Every living organism has always a desire to live a healthy long life and a fight for the survival has always been the main subject for all organisms. For this purpose, they strive for their best to enjoy a life with no or less problems using their innate and adapted immunity systems. Since the initial discoveries (Koch, 1876; Burrill, 1878), a wide array of microorganisms have been identified for their ability to harm animals or plants. Generally the capacity of an organism to keep invaders at a distance depends on the strength of innate and acquired resistance against the pathogens. Of the species under constant challenge, plants are one of the confronting species.
Despite the substantial advances in plant-pathogen interaction, recent characterization and cloning of many resistance genes in different plants, the global crop production and food supply to meet the needs of alarmingly increasing population of the world is still threatened by potential plant pathogens and pests which are significantly reducing crop yield and quality (Moffat, 2001; Garelik, 2002). Of many controls, chemical management provides effective protection but the nature has to compromise with its adverse effects such as environmental effects and the emergence of resistant pathogen strains. Moreover such controls are often beyond the means of farmers in many developing nations. Therefore much effort of the scientific community has always been towards understanding the natural resistance mechanisms in plants. The plant breeders have been exploiting this inexpensive, convenient and environmentally sound way to control plant diseases since many years (Agrios, 1988, Crute and Pink, 1996; Pink, 2002).
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This review of literature will cover the host-pathogen interaction, different classes of disease resistance genes, analysis of the genetic architecture of the resistance (R) genes, signalling and transduction pathways supposed to be involved in the activation of the resistance genes, and finally the strategies adopted by plants to generate recognition specificities against the new potential pathogens.
Plant pathogens deploy different strategies to attack plants e.g. necrotrophy, biotrophy or hemibiotrohpy (Keen, 1990; Long and Staskawicz, 1993; Hammond-Kosack and Jones, 1997; Dangl and Jones, 2001). During a necrotrphic invasion, pathogen get nutrition from the plant body after killing the plant cells usually by excreting toxins e.g. Pythium and Botrytis species. Some of necrotrophic pathogens have a broad range of hosts while others have narrow (Walton, 1996; Hammond-Kosack and Jones, 1997) but overall, necrotrophic invaders are less specialized (Agrios, 2005; Wit, 2007). By contrast, biotrophic and hemibiotrophic pathogens develop a friendly and give-and-take (symbiotic) relationship with their host. Biotrophs utilize host nutrients by keeping the host cells alive (Agrios, 1988) whereas hemibiotrophs can kill the surrounding cells in the later stages of infection (Hammond-Kosack and Jones, 1996; 1997). Some biotrophs live in the intercellular space between leaf mesophyl cells, while others feed on host through haustoria development (Voegele and mendgen, 2003; Ellis et al., 2006). Plant pathogens can affect plants in different ways and can cause significant economic losses in a managed ecosystem. They can have strong effects on plant fitness and can regulate the population sizes of the plant species as well (Rausher, 2001).
Conversely, plants being sessile cannot hide or escape themselves when invaded. Therefore, like other organisms, they are continuously under challenge by potential pathogens and are easily accessible to a wide array of pathogens (Zhang and Gassmann, 2003). Generally, most microbes access and infect plant body through direct penetration into the leaves and/or roots or these microbes enter into the plant body through wounds or natural openings e.g. stomata and other gaseous exchange pores. To be pathogenic, pathogens must have to breach many obstacles such as a cellulose-based rigid cell wall and plasma membrane. In the plasma membrane, they have to evade or confront extra-cellular or trasmembrane receptors (Chisholm et al., 2006).
The plants recognize the presence of a pathogen through extra-cellular or trasmembrane pattern recognition receptors (PRRs) which are specific against pathogen-associated molecular patterns (PAMPs) (Underhill and Ozinsky, 2003; Chisholm et al., 2006; Postel and Kimmerling, 2009). These PRs and PAMPs molecules are conserved between the pants and pathogens respectively, and are indispensable for the occurrence of resistance or susceptibility (Nürnberger et al., 2004). The generation of basal protection activated through the recognition of the PAMPs is known as PAMP-triggered immunity (PTI) (Zipfel et al., 2004; Chisholm et al., 2006). The pathogens have developed sophisticated ways to bypass the plant pattern recognition receptors (PPRs) using different strategies (Postel and Kimmerling, 2009). Most commonly, pathogens invade plants either by evading from PRs through the modification or acquisition of the PAMPs which are no longer recognized by plants (Gomez-Gomez et al., 1999; Chisholm et al., 2006) or suppressing the basal immunity through effector proteins (Alfano et al., 2004; Kim et al., 2005; Nomura et al., 2005; Block et al., 2008). To recognize the new pathogens evasions, plants have also evolved different up-to-date methods to combat against the pathogens having these niche PAMPs or effectors (Jones and Dangl, 2006). Such type of immunity developed as a result of effectors recognition is known as effector-triggered immunity (ETI). This type of immunity involves the direct or indirect recognition of the effectors or other proteins used to subvert the effect PAMP-triggered immunity (Postel and Kimmerling, 2009). All the above observations are based on the gene-for-gene hypothesis which was proposed by Flor in 1956 and 1971.
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Although, plants posses no circulatory system, antibodies, and B-or T-lymphocytes to protect themselves against pathogens (Martinon and Tschopp, 2005; Vivier and Malissen, 2005) yet they have developed strong multilayered defense system to cope with pathogens (Dangl and Jones, 2001; Holub, 2001; Jones, 2001). The understanding of evolution and generation of PAMPs and PRs along with the defense-related traits e.g. morphological structures, physiological responses, secondary metabolites, proteins and different enzymes will help researchers to develop durable resistance plant ecotypes and more sustainable pest management strategies (Tiffen and Moeller, 2006). However, the plant-pathogen interactions and plant protection can be divided into two lines.
First line of defense:
Plants have established very robust, sophisticated and effective mechanisms to recognize and respond to a variety of putative pathogens (Dangl and Jones, 2001; Zhen et al., 2006). During the first line of defense, plants attempt to restrict the growth of the pathogen at the pathogen ingress site through locally induced responses (Maleck and Lawton, 1998). These locally induced responses usually include a hypersensitive response (HR) i.e. a programmed cell death by rapid cell collapse in the infected and surrounding vicinity of the infection site (Holub et al., 1994; Morel and Dangl, 1997). The most robust biochemical changes associated with the hypersensitive response is the generation of the reactive species e.g. O2- and H2O2 (Dixon and Lamb, 1990; Dixon et al., 1994; Lamb and Dixon, 1997). The hypersensitive response is further followed by many rapid responses such as cross-linking of the cell wall of the infected tissues by callose deposition and production of antibiotics and antimicrobial enzymes e.g. chitinases and glucanases (Hammond-Kosack and Jones, 1996; Jones and Dangl, 1996; Maleck and Lawton, 1998; Zhen et al., 2006). Additionally, the prevalence of the cuticular wax layers, secondary metabolites, different toxin compounds like phytoalexins also play a significant role in providing the protection to the plants (Maleck and Lawton, 1998; Zeng et al., 2006; Zhen et al., 2006). However, some studies documented that these responses resulted in the reduction of pathogen growth but sometime were unable to provide a complete blockage against different pathogens (Broglie et al., 1991; Maher et al., 1994; Zhu et al., 1994). Therefore, it needs the activation of a broad spectrum and highly effective resistance system (Bent, 1996).
Second life of defense:
In addition to the outcome formed as a result of first of line defense, the production of many other products such as NO, Ca+2 and other ion fluxes, changes in protein phosphorylation and salicyclic acid (Dixon, 1994; Dangl et al., 1996; Hammond-Kosack and Jones, 1996; Métraux, 2001; Ryals et al., 1996; Delledonne et al., 1998; Durner et al., 1998) are thought to be involved in the initiation of signaling for the activation of some broad-spectrum, global and durable defense systems (resistance genes) against a wide array of pathogens (Bent, 1996; Dong, 2001; MacDowell and Woffenden, 2003; Durrant and Dong, 2004). The production and balance of the NO, RO, and SA is believed to be necessary for the complete occurrence of the hypersensitive response, and subsequent activation of the disease resistance genes (Klessing et al., 2000; Delledonne et al., 1998, 2001). Moreover, salicyclic acid (SA) production has been observed during both lines of defense thus it can be inferred that SA may play multiple roles like H2O2 generation (Laj et al., 1996) as well as HR stimulation (Tenhaken and Rubal, 1997).
During the second defense line, the most important response is the activation of different types of resistance (R) genes which provide protection against many pathogens including viruses, fungi, bacteria, oomycete nematodes and insects (Keen, 1990; Crute and Pink, 1996; Bent, 1996; Dangl and Jones, 2001). R gene-mediated resistance received more attraction and attention because it provides complete protection when activated and expressed timely. This system has no adverse environmental effect and provides pathogen specific protection. Moreover, economically it needs no inputs, and is a short period process from breeding point of view (McDowell and Woffenden, 2003).
Disease Resistance (R) Genes:
The disease resistance (R) genes are abundant in many plant species (Michelmore and Meyers, 1998). Many contemporary and previous studies suggest that almost virtually all plants have large number of disease resistance (R) genes to protect themselves from a wide array of pathogens (Dangl and Jones, 2001, Ding et al., 2007; Nemri et al., 2010). Over two hundred genes have been identified from the Arabidopsis thaliana ecotype Columbia, which were found to encode proteins similar to disease resistance proteins (Meyers et al., 2003; Kato et al., 2009). These resistance (R) genes belong to tightly linked multigene families with diversified functions (Hammond-Kosack and Jones, 1996; McDowell et al., 1998; Hulbert et al., 2001). Resistance (R) genes are the segments of the genome encoding proteins that can recognize avirulence (Avr) proteins and activate signaling cascade to circumvent pathogen ingress and allow resistance to occur (Baker et al., 1997; Hammond-Kosack and Jones, 1997; DeYoung and Innes, 2006; Jones and Dangl, 2006; Weaver et al., 2006). Plants with altered resistance genes showed susceptibility to most of pathogens (Kunkel et al., 1993; Bisgrove et al., 1994; Okubara et al., 1994; Innes, 1998; Maleck and Lawton, 1998). Many resistance (R) genes have been isolated, characterized and cloned from different plant species (Bent, 1996; Ellis et al., 2000) such as Arabidopsis thaliana (Jones et al., 1996; McDowell et al., 2000; Bergelson et al., 2001), Lactuca sativa (Meyers et al., 1998) flax (Lawrence et al., 1995; Dodds et al., 2000), tomato (Martin et al., 1993), rice (Wang et al., 1998), tobacco (Whitham et al., 1994) and Solanaceae (Couch et al., 2006).
Classification of resistance (R) genes:
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Since the plant disease resistance proteins are known to posses many domains (Fig. 1) like TIR (Toll/interleukin-1 receptor), CC (Coiled-coil), NBS (nucleotide-binding site), LRR (leucine-rich repeats), LZ (leucine-zipper), TM (transmembrane-domain) and PK (protein-kinase) (Meyers et al., 2002, 2003; Liu et al., 2007), therefore the prevalence of different domains make it very difficult and hard to classify these R proteins into proper groups. Nonetheless, Liu et al. (2007) divided these plant disease resistance (R) proteins into at least four main families which provide protection to the host against a wide array of pathogens (Staskawicz et al., 1995; Baker et al., 1997; Hammond-Kosack and Jones, 1997; Liu et al., 2007). These four main groups are enlisted below;
Nucleotide-binding site plus leucine-rich repeats (NBS-LRRs)
Receptor-like kinase (RLK)
Leucine-rich repeat transmembrance (LRR-TM)
Transmembrance coiled-coil TM-CC
The NBS-LRR gene family represents the largest class (table 1) of resistance genes (Kobe and Deisenhofer, 1993; Traut, 1994; Martin et al., 2003; Belkhadir et al., 2004). The annotation data of the Arabidopsis thaliana ecotype Columbia genome recognized 207 genes which showed characteristics of plant disease resistance proteins (Meyers et al., 2002, 2003). Of 207, 149 displayed homology to the "nucleotide binding site-leucine-rich repeat (NBS-LRR) class of resistance genes" (Meyers et al., 2003). These NBS-LRRs proteins characteristically encode NBS peptide of about 300 amino acids (Traut, 1994) sandwiched between a variable N-terminal TIR/CC-domain of about 200 amino acids and a highly variable C-terminal LRR-domain (10-40 tandemly arrayed short LRR motifs; Jones and Jones, 1997). The NBS-LRR proteins lack a recognizable signal peptide at its amino-terminus (Leister et al., 1996). On the basis of N-terminal domain, the NBS-LRR class of proteins can further be categorized into two or three subclasses (Pan et al., 2000; Steven et al., 2002; Meyers et al., 2005; Liu et al., 2007) as follows;
TIR-NBS-LRR (TNL proteins)
CC-NBS-LRR (CNL proteins)
The TIR-NBS-LRR (TNL) class includes genes that provide resistance to host against virus (Whitham et al., 1994), fungi (Lawrence et al., 1995; Anderson et al., 1997) and oomycetes including genes RPP5, RPP1A, RPP1B, and RPP1C in Arabidopsis (Parker et al., 1997; Botella et al., 1998). Similarly the second subclass "CNL proteins" mainly specify resistance to Pseudomonas syringe pathovars covering genes RPM1 (Grant et al., 1995), prf (Salmeron et al., 1996) and RPS2 (Bent et al., 1994; Mindrinos et al., 1994). The TNL family is nearly two times bigger than the CNL class in Arabidopsis but TNLs are more homogenous in occurrence than CNLs (Meyer et al., 2005). However, the third non-motif subgroup covers all the domains which represent either sequence similarity or functional similarity to the standard NBS-LRR proteins. This subclass of resistance proteins includes genes encoding proteins that are
Fig. 1 Members of resistance proteins (Liu et al., 2007)
The plant resistance proteins classified based on the presence of conserved domains, which contain 14 groups (a, b, câ€¦n) corresponding to different R protein types. Three novel R proteins (Xa13, Xa5 and Xa27) do not contain any conserved motifs of the known in R proteins. (NBS: nucleotide-binding site; LRR: leucine-rich repeat; TIR: Toll-interleukin-1 receptor; CC: coiled-coil; TM: transmembrane domain; PK: Protein Kinase; WRKY: WRKY domain; B-lectin: bulb-type mannose specific binding lectin domain).
Table 1. Arabidopsis NBS-LRR genes classes based on N-terminal proteins domain and genomic organization
a Numbers of genes contained in clusters are given in parentheses. (Richly et al., 2002)
predicted to be transmembrane (Cf gens of tomato; Hammond-Kosack and Jones; 1996) and with extracellular LRR domains (Xa21 genes of rice; Song et al., 1995; McDowell et al., 1998).
Modulation of phosphorylation state is one of the most important and common mechanisms that living organisms usually use to control their protein activities. Therefore, kinases and kinase-like receptors (RLKs) attracted more attention and have been studied extensively (Pawson, 1994). Plant receptor-like kinases (RLKs) belong to a superfamily of transmembrane proteins with variable N-terminal extracellular domains that vary in structure and C-terminal intercellular kinase catalytic domains (Torii, 2000, Shiu and Bleecker, 2001). Sequence and expression data of different expressed proteins show that these RLKs represent the largest gene family in Arabidopsis. Over 600 RLKs have been found in Arabidopsis which constitute nearly 2.5% of Arabidopsis protein encoding genes (Shiu and Bleecker, 2003). These results can be supported by (Dardick et al., 2006) where they found over two times (1,429) more RLKs than in Arabidopsis.
Since the establishment of roles of RLKs in many processes such as development, self incompatibility, and defense against pathogens (Clark et al., 1993; Stein et al., 1996; Torii et al., 1996; Li and Chory, 1997; Gomez-Gomez and Boller, 2000), many plant RLKs including a) tomato Pto encoding a Ser/Thr protein kinase (martin et al.,, 1993), b) Xa21 and Xa26, two Ser/Thr protein kinases (Song et al., 1995; Sun et al., 2004; Chen et al., 2006), and c) Rpg1, a barley RLK protein (Brueggeman et al., 2002), have been identified. However, in plant-microbe systems, these RLKs are of central importance in perception and transmission of external signals from pathogens (Dievart and Clark, 2004; Zhou et al., 1995; Salmero et al., 1996; Zhou et al., 1997; Sasssa et al., 2000; Wang et al., 2006 Doods et al., 2006).
However, the prevalence and role of these three families e.g. receptor-like kinases, leucine-rich repeat transmembrance or transmembrane coiled-coil domains is also significant (Liu et al., 2007). There are some genes which possess no obvious NBS domain in their architecture, thereby fall in a distinct group as non-NBS extracellular and/or transmembrane domains (Fig.1 & Table 1; Jones et al., 1994; Dixon et al., 1996; Meyers et al., 2002). There are many plausible explanations for the occurrence of these proteins. Most of the domains included in this group have interaction with the main LRR and NBS domains in one way or the other, and are encoded together in different combinations between LRR domain, transmembrace domain, and kinases encoded by many R genes (Meyers et al., 2002, 2003).
Structural and functional dissection of R gene motifs:
Sequence analyses accumulated a large number of structural domains of the disease resistance genes (see Fig. 1 & Table 1) some of the import structural domains are listed and briefly discussed below;
Coiled-coil domain (CC)
Necleotide-binding site (NBS)
Leucine-rich repeats (LRR)
The TIR domain is an N-terminal motif of many plant disease (R) resistance proteins which was originally characterized due to having a sequence homology to the cytoplasmic domain of the Drosophila Toll peptide and/or to the mammalian interleukin-1 receptor (Whitham et al., 1994; Lawrence et al., 1995; Meyers et al., 2005). The TIR-domain is comprised of three portions; a central part containing conserved residues (ranging from 135-160 amino acids) and two interfaces involved in mediating TIR domain interaction (Xu et al., 2000; Liu et al., 2007).
Since Toll/interleukin-1 receptors are of central importance in mediating the innate immunity in Drosophila and humans (Ausubel, 2005), therefore the plant TIR domain is believed to be implicated in signaling and activation of the defense responses against a wide array of pathogen proteins including bacterial lipoploysaccharides, microbial and viral effecter proteins, cytokines and growth factors (Xu et al., 2000). Moreover, many additional roles of TIR and TIR-like domains in stimulating the activated oxygen production, oxidative burst and maintenance of salicylic acid have been reported as well (Dangl et al., 1996; Hammond-Kosack, 1996; Ryals et al., 1996). This plant TIR domain is a unique characteristic of disease resistance genes of the dicotyledonous plants, and is lacking in the R genes of monocotyledonous plants (Pan et al., 2000; Meyers et al., 2003).
The Coiled-Coil domain (also called LZ) mainly occurs in the N-terminus of NBS-LRR genes and serves as oligomerization domain for a large number of proteins including structural proteins, motor proteins and transcription factors (Nooren et al., 1999). This Coiled-Coil domain has almost similar and conserved structure from viruses to plants and mammals. Sequence and expression analyses showed that these CC proteins are 5% of the total encoded proteins by a plant genome (Nooren et al., 1999). These CC proteins typically comprised of two or more alpha-helices that wrap around each other with a superhelical twist, and their structures are characterized by the heptad repeat sequences (abcdefg)n, where a & d denote hydrophobic residues, and e & g charged polar residues (Alber, 1992; Bent, 1996; Nooren et al., 1999). Likewise TIR domain, CC/LZ proteins are well known for their role in protein-protein interactions, homo-and hetrodimerization of many eukaryotic transcription factors (Nooren et al., 1999; Burkhard et al., 2001). Interestingly, the CC type is present in both dicotyledonous and monocotyledonous plant (Bai et al., 2002; Meyers et al., 2003; Monosi et al., 2004).
Nucleotide-binding site (NBS):
Most of the disease resistance (R) genes encoding LRR domain, also encode NBS domain, both of which make the largest class of R proteins. The NBS domains of the plant disease (R) genes show high degree of homology with NBS domains of cell death related genes (CED4) from Caenorhabditis elegans and Apaf-1, FLASH, CARD4, and Nod1 from humans (Van der Biezen and Jones, 1998; Arvind, 1999). The NBS domain is a key component of diverse proteins with three ATP or GTP binding motifs known as P-loop or kinase 1a, kinase 2 and kinase 3a. The P-loop motif with the consensus sequence GXXXXGK (T/S) is supposed to be involved in binding with phosphates and Mg21 ions (Saraste et al., 1990). The kinase 2, which require a phospho-transfer reaction, contains four consecutive hydrophobic amino acid followed by a conserved aspartate (D) residue, which coordinates the divalent metal ions on Mg-ATP. The kinase 3a motif is involved in binding purine or ribose and contains a conserved tyrosine (Y) or arginine (R) residue (Traut, 1994). Overall, this NBS domain is believed to be involved in cell growth, cytoskeletal organization, differentiation, apoptosis, vesicle transport, and defense, such as ATP synthase Î² subunits, ras protein, ribosomal elongation factors and adenylate kinase (Saraste et al., 1990; Traut, 1994; Dangl and Jones, 2001).
The NBS domain has central importance in the signaling of plant defense. Mutational, transgenic and functional studies and analyses showed that ATP binding site and P-loop of the NBS domains are of crucial importance in displaying the protection against these pathogens (Tameling et al., 2002). These results were later supported by Ade et al. (2007) during their study of amino acid substitution in P-loop motif of the NBS region of Arabidopsis RPS5 (D266E) and tobacco N (GK221, 222AA) (Mestre and Baulcombe, 2006) protiens, both of the replacements eliminated their activities.
Leucine-rich repeats (LRR):
The LRR domain, firstly recognized in the leucine-rich 2-glycoprotein, is repeats of motifs tandemly arrayed to the C-terminal of the NBS domain in many R proteins, and is considered to be responsible for pathogen protein recognition (Jones and Jones, 1997). Meyers et al. (2003) documented that these LRR domains are connected to NBS domain through an exon (with average size of approximately 300 bp which is conserved in many plant R proteins) in all TNL R proteins but this exon is missing in the CNL R proteins architecture. The LRR motif is usually 20-29 (with average 24; Kobe and Deisenhofer, 1994) amino acids long containing a conserved 11-residue sequence LxxLxLxxN/xxL where x can be any amino acid and L can also substituted by valine, isoleucine and phenylalanine) (Bostjan and Andrey, 2001). Bent et al. (1996) added that these hydrophobic and conserved regions (LxxLxLxxN/xxL) of LRR domains are more likely to be involved in assigning characteristic structure to the LRR proteins rather than in determining the functional specificity.
With the established roles of all LRR-containing proteins and their distinct subfamilies in mediating protein-protein interactions and protein-carbohydrate interaction during many important biological processes (Kobe and Deisenhofer, 1994; Kajava, 1998), such as enzyme-inhibition (RNase and RNase inhibitor), hormone-receptor interactions, cell adhesion and cellular trafficking (Bostjan and Andrey, 2001, Torii, 2004), the LRR domain of plant R proteins are considered as key mediators of plant-pathogen interaction (Kobe and Deisenhofer, 1994). It was proposed that these LRR domains are associated in binding with the ligand produced by pathogen (Jones and Jones, 1997; Dixon et al., 1996), and are the major determinant of resistance specificity (Hulbert et al., 2001). Domain swap and amino acid(s) substitution studies further strengthened the fact that these highly variable but conserved regions (LRRs) of R proteins are indispensable for the occurrence of resistance in the host and compatibility of interaction between plant and pathogen (Wulff et a., 2001; Van der Hoorn et al., 2001; Dodds et al.,, 2001).
Some studies reported that any change in the conserved region of the LRR is very crucial. Jia et al. (2000) were unable to found a resistance response by just substituting a single amino. Conversely, Zhou et al. (2006) refuted this notion. They documented that the 8 amino acids substitutions in the xxLxLxx motif brought no significant change in the resistance specificity (Zhou et al., 2006). More interestingly, Ade et al. (2007) found some results in which they observed that the LRR domain negatively affected activation of resistance genes by interacting with other domains (NBS) of R genes by blocking the signaling cascade.
Signaling Mechanisms of Disease Resistance (R) Genes:
Despite new advancements in the characterization of R genes, exact signaling pathway underlying these genes is poorly known. Preliminary studies showed that Calcium channels, phosphatases and kinases were found to be associated with R genes signal transduction (Scheel et al., 1998). The complete description of the signaling mechanism underlying the resistance genes is big challenge because R gene signaling is highly branched and partially redundant (Innes, 1998). Using genomic and mutational approaches, Innes (1998) observed that most of mutations/insertions in the potential signaling genes suppress more than one R genes, suggesting that most of the R genes share a common signaling pathway. In two different studies, it was also proposed that different R genes are either induced by different signaling pathways or through a common one (Freialdenhoven et al., 1994; Peterhänsel et al., 1997).
There are many preliminary reports that represent the involvement of either NBS or one of the TIR or coiled-coil/leucine-zipper domains in activating signal transduction of resistance proteins (Hommond-Kosack and Jones, 1997). The sequence comparison studies of plant TIR domain with Toll/interleukin-1 receptor of Drosophila and human respectively propose that plant TIR domain transduce the activation of R genes in a similar fashion to Toll/Interleukin-1 receptor of Drosophila or human (Hommond-Kosack and Jones, (1997). Since the redox reaction is the immediate and first response of mammalian innate immunity (Doke and Ohashi, 1988; Hommond-Kosack and Jones, 1996), the generation of reactive oxygen species (ROS) as a consequence of perceiving a signal from pathogen strengthens the above mentioned role for plant TIR domains (Doke and Ohashi, 1988; Hommond-Kosack and Jones, 1996; Meyers et al., 1993).
Careful investigations of the signaling components involved in the initiation of defense responses revealed intriguing results. Mutational studies and screening of the mutants led to the identification of many potential genes (NDR1, EDS1 and PAD4) required for the complete occurrence of resistance to bacteria, fungi, and oomycetes (Parker et al., 1996; Century et al., 1997; Rogers and Ausubl, 1997; Aarts et al., 1998; Zhou et al., 1998; Glazebrook et al., 1999; Nawrath and Métraux, 1999; Feys and Parker, 2000). Parker et al. (1996) and Century et al. (1995, 1997) identified and isolated EDS1 and NDR1 respectively as indispensable components of the Arabidopsis signaling pathways during their mutant screening. Evidences accumulated showed that a mutation in the NDR1 gene abolished resistance to RPS2 and RPS5 (Pseudomonas syringe) along with RPM1 gene-mediated (Perenospora parasitica) resistance but not RPS4 (Pseudomonas syringe) (Century et al., 1997). Conversely, eds1mutation caused susceptibility to RPP gene-mediated (Perenospora parasitica) resistant plants along with RPS4 (Pseudomonas syringe) whereas this mutation showed no effect on RPS2, RPS5 and RPM1specifed resistance. From these results it was inferred that NDR1 may encode a component needed for signal transduction and may interact either directly with many specific receptors (formed as a result of resistance gene products) to transduce a signal or serve as transporter/receptor for an elicitor signal (Century et al., 1997).
Similar results (see Table 2) were observed by Aarts et al. (1998) which indicate that the R genes strongly dependent on EDS1, showed very weak or no dependence on NDR1. Upon careful examination, it became obvious that the choice of activation of either of abovementioned signaling pathways (EDS1 or NDR1) depends upon the presence or absence of TIR and CC/LZ domains to the N-terminus of NBS-LRR R proteins (Rustérucci et al., 2001). They further added that the choice of activation of signaling pathways is independent of the pathogen type. Glazebrook et al. (1996) identified a third potential component PAD4, believed to be involved in signal transduction of disease resistance responses. Likewise EDS1, PAD4 was found to encode a lipase-like protein (Falk et al., 1999; Jirage et al., 1999) and was supposed to be implicated in salicyclic acid (SA) signaling pathway (Rustérucci et al., 2001).
Later on, Feys et al. (2001) studied and developed a direct relationship between EDS1 and PAD4 (see fig. 2) using mutational approaches. They demonstrated that both EDS1 and PAD4 are needed for the defense response to occur and these genes are the main regulators of SA abundance. It was observed that the mutant plants (eds1-2) displayed no HR response allowing a rapid pathogen ingress and mycelium to establish whereas mutants with pad4-2 strikingly produced HR response though this response can't stop the infection to develop. From their mutational analyses and results, they established facts that EDS1 is present upstream of the PAD4 in the genomic organization which either triggers early plant signals or recruits PAD4 to tranduce plant defense responses through the accumulation of the SA. Nonetheless, all these signaling components require further investigations.
F3 families, n
Col-gl 3 Ler-eds1-2
Col-gl 3 x Ws-eds1-1
Col-ndr1-1 3 x Ler
Col-ndr1-1 3 x Ler
Phenotypes of different RPP loci in combination with wild-type or mutant eds1or ndr1after inoculation of selected F3 families with P. parasitica
Figure 2. A proposed model for the roles of EDS1 and PAD4 in R gene mediated resistance (Feys et al., 2001).
Evolution of R genes:
The key and paramount components of evolutionary and developmental forces have always been the ability of plant defense system to detect and respond to potential pathogens (Kato et al., 2009). According to fossil records, the establishment of the first land plants occurred approximately 480 million years ago. However, molecular-clock estimates suggest that land plants evolved more than 700 million years ago (Heckman et al., 2001). As it is believed that the early land plants were established by the interaction with symbiotic fungal associations, it can be suggested that plants have coevolved with microbes since their first appearance on land (Gehrig et al., 1996). Furthermore, it can also be speculated that the evolution of land plants has been shaped by molecular interactions with epiphytic, symbiotic, and pathogenic microbes (Chisholm et al., 2006).
What were the ancestors of R genes and how they evolve is still obscure but there are some plausible explanations to these questions. Firstly, as it is evident that mammalian, yeast, insects and different homologues of these plant disease (R) genes from other species, have been found implicated in the endogenous signaling, development and/or cell adhesion, so it could be suggested that these proteins might be previously involved in the recognition or signaling of the plant growth and development (Hammond-Kosack and Jones, 1997). Their potential roles in plant growth and development can be further supported from the role of two Xa21proteins (similar to the extracellular LRR R protein) encoded by the Arabidopsis erecta and clavata genes determine floral organ shape and size (Torii et al., 1996) and are believed to be involved in communication between the adjacent cells (Hammond-Kosack and Jones, 1997). Secondly but less likely could be possible that the R genes of multi-cellular organisms might have evolved from progenitor R genes associated in pathogen recognition by their unicellular ancestors. This notion is further strengthened by the significant structural homology between the NBS/LRR class of R proteins and the human major histocompatibility complex (MHC) class II transcription activator (CIITA), and between the extracytoplasmic LRR class of R proteins and the mouse RP105 protein involved in B cell proliferation and protection against programmed cell death (Jones and Jones, 1996), which suggest that plant R genes and genes involved in mammalian immunity may have a common evolutionary origin (Jones and Jones, 1996; Hammond-Kosack and Jones, 1997).
Lastly, it has been observed that taxonomically distinct plant species provide recognition and protection against the same bacterial Avr genes (Whalen et al., 1988; Kobayashi et al., 1989). From this evidence, it can be inferred that either there has been preservation of an ancient specificity or this same recognitional specificity have evolved in the relevant pathogen in multisteps over a period of time (Hammond-Kosack and Jones, 1997). Currently from the availability of studies and systems for the isolation and characterization of additional plant R genes and their related gene families, it may be possible to determine which of these evolutionary scenarios is more likely.
Evolution of R gene specificity:
To enjoy a life at its best, organisms must be equipped with the best means of defense against pathogens like bacteria, fungi, and viruses otherwise these pathogens would replicate out of control inside those organisms which consequently lead to malfunctioning of many systems or even to death. Therefore organisms employ different types of defense to stop this happening. The opening of twenty-first century era with latest advances in genomics, proteomics, population genetics, evolutionary biology and bioinformatic tools has changed our biological views and developed new links between these sciences, ultimately increased our understanding about the interaction of host and pathogen and evolution of new mechanisms for their survival. Many evolutionary biologist have studied natural variation and its impact on the adaptation of the plants (Maloof, 2003; Ungerer et al., 2003; de Meaux et al., 2005; Juenger et al., 2005; Shindo et al., 2005; Weigel and Nordborg, 2005; Balasubramanian et al., 2006; Briggs et al., 2006; DeCook et al., 2006; Mitchell-Olds and Schmitt, 2006; Shindo et al., 2007). These evolutionary studies are of great significance for gaining better understanding of environmental adaptation and evolution of new plants.
According to gene-for-gene hypothesis (Flor, 1947, 1971), for each Avirulence gene in the pathogen, there is a virulence gene in the host (Plant or human). For an infection to be established, Avirulence ligand should interact with its receptor in the host and circumvent the activation of host defense response and system (Keen, 1990; De Wit, 1992). For this purpose, an arm race between the pathogen and the host to evolve novel Avirulence lignads and host systems to detect/recognize and response against these novel changes respectively has been observed by many researchers (Day, 1974; Keen, 1990; Ellis, 1993; Alfano and Collmer, 1996; Crute and Pink, 1996; Knogge, 1996; Hammond-Kosack and Jones, 1997; Dangl and Jones; 2001; de Wit et al., 2002). Since, evolutionary processes are the key determinant of the structure of genetic polymorphism (Kato et al., 2009) so it has been proposed that such forces are the basis of evolution of new varieties and populations within the same species (Luck et al., 2000; Dangl and Jones, 2001).
After the characterization of the genes responsible for resistance against many potential pathogens, determination/demonstration of the domains of R proteins residing resistance specificity was the next challenge which is still obscure. Because LRR domains of the resistance proteins are highly polymorphic in sequence (Botella et al., 1998; McDowell et al., 1998; Meyers et al., 1998; Ellis et al., 1999) so it was suggested that these are LRR domains that confer specificity against pathogen specificity (Jones and Jones, 1997). The presence of cytoplasmic and transmembrane LRR domains further supports their role in the determination of disease resistance specificities (Dangl and Jones, 2001). Some researchers suggest that in LRRs, the variable Î²-strand-Î²-turn motif of the LRR domains is more likely to be involved in the Avr-vr ligand recognition and interaction (Ellis et al., 1997, 2000).
Moreover, on the basis of mutations accumulated in the LRR region, it can be supposed that 1) these variable regions (LRR) are not involved in the generation of resistance gene specificity and therefore are accumulating mutations (neutral selection) or 2) they might be strictly engaged in determining R genes specificity. Later studies covering the rates of synonymous and non-synonymous amino acids substitutions in the LRR domains showed that these substitution are deleterious for gene specificity rejecting the 1st possibility (neutral selection; discussed above). They further documented that variation rate in the LRR domain (especially in the sequence coding for xxLxLxx) is higher than non-LRR regions, and this variation in the LRR region seems more likely to be responsible for the generation of specificities against pathogen ligands (Parniske et al., 1997; Botella et al., 1998; McDowell et al., 1998; Meyers et al., 1998; Noel et al., 1999; Wang et al., 1998). Sequence comparison (Ellis et al., 1995; Lawrence et al., 1995) and loss of function (addition or deletion) of LRR repeated units (Staskawicz et al., 1995) further supported the involvement of LRR domains in determining the resistance gene specificity. These results were refuted Hammond-Kosack and Jones (1997) where they found no change in the specificity after deleting 6 LRR units.
There are many proposed mechanisms through which plant has evolved different modifications in their genomes. The shuffling of DNA sequences has proven a powerful process for the generation of novelty in the resistance gene specificity. The most likely way of generating variation is believed to be governed by a combination of point mutations, duplication events following by recombination, deletion and insertion (Jones and Jones, 1997; Parker et al., 1997; Crameri et al., 1998; Ellis et al., 2000; Blanc et al. 2000; Meyers et al., 2003; Leister 2004; Sampedro et al. 2005; Kong et al. 2007Kato et al., 2009) and transposable elements (especially retrotransposon; Zhang and Wessler 2004; Bennetzen et al. 2005; Wang et al. 2006).
To gain a better understanding of how gene specificities evolve, detailed dissection and deep insights of disease resistance gene architecture and their existence in the genome would be of significant importance. Since it is evident that a trait/character can be the product of single gene or many genes (Mendelism), and these genes could be present in a tightly linked, unlinked or in both forms which could be on a single locus or many loci. In 1997, Hammond-Kosack and Jones proposed that there are many possible ways of existing genes and loci carrying these genes. Firstly, a single gene with distinct alleles each providing different recognition can exist on a single locus (e.g. flax L locus; Pryor, 1993). Secondly, it is also possible that a single locus may contain a single copy of R gene which is present only in the resistant plant but absent in susceptive one (e.g. RPM1 gene; Pryor, 1993). According to third possibility, a locus may comprised of closely linked tandemly arrayed gene homologues, each providing different specificities (e.g. flax complex M locus; Martin et al., 1993; Jones et al., 1994, 1995; Song et al., 1995; Dixon et al., 1996). Last but not the least possible way of containing disease resistance genes represents the loosely clustered formation of the R genes (1-2cm apart; Michelmore, 1995; Dixon et al., 1996; Kunkel, 1996; Holub, 1997).
Moreover, the genetic analysis of plant disease resistance genes showed that R genes at each locus are often highly polymorphic for resistance specificities (Botella et al., 1998; Hammond-Kosack and Jones, 1997; Ellis et al., 2000). Most of the R genes in complex loci are pseudogenes (Song et al., 1997; Bevan et al., 1998). Generally, it is assumed that at simple resistance loci where different R genes having specific borders and recombine normally, their number remain same or their chances to expand are limited. In this scenario, unequal intragenic recombination during cell cycle replication may result in the resistance gene diversity (Parniske et al., 1997). Expansion and clustering of disease resistance genes at complex loci suggest duplication as an initial force followed by unequal crossing over events i.e. deletion, insertion, inversion and translocation for the evolution of specificities (Herbers et al., 1992, 1996; Richter et al., 1995; Yang and Gabriel, 1995; Pryor, 1993; Sudapak et al., 1993; Ellis et al., 1995; Holub et al., 1997; Hulbert et al., 1997; Thomas et al., 1997; Young, 2000). Additionally, some researchers have suggested that alternative splicing of R genes could also be one of the reasons for the generation of new specificity (Whitham et al., 1994; Lawrence et al., 1995).
In addition to evolution of novel specificities, the final adaptability of these novel systems depends upon selection systems. Many researchers have established the fact that positive and diversifying selection has been the main force in shaping the evolution of plant systems and modifying changes in their genomes (Tiffer and Mullar, 2006) while others proposed the balancing or frequency-dependant selection for maintaining the diversity among resistance gene specificities (Tian et al., 2003; Caicedo and Schaal, 2004; Rose et al., 2004; Allen et al., 2004). Meyers et al. (2005) proposed that although balancing selection seems more likely the case for maintaining the polymorphism across disease resistance (R) genes but this arena needs further investigations and evidences.