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295 Defence signalling pathways in cereals Pietro Piffanelli, Alessandra Devoto and Paul Schulze-Lefert* The combination of mutational and molecular studies has shed light on the role of reactive oxygen intermediates and programmed cell death in cereal disease resistance mechanisms. Rice Rac1 and barley Rar1 represent conserved disease resistance signalling genes, which may have related functions in animals. The analysis of non-pathogenic Magnaporthe grisea mutants may provide novel tools to study host defence pathways. Addresses The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK *e-mail: [email protected] Current Opinion in Plant Biology 1999, 2:295–300 http://biomednet.com/elecref/1369526600200295 © Elsevier Science Ltd ISSN 1369-5266 Abbreviations 7TM seven transmembrane CHORD cysteine- and histidine-rich domain GPCR G-protein-coupled receptor HR hypersensitive response PCD programmed cell death R gene resistance gene REMI restriction enzyme mediated integration ROI reactive oxygen intermediates Introduction Despite the flurry of molecularly isolated plant resistance genes in dicot species [1–4], only fragmentary experimental data are available on resistance signalling pathways and even less is known about defence mechanisms. Little of the information derived from dicot species has been used so far to understand the molecular bases of attack and defence in cereal plant–pathogen interactions. Instead, advances in map-based cloning technologies [5–8] and transgene expression in cereals, both transient and stable [9–11], have made even complex monocot genomes amenable to molecular analysis. This has complemented the more traditional tools of Mendelian genetics and mutational studies. In this review we discuss some of the recent developments aimed at the genetic and molecular dissection of both pathogen and host signal transduction pathways in cereals. They reflect the variety and complexity of attack and defence strategies in monocot species and unveil novel insights into the interplay of resistance and cell death control. Attack, defence, and counter-defence: lessons from fungal pathogenicity mutants Unlike phytopathogenic bacteria, biotrophic and hemibiotrophic fungal pathogens must pass through a series of developmental transitions — including in many cases differentiation of intricate infection structures such as haustoria — before they can successfully colonise their hosts. One would expect non-pathogenic strains either to exhibit perturbations in these developmental programs or to affect more specialised steps, for example those which establish a communication with the host metabolism. In the past three years, the use of insertional mutagenesis, in particular restriction enzyme mediated integration (REMI) mutagenesis, has successfully identified components controlling pathogenicity of the cereal pathogen Magnaporthe grisea [12,13]. Perhaps unexpectedly, some of these pathogenicity mutants have also shed light on host defence mechanisms and signalling. M. grisea is a hemibiotrophic filamentous ascomycete that parasitises many grasses, including cereal crops such as rice, wheat, barley, and millet [14,15]. Appressorium formation is a key step during pathogenesis not only for Magnaporthe, but also for other ascomycete and basidiomycete plant pathogens. Appressoria are essential for host cell wall penetration, as they prepare for the transition from extracellular to invasive life style. In the mps1 non-pathogenic mutant [16••], early development in rice leaves is indistinguishable from the wild-type pathogenic strain, including appressorium formation. However, the mps1 strain fails to penetrate epidermal cells. Mps1 is highly sequence-related to Saccharomyces cerevisiae and Schizosaccharomyces pombe mitogen-activated protein kinases (Slt2 and Spm1, respectively) that play essential roles in the maintenance of cell wall integrity. Because Mps1 can rescue the yeast null mutant Slt2, the authors speculated that the wild-type protein exerts an analogous function in M. grisea by remodeling the appressorial wall to facilitate formation of a penetration hypha. Surprisingly, the mps1 mutation does not prevent infection of abraded leaf surfaces, suggesting that Mps1 is not required for invasive growth. Whether this latter observation indicates the existence of an Mps1-independent penetration mechanism, enabling invasive growth in the absence of an intact epidermal leaf surface, remains to be clarified. Despite aborted penetration attempts, the mps1 strain still retains its ability to induce actin re-assembly in attacked epidermal cells, an early and common host cell response to attempted fungal attack. There is direct experimental evidence that actin disassembly and re-assembly is a critical component of non-host resistance in barley, since cytochalasins, actin polymerisation inhibitors, enable a number of non-barley pathogens to penetrate cell walls and to establish either infection hyphae or haustoria [17,18]. Localized autofluorescence in the plant cells, directly subtending appressoria, is also induced by both wild-type and mutant mps1 strains. This cell-wall associated autofluorescence in the host cells is considered an early plant defence response [19]. It is a widespread phenomenon in interactions with 296 Biotic interactions phytopathogenic fungi and is likely to be the result of oxidative cross-linking of phenolics to the plant cell wall. Taken together, these observations demonstrate that at least a subset of early plant defence responses is triggered prior to host cell wall penetration. The M. grisea abc1 mutant, contrary to mps1, affects intracellular growth within epidermal cells, that is to say intracellular hyphae formation ceases shortly after successful penetration [20•]. The ABC1 protein belongs to the family of ABC carriers and shows highest sequence-relatedness to multi-drug resistance efflux pump transporters in yeast (PDR5 and CDR1). Strikingly, Abc1 transcription is strongly upregulated upon contact with the host epidermal cell. One interpretation is that ABC1 represents a counter-defence component against rice defence mechanisms by exporting anti-microbial compounds at early stages of pathogenesis. Contrary to yeast PDR5 and CDR1 mutants, however, the abc1 mutant does not exhibit altered sensitivity to a series of tested antifungal drugs including the rice phytoalexin sakuranetin. If ABC1 is indeed an efflux pump of host defence compounds, these must be widespread among cereals as the abc1 mutant exhibits arrested intracellular growth in both rice and barley. Alternatively, ABC1 is not part of a counter-defence mechanism, but may be involved in exporting small fungal molecules that could either act as toxins or function in reprogramming the host metabolism to the advantage of the fungus. In sum, the abc1 and mps1 mutants provide the first examples that fungal pathogenicity mutants can also be valuable tools to investigate host defence mechanisms. For example, they may be used in future experiments to dissect the temporal and spatial succession and the interplay between broad-spectrum defence mechanisms and race-specific resistance responses (see below). Recognition inside and outside the host cell Race-specific resistance is, as in dicots, a widespread phenomenon in cereal–pathogen interactions. In each case, cognate gene pairs in host (R genes) and pathogen (Avr genes) are essential components to trigger a resistance response. R gene products in monocots and dicots share the same structural features [21]. The major class contains an amino-terminal nucleotide binding site (NBS) and carboxy-terminal leucine-rich repeats (LRR) of various lengths. A second class comprises genes containing a kinase and/or a LRR domain [22]. Experimental and indirect evidence suggest that the highly variable LRRs in both classes of R genes have a major role in recognition of pathogen determinants encoded by Avr genes [23,24]. The molecular isolation of Xa1 and Xa21 in rice [25,26], each conferring Avr-dependent resistance to the bacterial leaf pathogen Xanthomonas oryzae pv. oryzae, provides the first example that a host plant can recognise determinants from the same pathogen via NBS–LRR and LRR–kinase type genes. Xa1, a member of the NBS–LRR class, is predicted to reside within the cytoplasm, whereas Xa21, a member of the LRR-kinase class, is likely to be a transmembrane protein with the LRRs exposed extracellularly. This suggests that recognition events of X. oryzae determinants can occur both extra- and intracellularly. As Avr genes are likely to function primarily as pathogenicity factors [27], this may suggest that X. oryzae can target both extra- and intracellular host factors to induce favourable growth conditions. It is an intriguing question whether the different sequence-deduced subcellular locations of Xa21 and Xa1 proteins result in different R gene triggered signalling pathways. A mutational approach has been initiated to identify genes required for Xa21 function (PC Ronald et al., personal communication). Nine fully susceptible and 15 partially susceptible lines of rice were recovered from 4,500 M2 families, following radiation and chemical mutagenesis of a rice genotype containing Xa21. All nine fully susceptible mutants contained deletions in the Xa21 gene. In contrast, none of the 15 partially susceptible lines showed detectable rearrangements in the R gene. It will be interesting to find out whether susceptibility in the latter class of mutants is caused by weakly defective alleles of Xa21, or is the result of mutations in other genes required for Xa21 function. As all candidate suppressor mutants of Xa21 exhibit only partial susceptibility, this may indicate that the resistance reaction branches rapidly downstream of Xa21, or that the downstream components are partially redundant. Alternatively, null alleles of the suppressors may be lethal. Transducing death signals Localised and rapid cell death at attempted infection sites is probably one of the most common host responses to pathogens in R gene-triggered resistance of higher plants. This hypersensitive reaction (HR) is believed to confine pathogen growth. Biochemically, the HR involves the coordinate activation of a complex series of events of which the generation of reactive oxygen intermediates (ROI) is one of the earliest to detect. Collectively, ROI include H2O2, O2•– and OH•, of which only H2O2 is relatively stable in solution. ROI are believed to drive downstream responses, including oxidative cross-linking of cell walls, accumulation of phenolics and phytoalexins, induction of a whole range of pathogenesis-related (PR) proteins, and the activation of proteases and protease inhibitors [28]. The end of this ‘biochemical inferno’ is the death of one or a cluster of host cells at the infection site. We are only now beginning to find answers to key questions: is host cell death the cause of or a consequence of R gene-triggered resistance? What are the regulatory components linking R gene-mediated pathogen recognition to the plethora of downstream responses? One focus of research has been to find out whether the HR is a form of programmed cell death (PCD). If this is the case, then, the first question becomes obsolete because resistance would have to be envisaged as an integral part of a genetic program destined to terminate in host cell death. Defence signalling pathways in cereals Piffanelli, Devoto and Schulze-Lefert One of the best characterized cereal systems used to study the genetic and molecular relationship between cell death and resistance is represented by the interaction between the obligate biotrophic fungus Erysiphe graminis f. sp. hordei and its natural host, barley. Obligate biotrophs may actively seek to suppress host cell death, as their life-cycle depends entirely upon host living tissues [29]. At least two genetically separable pathways control resistance to this pathogen [30] in barley leaves. The first is an R gene-triggered pathway which can be activated by different resistance alleles encoded at the Mla locus and by other unlinked powdery mildew R gene loci [31]. Each of these R genes requires at least two additional genes, Rar1 and Rar2, to execute the resistance response [32]. The second resistance pathway also requires at least three gene functions: Mlo, Ror1, and Ror2. Unlike the first, in which race-specific resistance is triggered by dominant R genes, this second pathway is mediated by recessive mlo alleles, each conferring broad-spectrum resistance to all tested powdery mildew isolates [33]. A conserved cell death component in plants and animals? Rar1 was recently isolated using a map-based cloning approach ([7], T Lahaye et al., unpublished data). The deduced sequence of the 25.5 kDa RAR1 protein uncovered a novel 60 amino acid domain, designated CHORD (cysteine- and histidine-rich domain). CHORD is arrayed in a tandem repeat in RAR1 and maybe involved in metal ion co-ordination. Surprisingly, CHORD-containing proteins are not only conserved in all tested higher plant species, but also in protozoa and metazoa, including Drosophila melanogaster, the nematode Caenorhabditis elegans, and Homo sapiens. To test the function of the CHORD-containing protein in the nematode C. elegans, the corresponding single-copy gene, chp, was silenced by RNA interference (RNAi). Silencing of chp revealed semisterility, embryo lethality and gonad hyperplasia. The latter phenotype is reminiscent of ced-3 and ced-4 cell death execution mutants [34]. PCD in the germline and soma of C. elegans is known to depend upon the same execution machinery (ced-3, ced-4, and ced-9), but to be controlled by distinct sets of regulatory genes. This suggests an essential function for chp in the nematode. Whether the gonad hyperplasia phenotype in the silenced chp nematodes also indicates an involvement of the gene in the control of germline PCD remains to be tested. The significance of Rar1 is based on its requirement for the function of multiple R genes, indicating a convergence point in race-specific resistance. Interestingly, susceptible rar1 mutants compromise an R gene-triggered host cell death response including the attacked epidermal and subtending mesophyll cells. Moreover, Rar1-dependent and R gene-specified resistance coincides with a biphasic H2O2 accumulation of which only the second wave is compromised in susceptible rar1 mutant plants. Thus, Rar1 appears to function downstream of R gene-mediated 297 pathogen recognition, but upstream of the second wave of H2O2 accumulation. None of the Rar1-dependent R genes have been isolated from barley. However, the above-mentioned structural similarities between R genes in monocot and dicot species makes it likely that these will have a similar structure to the intracellular NBS–LRR or the extracellular LRR classes. To date, only two other genes representing convergence points in race-specific resistance to pathogens have been characterised at the molecular level. Both are from the dicot Arabidopsis: NDR1 [35] encodes a small and possibly membrane-anchored protein with unknown function, and EDS1 [36] is predicted to be an intracellular protein containing three sequence motifs characteristic of lipases. RAR1, NDR1, and EDS1 differ from each other in that the Arabidopsis proteins are plant-specific, whereas homologues of barley Rar1 exist in all eukaryotic species examined so far, except yeast. If the gonad hyperplasia phenotype, induced by silencing of the Rar1 homologue chp in C. elegans is indeed a consequence of a deregulated germline PCD, then CHORD proteins may share cell death control functions in plants and animals. Alternatively, the sequence-conserved Rar1 homologues control distinct cellular functions in plants and animals. Similar to human macrophages or neutrophils, ROI are believed to function in plant defence by either directly killing the pathogen, or indirectly by triggering a cell death program [37]. Homologues of gp91phox, a component of the H2O2-generating complex in mammalian neutrophils, were recently identified in both monocot and dicot plants [38–40]. Support for a functional role of this H2O2-generating complex in pathogen defence derives from the expression of constitutively active and dominant-negative forms of the small GTP-binding protein OsRac1 (K Shimamoto et al., personal communication), a rice homologue of the human Rac. The human RAC protein is one out of three cytosolic factors which, together with two membrane-bound proteins — P22phox and the above mentioned GP91phox, define the neutrophil multicomplex enzyme NADPH oxidase [41]. Rice contains three sequence-related genes of human Rac, designated OsRac1 to OsRac3, each revealing about 60% amino acid sequence identity to the human counterpart (K Shimamoto, personal communication). Recombinant OsRAC1 was shown to have both GTP-binding and GTPase activities. Transgenic expression of a constitutively active form of OsRAC1 led to the formation of discrete spontaneous leaf necrosis in young and adult plants. Transgenic expression of a dominant-negative form of OsRAC1 significantly compromised R-gene triggered resistance upon challenge with an avirulent M. grisea isolate, though not completely. Because the constitutively active form of OsRAC1 was also shown to mediate a constitutive and DPI (diphenyleniodonium)-sensitive H2O2 accumulation in suspension cultures derived from transformed rice calli, the authors conclude that the OsRAC1 variant acts specifically through 298 Biotic interactions an altered activity of the NADPH-dependent oxidase complex. Together with the compromised second wave of H2O2 accumulation in partially susceptible barley rar1 mutants, the experimental data suggest that in R gene-triggered pathogen resistance at least some of the H2O2 accumulation is catalyzed by a cereal NADPH-dependent oxidase complex. A plant-specific death pathway intertwined with pathogen resistance Molecular and genetic analysis of the mutation-induced and broad-spectrum mlo-mediated resistance to the powdery mildew fungus in barley underlines the intimate link between pathogen defence mechanisms and cell death control in plants. Wild-type Mlo encodes the prototype of a novel class of plant-specific integral membrane proteins [5]. Molecular analysis of susceptible Mlo and a number of resistant mlo genotypes confirmed that resistance is caused by a lack of the wild-type gene. We have recently shown that MLO resides in the plasma membrane of leaf cells and it is membrane-anchored via seven transmembrane helices so that the amino-terminus is located extracellularly and the carboxy-terminal tail intracellularly. The wild-type protein functions in a cell-autonomous fashion as, in mlo genotypes, transient expression of Mlo in single epidermal leaf cells is sufficient to confer susceptibility to the fungus Erysiphe graminis f. sp hordei [10]. Following a genome-wide search for Mlo sequence-related genes in Arabidopsis thaliana, we estimate the presence of approximately 35 Mlo family members, each sharing common gene structure and predicted protein topology. This genome-wide analysis in Arabidopsis also suggests that Mlorelated genes represent the only abundant gene family encoding 7TM proteins in higher plants. The genetic diversification of Mlo family members within a single species, their topology and subcellular localization are reminiscent of the most abundant class of 7TM receptors in C. elegans and H. sapiens, the G-protein-coupled receptors (GPCRs) [42]. These receptors relay extracellular signals through ligand binding into amplified intracellular responses via activation of the α subunit of heterotrimeric G proteins [43,44]. Mlo family members do not share significant sequence similarities with GPCRs, but this does not preclude MLO being a GPCR, as mammalian GPCR subfamilies often exhibit negligible amino acid sequence relatedness with each other [45]. Determining whether MLO functions like a GPCR or through a novel mechanism remains one of the future challenges. An apparent pleiotropic effect in mlo plants — the developmentally-controlled leaf necrosis even under axenic conditions [46] — suggests a possible link between cell death control and pathogen resistance. Resistance mediated by mlo is dependent upon at least two additional genes, Ror1 and Ror2, as double mutants mlo ror1 and mlo ror2 are susceptible to powdery mildew attack [33]. Similarly, developmentally-controlled lesion formation in mlo genotypes is dependent on Ror1 and Ror2 as both double mutant geno- types show a reduced number of spontaneous leaf lesions. Thus, the same three genes that control broad-spectrum resistance to the fungus also control spontaneous leaf cell death [30]. Therefore, we re-examined previous observations that leaf epidermal cells resist fungal attack in mlo plants without an accompanying host cell death. Host cells retain viability even 30 hours after microscopically detectable arrest of fungal germlings within the host cell wall. Thereafter, however, clusters of mesophyll cells, subtending attacked epidermal cells, undergo a cell death response. This pathogen-triggered cell death in mlo genotypes is also dependent on Ror1 and Ror2 since it is compromised in both mlo ror1 and mlo ror2 double mutants (J Orme, personal communication). This suggests that signals emanate from the attacked but non-penetrated epidermal host cell into subtending mesophyll tissue, leading in the latter to the execution of a cell death program. Thus, although the mesophyll cell death can be spatially and temporally separated from the mlo-mediated resistance reaction, both responses are clearly genetically intertwined. This may suggest that Mlo has a primary function in the negative control of leaf cell death which converges, via Ror1 and Ror2, in a pathogen defence pathway. Conclusions Pathogen defence and pathogenicity mechanisms can define opposite sides of the same coin. This is reflected by a unique collection of non-pathogenic M. grisea mutants whose analysis is uncovering both general host defence responses and pathogen counter defence mechanisms. Disease resistance components Rar1 and OsRac1 provide the first molecular insights into the signalling of R-genetriggered resistance in cereals. Both proteins are conserved between plants and animals. However, more experimental evidence is needed to critically examine a possible functional overlap of the plant genes and their animal counterparts. Currently, data identify ROI as critical signalling molecules and PCD as the underlying theme in R gene-mediated resistance responses to biotrophic and hemibiotrophic cereal fungi. The interconnection between pathogen resistance and cell death is also underlined by the genetic and molecular dissection of broad-spectrum mlo-controlled resistance. In contrast to Rar1 and OsRac1, Mlo homologues are only found in plants, indicating the existence of another, possibly plant-specific, PCD control mechanism. Future comparative functional analysis of Mlo, Rar1 and OsRac1 homologues should reveal if wiring diagrams in pathogen resistance and cell death control are conserved between monocots and dicots. Acknowledgements The authors are grateful to K Shimamoto, PC Ronald, T Lahaye, K Shirasu and J Orme who made available unpublished information to assist the preparation of this review. Many thanks to all members of the P ShulzeLefert lab for helpful discussions and suggestions, and to R Innes for critical reading of the review. Research work in P Shulze-Lefert’s lab is supported by grants from the European Union (TMR program — Biotechnology), the Gatsby Foundation and from the Biotechnology and Biological Sciences Research Council. Defence signalling pathways in cereals Piffanelli, Devoto and Schulze-Lefert References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 299 20. Urban M, Bhargava T, Hamer JE: An ATP-driven efflux pump is a • novel pathogenicity factor in rice blast disease. EMBO J 1999, 18:512-521. 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