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Plant disease resistance (R) genes encode proteins that serve to sense the invasion of viral, bacterial or fungal pathogens and subsequently trigger a series of downstream immune responses, thereby playing a critical role in the plant innate immune system (Dangl & Jones, 2001). There are two major classes of immune receptors in plants: nonspecific transmembrane pattern recognition receptors (PRRs), for example, receptor-like kinases (RLKs), and specific cytoplasmic receptors, for example, the nucleotide-binding site–leucine-rich repeat (NBS-LRR) proteins. Proteins in the former class recognize conserved pathogen-associated or microbe-associated molecular patterns (PAMPs or MAMPs), whereas those in the latter class recognize pathogen-specific effectors (Chisholm et al., 2006; Jones & Dangl, 2006; Maekawa et al., 2011). In 1992, Johal & Briggs isolated the first plant R gene, Hm1, from maize (Zea mays). Since then, > 70 R genes have been cloned. Among them, NBS-LRR genes comprise the largest class and account for more than half of plant R genes (McHale et al., 2006).
NBS-LRR genes are characterized by encoding an N-terminal variable domain, a central nucleotide-binding site (NBS) domain, and a C-terminal leucine-rich repeat (LRR) domain. Based on whether they also encode an N-terminal Toll/interleukin-1 receptor (TIR) domain, NBS-LRR genes can be further divided into two subclasses, the TIR subclass and the nonTIR subclass (Meyers et al., 1999). In addition to their different domain architectures, NBS-LRR genes from these two subclasses also differ considerably in their phyletic distribution and downstream signaling pathways, suggesting possible functional divergence between them (Aarts et al., 1998; Tarr & Alexander, 2009). In the plant innate immune system, receptors encoded by NBS-LRR genes are localized in the cytoplasm and can specifically recognize viral effectors secreted by pathogens, either directly or indirectly; this recognition subsequently activates a complex downstream signaling pathway usually leading to rapid local cell death, termed the hypersensitive response (HR), around infected sites (Chisholm et al., 2006). Genome-wide investigations conducted in Arabidopsis thaliana, Oryza sativa, Medicago truncatula, Vitis vinifera, and Populus trichocarpa revealed that there are generally hundreds of NBS-LRR genes in plant genomes, reflecting the important roles of these genes (Meyers et al., 2003; Zhou et al., 2004; Ameline-Torregrosa et al., 2008; Yang et al., 2008). However, NBS-LRR genes are often polymorphic between individuals of a host population, and the complete set of these genes defines the repertoire for the detection of polymorphic pathogen effectors (Bakker et al., 2006; Zhang et al., 2009; Maekawa et al., 2011). It is therefore of particular interest to elucidate the origin and evolutionary history of this important gene family and also the evolutionary relationship between TIR- and nonTIR-NBS-LRR genes.
There is another line of plant defenses represented by the LRR-RLK transmembrane receptors which are localized on the plant cell surface. Containing an extracellular LRR domain and an intracellular Pkinase domain, LRR-RLKs are typically able to recognize highly conserved PAMPs, such as flagellin, lipopolysaccharides (LPSs), cold-shock protein, chitin, and β-glucans (Nurnberger & Brunner, 2002). These molecular patterns are often common components that participate in important functions for various microbial species (pathogenic or not). Therefore, resistance driven by LRR-RLKs is generally non-host-specific and may represent a more basal defense strategy. If so, one unanswered question about the plant innate immune system is whether LRR-RLK genes have a more ancient origin than NBS-LRR genes.
Analogous to the case for plants, animal innate immune systems also rely on two groups of receptors, the transmembrane Toll-like receptors (TLRs) and cytosolic Nod-like receptors (NLRs) (Ausubel, 2005). TLRs are characterized by an extracellular LRR domain and an intracellular TIR domain, whereas NLRs often demonstrate a tripartite domain architecture consisting of a variable N-terminal domain, a central NACHT domain and also a C-terminal LRR domain, like NBS-LRR proteins in plants (Ausubel, 2005; Staal & Dixelius, 2007). In addition, NBS-LRR and NACHT-LRR proteins are members of the signal-transduction ATPases with numerous domains (STAND) class of ATPases (Leipe et al., 2004; Rairdan & Moffett, 2007; Maekawa et al., 2011) which share similar biological functions and protein structures; however, the NACHTs have been placed in a separate phylogeny, suggesting different evolutionary histories of these two domains (Leipe et al., 2004; Rairdan & Moffett, 2007; Maekawa et al., 2011). Therefore, it is interesting to ask: what forces drove such striking similarities between the plant and animal innate immune systems? Did they come from the same common ancestor (divergent evolution) or did they evolve independently (convergent evolution), as suggested by a number of reviews (Ausubel, 2005; Rairdan & Moffett, 2007; Staal & Dixelius, 2007)?
The recent avalanche of whole-genome data from diverse species offers us an unprecedented opportunity to explore this series of questions via comparative analysis at the genomic level. In this study, we sampled 38 representative genomes, covering all major kingdoms of organisms (eubacteria, archaebacteria, fungi, protists, plants and animals) and identified all homologous genes of NBS-LRR, LRR-RLK, TLR, and also NLR genes in these genomes. For these well-defined immune receptor genes, we conducted a set of comparative analyses on their phyletic distribution, domain architecture, phylogenetic topology, and conserved motif evolution patterns. These data shed light on the origin and history of NBS-LRR genes and their evolutionary relationship with LRR-RLK genes in plants, and also those of the TLR and NLR genes in animals. In addition to R genes, a number of important chaperones and regulators involved in the plant immune response have been identified through genetic screens; these include WRKY in the LRR-RLK defense pathway and SGT1, RAR1, EDS1, PAD4, SAG101, HSP90 and SID2 in the NBS-LRR defense pathway (Rusterucci et al., 2001; Azevedo et al., 2002; Hubert et al., 2003; Leister et al., 2005; Eulgem & Somssich, 2007). Because of the essential functions of these signaling components for plant immunity, they were also incorporated in our study.
The clarification of the evolutionary relationships and timelines of plant R genes, especially NBS-LRR genes, in this study paves the way for elucidation of how plant disease resistance originated and evolved, and how this evolutionary innovation helped plants to survive and thrive in diverse terrestrial habitats. In addition, the findings of our study provide a good example of how recruiting and reorganizing of pre-existing building blocks can lead to functional innovation, thus shedding new light on the origin of evolutionary novelties.
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Fig. S1 Phyletic distribution pattern of characteristic domains within other essential signaling components in the plant disease resistance response pathway.
Fig. S2 Evolution of motif combinatorial patterns within the key domains of some essential genes in plant disease resistance response pathways.
Table S1 Whole-genome data used in this study
Table S2 Current available sequence data of nine early plant lineages from NCBI Genbank
Table S3 The characteristic domains within plant immune receptor genes and other essential signaling components in plant and animal innate immune systems
Table S4 Pfam and BLASTP analysis for candidate nucleotide-binding site (NBS) domain encoded in early plant lineages
Notes S1 Sequence and alignment files.
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