Sentinels of Disease. Plant Resistance Genes

Act 2, SCENE I. King Henry VI, William Shakespeare (Enter a Sergeant with two Sentinels) Sergeant speaks: “Sirs! take your places and be vigilant: If any noise or soldier you perceive Near to the walls, by some apparent sign Let us have knowledge at the court of guard.“ Successful defense against an enemy requires perception of his whereabouts. In the last few years, much progress has been made in delineating the plant molecular sentinels that participate in pathogen identification. This ability is encoded by genetically “hard-wired” information, and is called “innate immunity”. It draws its origins from a phylogenetically ancient form of immunity that is common to all Metazoa and Viridiplantae. However, the rapid evolution of plant innate immunity genes has led to massive gene diversification. The appreciation of this diversification offers challenging prospects in understanding the forces that have shaped multicellular innate immunity and underlines the necessity of furthering our understanding by examining multiple plant systems. Strategies of self and non-self recognition in innate and adaptive immunity. The shaded boxes represent infected cells. In animals, specialized cells are sources for acquired immunity and they spread throughout the organism (arrows). The genomic information content (bp) devoted to genes of acquired immunity is represented by three recombinatorial systems as exemplified in humans by the following: T-cell receptors (TCR), major histocompatibility complex (MHC), and the different antibody classes (V). The major potential pattern recognition genes in plants are shown in Figure 2. Gene number estimates are for Arabidopsis and include NBS/LRR-type (150), LRR kinase-type (174), Cf-type (30), and Pto-type (70). Estimates of genomic sequence dedicated to each gene type are 5 × 103 for NBS/LRR and LRR-kinase and 2 × 103 for the rest. Pattern receptors are built from domains common to plants and animals. Upper, Domains have been conceptually divided into five functional categories. A, LRR, CC, and a kinase are domains that can function as pattern receptors. B, TIR, CC and caspase-activating recruitment domains (CARD) are likely involved in signal transduction by homo- or heterodimerization with acceptor molecules. C, NBD common to Nod and plant TIR/CC-NBD may serve a nucleotide-dependent switch function. D, The transmembrane motif functions to anchor attached domains or participate in transmembrane signal transfer and may also contain endocytosis signals for signal attenuation as has been found in the tomato Ve Verticillium R-gene (Kawchuk et al., 2001). E, The kinase domain probably participates in signal transduction. Lower, Known examples of domain combinations are illustrated. What are the biophysical properties of the LRR domain that favored its choice as the pattern receptor for sentinels? LRR structures mediate protein-protein interaction and are the major determinants of recognition specificity. The protein structure solved by the crystal structure of the porcine ribonuclease inhibitor (RI) has served as a rough structural platform for conceptualizing what the distantly related plant LRR may look like (Kobe and Deisenhofer, 1994). The 15 LRRs of RI are composed of an inner “solvent-exposed” surface rim comprising β-sheets connected by an outer rim of α-helical segments. In RI, the β-sheets are stabilized by a ladder of hydrogen bonds between conserved cysteines and Asn side chains. The α-helical segments force a curvature on the molecule so that it comes nearly full circle leaving a 60° opening. It is through this opening that the ribonuclease interacts with the solvent surface. Plant LRR can contain many fewer repeats; for example, seven as found in the sugar beet (Beta vulgaris) nematode Hs1pro1 R-gene (Cai et al., 1997), or many more than 15 repeats, which is usually the case. Overabundant LRR repeats in a domain would seemingly force complete circle closure. However, the plant α-helical regions tend to be much less conserved (they are shorter or nonexistent). The LRR domain structure must therefore deviate from RI in a manner that would generate a less constrained flexible domain stabilized by the hydrogen bonds between the β-sheets. Specificity between LRR domains and their potential ligands has been inferred in animal TIR-LRR by the finding that Toll-Like Receptor 4 (TLR4) immunity genes from different species can each impart the particular species-specific sensitivity to pharmacologically different lipopolysaccharide structures (Poltorak et al., 2000). The tomato (Lycopersicon esculentum) Cf-like R-genes confer resistance to infection by the biotrophic leaf mold pathogenCladosporium fulvum and contains an extracellular LRR domain. By precise domain swapping, a number of LRRs could be shown to be essential for both Cf-4 and Cf-9 function (Van Der Hoorn et al., 2001; Wulff et al., 2001). Direct evidence for interaction of LRR domain with avirulence factors is based on the finding that a single amino acid difference in the LRR domain distinguished susceptible and resistant alleles of the fungalMagnapporthe grisea rice R-gene (Bryan et al., 2000). In this case, by using the yeast (Saccharomyces cerevisiae) two-hybrid system, the recombinant LRR domain of the resistant allele could be shown to directly interact with its avirulence factor while the susceptible allele displayed a much weaker interaction (Jia et al., 2000). Interestingly, this system gives us a glimpse of the complex biology involved in plant receptor/avirulence factor interactions. The avirulence factor gene from M. grisea (AVR-Pita) encodes for a pre-propeptide that has the features of a metalloprotease. The processed form is then transferred by an unknown mechanism from the fungus into the plant cell. If solvent-exposed regions of the LRR domain play a role in interaction with the pathogen avirulence factor, they may display high mutability. Detecting adaptive evolution is carried out by estimating base changes needed to generate amino acid changes, i.e. comparing the number of substitutions per synonymous site (Ks) with the number of substitutions per nonsynonymous site (Ka; Li, 1993). Ks is expected to exceed Ka when mutations that generate amino acid change in a particular gene are deleterious to an organism's fitness. However, if mutations in an area of a particular gene are advantageous for the organism, natural selection favors sequence diversification, and a Ka/Ks ratio larger than 1 will emerge. A survey of plant R-genes shows that, generally, the N-terminal CC structures as well as the NBD show low average Ka/Ks ratio. In contrast, positive selection (high Ka/Ks ratio) was detected in the predicted β-strand region of the LRR domain in many R-gene analogs (for review, see Bergelson et al., 2001). What dictates the elevated Ka/Ks ratio? Is it entirely due to high selection pressure applied to the basal mutation rate? Or do perhaps the solvent-exposed LRR regions also have a propensity for hypermutation, a phenomenon that has been detected for immunoglobulin genes? TIR domains are a common link between animal and plant innate immunity (Kimbrell and Beutler, 2001). In plant NBD-LRR R-genes, the TIR motif appears at the N terminus, whereas in animals the TIR domain is at the carboxyl end of a single-pass transmembrane receptor (Fig.1). Structural analysis of the diverged TIR domains of human TLR2 and TLR4 reveals a relatively conserved surface for binding to MyD88 (Xu et al., 2000). MyD88, a signal adapter molecule, is essential for signal transduction of the immune response. The idea of surface conservation of TIR is important because it may explain how divergent TIR-containing genes can signal through the apparently singular MyD88 adapter. Database searches have yet to reveal a plant MyD88 homolog; however, the finding that mutations in enhanced disease susceptibility locus 1 (EDS1) compromise the articulation of distinct R-genes of the TIR-NBD-LRR class argues for signal funneling through a common intermediate (Aarts et al., 1998). The TIR domain is essential for tobacco (Nicotiana tabacum) N-gene-mediated tobacco mosaic virus resistance, and amino acids that affect Drosophila melanogaster and human TIR-dependent signaling cause either partial or full loss of N-gene function (Dinesh-Kumar et al., 2000); for example, N-gene D46 resides in a position consistent with the conserved human TLR2 surface. Mutation D46H completely eliminates function, whereas the D46Y substitution that is the normal state for the human interleukin-1 receptor had no effect. Interestingly, partial loss-of-function mutations can act as dominant negative mutations by promoting systemic hypersensitive response in the wild-type N-gene background, a result that also argues for the involvement of sentinels in higher order complexes. Unexpectedly, the plant TIR domain may contribute to determining R-gene specificity. In flax rust resistance, swapping the TIR domains of L6 and L7R-genes switches their specificity (Luck et al., 2000). This, together with evidence for diversifying selection in the TIR region of the flax rust R-genes, may also indicate that pattern recognition operates as a complex. Phylogenetic trees of TIR domains reveal a clear division between animal and plant taxa (Kimbrell and Beutler, 2001). However, the low identity of amino acid sequence between plant and animal TIR domains (less then 20%) obviates facile structural comparison. The animal taxa are further divided into at least two independent groups, Toll-like and Interleukin receptor-like. The plant TIR family as potentially represented in the Arabidopsis genome can likewise be divided into a few TIR phylogenetic groups. One group of over 100 genes includes TIR domains as part of NBD or as part of NBD-LRR sequences. Another group of more than 30 genes is composed of solitary TIR domains or TIR domains juxtaposed to other domains of unknown function (N. Kaplan-Levy and R. Fluhr, unpublished data). The sequence similarity between plant and animal TIR domains suggests a common unicellular ancestor. Indeed, sensitive “SMART” searches of current databases, which use reiterative sequence alignment together with a broad definition of conserved polypeptide structural elements, have revealed distant TIR homologies in prokaryotes as well (http://smart.embl-heidelberg.de/; Schultz et al., 2000). Surprisingly, despite their obviously ancient origin, TIR domains have not been detected in any of the cereal genomes, their function apparently replaced by CC domains (Meyers et al., 1999; Pan et al., 2000b). CCs are an oligomerization motif of helical structures that are made up of bundles containing two to five helices. A typical CC structure shows a heptad repeat where the seven positions are labeled a through g. Residues a and d tend to be hydrophobic, and the residues at the e and g positions are charged or polar. A large subset of eudicot and cereal NBD-LRR genes can be shown to contain general CC domains in their N-terminal region with over 95% probability (Pan et al., 2000b). In this context, they may serve the function of adapter TIR-like motifs (Fig. 1). However, alternative functionality is suggested in the case of the RPW8 Arabidopsis R-gene (DA2-type, Fig. 2), responsible for broad-spectrum resistance to mildew. It contains CC domains that appear at the C-terminal of a predicted transmembrane domain or signal peptide domain (Xiao et al., 2001). In this case, the CC domain may be analogous to the function played by the LRR domain and could act directly in pattern recognition of an avirulence factor common to many mildew races. Alternatively, it may play an accessory role facilitating other pattern recognition molecules to function. RPW8 is encoded by a small gene family of only five linked members, but due to the nature of CC domains, true assessment of their potential numbers in plant genomes will require sophisticated database mining. The tomato resistance protein kinase encoded by Ptoshows remarkable similarity to interleukin-1 receptor-associated kinase and Pelle kinases that act downstream in animal TIR-LRR-directed immune response (for review, see Cohn et al., 2001). Autophosphorylation competence and the Pto activation domain were obligatory for interaction of Pto and its cognate avirulence factor (Sessa et al., 2000). Importantly, the function of Pto requires the presence of Prf gene, an NBD-LRR-type gene (Salmeron et al., 1996). Conserved activation domains delineate numerous but distant putative Pto homologs in Arabidopsis (Fig. 1); some of them appear together with other functional domains in receptor-like kinases. In functional analogy to Pto, the Arabidopsis avrPphB-susceptible kinase is necessary forResistance to Pseudomonas syringae 5 function but not forResistance to P. syringae subsp. maculicola 1 orResistance to P. syringae 2-type NBD-LRR resistance function (Swiderski and Innes, 2001). Sequence signatures of the Pto activation domain together with the presence of sequence insertions/deletions helped define nine Pto-like phylogenic families. The clades were made up of sequence from different plant families suggesting ancient origin for Pto in the genus Solanum(Vleeshouwers et al., 2001). Indeed, orthologous Pto genes of Lycopersicon pimpinellifolium and Lycopersicon hirsutum interact with the same avirulence factor and confer resistance (Riely and Martin, 2001). Cross-species maintenance of R-gene functionality may be due to the existence of persistent pathogens that help maintain ancient disease lineages. Interestingly, L. esculentum accessions examined contained no Pto ortholog showing that Pto kinases, like their cognate NBD-LRR, are either part of metabolically dispensable pathways or are functionally redundant. Ser/Thr kinase domains are also found as part of A1DE-type receptors (Fig. 2). These domains play a role in signal transduction as was shown exquisitely by switching the extracellular LRR and transmembrane domains of the Arabidopsis brassinosteroid receptor kinase BRI1 with the Ser/Thr kinase domain of the rice XA21 R-gene. The resultant transgene promoted brassinosteroid-dependent resistance signaling (He et al., 2000). Shiu and Bleecker (2001) have shown that all the kinase domains of the A1DE-type receptors belong to a single monophyletic group. The closest eukaryotic homolog to the plant receptor kinase family was found to be the D. melanogasterkinase Pelle, similar to that found for Pto. NBDs are characterized by several sequence motifs found in animal ATP- and GTP-binding proteins including the Ras superfamily and the caspase pathway-related Ced-4 and Apaf-1 animal genes (Li et al., 1997). In animals, the genes regulate the activity of proteases that can initiate apoptotic cell death. As defense mechanisms in plants include apoptotic-like hypersensitive responses, the appearance of these homologies in this context is particularly intriguing. By analogy to Ras, NBD may serve as a switch function moderating the inter- or intramolecular activity of the polypeptide. Alternative structural modeling of a subset of the NBD region revealed homology to the receiver domain of His-Asp phosphoproteins typical of prokaryotic signaling pathways (Rigden et al., 2000). This would argue for NBD participation in phosphorelay as opposed to actual nucleotide binding. Direct biochemical studies with this motif are lacking. Interestingly, the Arabidopsis RPP5 NBD domain was shown to interact in the yeast two-hybrid system with RelA/SpoT gene homologs. InEscherichia coli these genes are involved in p/pppGpp stress effector molecule signaling (van der Biezen et al., 2000). Whether this implies a similar function in planta is not known. A systematic approach to elucidate regions critical for activity was carried out in the N-gene NBD region. Mutations in the conserved nucleotide binding site, e.g. glycines or Lys (G216 and K222), led to loss of N-gene action but also interfered in a negative dominant fashion when the mutant transgene was present in the normal N-gene background (Dinesh-Kumar et al., 2000). These results are consistent with that detected in other NBD-containing proteins and point to similar mechanisms of action. For example, mutations in the conserved kinase 2 position at D301 leads to complete loss of function. The equivalent mutations of the conserved Asp in CED4 disrupt the oligomerization process that is necessary for activating downstream et al., 1998). In to the diverged and LRR domains that NBD-LRR resistance all NBD domains contain conserved of sequence can be divided into two distinct due to their different conserved (Meyers et al., 1999; Pan et al., 2000b). One group is found linked to TIR in its N-terminal region. This group is from cereal and be from cereal genomes (Pan et al., 2000b). The other which also contains particular signatures in the is linked to CCs in its N-terminal region. This group is present as a superfamily of genes in both eudicot and As and were shown to or multiple R-gene in Arabidopsis (Aarts et al., the of a group of R-gene analogs implies of signal transduction pathways between these two plant of NBD-LRR-type disease R-gene homologs. The shows that in plant R-gene evolution involved domain and by massive gene and gene diversification. The two major are called and The genes Fig. were probably from cereal genomes to their NBD-LRR can in large of that of of R-gene homologs. In the Arabidopsis of the detected NBD sequence appear as of more than gene an idea of the genome of R-genes, NBD analogs have been and by in et al., cereal genomes et al., and et al., Pan et al., and 2001). of NBD was found to be of in the genome would serve as a of due to and gene R-gene evolution could a pattern of rapid positive selection of combinations of selection pressure may gene by of It is of that related show the Ka/Ks suggesting rapid selection in the alleles et al., 2001). the rapid evolution of R-genes due to it is of to the of these genes to general genome conservation of despite has been in together with some R-gene loss (Pan et al., loss of but together with the of more was for cereal R-genes and their homologs and may indicate more rapid sequence in the et al., 1998). The that completely the more conserved R-genes is consistent with their rapid of R-gene It is important to in that when are is no to functional of species the family can conserved structural but show completely different disease specificity. For example, resistance in tomato to resistance to tobacco mosaic virus in on and the tomato R-gene to a resistance on 4 et al., and 2001). However, at the of related a of functional conservation can be This was by directly examining a of the complete Lycopersicon genome into a L. esculentum independent resistance were detected with between and two out of show common origins by in the same in more than species et al., 2001). Whether these represent true orthologous sequence is of and to be What then is the in genomic of than furthering our understanding of how resistance has been shaped on the species at the family will for and of of in the of potential non-self ligands to common pattern recognition is shown by the Arabidopsis LRR receptor kinase and 2000). In this case, the pattern of a conserved amino acid domain in is to an response et al., of the avirulence factors characterized to each However, the that a single R-gene can more than avirulence factor, as in the case of to P. syringae subsp. maculicola 1 et al., and which resistance to both and in tomato et al., argues for structural that may have been Another that would the for R-gene is the and 2001). 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