In plant–parasite systems, pathogenicity has been most often related and analysed in GFG interactions, first described in the flax – flax rust (Me. lini) system (Flor, 1955). In GFG interactions, plant resistance proteins (R proteins) recognize corresponding proteins of the pathogen, named avirulence (Avr) factors, either through direct R–Avr protein–protein interaction or indirectly through detection of changes in the host targets of Avr proteins, the so-called guardee proteins in the guard model (Jones and Dangl, 2006; McDowell and Simon, 2006). The recognition of the Avr factor by the host triggers defence responses leading to limitation of the spread of the pathogen from the infection site, often associated with localized host cell death or hypersensitive response (HR). In the absence of the Avr allele in the parasite or if the host has not have the resistance R allele, the parasite is not recognized by the host, resistance is not triggered and the host is infected. Accordingly, a key feature of the GFG model is that universal pathogenicity occurs, i.e. there are parasite genotypes able to infect all host genotypes (Agrawal and Lively, 2002).
Variability of avirulence genes
Avirulence factors were first identified in viruses, following the development of reverse genetic approaches for RNA viruses in the early 1980s. The first Avr factor identified in a plant pathogen was the capsid protein (CP) of Tobacco mosaic virus (TMV): reverse genetics experiments showed that the CP determined the elicitation of the HR defence response triggered by the N′ resistance gene in Nicotiana spp. (Knorr and Dawson, 1988; Saito et al., 1987). Since then, virus-encoded proteins with each possible role in the virus life cycle have been shown to act as Avr factors (Maule et al., 2007). For instance, within the genus Tobamovirus, the helicase domain of the RNA-dependent polymerase (RdRp) of TMV is the Avr factor for the N gene in Nicotiana, and the movement protein of Tomato mosaic virus is the elicitor of the Tm2- and Tm22-encoded HR reaction of tomato, in addition to the above-mentioned TMV CP and N′ gene (Meshi et al., 1989; Padgett et al., 1997; Weber and Pfitzner, 1998). Within the genus Potyvirus, the NIa protease of PVY is the elicitor of Ry in potato (Mestre et al., 2003), the P3 protein of Soybean mosaic virus elicits Rsv1 in soybean (Hajimorad et al., 2005), or the cylindrical inclusion helicase of TuMV elicits TuRB01 of Brassica (Jenner et al., 2000). All these proteins interact with host factors and are required for completion of the virus life cycle within the infected host and, thus, can be considered as pathogenicity effectors.
Much progress has been made also in studies of the molecular genetics of avirulence in bacterial pathosystems, particularly in interactions with Arabidopsis (McDowell and Simon, 2006). Several bacterial Avr proteins, delivered into the plant cell using type III secretion systems, have been shown to be pathogenicity effectors, for instance by having enzymatic activity and modifying host proteins, and mutations in Avr genes impaired infectivity or multiplication in susceptible hosts (Grant et al., 2006). By contrast, few fungal Avr genes have been cloned thus far, most of them encoding novel proteins (Fudal et al., 2007) with no obvious function. In a few cases the hypothesis of a function as effectors is strongly supported, for example: the barley powdery mildew AVRk1 and AVRa10 genes, which directly contribute to infection success (Ridout et al., 2006); Avr2 and Avr4 from Fulvia fulva (anamorph: Cladosporium fulvum), which protect the fungus from the action of the defence mechanisms of the plant (Rooney et al., 2005; van den Burg et al., 2006); and SIX1 of Fusarium oxysporum, deletion of which leads to a reduced virulence on susceptible lines (Rep et al., 2005). In addition, a pathogenicity effector role may be assumed based on evidence for evolutionary conservation of Avr genes in fungi and oomycete species (Skamnioti and Ridout, 2005). The genomics of filamentous fungi has made great advances in recent years (Weld et al., 2006), and the genomes of several phytopathogenic fungi have been sequenced or will be so imminently (Xu et al., 2006). This will facilitate functional studies of Avr proteins and the identification of additional Avr genes based on similarity with known avirulence effectors (Tyler et al., 2006).
In GFG interactions, host–pathogen co-evolution will lead to pathogens altering their Avr factors to avoid R-dependant recognition as well as the host evolving new specificities in their R proteins to identify the corresponding Avr factors. There is ample evidence for allelic polymorphisms at R and Avr loci in plants and pathogens, respectively (e.g. Parker and Gilbert, 2004; Thrall et al., 2001). It has been argued that the mechanism of recognition of Avr by R will determine Avr evolution. Thus, direct recognition of Avr by R can lead to relatively rapid evolution of new virulence phenotypes by alteration of the Avr structure without affecting its virulence role (Van der Hoorn et al., 2002). A direct physical interaction between Avr and R proteins has been shown for the AvrPita–Pi-ta pair in the rice blast fungus, Magnaporthe grisea, and rice (Jia et al., 2000). According to predictions, the AvrPita proteins from natural isolates of Ma. grisea virulent on Pi-ta plants differ from one another by several mutations (Orbach et al., 2000). In the flax—flax rust system, in which a direct Avr–R interaction has also been shown, diversifying selection has led to extreme levels of polymorphism at the AvrL567 locus in different rust strains, leading to qualitative differences in recognition specificity by the corresponding R genes (Dodds et al., 2006). Diversifying selection and high levels of polymorphism were also reported for the Atr13 and Atr1 loci of Hy. parasitica and the corresponding RPP13 and RPP1 resistance loci of Arabidopsis, respectively (Allen et al., 2004; Rose et al., 2004). Differential recognition of Atr1 alleles by RPP1 alleles has been shown (Rehmany et al., 2005), and it has been suggested that the encoded proteins might interact directly (Jones and Dangl, 2006). Many analysed Avr–R systems seem to conform to the guard model of indirect recognition, where the R protein recognizes changes in the virulence target after interaction with Avr. While direct recognition would lead to relatively rapid evolution of new virulence phenotypes, it has been argued that indirect recognition can lead to balancing selection in Avr and R. If guardee proteins are virulence targets for the pathogens, and the guard protein (i.e. the R protein) recognizes changes in the guardee due to interaction with Avr, resistance could not be circumvented by mutations in Avr without affecting its virulence functions (Van der Hoorn et al., 2002). Hence, purifying selection is predicted to act on Avr. Alternatively, the pathogen could overcome host detection by discarding the Avr gene, if its function can be provided by other genes of the pathogen. This situation will result in the presence of ancient polymorphisms in R genes, and is well exemplified by the Arabidopsis–Ps. syringae system (Mauricio et al., 2003; Stahl et al., 1999). According to the predictions above, evidence of purifying selection has been reported for several families of type III effectors of this bacterium, although there is also evidence for diversifying selection in domains of some gene families (Rohmer et al., 2004). Examples of fungal systems that conform to a model of indirect interaction are the R/Avr gene pairs Cf-2/Avr2 and Cf-9/Avr9 of tomato and Fu. fulva (Rivas and Thomas, 2005). Avr-2 is a cystein protease inhibitor, inhibiting the tomato cystein protease Rcr3, which is guarded by Cf-2 (Rooney et al., 2005). According to predictions, Fu. fulva races virulent on Cf-9 have large deletions in Avr9 or express truncated proteins, and races virulent on Cf-2 arise due to single insertion–deletions that generate truncated proteins (Rivas and Thomas, 2005). Thus, available data on the evolution of Avr genes in cellular pathogens agree with predictions according to the recognition mechanism of Avr by R.
In plant–virus interactions, data do not support differences in Avr evolution linked to the mode of Avr–R recognition. In the tobacco–TMV system, the p50 helicase domain of the RdRp is necessary for N oligomerization and activity (Mestre and Baulcombe, 2006) and p50 directly interacts with the TIR domain of N (Burch-Smith et al., 2007). However, no diversifying selection in p50 has been described. Rather, evidence supports strong negative selection on p50, as avr on N is extremely rare, occurring only in Obuda mosaic virus, a tobamovirus species with a restricted geographical distribution (García-Arenal and McDonald, 2003). Rx resistance of potato to Potato virus X (PVX) is elicited by the virus CP, and requires the interaction of Rx with a Ran GTPase activating protein, although this interaction does not fit the guard hypothesis (Tameling and Baulcombe, 2007). Virulence on Rx is conferred by mutations at two positions in PVX CP, but mutants leading to resistance breaking were shown to be selected against (Goulden et al., 1993). In nature only one strain of PVX, PVX-HB, has been described to be pathogenic on Rx with, again, a limited geographical distribution (see García-Arenal and McDonald, 2003). In addition, engineered mutations on Rx change its recognition pattern expanding it to new PVX strains and new viruses (Farnham and Baulcombe, 2006). Therefore, data suggest that diversification on PVX CP and Rx can be constrained by fitness penalties. Elicitation of HRT-resistance in Arabidopsis by the CP of Turnip crinkle virus (TCV) requires interaction with a NAC protein, according to the guard model (Ren et al., 2000). Virulence on HRT has not been found in TCV, although this has not been explored extensively. Regardless, at odds with the N and Rx resistances, which have been extensively used in tobacco and potato cultivars for decades, resistance to TCV is not frequent in Arabidopsis accessions (Dempsey et al., 1997), and it is not known if TCV is an important pathogen exerting a selection pressure on Arabidopsis wild populations.
Thus, the hypothesis that the mechanisms of Avr recognition by the host plant determine Avr evolution (Van der Hoorn et al., 2002) is supported by evidence from some host–cellular parasite systems, but not from virus–host systems. More evidence both from cellular and viral parasites is needed to test its general validity.
The processes resulting in evolution of Avr genes also differ between viruses and cellular parasites. Changes in recognition of viral Avr by R proteins depend of one or a few amino acid substitutions (Harrison, 2002; Maule et al., 2007). Available data concern RNA viruses, which have spontaneous mutation rates several orders of magnitude higher than DNA-based microbes (Drake and Holland, 1999; Malpica et al., 2002), so that a rapid generation of mutants should be expected. Hence, selection of avr in plant RNA viruses seems to be countered by intrinsic or extrinsic factors. In contrast, for fungi and oomycetes there is evidence that Avr genes are selected for high mutability and vary according to multiple mechanisms. Reported mechanisms resulting in conversion of Avr to avr include point substitutions, insertions and deletions, as in Avr-Pita of Ma. grisea (Orbach et al., 2000); mutations leading to truncation of the encoded protein, as with the frame shift mutations and point mutations resulting in premature stop codons described in Avr2, AVRk1 and AVRa10 of Fu. fulva and Bl. graminis f. sp. hordei (Luderer et al., 2002; Ridout et al., 2006); or deletions of large fractions of the Avr gene, as reported for NIP1 of Rh. secalis (Schúrch et al., 2004). Alleles of this gene and also of Atr13, Avr3a and AvrL56 7 from Hy. parasitica, Ph. infestans and Me. Lini, respectively, are subject to diversifying selection (Allen et al., 2004; Armstrong et al., 2005; Dodds et al., 2006). The genomic context can influence the high potential of virulence/avirulence genes to mutate. Thus, in Ma. grisea, Avr-Pita is located close to a telomere (Orbach et al., 2000). Transposable elements can also have a role in Avr gene expansion and diversification, by disrupting the expression of Arv genes or by hitchhiking the sequences nearby when they multiply and proliferate in the genomes. For example, the barley powdery mildew AVRk1 and AVRa10 genes pertain to a gene family with more than 30 homologues in the fungal genome that is closely associated with sequences homologous to the retrotransposon CgT1 (Ridout et al., 2006), and isolates with point mutations that cause a frame shift and fusion with a CgT1 sequence in both Avr genes are virulent. In addition, in Ma. grisea, insertion of the Pot3 transposon into the promoter of Avr-Pita, or of the retrotransposon MINE in the avirulence gene ACE1 resulted in gain of virulence (Fudal et al., 2005; Kang et al., 2001). The pathogenicity islands described in prokaryotes are groups of clustered genes that can undergo rapid, radical changes, frequently flanked by transposable elements, which may contribute to their proliferation in the genome (Kim and Alfano, 2002). Similarly, two miniature impala transposable elements flank the SIX1 gene from Fu. oxysporum, and can be considered as characteristic of such pathogenicity islands (Rep et al., 2005). Genetic or molecular evidence of clusters of Avr genes that may be interspersed with various transposable elements have been described in Phytophthora (Jiang et al., 2005, 2006), Bl. graminis f. sp. hordei (Brown and Jessop, 1995; Jensen et al., 1995; Ridout et al., 2006) and Leptosphaeria maculans (Balesdent et al., 2002; Fudal et al., 2007).
Therefore, the evolvability of Avr genes seems to be different for viruses and mycelial parasites. This difference could be related to different costs of pathogenicity in the two parasite groups. The cost of pathogenicity, which is treated in the next section, is a major and most debated question in host–parasite co-evolution.
Costs of pathogenicity
Much theory on host–parasite co-evolution is based on GFG systems in plants. The classical model of GFG co-evolution was proposed by Leonard in 1977, and has set the ground for later ones (Agrawal and Lively, 2002; Frank, 1994, 2000; Leonard and Czochor, 1980; Parker, 1994, 1996; Pietravalle et al., 2006; Thrall and Burdon, 2002). Leonard's model is based on indirect frequency-dependent selection, in which host allele frequencies determine those of the pathogens and vice versa. Under this model, non-trivial equilibria in the frequency of R and avr (i.e. equilibria in which gene frequencies are different from 0 or 1) require that both resistance and pathogenicity have a fitness cost for the host and the pathogen, respectively. Tellier and Brown (2007) have shown that the equilibrium predicted under this model is unstable, and requires that the cost of pathogenicity (commonly referred to as the cost of virulence) for the parasite is much smaller than the cost of not infecting a resistant plant, assumed to be about 1 in a GFG system. A condition for stable equilibriium is that, in addition to indirect frequency-dependent selection, negative direct frequency-dependent selection on resistance and avirulence occurs, i.e. that the effect of an allele for resistance or virulence on fitness decreases as its frequency in the population increases (Tellier and Brown, 2007). Different sets of factors can result in direct frequency-dependent selection. The solution of the various models proposed by Tellier and Brown (2007) under different scenarios requires fitness costs of both resistance and pathogenicity and, importantly, that these costs are small (less than 10%). Because of their importance on host–pathogen co-evolution, and on pathogenicity management, much effort has been devoted to the analysis of the costs of resistance in hosts and of pathogenicity in parasites, with conflicting results. We will not deal here with the costs of resistance, and direct the reader to reviews of this subject (Bergelson and Purrington, 1996; Bergelson et al., 2001; Brown, 2003; Mauricio, 1998), but will limit our discussion to the costs of pathogenicity.
An approach to evaluate fitness costs of pathogenicity has been to analyse the dynamics of avr genes in field populations of parasites. Assuming the cost of pathogenicity, there will be a decline in avr frequency in the absence of the corresponding R gene, and thus unnecessary pathogenicity will be selected against. Data from studies with fungi provide conflicting results (Leach et al., 2001; McDonald et al., 1989). For instance, selection against avrXa7 in Ma. grisea in the absence of the corresponding R gene suggests a cost for this pathogenicity factor (Vera Cruz et al., 2000). In addition, selection against unnecessary avr on Sr6 resistance in Puccinia graminis f. sp. tritici in Australia, or against unnecessary avr on Mla6 resistance of Bl. graminis f. sp. hordei in the UK, allowed Grant and Archer (1983) to estimate selection coefficients of 4–6% for both systems. By contrast, unnecessary avr genes are present in populations of Bl. graminis fsp. hordei and tritici, and avr did not segregate with fitness traits (Bronson and Ellingboe, 1986; Brown and Wolfe, 1990). Also, unnecessary avr on R5, R9 and R10 resistance of lettuce increased in Swedish populations of Bremia lactucae although these genes were not used in lettuce cultivars in Sweden, suggesting no cost of pathogenicity (McDonald et al., 1989). We are not aware of analyses of the frequency of unnecessary avr in virus populations. However, there is ample evidence from several different systems that genotypes pathogenic on a particular R gene do not become prevalent in the virus population even with extensive use of cultivars with that R gene, so that resistance has been effectively durable (García-Arenal and McDonald, 2003). In fact, resistance to viruses is durable more often than not, in spite of pathogenic genotypes being reported in the field or in the laboratory (García-Arenal and McDonald, 2003). This is in clear contrast to the short life of most resistance factors deployed against fungi or bacteria (McDonald and Linde, 2002), suggesting higher costs of pathogenicity for viruses than for cellular plant pathogens. Data from field populations, however, should be viewed with caution, as selection against unnecessary avr genes could be countered by a number of factors. Most studies from natural populations do not check for conditions necessary for selection being the major determinant of avr frequency. For instance, selection on avr could be countered by random genetic drift due to small effective population sizes associated with population bottlenecks during the parasite life cycle, spatial or host-associated structure of its population, etc. In addition, linkage disequilibrium may be important, and unnecessary avr genes could be maintained in the population by hitchiking with alleles at other loci that determine higher parasite fitness. Linkage disequilibrium could be particularly important on plant parasitic fungi or viruses, in which sexual processes may be limited.
Another approach to analyse pathogenicity costs has been experimental. Evidence for a possible cost of pathogenicity came from mutagenic experiments with rusts of flax (Me. lini) and wheat (Pu. graminis f. sp. tritici) in which a correlation between the disruption of pathogenicity by mutations and a decrease in fitness was found (Flor, 1958; Luig, 1979). However, the effects on fitness due to second-site mutations could not be discarded. Experimental evidence for the costs of avr in viruses is more abundant. Different mutations have been described in the CP of TMV that disrupt the elicitation of N′ (Culver et al., 1994). Nevertheless, these mutations cause incorrect folding of the CP and, hence, are expected to have a fitness penalty. Also, all experimental mutants in the protease domain of the NIa protein of Potato virus Y (PVY), the Avr factor for Ry-resistance, resulted in virulence. However, no field isolate of PVY has been described to overcome Ry as elicitation of Ry seems to require a functional protease domain, which is also necessary for viability of the virus (Mestre et al., 2003). Specific experiments to estimate avr costs have been reported for genotypes of Raspberry ringspot virus overcoming Irr resistance in raspberry, which have a decreased transmission both by nematodes and through the seed in alternative hosts (Hanada and Harrison, 1977; Murant el al., 1968). For Pepper mild mottle virus genotypes overcoming L3 resistance in pepper, competition experiments with Avr genotypes on susceptible pepper allowed an estimate of the fitness of avr genotypes relative to Avr of about 0.6 (A. Fraile et al., unpublished data). Similarly, M. Molina et al. (personal communication) found a high penalty for avr on L3, but the analyses of chimeras between Avr and avr genotypes showed that the penalty was only in part determined by mutations in the CP (the Avr factor for L3; Berzal-Herranz et al., 1995) and that other genomic regions also determined the fitness of avr genotypes. For TuMV, genotypes overcoming TuRB01 resistance in rape were out-competed by Avr ones in susceptible hosts. Importantly, assays included engineered avr mutants with no second-site mutations, and thus provide unequivocal evidence for a cost of pathogenicity due to a pleiotropic effect of the avr mutation (Jenner et al., 2002b). Similarly, Jenner et al. (2002a) reported fitness costs and a high rate of reversion for mutations in TuMV resulting in avr on a second resistance gene, TuRB04. From the data in Jenner et al. (2002a,b) the fitness of avr mutants relative to Avr ones, on both genes, can be estimated to be about 0.50. It is worth noticing that these pathogenicity costs are much higher than assumed in theoretical models of GFG co-evolution (Tellier and Brown, 2007), and, hence, may violate model assumptions.
Therefore, the scenario is that pathogenicity costs may be small, if any, for mycelial pathogens, where no unequivocal evidence of costs has been reported, while costs may be quite high for viruses. This important difference between both groups of parasites could be explained by a variety of factors. It has been hypothesized that effective population sizes are smaller for plant viruses than for plant parasitic fungi (Harrison, 1981). Indeed, small effective sizes, several orders of magnitude below the census, have been estimated for population bottlenecks during viral colonization of the host plant or during horizontal transmission (Ali et al., 2006; French and Stenger, 2003; Sacristán et al., 2003). Estimates of effective population sizes for fungi are rare, but indicate no such gross difference with census sizes (Leslie and Klein, 1996; Zhan et al., 2001).
A major determinant for the difference in pathogenicity costs between fungi and viruses could be the nature of their genome. In the genomes of RNA viruses there are few neutral sites, and most mutations, including nucleotide substitutions, are deleterious (Sanjuán et al., 2004a). The small genomes of RNA viruses are tightly packed with information: there is overlapping of coding and regulatory sequences and of different coding sequences, and the few encoded proteins perform different functions in the virus life cycle, imposing different selection pressures on the corresponding genes (García-Arenal et al., 2001). In addition, epistatic interactions occur among and within genes (Escriu et al., 2007; García-Arenal et al., 2001; Martin et al., 2005; Sanjuán et al., 2004b), which limit the variability of viral proteins. Thus, the plasticity of plant virus genomes could be low, in spite of high mutation and recombination rates (Drake and Holland, 1999; Froissart et al., 2005; Malpica et al., 2002), and mutation to pathogenicity would have high costs. This would not be case for fungi, in which genome complexity allows for high levels of redundancy, alternative metabolic pathways and multiple regulatory elements that could reduce the effects of mutational perturbations. Accordingly, the fitness effects of mutations are much smaller in eukaryotes, including fungi, than in RNA viruses (Sanjuán and Elena, 2006). As mentioned above, multigene families for Avr factors have been described, which would result in few penalties for mutation in an individual gene or even for its whole loss. In agreement, frameshift mutations and deletions are common mechanisms for variation of fungal Avr genes, as pointed out above. Hence, differences in genome size, structure and plasticity may determine differences in the evolution of pathogenicity of viruses and cellular pathogens.