type plants support the growth of sporulating mildew colonies on the leaf surface, mlo mutant plants are immune to fungal attack. The photograph was taken 6 d post spore inoculation.

fungal and bacterial pathogens, suggesting that wild-type EDR1 is unlikely to serve as a pathogen “docking molecule” but is involved further downstream as negative regulator in disease resistance signalling [26]. Only induced resistant mutants are molecularly characterized to date, including defective alleles of barley Mlo [18], Arabidopsis EDR1 (encoding a putative mitogen-activated protein kinase kinase kinase, MAPKKK, [27]), and PMR6 (encoding a putative pectate lyase, [21]). One of the best-studied examples discussed here in greater detail is the 7-TM MLO protein in barley (Hordeum vulgare), which is essential for successful infection of the crop with the ascomycete powdery mildew fungus, Blumeria graminis f sp. hordei (Bgh, Fig. 1).

4. Breaching the walls: pathogenesis of powdery mildew fungi Powdery mildew is a common and widespread plant disease that is caused by obligate biotrophic ascomycete fungi called Erysiphales. Other genera of this fungal phylum are of clinical relevance, since they represent the causal agents of major human fungal diseases (e.g. Aspergillus ssp. → aspergillosis, Candida ssp. → candidiasis). The name “powdery mildew” reflects the powdery tarnish on the plant surface and the trickling spores that are the result of the nonsexual reproductive phase of the fungus. The Bgh infection cycle starts with the landing of a wind-blown haploid conidiospore on a barley leaf. After about 1 h, a primary germ tube emerges at one pole of the oval-shaped spore (Fig. 2). This is thought to contribute to recognition of the host surface, and firm physical attachment to the leaf, as well as to gain access to host water. During the following hours, a second germ tube emerges on the opposite side of the spore, elongates on the

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leaf surface, and forms at its end a swollen structure, the appressorium (Fig. 2). Approximately 12 h after spore landing, the fungus attempts to breach the host cell wall beneath the appressorium by means of a penetration peg. In compatible interactions (interactions leading to successful colonization), about 50–70% of germinated spores succeed penetration attempts, depending on environmental conditions and genetic variation in particular host lines and Bgh isolates. In case of successful cell wall penetration, the fungus establishes the haustorium, a specialized infection structure with finger-like protrusions, by about 24–h post inoculation (Fig. 2). The haustorium invaginates the host plasma membrane and is thought to serve as a “feeding” organ for nutrient uptake. Subsequent to successful establishment of the haustorium, growth of aerial hyphae takes place on the epidermal surface, and neighbouring cells may be attacked to establish further haustoria, leading ultimately to a fine web of white mycelium representing a mildew colony. After about 4–5 d of hyphal growth, short erect sporangiophores carrying chains of 5–10 conidiospores are formed, and subsequently massive amounts of conidiospores are released and wind-blown to initiate a new infection cycle. This asexual spread is the predominant mode of propagation during the warm summer months. Sexual reproduction takes place towards the end of the season, generating so-called cleistothecia that are overwintering structures. In the cleistothecium, haploid ascospores are generated by caryogamy and meiosis, which takes place in the so-called ascus. Upon release from the ascus, ascospores behave like conidiospores and are able to start new rounds of infection.

5. Recessive barley mlo alleles: blockbusters of powdery mildew resistance Resistance to Bgh is often inherited as dominant trait. Intracellular CC-NB-LRR innate immunity proteins, each recognizing a cognate isolate-specific fungal effector component, mediate known examples of this resistance [28]. Resistance mediated by recessive mlo alleles is race nonspecific, i.e. it is effective against all known isolates of the fungal pathogen. However, mlo alleles are ineffective against other fungal diseases including barley leaf rust (Puccinia striiformis), stripe rust (Puccinia hordei), scald (Rhynchosporium secalis), and the take-all fungus (Gaeumannomyces graminis) [29]. This would be consistent with the idea of Mlo encoding a pathogen species-specific compatibility factor. Interestingly, mlo mutants show enhanced disease susceptibility to the hemibiotrophic rice blast fungus, Magnaporthe grisea, and to the necrotrophic fungus Bipolaris sorokiniana [30,31]. This shows that wild-type Mlo influences sensitivity to more than one pathogen species in opposite directions. As demonstrated in Arabidopsis, different defence pathways in plants can be mutually inhibitory (reviewed in [32]). Therefore, elevated powdery mildew resistance in mlo mutants might render other resistance pathways that are effective

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Fig. 2. Scheme of the asexual life cycle and pathogenesis of the barley powdery mildew fungus. The scheme represents the chronological order of events after the landing of a conidiospore on a barley wild-type (Mlo) leaf. Black rectangles symbolize longitudinal sections of epidermal cells. Formation of the primary germ tube, the secondary germ tube, and the appressorium occurs within approximately 12 h after spore landing on the leaf surface. At about 24–h post inoculation, approximately 50–70% of the attacking sporelings succeed in differentiating a haustorium for nutrient uptake. This is essential for subsequent growth of secondary hyphae and asexual propagation via sporulation (~5 d post inoculation). In approximately 30–50% of penetration attempts, Bgh growth ceases coincident with the process of cell wall penetration. Microscopic photographs illustrate prominent stages of fungal development as top-view. Fungal structures were stained with Coomassie blue. CWA, cell wall apposition.

against other pathogens such as M. grisea less effective. The effects of Mlo on these different pathogens may be indirectly related to pathogen lifestyles. Blumeria is an obligate biotrophic pathogen that thrives on living host cells only, whereas M. grisea and B. sorokiniana kill host cells during compatible infections [31,33]. Thus, Mlo may serve the purpose of ensuring a balance between mutually inhibitory defence responses to different pathogen species. Perhaps Bgh targets MLO, in analogy to the human chemokine receptors, to corrupt a plant defence pathway.

6. Cell wall appositions: another brick in the wall Plants containing mlo resistance alleles support efficient Bgh spore germination and full differentiation of primary and secondary germ tubes. Further development of fungal structures ceases during the subsequent cell wall penetration stage. Abortion of fungal attack is tightly linked to local host cell wall remodelling and synthesis processes directly beneath fungal appressoria, resulting in the formation of ringshaped cell wall appositions (CWAs; also called papillae; Fig. 2). The tight link between mlo resistance and cessation of Bgh growth during CWA formation raises questions whether CWAs play a causal role in the resistance reaction.

The molecular organization of papillae is still poorly understood, but a major constituent is the carbohydrate polymer b-1,3 glucan (callose). Papillae are deposition sites for small plant-derived molecules like phenolic compounds and proteins such as extracellular peroxidases [34,35]. In addition, a sustained accumulation of reactive oxygen species (ROS) is observed at CWAs that coincides with attempted fungal invasion [36]. The CWA-associated ROS are likely to serve as catalysts for oxidative cell wall cross-linking processes and may also exert direct antimicrobial effects. Consistent with this, CWAs become resistant to cell wall degrading enzymes, unlike adjacent cell walls, and the deposited phenolic compounds become resistant to alkaline extraction during CWA maturation, which is indicative of rapid dynamic changes of chemical bonding types in the wall appositions [37]. Together, this suggests a role of papillae in structural cell wall reinforcement against fungal ingress. However, papillae formation also takes place at interaction sites that are successfully penetrated by Bgh germlings in Mlo wild-type plants [38]; Fig. 2. Although it has been noted that the process of cell wall remodelling initiates earlier, supports higher levels of ROS and faster cross-linking of phenolics in resistant mlo compared to susceptible Mlo plants, this is not conclusive evidence for a direct (negative) role of Mlo in the CWA fabrication process. The fungus may have a means to sup-

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press the accumulation of ROS, which is less effective if fungal growth ceases early. 7. Barley MLO: a structural GPCR analog required for mildew pathogenesis The first described mlo mutant was induced by X-rays in 1942 [39]. Subsequently, many radiation- or chemically induced mlo mutants originating from various domesticated lines were described and each displays broad-spectrum resistance to all tested Bgh isolates [40]. Isolation of the Mlo gene and characterization of mlo resistance alleles fully supported that resistance is due to loss of function of the wild-type allele [18]. The mlo-11 resistance allele is noteworthy because it arose spontaneously and is present in Ethiopian barley landraces found in a region of high rainfall and high pathogen pressure [40]. Unlike all other characterized mutation-induced mlo alleles that affect coding sequences, a repeat structure in the Mlo promoter leads to drastically reduced MLO transcript levels (unpublished results). This is reminiscent of the naturally occurring promoter mutation in the Duffy chemokine receptor mediating resistance against malaria (see above). It is tempting to speculate that the mlo-11 polymorphism had a selective advantage in wild barley populations in the Ethiopian highlands and was potentially maintained during barley domestication. The wild-type gene encodes a novel plant-specific protein of ~60 kDa harbouring seven hydrophobic stretches. Scanning N-glycosylation mutagenesis and MLO-Lep fusions demonstrated that the hydrophobic stretches form transmembrane helixes, anchoring the protein in membranes [41]. Using the same techniques, the amino terminus of the protein was found to be located on the extracellular side of the plasma membrane, whereas the carboxyl terminus faces the cytoplasmic side (Fig. 3). Thus, the topology of MLO is identical to chemokine and other GPCRs in animals. Fractionation of leaf microsomal vesicles and immunoblotting localized the protein in the plasma membrane [41]. In the near full-size genomes of the model plant species rice (Oryza sativa) and Arabidopsis thaliana, 11 and 15 Mlo gene family members were respectively identified, demonstrating that Mlo genes are organized as gene families of moderate size ([42] and unpublished results). In Arabidopsis, Mlo genes are dispersed on all five chromosomes without evidence of extensive clustering. Inspection of publicly available DNA databases revealed that Mlo genes are present in all tested land plant species, including ancient species like mosses, but apparently not in algae. In addition, no Mlo-like sequences or domains were found in bacterial, fungal, or animal genomes (genomic and EST databases). This suggests that Mlo genes are plantspecific and that the evolution of Mlo genes possibly coincided with the development of the first land plants [42]. All deduced MLO proteins are of comparable size (492–593 amino acids), possess the same predicted scaffold topology including 7-TM domains, and share few (<10%) seemingly invariant amino acids [42]. On average, members

Fig. 3. A model for MLO function. The 7-TM MLO protein (represented by the black line; dark grey boxes symbolize transmembrane helices) is localized in the plasma membrane (light grey rectangle). Positions of amino acid changes in mlo mutants (mlo-10, mlo-27, and mlo-29) leading to stable protein variants are indicated by red circles designated with the respective number of the mutant allele. Upon biotic stress cues (e.g. powdery mildew attack), the resulting rise in free cytosolic calcium ions ([Ca2+]cyt) may promote CaM binding to MLO, whereas an as yet unknown Bgh-derived ligand (L) might bind to extracellular regions of MLO such as extracellular loop 1. One or more as yet unknown proteins (X) may bind to intracellular loops. MLO could serve a dual role as defence modulator and docking molecule (e.g. via ligand L) that is essential for successful pathogenesis.

exhibit 45% identity and 70% similarity at the amino acid level. However, two regions within the protein are unusual with respect to amino acid sequence and length variation. The first stretch is located within extracellular loop 1, between two cysteine residues that are invariant throughout the MLO protein family. The second region is represented by the distal part of the cytoplasmic carboxyl terminus [42]. Thus, subcellular localization and the high sequence diversification within the Mlo family are reminiscent of GPCRs from metazoans. In contrast to animals, the model plant Arabidopsis appears to contain only a single gene (GCR1) encoding a protein with limited sequence relatedness to GPCRs. Likewise, single canonical Ga and Gb subunits, and possibly two Gc subunits are present in the Arabidopsis genome [43]. Since no coupling of heterotrimeric G-protein subunits to GCR1 has been demonstrated to date, MLO proteins remain candidates for G-protein binding although they exhibit no significant sequence relatedness to currently known GPCR classes (GPCR subclass identifier; http://www.soe.ucsc.edu/ research/compbio/gpcr-subclass/; [44]). A potential involvement of heterotrimeric G-proteins in MLO function was tested using genetic and pharmacological approaches. Transient gene expression of constitutive active and dominant negative mutant variants of the single copy barley Ga subunit in single cells did not alter the powdery mildew infection phenotypes in susceptible barley Mlo wild-type or in resistant mlo mutant backgrounds [45]. In addition, application of the G-protein modulating compounds mastoparan (Mas-7) or cholera toxin had no effects on infection phenotypes [45]. Although this strongly suggests that MLO-dependent pathogenesis functions independently of heterotrimeric G-proteins, this remains a possibility for other yet unknown MLOdependent processes.

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An interaction between MLO proteins and calmodulin (CaM) was initially discovered by an in vitro screen for novel CaM-interacting proteins [46]. CaM is a small approximately 17-kDa cytoplasmic protein that is highly conserved among eukaryotic organisms and acts as a calcium sensor. It harbours four domains, called EF hands, each of which binds one Ca2+ ion. Upon Ca2+ binding, the conformation of CaM changes and allows binding to target proteins to modulate their function. The calmodulin-binding domain (CaMBD) of MLO is located in the proximal part of the C-terminal cytoplasmic tail (Fig. 3). The CaMBD is conserved throughout the MLO family, suggesting that CaM binding is a general feature of MLO proteins [45,46]. Indeed, in vitro Ca2+dependent binding of CaM to the CaMBD of MLOs was demonstrated for barley and rice MLO proteins using gel overlay assays. Likewise, several Arabidopsis MLOs and CaM were found to interact in a yeast split-ubiquitin assay that is suitable to probe physical interactions between integral membrane and cytoplasmic proteins ([45,46] and unpublished results). The in vivo role of CaM in barley MLOmediated defence modulation was examined by expression of Mlo variants harbouring single amino acid mutations in the CaMBD in an mlo mutant background. These CaMBD mutants showed halved activity compared to wild-type Mlo in the single-cell complementation assay, suggesting that CaM is an activator of MLO [45]. Increases in intracellular calcium levels appear to be a general and immediate early response of attacked host cells in plant-pathogen interactions (reviewed in [47]). While MLO activation by CaM demonstrates a role for a plant calcium sensor in pathogenesis, pharmacological as well as genetic evidence suggests that Ca2+ sensors also serve as key components in the signalling of plant defence responses [48]. Interestingly, evidence for CaM binding was recently also reported for members of the opioid and metabotropic glutamate GPCR families, suggesting that CaM binding can modulate the activity of several unrelated 7-TM proteins [49,50]. Molecular analysis of barley mlo resistant mutants revealed a subset of 12 alleles that result in minor sequence alterations including single amino acid substitutions (at 10 different sites) or small in-frame deletions (two). The majority of these mutant MLO proteins are either fully or partially unstable, whereas three mutant variants, each affecting residues in cytoplasmic loops, accumulate in the plasma membrane like wild-type MLO ([12,51] and unpublished results). It seems likely that the amino acid changes resulting in stable MLO mutant variants compromise protein-protein interactions (Fig. 3). Since these mutations are outside of the CaMBD and compromise MLO activity to a greater extent compared to the CaMBD mutations, the substitutions in the cytoplasmic loops may affect interactions with additional, as yet unidentified, factors besides CaM or heterotrimeric G-proteins. Indirect evidence may also suggest a functional contribution of extracellular loops to MLO defence modulation. Site-directed mutagenesis of the four invariant extracellular cysteine residues to alanine resulted in each case in a

complete loss of MLO function, and two of the mutant variants accumulated like MLO wild-type (unpublished results). This suggests that extracellular parts, possibly fixed by disulphide bridges in a higher order structure, contribute to MLO function during powdery mildew pathogenesis (Fig. 3). Thus, it is conceivable that extracellular domains of MLO participate in interactions with yet unidentified plant- and/or pathogen-derived molecules (Fig. 3). Interestingly, overexpression of barley Mlo in Mlo wildtype plants leads to super-susceptibility, rendering almost every attacked leaf epidermal cell susceptible to Bgh germlings [45]. This shows that the amount of the wild-type protein is rate-limiting for pathogenesis and is consistent with a potential role of MLO as pathogen “docking molecule”, since a higher density of MLO may increase the probability for successful docking. Heterologous expression of barley Mlo in Arabidopsis mutants that are partially compromised in non-host resistance to Bgh (these mutants permit the establishment of Bgh haustoria structures but do not support hyphal growth on the leaf surface or sporulation; V. Lipka and P. S.-L., unpublished results) and the analysis of Arabidopsis T-DNA insertion mutants containing disrupted Mlo homologues is expected to shed further light on the role of MLO proteins in powdery mildew pathogenesis. 8. Suppressor mutants of mlo resistance: bypass routes for pathogenesis? Re-mutagenesis of powdery mildew resistant mlo barley plants resulted in the isolation of a few partially susceptible suppressor mutants. The mutants identified two unlinked complementation groups, designated Ror1 and Ror2 (required for mlo-specified resistance; [52]). Mutations in Ror1 or Ror2 permit Bgh sporelings to penetrate CWAs successfully at approximately 20% of interaction sites and this correlates with a reduction of CWA-associated ROS levels [51,52]. The existence of partially susceptible ror mutants in the presence of an mlo null mutation demonstrates that MLO is not an absolute requirement for powdery mildew pathogenesis. Mutations of either ror1 or ror2 in the genetic background of wild-type Mlo plants result in supersusceptibility, demonstrating that Ror genes do not have a specific function in mlo resistance but act as positive regulatory components of a basal Bgh defence mechanism. Thus, if MLO serves as docking receptor during pathogenesis, mutations in Ror genes may open a bypass route for pathogenesis. The Ror2 gene has been recently isolated and encodes a t-SNARE (syntaxin) that is localized in the plasma membrane (unpublished results). t-SNAREs are conserved in animals and plants and control intracellular vesicle trafficking [53]. Strikingly, in Arabidopsis, a plant that is not a host for Bgh in nature, mutations in the candidate Ror2 ortholog dramatically enhanced Bgh penetration rates, demonstrating a conserved role of the syntaxin in Bgh defence (V. Lipka and P. Schulze-Lefert unpublished results). Thus, it appears that vesicle transport processes play a role in basal Bgh defence in

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its host plant barley and in limiting host range in non-host plants. Since vesicles containing ROS were shown to congregate in attacked epidermal cells at sites subtending Bgh appressoria [54], it is tempting to speculate that the vesicles contribute to CWA formation and that the ROR2 syntaxin may control this process. Conceptually, Bgh may have exploited Mlo to bypass this basal defence pathway in barley. Another candidate in the Ror defence pathway appears to be RacB, encoding a small GTP-binding protein of the ROP (Rho GTPase of plants) family. Silencing of HvRacB by double-stranded RNA interference (dsRNAi) in susceptible Mlo wild-type plants resulted in a 44% reduction of penetration efficiency, whereas expression of a constitutive active RACB variant led to super-susceptibility ([55] and H. Schultheiss and R. Hückelhoven, unpublished results). Since partial susceptibility in mlo ror1 and supersusceptibility in Mlo ror1 mutants was not altered upon HvRacB silencing, an intact Ror1 gene seems to be a requirement for RACB function in Bgh defence. Formally, this places RacB as a negative regulator upstream of Ror1 in a positive regulatory pathway for basal defence.

7-TM protein) results in disease resistance to the respective pathogen. While the 7-TM protein is, in the case of HIV, a polypeptide with a known function for the host, namely the chemokine GPCRs CCR5 and CXCR4, a function of barley MLO in non-infected wild-type plants may be related to leaf senescence (see above). Medium-sized gene families encode both plant MLOs and animal chemokine receptors CCR5 and CXCR4, and presence of a specific member of each family is required for the establishment of disease. Likewise, susceptibility in the presence and resistance in the absence of particular 7-TM protein isoforms appears to be restricted to a particular pathogen. In barley, Mlo orthologs from closely related species like wheat and rice, but not other homologues, can complement mlo mutants and restore susceptibility [46,62], whereas the murine mCCR5 chemokine receptor cannot substitute for the human CCR5 ortholog (hCCR5; [13]). Naturally occurring polymorphisms in chemokine receptor genes as well as in Mlo give rise to disease-resistant subpopulations, possibly reflecting selective advantages of the respective polymorphisms. During pathogenesis, signalling via heterotrimeric G-proteins is neither required for the chemokine co-receptor nor for MLO.

9. Losing control: deregulated leaf cell death in mlo mutants

While extensive experimental analysis revealed the importance of extracellular domains for CCR5 and CXCR4 co-receptor function, a contribution of intracellular domains is not documented so far. In contrast, both extracellular as well as intracellular domains appear to be required for MLO function during pathogenesis. This may indicate that MLO does not function as a mere receptor for pathogenesis but also suggests the involvement of intracellular components interacting with MLO such as CaM and other proteins (Fig. 3). Although the docking model is consistent with the observation that mlo resistance appears to be restricted to a single pathogen species, the fact that wild-type Mlo influences sensitivity to more than one pathogen species in opposite directions is strong evidence for a role beyond a “docking molecule” function. Thus, the powdery mildew fungus may target MLO for defence suppression but not for docking. This might be accomplished by (pathogen-derived) ligands that bind to extracellular domains of MLO (Fig. 3). Alternatively, it remains possible that the fungus does not directly target MLO but that the wild-type protein acts as a plant intrinsic defence modulator ensuring a balance between mutually inhibiting defence responses. In the absence of defence suppression in mlo mutants, host defence responses might become either accelerated or more effective, thus preventing fungal infection at an early stage of Bgh development before haustorium formation. Finally, Bgh may target MLO for “docking” and concomitant defence suppression. In this scenario, Bgh performs “docking” and subversion of the host’s defence system in a single step. In the future, it may become possible to develop compounds that bind to extracellular domains of MLO and selectively block fungal assault, similarly to the development of drugs that impede chemokine receptor usage by HIV-1 [63].

Does MLO have a role in biological processes that are not directly related to plant microbe interactions? Homozygous mutant mlo plants exhibit developmentally controlled chlorotic and necrotic leaf spotting that occurs even in axenically (pathogen-free) grown plants but is absent in resistant seedlings [56–58]. Unlike many other so-called “disease lesion mimic mutants” that show constitutive expression of defence-related genes, there is no evidence for constitutive defence responses in barley mlo mutants [59,60]. The premature cell death in leaves of mlo plants resembles accelerated leaf senescence, since it is associated with an earlier removal of chlorophyll a and b leaf pigments compared to Mlo wildtype plants [51]. The genetic background determines the occurrence and amount of spontaneous cell death in mlo mutants. Reduced grain yield and size (both by about 4%) due to this pleiotropic effect has hampered the agricultural use of mlo resistance for a long time [40,57]. Breeding of mlo resistance alleles into suitable genetic backgrounds finally resulted in broad-spectrum resistant varieties almost lacking any yield penalty. The durability of mlo resistance led to a widespread use of these varieties, covering currently about 50% of the spring barley acreage in Europe [61]. 10. A synopsis of human chemokine receptor and barley MLO functions during pathogenesis A comparative analysis of the role of chemokine coreceptors during HIV infection and MLO function in Bgh infection shows striking parallels. In both cases, absence of a host 7-TM protein (or presence of a mutant variant of the host

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Acknowledgements We wish to thank Luke Ramsay and David Marshall (Scottish Crop Research Institute, Invergowrie, UK) for encouraging us to compare plant MLO and animal chemokine receptor functions in pathogenesis. We are grateful to our colleague Quian-Hua Shen for providing a microscopic photograph for Fig. 2. We also thank R. Hückelhoven (IPAZ, University of Gießen, Germany) for communicating unpublished results.

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