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288 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 Biology 1998, 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, LRR-TM-PK, LRRTM 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. Hs1pro-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. An Arabidopsis homologue (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 Xa21 gene from rice confers resistance to Xanthomonas oryzae pv. oryzae (Xoo) and encodes a protein with the LRR-TM-PK signature in which the LRR is predicted to be extracellular [4]. So far, Xa21 is the only R protein in this class. PCR-based methods could provide the means to clone more R genes in this class, however, this is clearly not a direct route now that several other plant genes have been cloned which encode LRR-TM-PK proteins apparently not involved in pathogen resistance. These include developmental genes like ERECTA [5] and CLAVATA1 [6] of Arabidopsis and SERK [7] of carrot, and the gene encoding a putative brassinosteroid receptor (BRI) [8] of Arabidopsis. Thus, this class of protein fulfils several roles in plants. The LRR domain might detect extracellular ligands of various types, including potentially Structure and function of proteins controlling strain-specific pathogen resistance in plants Ellis and Jones 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 gene avrXa1, 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 corresponding avr genes, avrXa1 and avrXa2, from Xoo is 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 289 [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 cloned NDR1 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 Database searches have consistently failed to identify NBS-LRR organisation in bacteria, yeast or animal genomes. Recently, however, clear and extensive amino acid sequence similarity in the NBS domain, including motifs that were previously thought to be common only to plant NBS-LRR proteins, has been detected in nematode CED-4 and mammalian Apaf-1 proteins — activators of the apoptotic proteases CED-3 and caspase-9 respectively [29•,30••]. One common, but not necessarily universal, feature of NBS-LRR function is the occurrence of hypersensitive cell death at infection sites during resistance reactions. The similarity between this response and animal apoptosis has often been remarked upon, so this structural similarity between the NBS-LRR proteins and CED-4 and Apaf-1 is intriguing. Experimental evidence for nucleotide binding at the NBS site has been obtained for CED-4 [30••] and for the RPS2 resistance protein (A Bent, personal communication), suggesting a functional similarity. CED-4 and Apaf-1 appear to act as adaptor molecules with a central NBS domain and an amino-terminal effector domain [30••,31]. The carboxy-terminal receptor domain of Apaf-1 is composed of repeating amino acid motifs, designated WD-40 repeats, involved in protein–protein interactions (analogous to the LRR domain) [31]. Interestingly, both CED-4 and Apaf-1 appear to activate their target proteases (CED-3 and caspase-9 respectively) by heterodimerisation of homologous amino-terminal domains present in both the activators and the proteases [30••,32]. This is reminiscent of the interaction between homologous domains of MyD88 290 Plant–microbe interactions 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 tomato Cf-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 rice Xa21 locus (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 resistance 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 in Drosophila where 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 recognition. In the interaction between the tomato Pto and Pseudomonas syringae AvrPto proteins, domain swap experiments between Pto and 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 of P. syringae carrying the avrPto gene [9,10]. In the second system, also in tomato, 11 homologous genes at the Cf-4/9 locus for Cladosporium fulvum resistance 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 homologues revealed variation originating from mutation, segmental 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 parallel β-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. In the third system, the predicted protein sequences of 13 active alleles of the flax L gene (TIR-NBS-LRR) conferring rust resistance [44•] are over 90% identical but show variation over the entire length of the protein. The greatest sequence variation is observed in the LRR region. The same three sources of variation acting on the Cf genes also appear to operate for LRR domains of the L alleles. Selection seems to be acting in a similar way to favour diversification in the potential strand regions and conservation in the nonstrand regions of the LRR Structure and function of proteins controlling strain-specific pathogen resistance in plants Ellis and Jones domain. Sequence comparisons and interallelic domain swap experiments, however, also indicate that the aminoterminal TIR domain makes important contributions to allelic specificity in addition to the LRR domain (J Ellis et al., unpublished data). 4. Song W-Y, Wang G-L, Chen L-L, Kim H-S, Pi L-Y, Holsten T, Gardner J, Wang B, Zhai W-X, Zhu L-H et al.: A receptor kinaselike protein encoded by the rice disease resistance gene, Xa21. Science 1995, 270:1804-1806. 5. Torii KU, Mitsukawa N, Oosumi T, Matsuura Y, Yokoyama R, Whittier RF, Komeda Y: The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucinerich repeats. Plant Cell 1996, 8:735-746. 6. Clark SE, Williams RW, Meyerowitz EM: The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis. Cell 1997, 89:575-585. 7. Schmidt EDL, Guzzo F, Toonen MAJ, de Vries SC: A leucinerich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos. Development 1997, 124:2049-2062. 8. Li J, Chory J: A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 1997, 90:929-938. 9. Scofield SR, Tobias CM, Rathjen JP, Chang JH, Lavelle DT, Michelmore RW, Staskawicz BJ: Molecular basis of gene-forgene specificity in bacterial speck disease of tomato. Science 1996, 274:2063-2065. 10. Tang XY, Frederick RD, Zhou JM, Halterman DA, Jia YL, Martin GB: Initiation of plant disease resistance by physical interaction of AvrPto and Pto kinase. Science 1996, 274:20602063. Conclusions The number of cloned R genes is rapidly increasing and genetic and molecular studies have identified several downstream signalling components in resistance [27,28•,45•]. One example of a strain-nonspecific resistance 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 1. Hammond-Kosack KE, Jones JDG: Plant disease resistance • genes. Annu Rev Plant Physiol Plant Mol Biol 1997, 48:575-607. An extensive review of cloned plant disease resistance genes and comparison of their products. 2. •• Bogdanove AJ, Kim JF, Wei Z, Kolchinsky P, Charkowski AO, Conlin AK, Collmer A, Beer SV: Homology and functional similarity of an hrp-linked pathogenicity locus, dspEF, of Erwinia amylovora and the avirulence locus avrE of Pseudomonas syringae pathovar tomato. Proc Natl Acad Sci USA 1998, 95:1325-1330. The function of pathogen avirulence genes has been enigmatic. Several examples among plant bacterial pathogens have indicated that avirulence gene mutants have decreased virulence. This paper describes two dspEF genes in the apple fireblight pathogen Erwinia that encode factors necessary for virulence. When transferred and expressed in Pseudomonas syringae, however, they function as avirulence genes and induce defence responses in specific resistant lines of soybean. Avirulence genes with high sequence similarity are also identified in P. syringae and these genes can complement dspEF mutants in Erwinia. These data provide strong support for the view that avirulence genes encode virulence factors for which host genotypes have evolved specific surveillance mechanisms. Furthermore, they suggest the intriguing possibility of isolating resistance genes from soybean which have the appropriate specificity to protect apple (for which no fireblight R genes are known) from this disease. 3. Cai D, Kleine M, Kifle S, Harloff H-J, Sandal NN, Marcker KA, Klein-Lankhorst RM, Salentijn EMJ, Lange W, Stiekema WJ et al.: Positional cloning of a gene for nematode resistance in sugar beet. Science 1997, 275:832-834. 291 11. • Yoshimura S, Yamanouchi U, Katayose Y, Toki S, Wang Z-X, Kono I, Kurata N, Yano M, Iwata N, Sasaki T: Expression of Xa1, a bacterial blight-resistance gene in rice, is induced by bacterial inoculation. Proc Natl Acad Sci USA 1998, 95:1663-1668. The map-based cloning of a second blight resistance gene in rice is described and the amino acid sequence predicted. The predicted product contains six near perfect 93 amino acid repeats in the LRR region and Xa1 expression is induced by infection and wounding. Whether any other R genes are induced has not been addressed in any detail in previous publications. 12. Jones DA, Jones JDG: The role of leucine-rich repeat proteins • in plant defences. Adv Bot Res 1997, 24:89-167. An extensive review of plant LRR proteins and discussion of features that may be used to predict whether these proteins are intracellular or extracellular. 13. • Parker JE, Coleman MJ, Szabo V, Frost LN, Schmidt R, van der Biezen E, Moores T, Dean C, Daniels MJ, Jones JDG: The Arabidopsis downy mildew resistance gene RPP5 shares similarity to the Toll and Interleukin-1 receptors with N and L6. Plant Cell 1997, 9:879-894. Like [16•] this paper describes a deletion event involving repeated sequence elements in the leucine-rich repeat region and indicates that these events may play a role in the evolution of the genes. 14. Baker B, Zambryski P, Staskawicz B, Dinesh-Kumar SP: Signaling in plant-microbe interactions. Science 1997, 276:726-733. 15. Rock FL, Hardiman G, Timans JC, Kastelein RA, Bazan JF: A family of human receptors structurally related to Drosophila Toll. Proc Natl Acad Sci USA 1998, 95:588-593. 16. • Anderson PA, Lawrence GJ, Morrish BC, Ayliffe MA, Finnegan EJ, Ellis JG: Inactivation of the flax rust resistance gene M associated with the loss of a repeated unit within the leucinerich repeat coding region. Plant Cell 1997, 9:641-651. A description of the TIR-NBS-LRR (Toll and Interleukin 1 receptor-nuclear binding site-leucine rich repeat) flax rust resistance gene M and of mutants that arise by intragenic events involving two direct repeats in the LRR. It compares 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: The dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 1996, 86:973-983. 292 Plant–microbe interactions 18. Williams MJ, Rodriguez A, Kimbrell DA, Eldon ED: The 18-wheeler mutation reveals complex antibacterial gene regulation in Drosophila defense. EMBO J 1997, 16:6120-6130. 19. Medzhitov R, Preston-Hurlburt P, Janeway CA: A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997, 338:394-397. 20. Yang J, Steward R: A multimeric complex and the nuclear targeting of the Drosophila rel protein dorsal. Proc Natl Acad Sci USA 1997, 94:14524-14529. 21. Muzio M, Ni J, Feng P, Dixit VM: IRAK (Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling. Science 1997, 278:1612-1615. 22. 30. •• Chinnaiyan AM, Chaudhary D, O’Rourke K, Koonin EV, Dixit VM: Role of CED-4 in the activation of CED-3. Nature 1997, 338:728-729. This paper was the first to draw attention to the amino acid similarities between the NBS regions of the nematode CED-4 protein and the L6 flax rust resistance protein. 31. Zou H, Henzel WJ, Liu XS, Lutschg A, Wang XD: Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 1997, 90:405-413. 32. Li P, Nijhawan D, Budihardjo J, Srinivasula SM, Ahmad M, Alnemri ES, Wang XD: Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997, 91:479-489. Volpe F, Clatworthy J, Kaptein A, Maschera B, Griffin A-M, Ray K: The IL1 receptor accessory protein is responsible for the recruitment of the interleukin-1 receptor associated kinase to the IL1/IL1 receptor I complex. FEBS Lett 1997, 419:41-44. 33. Botella MA, Coleman MJ, Hughes DE, Nishimura MT, Jones JDG, Sommerville SC: Map locations of 47 Arabidopsis sequences with similarity to disease resistance genes. Plant J 1997, 12:1197-1211. 23. Wesche H, Henzel WJ, Shillinglaw W, Li S, Cao ZD: MyD88 — an adapter that recruits IRAK to the IL-1 receptor complex. Immunity 1997, 7:837-847. 34. Leister D, Ballvora A, Salamini F, Gebhardt C: A PCR-based approach for isolating pathogen resistance genes from potato with potential for wide applications in plants. Nat Genet 1996, 14:421-429. 24. Salmeron JM, Oldroyd GED, Rommens CMT, Scofield SR, Kim HS, Lavelle DT, Dahlbeck D, Staskawicz BJ: Tomato Prf is a member of the leucine-rich repeat class of plant disease resistance genes and lies embedded within the Pto kinase gene cluster. Cell 1996, 86:123-133. 35. Yu YG, Buss GR, Saghai-Maroof MA: Isolation of a superfamily of candidate disease resistance genes from soybean based on a conserved nucleotide binding site. Proc Natl Acad Sci USA 1996, 93:11751-11756. 36. Kanazin V, Marek LF, Shoemaker RC: Resistance gene analogues are conserved and clustered in soybean. Proc Natl Acad Sci USA 1996, 93:11746-11750. 37. Leister D, Kurth J, Laurie DA, Yano M, Sasaki T, Devos K, Graner A, Schulze-Lefert P: Rapid reorganization of resistance gene homologues in cereal genomes. Proc Natl Acad Sci USA 1998, 95:370-375. 38. Jia Y, Loh Y-T, Zhou J, Martin GB: Alleles of Pto and Fen occur in bacterial speck-susceptible and fenthion-insensitive tomato cultivars and encode active protein kinases. Plant Cell 1997, 9:61-73. 25. • Ori N, Eshed Y, Paran I, Presting G, Aviv D, Tanksley S, Zamir D, Fluhr R: The I2C family from the wilt disease resistance locus I2 belongs to the nucleotide binding, leucine-rich repeat superfamily of plant disease resistance genes. Plant Cell 1997, 9:521-532. A family of R genes of the NBS-LRR class encoding Fusarium oxysporum f.sp lycopersici resistance is described. The authors show that antisense technology can be used to confirm that a cloned R gene candidate belongs to the the gene cluster thought to contain one or more genes necessary for a particular resistance specificity. This will be an important initial tool for identification of gene families involved in resistance in complex genomes containing many R gene families. 26. • Lagudah ES, Moullet O, Appels R: Map-based cloning of a gene sequence encoding a nucleotide-binding domain and a leucine-rich region at the Cre3 nematode resistance locus of wheat. Genome 1997, 40:650-665. The first indication that a gene of the NBS-LRR class can encode resistance to plant parasitic nematodes. 27. Parker JE, Holub EB, Frost LN, Falk A, Gunn ND, Daniels MJ: Characterization of eds1, a mutation in Arabidopsis suppressing resistance to Peronospora parasitica specified by several different RPP genes. Plant Cell 1996, 8:2033-2046. 28. • Century KS, Shapiro AD, Repetti PP, Dahlbeck D, Holub E, Staskawicz BJ: NDR1, a pathogen-induced component required for Arabidopsis disease resistance. Science 1997, 278:19631965. This paper demonstrates the power of mutation to identify genes necessary for resistance and the power of map-based cloning in Arabidopsis. The NDR protein has no close homologue in the present databases and is predicted to be a membrane spanning protein. Mutations in NDR1 cause loss of action of several different R genes. 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 Biol 1998, 8:R226R227. 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 activators 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. 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 the Cf-4/9 locus. Cell 1997, 91:821-832. This paper provides the complete molecular description of a complex disease resistance locus in three different genotypes and provides a multicomponent 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 express Cladosporium resistance with a different developmental program and specificity. 40. • Song W-Y, Pi L-Y, Wang G-L, Gardner J, Holsten T, Ronald PC: Evolution of the rice Xa21 disease resistance gene family. Plant Cell 1997, 9:1279-1287. This paper provided the first insight into a complex R gene locus and indicates that many genes at such loci are pseudogenes inactivated by transposable elements. 41. Bevan M, Bancroft I, Bent E, Love K, Goodman H, Dean C, Bergkamp R, Dirkse W, van Staveren M, Stiekema W et al.: Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana. Nature 1998, 391:485-488. 42. Lemaitre B, Reichhart J-M, Hoffmann JA: Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. Proc Natl Acad Sci USA 1997, 94:14614-14619. 43. • Thomas CM, Jones DA, Parniske M, Harrison K, Balint-Kurti P, Hatzixanthis K, Jones JDG: Characterization of the tomato Cf-4 gene for resistance to Cladosporium fulvum identifies sequences that determine recognitional specificity in Cf-4 and Cf-9. Plant Cell 1997, 9:2209-2224. 29. • Structure and function of proteins controlling strain-specific pathogen resistance in plants Ellis and Jones The comparison of two resistance genes, Cf-4 and Cf-9, for Cladosporium resistance provides evidence about the regions in Cf proteins that control specificity of pathogen race recognition. 44. • Ellis J, Lawrence G, Ayliffe M, Anderson P, Collins N, Finnegan J, Frost D, Luck J, Pryor T: Advances in the molecular genetic analysis of the flax–flax rust interaction. Annu Rev Phytopathol 1997, 35:271-291. An extensive review of recent activity in the analysis of the flax–flax rust interaction, which was the host–pathogen system that served as the basis for Flor’s gene-for-gene hypothesis, and a description of the processes used for isolating the L6 and M rust resistance genes. 45. • Zhou J, Tang X, Martin GB: The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related proteins. EMBO J 1997, 16:3207-3218. 293 The first description of a potential molecular link between a disease resistance receptor and expression of genes involved in defence responses. 46. • Buschges R, Hollricher K, Panstruga R, Simons G, Wolter M, Frijters A, van Daelen R, van der Lee T, Diergaarde P, Groenendijk J, Topsch S et al.: The barley Mlo gene: a novel control element of plant pathogen resistance. Cell 1997, 88:695-705. While the scope of our review was restricted to gene-for-gene resistance, special attention is also drawn to this recent paper. Recessive mutants of the Mlo gene are used extensively, especially in Europe, to protect barley from the fungal disease, mildew. The Mlo gene is unusual in that, in contrast to gene-for-gene resistance which is usually effective against some but not all strains of a pathogen species, no barley mildew strains are known in nature that overcome this resistance. It encodes a novel protein with at least six predicted membrane spanning helices and none of the structural signatures of R proteins discussed in our review. The identification of isologues in rice and Arabidopsis raises the possibility that this form of resistance can be extended to other plant species.