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Necrotrophic fungal pathogens secrete a variety of necrotrophic effectors (NEs) (syn: host-specific/selective toxins) that interact in a gene-for-gene manner with host susceptibility genes (Oliver & Solomon, 2010). Typical plant defense responses involve the induction of pathogenicity-related genes, which leads to the production of antimicrobial compounds, the accumulation of reactive oxygen species and localized cell death. This defense response is known as the hypersensitive response (HR) ( Jones & Dangl, 2006). A growing body of evidence supports the hypothesis that necrotrophic pathogens have taken advantage of the HR, using small secreted proteins or secondary metabolites to activate HR preceding fungal growth (Effertz et al., 2002; Liu et al., 2009, 2012; Lorang et al., 2012). This group of molecules is collectively referred to as NEs.
Effectors are a class of pathogen proteins or metabolites whose function is to alter or suppress the host's normal immune response. Three NEs have been described for the fungal wheat pathogen Phaeosphaeria nodorum (Friesen et al., 2006; Liu et al., 2009, 2012). Each of these NEs is a small, secreted protein that displays a presence/absence polymorphism in natural field populations. SnTox1 is a cysteine-rich protein that was shown to exhibit significant diversifying selection (Liu et al., 2012). SnTox3 has no known homology to any proteins available in public databases (Liu et al., 2009). SnToxA has limited homology to a prokaryotic gene and also exhibits significant diversifying selection (Stukenbrock & McDonald, 2007). Transformation with any of the three NEs into a nonpathogenic fungal isolate was sufficient to induce necrosis on susceptible wheat (Triticum aestivum) cultivars (Friesen et al., 2006; Liu et al., 2009, 2012). Clamped homogeneous electric field (CHEF) gel analysis indicates that each NE is located on a different chromosome and each gene is located on a different scaffold in the genomic assembly (Hane et al., 2007; Liu et al., 2012).
While there is a growing list of shared properties associated with effectors (reviewed in Kamoun, 2007 and Stergiopoulos & de Wit, 2009), very little is known about the evolutionary origins of effector-encoding genes. Recent genome sequencing has revealed families of what have been termed core effector proteins within the Dothideomycetes (Stergiopoulos et al., 2010, 2012). Identifying these core effectors relies on conservation of homologous elements within the effector proteins. The three characterized NEs of P. nodorum share no homology with any proteins for any fungal species available in GenBank and they do not appear to represent core effector proteins. Population genetic studies of effector loci have provided important insights into the evolutionary processes that affect NE loci within a species. A population genetic analysis of the NIP1 gene in Rhynchosporium commune showed that alteration of the effector protein sequence or deletion of the effector allele could lead to virulence (Schürch et al., 2004). Studies on the obligate biotroph of flax (Linum usitatissimum), Melampsora lini, revealed high levels of nonsynonymous substitutions at the AvrL567 locus and sectional insertions/deletions at the AvrM locus that alter or abolish recognition by their corresponding resistance (R) genes (Dodds et al., 2006; Ellis et al., 2007). Virulence alleles in Leptosphaeria maculans were attributed to both deletion of the AvrLm6 locus and introduction of early stop codons by repeat-induced point mutation (RIP) (Fudal et al., 2009; Van de Wouw et al., 2010).
For NEs, deletion of the effector gene leads to loss of the virulent phenotype on hosts with compatible genetic backgrounds. To date, effector studies have focused on individual genes or small groups of effector genes in a small number of individuals (Dodds et al., 2006; Barrett et al., 2009; Liu et al., 2009; Chuma et al., 2011; Liu et al., 2012), though it is clear that fungal populations are large and capable of harboring high levels of NE diversity. As more effector genes are discovered and characterized, a key question has become: how did this class of genes originate within fungal pathogens? Understanding the evolutionary origins of these genes could provide significant insights into the mechanisms involved in pathogen emergence and host specificity.
The horizontal transfer of SnToxA from P. nodorum to Pyrenophora tritici-repentis is thought to have led to the emergence of P. tritici-repentis as the tan spot pathogen on wheat (Friesen et al., 2006). Detection of this horizontal gene transfer (HGT) event was made possible by the high sequence similarity between the SnToxA alleles found in P. nodorum and the PtrToxA allele found in P. tritici-repentis. It remains unknown if the NEs present in P. nodorum are the result of a long co-evolutionary process between the pathogen and its hosts, or alternatively if the NEs were acquired more recently via other mechanisms such as horizontal transfer or interspecific hybridization.
This study focuses on the population genetics and evolutionary history of SnTox1, SnTox3 and SnToxA in P. nodorum and its closest known relatives. We assessed the global distribution and geographic diversity for all three effectors. We determined the presence or absence of each gene in over 1000 global isolates using both PCR and Southern hybridization. We calculated the frequency of each NE over spatial scales ranging from fields to continents and generated multi-effector genotypes to determine if selection was operating on the combination of NEs. To gain insight into the ancestral origin of these NEs in P. nodorum, we assessed the presence or absence of each NE in eight recently described sister species. Finally, we sequenced each NE in several hundred global strains to compare NE sequence diversity with previously published population genetic studies based on neutral markers.
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Phylogenetic analysis of the Phaeosphaeria species complex revealed that only two out of nine closely related species carry the NEs SnTox1, SnTox3 and SnToxA. All three NEs are diverse at the amino acid level with a high proportion of population-specific sequence alleles. Rarefaction analysis indicated that the center of diversity for each NE did not correspond with previous population genetic studies that identified the highest levels of diversity at neutral genetic loci in Iran (McDonald et al., 2012).
Our analyses showed that the likelihood of an individual carrying one of the three NEs is population dependent. Phaeosphaeria nodorum is a sexual pathogen with a large effective population size that exhibits high levels of gene flow over continental scales (e.g. between Oregon, Texas and New York within North America; Keller et al., 1997; Stukenbrock et al., 2006). Based on gene flow estimates from neutral microsatellite loci, the expected frequencies of NEs in pathogen populations are not expected to differ among populations within a continent. Instead, we found significant differences in NE frequencies among many field populations that did not differ for neutral markers (Table S1). We believe that the differences in NE frequency among populations reflect differences in the frequencies of the corresponding host NE sensitivity genes among regions. As already shown for SnToxA and the corresponding host sensitivity protein Tsn1, we hypothesize that the activity of SnTox1 and SnTox3 depends upon an interaction with a corresponding host sensitivity protein which is present in some wheat cultivars but absent in others (Friesen et al., 2006; Liu et al., 2009, 2012). Unless there is a secondary virulence function, in the absence of a host sensitivity protein, pathogen strains carrying the corresponding NE have no fitness advantage and the NE gene is expected to be essentially neutral and subject to genetic drift (Tan et al., 2012).
This highlights one of the main challenges associated with controlling globally disseminated pathogens with a high capacity for gene flow. Susceptible cultivars planted within the dissemination range of pathogen populations carrying an NE could rapidly select for pathogen populations carrying the NE. Thus, breeding efforts should be coordinated across large geographic regions to eliminate known susceptibility genes and reduce the frequencies of NEs at the continental scale. This type of effort is now underway in Australia to eliminate the Tsn1 gene that encodes susceptibility to SnToxA and the sensitivity locus for Tox3 (Oliver & Solomon, 2010; Waters et al., 2011).
A similar study that illustrated the dynamics of multiple effector loci in large natural populations was recently completed using the flax rust pathogen Melampsora lini. Thrall et al. (2012) found dramatic fluctuations in the frequency of M. lini avirulence alleles across multiple loci and they were able to correlate these fluctuations with the susceptibility of the corresponding host populations. Their analyses show how rapidly the genotype frequencies of host and pathogen can change in a gene-for-gene system experiencing antagonistic co-evolution. While we did not measure the sensitivity of the host in each field population, the large differences in local NE frequency despite high levels of neutral gene flow suggest that there is very strong selection operating on NEs at the field level.
Despite significant differences in NE frequency between field populations, the distribution of multi-effector genotypes within all but one of the 16 field populations did not differ from the expectation under random mating of neutral markers (Table 2, Fig. 2). Traditionally, pathogens carrying particular combinations of avirulence or effector genes are classified into races (Barrett et al., 2009), analogous to the multi-effector genotyping conducted in this study. The ‘cost of carrying’ is believed to drive the loss or alteration of the avirulence gene. Our finding of random associations among effector alleles within a population suggests that there is little fitness cost associated with carrying particular combinations of NEs. We hypothesize that the ‘carrying cost’ of these effector genes is low in the absence of the corresponding host sensitivity allele. This finding is also consistent with the hypothesis that host cultivar is the main determinant of NE frequency in these pathogen populations.
The haplotype networks presented in Fig. 3 show a prevalence of nonsynonymous mutations. It was reported previously that SnToxA and SnTox1 are under significant positive selection (Friesen et al., 2006; Liu et al., 2012), while SnTox3 did not show evidence of positive selection (Liu et al., 2009). The detection of positive selection operating on protein-coding genes with unknown function has become a powerful tool for identifying effectors in fungal genomes (Ma & Guttman, 2008; Raffaele et al., 2010; Stukenbrock et al., 2011; Saunders et al., 2012). It is often assumed that the higher rates of nonsynonymous substitutions exhibited by pathogen effector genes reflect diversifying selection favoring novel effector variants that are not recognized by plant R proteins. Dodds et al. (2006) performed experiments that supported this assumption in the gene-for-gene interactions between M. lini and flax, where specific amino acid changes in Avr proteins altered or abolished recognition by their corresponding R proteins. For necrotrophic pathogens, it was hypothesized that the inverse process was operating, whereby higher rates of nonsynonymous substitution within NE proteins resulted from the pathogen tracking changes in the host susceptibility alleles (Stukenbrock & McDonald, 2007). An alternative hypothesis to explain the higher rates of nonsynonymous substitution seen in NEs is that positive selection has favored mutant NE alleles that increase pathogen fitness through a quantitative increase in virulence.
This alternative hypothesis is supported by recent experimental studies showing that the most frequent SnToxA protein variant is significantly more active against identical wheat Tsn1 alleles hypothesized to increase pathogen fitness (Tan et al., 2012). The haplotype networks of both SnTox1 and SnTox3 exhibit two or more frequent and widely distributed protein variants that differ at two or more amino acid positions. We hypothesize that the most common protein variants in these networks induce significantly more necrosis than the less common protein variants in the network. Experimental testing of this hypothesis is now underway.
There has been a rapid expansion of literature describing fungal effectors as small, secreted proteins or small metabolites that interact with the host to suppress or alter the immune response (reviewed by Kamoun, 2007; Hogenhout et al., 2009; Stukenbrock & McDonald, 2009; de Wit et al., 2009). As more effectors are identified and characterized, an important question has become: what are the evolutionary origins of these effectors and how did pathogens acquire them? For some filamentous plant pathogens, acquisition of effectors appears to be through the horizontal transfer of conditionally dispensable chromosomes (Hatta et al., 2002; Oliver & Solomon, 2008; Akagi et al., 2009; Ma et al., 2010). Within the Dothideomycetes, families of functionally conserved effector genes analogous to Ecp2 and Avr4 in the tomato (Lycopersicon esculentum) pathogen Cladosporium fulvum have been identified (Stergiopolous et al., 2010; Stergiopolous et al., 2012). The SnTox1 protein shares local similarity with the chitin-binding domain found in Avr4, but otherwise the proteins appear to be unrelated (Liu et al., 2012). Among more distantly related organisms, the rapid increase in genome sequences has led to the detection of HGT events involving single genes across kingdoms (Richards et al., 2011). These HGT events were identified as a result of high homology among gene sequences. With the exception of SnToxA found in P. tritici-repentis, the NEs of P. nodorum do not show homology with any known proteins in other organisms, making it impossible to reconstruct the evolutionary history of these proteins. Though all three proteins require a host gene to induce symptoms and show similar expression profiles during infection, they are not homologous with each other. Because of the absence of homologous sequences outside of P. nodorum, we relied on diversity measurements from large population samples coupled with coalescent analyses of the Phaeosphaeria species complex to infer some aspects of the evolutionary history of these genes.
As shown in Fig. 5, the last highly supported (posterior probability = 1) recent common ancestor of P. nodorum is shared with six sister species. Among these seven species, SnToxA, SnTox1 and SnTox3 were found only in P. nodorum and Pat1. The sequences of the NE genes found in Pat1 indicate that they were acquired directly from P. nodorum via hybridization, as discussed below. Taken together, our findings are consistent with at least two different evolutionary scenarios that could explain the origins of NEs within the Phaeosphaeria species complex. In one scenario, all three NEs were present in a common ancestor of the nine characterized Phaeosphaeria species and were then lost or became highly diverged in seven of these species. Under this scenario, strong selection operating on these genes generated high sequence divergence and explains our inability to detect homologous genes in the other species. We used low-stringency hybridization conditions to test this hypothesis, but could not detect homologous sequences by hybridization. Another possibility is that a presence/absence polymorphism exists for each NE in all nine species and the isolates included in the analysis were missing all three NEs. Under a second scenario, all three NE genes in P. nodorum were acquired horizontally from an unknown donor or series of donors. The geographic distribution of diversity for each NE is consistent with three separate HGT events.
McDonald et al. (2012) provided evidence of hybridization between P. nodorum and Pat1 based on sequence analysis of the β-tubulin locus. All of the effector sequence alleles found in Pat1 were also found in P. nodorum, although the two species differed significantly for conserved housekeeping genes. For SnTox3 and SnToxA, the shared alleles were also the most frequent alleles found in P. nodorum. This NE sequence data provide additional support for the hypothesis that hybridization occurred between P. nodorum and Pat1. Based on these findings we postulate that all three effectors in Pat1 were acquired from P. nodorum via inter-specific hybridization, though the extent and nature of this hybridization require further investigation.
This population genetic study is only the second to compare neutral marker diversity with diversity at effector loci to infer the evolutionary history of effector genes. The earlier population study on the barley (Hordeum vulgare) scald pathogen Rhynchosporium commune found that the highest diversity for neutral marker loci, including DNA sequences, restriction fragment length polymorphisms and microsatellites, corresponded with the highest diversity for the NIP1 effector gene in Scandinavia (Schürch et al., 2004; Brunner et al., 2007), indicating that NIP1 shared the same evolutionary history, and probably the same common ancestor, as the other R. commune genes. In contrast, the lack of geographic correspondence between toxin diversity and neutral marker diversity provides strong evidence that the NEs of P. nodorum do not share the same evolutionary history as the neutral loci.
The geographic region that harbored the highest sequence diversity was different for each effector gene. The highest diversity for SnToxA was found in South Africa, for SnTox3 in North America and Australia, and for SnTox1 in Europe. The finding of higher effector diversity in ‘New World’ populations, where wheat cultivation began only during the last few hundred years following the arrival of European colonists (South Africa c. 350 yr ago, Australia c. 200 yr ago and North America c. 500 yr ago), suggests that each of these effectors may be under strong regional selection or alternatively may have different geographic origins representing three separate HGT events. None of the populations harboring the highest effector diversity overlapped with the hypothesized center of origin of P. nodorum in the ancient Fertile Crescent. The original source of the three effector genes remains unknown, but the SnToxA protein has a domain consistent with a prokaryotic origin (Friesen et al., 2006). McDonald et al. (2012) presented evidence that P. nodorum existed as a species before the domestication of wheat, probably as a fungal endophyte on grasses. We propose that these NE genes, whether inherited from an unknown common ancestor or acquired horizontally, enabled P. nodorum to emerge from this species complex as the dominant, specialized pathogen on wheat. Hybridization with its sister species Pat1 enabled the transfer of these genes to a new fungal species, resulting in the emergence of another damaging, though closely related, pathogen.