Structure and function of proteins controlling strain-specific
pathogen resistance in plants
Jeff Ellis
∗
and David Jones
†
Recently recognised structural and amino acid sequence similarities between plant disease resistance (R) proteins and animal proteins such as Apaf-1 and CED-4 are providing conceptual models for resistance protein function. Data from extensive DNA sequencing of resistance gene families are indicating that the leucine-rich repeat motif is an important determinant of gene-for-gene specificity and that intergenic DNA sequence exchange is a major contributor to R gene diversity.
Addresses
∗CSIRO Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia; e-mail: [email protected]
†Research School of Biological Sciences, Australian National
University, PO Box 475, Canberra ACT 2601, Australia; e-mail: [email protected]
Current Opinion in Plant Biology1998,1:288–293 http://biomednet.com/elecref/1369526600100288
Current Biology Ltd ISSN 1369-5266 Abbreviations
avr avirulence IL-1 interleukin-1 LRR leucine-rich repeat NBS nucleotide binding site PK protein kinase R resistance
TM transmembrane domain TIR Toll and IL-1 receptor
Introduction
Highly polymorphic plant genes (R genes) that control specific disease resistance [1•], encode proteins in several classes, some of which bear the hallmarks of innate immunity and cell death proteins in animals. These proteins function in surveillance systems for pathogens, probably detecting pathogen-encoded avirulence gene products which in other contexts may function as virulence factors (see [2••] for an excellent example).
Recent reviews (e.g. [1•]) have extensively covered the comparisons and classification of predicted plant disease resistance gene products (R proteins) which incorporate several common structural themes: nucleotide binding sites (NBSs), leucine-rich repeats (LRRs), transmembrane domains (TMs) and serine/threonine protein kinases (PKs). These are combined in several arrangements with the following signatures, NBS-LRR, TM-PK, LRR-TM and PK. An additional and novel class containing the Hs1pro-1 nematode resistance protein is discussed in this review. The largest number of characterised R proteins are in the NBS-LRR class and so far, proteins in this
class have no other known functions apart from disease resistance. Similarities between the NBS domain of these proteins and animal proteins regulating apoptosis (detailed below), however, suggest that researchers be mindful of the possibility that some NBS-LRR proteins may function in plant developmental processes involving programmed cell death. One example of a ‘multiskilled’ family of proteins is the LRR-TM-PK class. Although Xa21 is the only resistance protein in this class, several plant protein ‘relatives’ involved in developmental control and hormone perception have now been identified, suggesting that Xa21 may have been ‘recruited’ for pathogen detection.
Hs1
pro-1: an unusual resistance gene
The predicted protein encoded by the recently cloned Hs1pro-1 gene for nematode resistance in sugar beet was reported to have an LRR-TM signature [3], but our analysis indicates that it does not appear to fit this or any other of the existing R protein structural signatures. For example, database searches show no homology to any previously analysed R gene or LRR protein. Additionally, the proposed signal peptide and TMs of Hs1pro-1 are very short and contain a number of charged or polar residues inconsistent with such roles. Furthermore, the proposed LRR domain lacks the features of any LRR consensus that has been described to date, not only with respect to the spacing of the leucines, but also regarding the absence of distinctive non-leucine residues. AnArabidopsishomologue (GenBank accession G2088653) has been described that was predicted to have a more extensive amino-terminal domain than Hs1pro-1, but this shows no better similarity to the LRR-TM signature. Hs1pro-1 seems likely, therefore, to represent the first member of an entirely new class of R genes encoding a novel cytoplasmic proteins.
Xa21: relatives in different professions
the product of the avrXa21 avirulence gene of Xoo. The involvement of the LRR region of this class in ligand recognition, however, has not been experimentally demonstrated and so it is possible that the PK domain of Xa21, like that of the tomato bacterial resistance protein Pto which contains only the PK domain [9,10], detects the corresponding Avr protein.
Xa1, a second rice gene for resistance to Xoo strains carrying avirulence geneavrXa1, has recently been cloned and in contrast to Xa21, found to encode a NBS-LRR protein [11•]. Thus, two unrelated classes of R protein are used in rice to perceive the same pathogen species (but different avr genes!). The NBS-LRR proteins are predicted to be intracellular, whereas the LRR domain of Xa21 is predicted to be extracellular [12•]. The LRR domains of Xa1 and Xa21, assumed to be involved in avr ligand binding, are therefore in different subcellular locations. The cloning and comparison of the correspondingavrgenes,avrXa1andavrXa2, from Xoois keenly anticipated to determine whether the nature and delivery of the avr ligands reflects these locations.
NBS-LRR resistance proteins: strength in
numbers
The NBS-LRR class is by far the largest group of resistance proteins. Two subgroups within the NBS-LRR class have been recognised by the presence or absence of an amino-terminal region (TIR domain) with amino acid sequence similarity and predicted structural similarity [13•,14,15] to the cytoplasmic signalling domains of the Toll and interleukin-1 (IL-1) receptor. The first subgroup (TIR-NBS-LRR) includes N (tobacco mosaic virus resistance, [1•]), L6 (flax rust resistance [1•]), M (flax rust resistance [16•]) and RPP5 (downy mildew resistance [13•]). Furthermore, the involvement of plant and animal TIR proteins in defence processes [15–19] indicates that they may have their origins in a very ancient defence mechanism. Recent advances in understanding the role of Toll and IL-1R in defence processes provide additional clues about the role of the plant TIR domain. In Toll/IL-1R signalling, the cytoplasmic TIR domains are involved in the activation of the PKs Pelle/IRAK and IRAK-2 via the protein intermediaries Tube/IL-1AcP and MyD88 [20–23]. Interestingly, MyD88 has an adaptor protein with an amino-terminal domain similar to those of IRAK and IRAK-2 and a carboxy-terminal TIR domain similar to those of IL-1R and IL-1AcP. Heterodimerisation via the TIR domains on one hand and the amino terminal domains on the other, might enable MyD88 to act as an adaptor linking the upstream and downstream components of the signal transduction process in IL-1R signalling [21–23]. TIR domains, however, have yet to be detected among any plant proteins other than the TIR-NBS-LRR class.
The second subgroup, which lacks the TIR region, includes the bacterial resistance proteins RPS2, RPM1
[1•], Xa1 [11•], Prf [24], the fungal resistance protein I2 [25•] and the possible nematode resistance protein Cre3 [26•]. Of this subgroup, RPS2, RPM1 and Prf are predicted to contain an amino-terminal leucine zipper (LZ), a motif frequently involved in protein dimerization. Whether this motif is functional in dimerisation of R proteins has not yet been reported and no homologous animal system with extended similarity in the LZ domain has emerged upon which to model the function of this domain. The interaction between this domain and the LZ domain of a downstream signalling component in a pathway distinct to that used by the TIR-NBS-LRR subclass seems plausible, however. Recent genetic evidence indicates that proteins in these two classes signal through different pathways. In Arabidopsis, proteins in the TIR class signal via a pathway that includes EDS1 ([27]; JE Parker, personal communication) and proteins in the LZ class signal through a pathway that includes the recently clonedNDR1 gene ([28•]; BJ Staskawicz, personal communication). Both NDR1 and EDS1 were identified by mutation screens for loss of disease resistance [27,28•]. There is no apparent correlation between pathogen type (virus, fungus, bacterium etc.) and the NBS-LRR subclass used by plants to detect these pathogens.
NBS-LRRs: the nucleotide binding domain,
an apoptosis connection and a model for
function
and other components of the IL-1R system discussed above and encourages a search for plant proteins that may interact through homology with the TIR or LZ domains of resistance proteins.
Many NBS-LRR plant genes have been identified through plant genome sequencing projects [33] and by PCR using redundant primers based on NBS motifs [34–37]. Programmed cell death is used in plant developmental processes and the question arises whether any of these newly identified NBS-LRRs lacking ascribed resistance function may have a role in development rather than resistance.
Resistance genes at complex loci
Genetic mapping of resistance gene specificities has indicated that they frequently cluster at complex loci. Sequence data now show that such loci represent all classes of R genes except Hs1pro-1, and contain tandem arrays of closely related genes [25•,38,39••,40•]. The tomatoCf-4/9 locus (LRR-TM class) from two lines (Cf4 and Cf9) and the tomato Pto locus (PK class) contain multiple genes, most of which appear to be functional [38], while the riceXa21locus (LRR-TM-PK class) [40•] and the Arabidopsis RPP5 locus (TIR-NBS-LRR class) from ecotype Col-O [41] contain a high proportion of pseudogenes, with several carrying transposons, deletions or frame shifts. These arrays of paralogous genes may provide a means of maintaining favourable haplotypes with multiple specificities or may act as reservoirs of sequence diversity for generating new specificities by intergenic sequence exchange. In contrast, members of the LRR-TM-PK class mentioned above as developmental regulators or hormone receptors occur as single genes at simple loci. The latter proteins are likely to be focused on single ligands that are evolutionarily stable in contrast to rapidly changing pathogen ligands. Simple R gene loci also exist and in at least one case, the L rust resistance locus in flax, diversity is provided by a multiply allelic series. In a number of cases, however, such as RPS2 [1•], RPM1 [1•], and Xa1 [11•], the R genes exist as single genes at simple loci with only a single recognitional specificity. Furthermore, complexity at a particular locus can be variable; for example, only a single gene occurs at the Cf-4/9 locus in the susceptible Cf0 line of tomato [39••], probably reflecting deletion events resulting from unequal crossing over at an ancestral complex locus.
Resistance gene specificity
All of the R genes discussed in this review show a high level of recognitional specificity, that is, they provide re-sistance against only some strains of a particular pathogen species. This is principally the result of a high level of polymorphism among pathogen avirulence genes which encode the probable ligands of the resistance protein receptors. This contrasts with our current perception of the innate immunity systems inDrosophilawhere specificity is manifest against common ligands within pathogen classes,
for example, Gram-positive or Gram-negative bacteria or fungal pathogens [42].
Three systems are currently providing insight into the molecular basis of specificity in pathogen strain recog-nition. In the interaction between the tomato Pto and Pseudomonas syringaeAvrPto proteins, domain swap exper-iments betweenPtoand another closely related gene,Fen, at the complex Pto locus and yeast two-hybrid analysis of the chimeric genes have identified a small region of Pto involved in the interaction with AvrPto and thus also involved in the determination of specificity towards races ofP. syringae carrying theavrPtogene [9,10]. In the second system, also in tomato, 11 homologous genes at the Cf-4/9 locus forCladosporium fulvumresistance have been sequenced [39••]. Comparison of the predicted amino acid sequences shows conservation in the carboxy-terminal halves of the proteins, which include about a third of the LRRs; an extreme example being complete identity in the carboxy-terminal halves of the Cf-4 and Cf-9 proteins [43•]. This implies that the specificity of recognition of avirulence ligands is determined by the LRRs of the variable amino-terminal regions. These comparisons also show that variation within the amino-terminal LRR region occurs predominantly in regions predicted to form a solvent-exposed parallel sheet and thought to provide a platform for the presentation of nonconserved residues for specific interaction with protein ligands [12•].
Analysis of the nucleotide sequence of these 11 homo-logues revealed variation originating from mutation, seg-mental exchange between adjacent homologues within tandem arrays (either by repeated rounds of unequal exchange or by gene conversion) and duplication or deletion of complete LRR units; for example, Cf-4 differs from Cf-9 by a precise deletion of two complete LRRs [43•]. The predicted parallelβ-sheet arrangement of LRRs means that a given amino acid has both a horizontal context — the neighbouring amino acids within its own LRR unit, and a vertical context — amino acids in a similar position in the two flanking LRRs. Segmental exchange, although it may have had a limited role in producing novel combinations in the horizontal context, would seem to have had a more important role in producing novel combinations of amino acids in the vertical context. Deletion and duplication of LRRs would have the same effect.
domain. Sequence comparisons and interallelic domain swap experiments, however, also indicate that the amino-terminal TIR domain makes important contributions to allelic specificity in addition to the LRR domain (J Elliset al., unpublished data).
Conclusions
The number of cloned R genes is rapidly increas-ing and genetic and molecular studies have identified several downstream signalling components in resistance [27,28•,45•]. One example of a strain-nonspecific resis-tance gene, mlo, has been cloned [46•] and analysis and engineering of the homologues in other plant species may lead to further examples of ‘broad spectrum resistance’. Basic information concerning how these genes function is appearing more slowly, but genomic and comparative studies are providing leads. In only one example (Avr Pto and Pto [9,10]) has evidence of direct interaction between a resistance and avirulence protein been reported, in spite of the fact that several other cloned bacterial and fungal avirulence genes corresponding to cloned resistance genes are available. The possibility remains that these interactions will involve one or more additional host proteins as in the animal apoptosome complex which involves Apaf-1, Bcl-2 and Caspase 3 [29•]. Many avirulence genes corresponding to cloned resistance genes are yet to be cloned and no X-ray crystallographic analysis of R proteins has been reported. Thus several critical areas of ignorance are ripe for illumination in the near future.
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
•
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••
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•
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•
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A description of the TIR-NBS-LRR (Toll and Interleukin 1 receptor-nuclear binding site-leucine rich repeat) flax rust resistance geneMand of mutants that arise by intragenic events involving two direct repeats in the LRR. It com-pares M with L6, two highly related products with different rust resistance specificity, the former being encoded by a complex tandem array of genes and the latter by a single gene with multiple alleles at a simple locus. 17. Lemaitre B, Nicolas E, Michaut L, Reichhart J-M, Hoffmann JA:
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25. Ori N, Eshed Y, Paran I, Presting G, Aviv D, Tanksley S, Zamir D, Fluhr R:TheI2Cfamily from the wilt disease resistance locusI2belongs to the nucleotide binding, leucine-rich repeat superfamily of plant disease resistance genes.Plant Cell1997, 9:521-532.
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•
29. Van der Biezen EA, Jones JDG:The NB-ARC domain: a novel signalling motif shared by plant resistance gene products and regulators of cell death in animals.Curr Biol1998,8 :R226-R227.
This paper draws attention to the extremely strong amino acid sequence similarity between the nucleotide binding site (NBS) regions of NBS-LRR (leucine-rich repeat) resistance proteins and CED-4 and Apaf-1, the activa-tors of apoptotic proteases, and provide an interesting model for resistance protein function whereby NBS-LRR proteins are members of an inactive cell death-controlling protein complex (apoptosome), possibly including a capase and Bcl-2 homologue, whose activation is triggered by specific (avr encoded) pathogen signals. This model lights the fuse of an idea, and time will tell whether it sets off a bang or a whimper.
••
30. Chinnaiyan AM, Chaudhary D, O’Rourke K, Koonin EV, Dixit VM: Role of CED-4 in the activation of CED-3.Nature1997, 338:728-729.
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38. Jia Y, Loh Y-T, Zhou J, Martin GB: Alleles ofPtoandFenoccur in bacterial speck-susceptible and fenthion-insensitive tomato cultivars and encode active protein kinases.Plant Cell1997, 9:61-73.
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39. Parniske M, Hammond-Kosack KE, Golstein C, Thomas CM, Jones DA, Harrison K, Wulff BBH, Jones JDG:Novel resistance specificities result from sequence exchange between tandemly repeated genes at theCf-4/9locus.Cell1997,91:821-832. This paper provides the complete molecular description of a complex dis-ease resistance locus in three different genotypes and provides a multicom-ponent comparison of gene and protein sequences for the elucidation of possible evolutionary processes and the basis of gene-for-gene specificity. It also uncovers the hitherto unknown presence within the locus of several additional genes that expressCladosporiumresistance with a different de-velopmental program and specificity.
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40. Song W-Y, Pi L-Y, Wang G-L, Gardner J, Holsten T, Ronald PC: Evolution of the riceXa21disease resistance gene family. Plant Cell1997,9:1279-1287.
This paper provided the first insight into a complex R gene locus and indi-cates that many genes at such loci are pseudogenes inactivated by trans-posable elements.
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•
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The first description of a potential molecular link between a disease resis-tance receptor and expression of genes involved in defence responses.
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