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 Rar1represent conserved disease resistance signalling genes, which may have related functions in animals. The analysis of non-pathogenic
Magnaporthe griseamutants may provide novel tools to study host defence pathways.
Addresses
The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
*e-mail: schlef@bbsrc.ac.uk
Current Opinion in Plant Biology1999, 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
Rgene 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 mole-cular 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 compo-nents 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. griseais a hemibiotrophic filamentous ascomycete that parasitises many grasses, including cereal crops such as rice, wheat, barley, and millet [14,15]. Appressorium for-mation is a key step during pathogenesis not only for
Magnaporthe, but also for other ascomycete and basid-iomycete plant pathogens. Appressoria are essential for host cell wall penetration, as they prepare for the transition from extracellular to invasive life style.
In the mps1non-pathogenic mutant [16••], early develop-ment in rice leaves is indistinguishable from the wild-type pathogenic strain, including appressorium formation. However, the mps1strain fails to penetrate epidermal cells.
Mps1is 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. griseaby 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 Mps1is not required for inva-sive growth. Whether this latter observation indicates the existence of an Mps1-independent penetration mecha-nism, enabling invasive growth in the absence of an intact epidermal leaf surface, remains to be clarified.
Despite aborted penetration attempts, the mps1strain 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 evi-dence that actin disassembly and re-assembly is a critical component of non-host resistance in barley, since cytocha-lasins, actin polymerisation inhibitors, enable a number of non-barley pathogens to penetrate cell walls and to estab-lish 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
mps1strains. 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
Defence signalling pathways in cereals
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 abc1mutant, contrary to mps1, affects intra-cellular growth within epidermal cells, that is to say intracellular hyphae formation ceases shortly after success-ful penetration [20•]. The ABC1 protein belongs to the family of ABC carriers and shows highest sequence-relat-edness to multi-drug resistance efflux pump transporters in yeast (PDR5 and CDR1). Strikingly, Abc1transcription is strongly upregulated upon contact with the host epider-mal cell. One interpretation is that ABC1 represents a counter-defence component against rice defence mecha-nisms 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 bar-ley. 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 abc1and mps1 mutants provide the first exam-ples that fungal pathogenicity mutants can also be valuable tools to investigate host defence mechanisms. For exam-ple, 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 phe-nomenon in cereal–pathogen interactions. In each case, cognate gene pairs in host (R genes) and pathogen (Avrgenes) 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 car-boxy-terminal leucine-rich repeats (LRR) of various lengths. A second class comprises genes containing a kinase and/or a LRR domain [22]. Experimental and indi-rect evidence suggest that the highly variable LRRs in both classes of Rgenes have a major role in recognition of pathogen determinants encoded by Avrgenes [23,24]. The molecular isolation of Xa1 and Xa21in 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 viaNBS–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 pro-tein with the LRRs exposed extracellularly. This suggests that recognition events of X. oryzaedeterminants can occur both extra- and intracellularly. As Avr genes are likely to function primarily as pathogenicity factors [27], this may suggest that X. oryzaecan target both extra- and intracellu-lar host factors to induce favourable growth conditions.
It is an intriguing question whether the different sequence-deduced subcellular locations of Xa21and 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 Rgene. It will be interesting to find out whether susceptibility in the lat-ter 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 Rgene-triggered resistance of higher plants. This hypersensitive reaction (HR) is believed to confine pathogen growth. Biochemically, the HR involves the co-ordinate 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 H2O2is relatively sta-ble 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 Rgene-triggered resistance? What are the regulatory components linking R
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 active-ly 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-trig-gered pathway which can be activated by different resistance alleles encoded at the Mlalocus and by other unlinked powdery mildew Rgene loci [31]. Each of these
Rgenes 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 func-tions: Mlo, Ror1, and Ror2. Unlike the first, in which race-specific resistance is triggered by dominant Rgenes, this second pathway is mediated by recessive mloalleles, 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 uncov-ered 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 pro-teins are not only conserved in all tested higher plant species, but also in protozoa and metazoa, including
Drosophila melanogaster, the nematode Caenorhabditis ele-gans, 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 chprevealed semi-sterility, embryo lethality and gonad hyperplasia. The latter phenotype is reminiscent of ced-3and ced-4cell 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 chpnematodes also indicates an involvement of the gene in the control of germline PCD remains to be tested.
The significance of Rar1is 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 sub-tending mesophyll cells. Moreover, Rar1-dependent and R
gene-specified resistance coincides with a biphasic H2O2 accumulation of which only the second wave is compro-mised in susceptible rar1 mutant plants. Thus, Rar1
appears to function downstream of R gene-mediated
pathogen recognition, but upstream of the second wave of H2O2accumulation.
None of the Rar1-dependent Rgenes have been isolated from barley. However, the above-mentioned structural sim-ilarities 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 mem-brane-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 Rar1exist in all eukaryotic species examined so far, except yeast. If the gonad hyperplasia phenotype, induced by silencing of the Rar1homologue chpin C. ele-gans 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 Rar1homologues control distinct cel-lular 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 -gener-ating 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 multicom-plex enzyme NADPH oxidase [41]. Rice contains three sequence-related genes of human Rac, designated OsRac1
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-trig-gered 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 pow-dery mildew fungus in barley underlines the intimate link between pathogen defence mechanisms and cell death control in plants. Wild-type Mloencodes the prototype of a novel class of plant-specific integral membrane proteins [5]. Molecular analysis of susceptible Mloand 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 extracellular-ly and the carboxy-terminal tail intracellularextracellular-ly. The wild-type protein functions in a cell-autonomous fashion as, in mlogenotypes, transient expression of Mloin 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 approxi-mately 35 Mlo family members, each sharing common gene structure and predicted protein topology. This genome-wide analysis in Arabidopsis also suggests that Mlo -related 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 rem-iniscent 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 viaactivation of the αsubunit of heterotrimeric G proteins [43,44]. Mlofamily members do not share sig-nificant 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 mecha-nism remains one of the future challenges.
An apparent pleiotropic effect in mloplants — the devel-opmentally-controlled leaf necrosis even under axenic conditions [46] — suggests a possible link between cell death control and pathogen resistance. Resistance mediat-ed by mlois dependent upon at least two additional genes,
Ror1and Ror2, as double mutants mlo ror1and mlo ror2are susceptible to powdery mildew attack [33]. Similarly, devel-opmentally-controlled lesion formation in mlogenotypes is dependent on Ror1and Ror2as 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 observa-tions 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, sub-tending attacked epidermal cells, undergo a cell death response. This pathogen-triggered cell death in mlo geno-types is also dependent on Ror1 and Ror2 since it is compromised in both mlo ror1and mlo ror2double mutants (J Orme, personal communication). This suggests that sig-nals emanate from the attacked but non-penetrated epidermal host cell into subtending mesophyll tissue, lead-ing 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 OsRac1provide the first molecular insights into the signalling of R -gene-triggered resistance in cereals. Both proteins are conserved between plants and animals. However, more experimental evidence is needed to critically examine a possible func-tional overlap of the plant genes and their animal counterparts. Currently, data identify ROI as critical sig-nalling 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 Rar1and 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,
Rar1and OsRac1homologues should reveal if wiring dia-grams in pathogen resistance and cell death control are conserved between monocots and dicots.
Acknowledgements
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