Las Relaciones con Instituciones y Agentes Internacionales

Plant NB-LRR proteins share structural and mechanistic similarities with members of the NOD-like receptor (NLR) family that function in innate immune

104 M.A. Sacco and P. Moffett

Fig. 5.1. Multi-domain structures of the proteins encoded by major classes of dominant R genes. Representative receptor-like proteins (RLPs) and receptor-like kinases (RLKs) are shown, which associate with the plasma membrane through a transmembrane (TM) domain and have both intra- and extracellular moieties as described in the text. Note the two protein products predicted to be encoded by the different splice variants of RLM3. The two main classes of NB-LRR proteins are depicted schematically showing the NB, ARC and LRR domains. Proteins in the non-TIR class have a high degree of variability at the amino terminus compared to the TIR class, with various amino-terminal domains encoding CC, BED, SD domains, or no assigned structure (X). Alternatively, some proteins of this class have no domain N-terminal to the NB domain (represented by a small rectangle).A subset of proteins within the TIR-NB-LRR family have been found with additional C-terminal domains

homologous to WRKY transcription factors or may have a large domain of unknown structure (X) with possible nuclear localization signals (NLS) such as in the RPS4 protein.

Disease Resistance Genes: Form and Function 105 responses in animals (Rairdan and Moffett, 2007). Plant NB-LRR proteins are so named because they possess central nucleotide-binding (NB) and carboxy-terminal LRR domains (Fig. 5.1). The LRR domains of the NB-LRR class, however are evolutionarily distinct from the extracellular LRR domains found in receptor-like R proteins, with a consensus of LxxLxxLxxLxLxx(N/C/T)x(x) LxxIPxx (Jones and Jones, 1997; Kajava, 1998). The NB-LRR proteins can be classified into separate clades that have been defined in part by the domain encoded at their amino termini. The primary subdivisions of NB-LRR proteins are based on whether or not the amino-terminal domain shares homology with the cytoplasmic domain of the animal Toll and interleukin-1 receptors (TIR domain; TIR-NB-LRR class) (Whitham et al., 1994). TIR-NB-LRR proteins appear to all belong to a single ancient clade, whereas those NB-LRR lacking a TIR domain appear to show greater diversity in the domains present at their amino termini and can be grouped into at least four different clades (Meyers et al., 1999). Since many of the first non-TIR NB-LRR proteins identified were predicted to encode a coiled-coil (CC) domain at their amino terminus, this class is historically referred to as the CC-NB-LRRs regardless of whether or not they actually conform to CC prediction programmes. The difference between the TIR and CC (non-TIR) NB-LRR proteins however is best defined by consensus motifs present in the NB and ARC domains that show distinct variations between the two classes of NB-LRR proteins (Meyers et al., 1999;

Cannon et al., 2002).

Over 60 R genes of defined specificity encoding NB-LRR proteins have been cloned from a variety of plant species (Tables 5.3 and 5.4). However, bioinformatic analyses of sequenced plant genomes and expressed sequence tags have revealed the presence of vast numbers of genes encoding NB-LRR proteins. In the Arabidopsis thaliana ecotype Columbia-0, 149 genes encode NB-LRR proteins, 94 of which have an amino-terminal TIR domain (Meyers et al., 2003). The number of genes encoding NB-LRR proteins is even larger in plants with larger genomes, with 333 non-redundant genes identified in the incomplete draft sequence of Medicago truncatula, and 399 in black cottonwood, Populus trichocarpa (Tuskan et al., 2006; Ameline-Torregrosa et al., 2008; Kohler et al., 2008). From the genome of a Pinot Noir variety of grape (Vitis vinifera), 233 genes encoding NB-LRR proteins have been identified (Velasco et al., 2007), whereas the genome of a Cabernet Sauvignon variety appears to encode a considerably larger number of NB-LRR proteins (Moroldo et al., 2008). In rice, the number of genes encoding NB-LRR proteins numbers is in excess of 400 genes (Monosi et al., 2004; Zhou et al., 2004), none of which are predicted to encode TIR-NB-LRR proteins (Bai et al., 2002).

Like Arabidopsis, the grape R genes encode both CC-NB-LRR and TIR-NB-LRR proteins (Velasco et al., 2007); however, there has been differential amplification of CC versus TIR classes. On the other hand, the Populus genome shows expansion of a class of NB-LRR proteins with an amino-terminal zinc-finger DNA-binding homology domain, known as a BED finger domain, a structure that is absent from the Arabidopsis genome but conserved in the rice Xa1 and two Xa1-like proteins (Bai et al., 2002; Kohler et al., 2008). In contrast to the large repertoires of NB-LRR genes identified in

106 M.A. Sacco and P. Moffett

Arabidopsis, rice, poplar, grape and Medicago, the papaya genome has a mere 58 NB-LRR genes with a predominance of CC-NB-LRRs (Ming et al., 2008).

In addition to genes with structures resembling NB-LRR proteins, a number of genes in several genomes have been identified encoding proteins with alternate domain configurations (Fig. 5.1). One example with additional domains and known resistance function is the protein RRS1-R from Arabidopsis that recognizes the PopP2 protein from the bacterium Ralstonia solanacearum, and has the structure TIR-NB-LRR-NLS-WRKY. The latter domain encodes a nuclear localization signal (NLS) and resembles a family of transcription factors sharing an amino acid signature motif (WRKY), some of which have been implicated in disease resistance signalling (Deslandes et al., 2002). The alternatively configured proteins are not abundant or conserved between plant genomes and it is unclear whether all are functional genes or whether some may represent pseudogene remnants of recombination events. Plant genomes also encode proteins without LRR domains, consisting of CC-NB, TIR and TIR-NB configurations (Bai et al., 2002; Meyers et al., 2002, 2003). These proteins are present even in rice, which lack TIR-NB-LRR proteins (Bai et al., 2002), suggesting that they may have an evolutionarily conserved function such as acting as signalling adapters analogous to the mammalian TIR-containing immune adapter proteins MyD88 and Mal (Jebanathirajah et al., 2002; Meyers et al., 2002). At the same time, resistance functions have been described for two Arabidopsis loci with unusual domain arrangements, and these are discussed below in the section ‘5.7 Atypical Dominant R Genes’.

An interesting observation can be made by comparing the entire NB-LRR gene complements of the sequenced plant genomes, as well as the growing sequence collections for R gene candidates amplified by PCR from many other plant species. In addition to the diversity of R gene structures within a given plant genome, there is considerable diversity in how different gene families have expanded and evolved in independent plant lineages. The most dramatic of these is the expansion or loss of the TIR-NB-LRR class. In Arabidopsis, these are the most numerous, while this NB-LRR class has been lost in the monocot genome. Detection of TIR-NB-LRR genes in pine species, however, demonstrates the antiquity of this class of genes, which must have existed in an ancestral plant before the divergence of gymnosperms and angiosperms (Liu and Ekramoddoullah, 2003). Furthermore, attempts to amplify genes of the TIR class from sugarbeet have failed so far (Tian et al., 2004), suggesting that this gene family was lost independently in two distant plant lineages. Thus, genes encoding NB-LRR genes may expand and diversify differentially upon speciation, explaining why the repertoires of genes encoding NB-LRRs from unrelated species are so different.

Exploration of the genomic distribution of R gene candidates and isolation of resistance loci has shown that the NB-LRR proteins often exist as clusters.

These clusters are thought to be generated by ancient duplication events and divergence accompanied by selection for new recognition specificities (reviewed in Michelmore and Meyers, 1998). Additional divergence and variation in gene copy numbers at a given locus are likely to have arisen by unequal

crossing-Disease Resistance Genes: Form and Function 107 over events. The shared origin and tight linkage of paralogous R genes in a locus allows for coordinated regulation of their transcriptional activity, a charac-teristic shown recently for the Arabidopsis genes found within the RPP5 locus (Yi and Richards, 2007).

While R genes that are closely related (either by common descent in closely related species, or by duplication within a locus) may be highly similar, the pathogens recognized by the different paralogues may be very different (Grube et al., 2000). This is exemplified well by the Rx and Gpa2 genes from potato that are located in the same R gene cluster, but confer resistance against a virus and nematode, respectively (Bakker et al., 2003). In addition to the diversity afforded by different genes within a locus, there are also examples of considerable allelic diversity of a single R gene. In some cases, this allelic diversity matches similar variation of the pathogen Avr gene, a relationship illustrated by the highly polymorphic Mla locus of barley and the corresponding Avr genes from different isolates of the powdery mildew-causing Blumeria graminis f. sp. hordei (Halterman and Wise, 2004). The allelic diversity of an R gene has the potential to specify recognition of different pathogens as well, a circumstance documented for three R genes (HRT, RCY1 and RPP8) that are in fact alleles of the same locus from different Arabidopsis ecotypes that confer resistance to viruses from two different genera, TCV and CMV, and to the oomycete Hyaloperonospora parasitica, respectively (Cooley et al., 2000; Takahashi et al., 2002). In addition to different alleles from an R gene conferring distinct recognition specificities, examples have also been found where a single allele provides recognition of multiple Avr determinants. In one scenario, specificity may be directed towards distinct Avr effectors that originate from the same species of pathogen, such as the recognition by Arabidopsis RPM1 of two Pseudomonas syringae effectors, AvrRPM1 and AvrB (Grant et al., 1995). Likewise, a single R gene can mediate recognition of effectors from different types of organisms, as seen by the resistance mediated by Mi-1 gene against a root-knot nematode, a white fly and the potato aphid (Rossi et al., 1998). Contrasting the diversity at a single locus is the convergent evolution observed for the Arabidopsis RPM1 and the soybean Rgp1-b genes that both recognize the P. syringae effector AvrB and encode two CC-NB-LRR proteins with limited sequence similarity and likely distinct ancestral origins (Ashfield et al., 2004). In addition, the Arabidopsis Tao1 gene, which encodes a TIR-NB-LRR, responds weakly to AvrB (Eitas et al., 2008).