Genetic diversity, recombination and cryptic clades in Pseudomonas viridiflava infecting natural populations of Arabidopsis thaliana

Species-level genetic diversity and recombination in bacterial pathogens of wild plant populations have been nearly unexplored. Pseudomonas viridiflava is a common natural bacterial pathogen of Arabidopsis thaliana, for which pathogen defense genes and mechanisms are becoming increasing well known. The genetic variation contained within a worldwide sample of P. viridiflava collected from wild populations of A. thaliana was investigated using five genomic sequence fragments totaling 2.3 kb. Two distinct and deeply diverged clades were found within the P. viridiflava sample and in close proximity in multiple populations, each genetically diverse with synonymous variation as high as 9.3% in one of these clades. Within clades, there is evidence of frequent recombination within and between each sequenced locus and little geographic differentiation. Isolates from both clades were also found in a small sample of other herbaceous species in Midwest populations, indicating a possibly broad host range for P. viridiflava. The high levels of genetic variation and recombination together with a lack of geographic differentiation in this pathogen distinguish it from other bacterial plant pathogens for which intraspecific variation has been examined.     

The role of pectate lyase and the jasmonic acid defense response in Pseudomonas viridiflava virulence

Pseudomonas viridiflava is a common pathogen of Arabidopsis thaliana in wild populations, yet very little is known about mechanisms of resistance and virulence in this interaction. We examined the induced defense response of A. thaliana to several strains of P. viridiflava collected from this host by quantifying the expression of PR-1 and LOX2/PDF1.2, which serve as markers for induction of the salicylic and jasmonic acid (JA) pathways, respectively. Growth of these strains then was assessed on Col-0, the fad3/7/8 and coil-1 mutants deficient in JA- and ethylene (ET)-induced defense responses, and the sid2-1 mutant deficient in salicylic acid-induced defense responses. All strains of P. viridiflava induced high expression of LOX2 and PDF1.2 on Col-0. In contrast, PR-1 expression was delayed and reduced relative to PDF1.2 expression. Additionally, three of four P. viridiflava strains were more virulent on fad3/7/8 relative to Col-0, whereas all strains were more virulent on coil-1 relative to Col-0, indicating that P. viridiflava generally may be suppressed by JA/ET-mediated defense responses. In contrast, no increase in the growth of P. viridiflava strains was observed in the sid2-1 mutant relative to Col-0. Parallel experiments were performed with the closely related P. syringae pv. tomato for comparative purposes. In addition, we assessed the role of pectate lyase and the alternative sigma factor HrpL in P. viridiflava virulence on A. thaliana and found that pectate lyase activity is correlated with virulence, whereas the removal of pectate lyase or HrpL significantly reduced virulence.     

Pseudomonas viridiflava and P. syringae--natural pathogens of Arabidopsis thaliana

We report the isolation and identification of two natural pathogens of Arabidopsis thaliana, Pseudomonas viridiflava and Pseudomonas syringae, in the midwestern United States. P. viridiflava was found in six of seven surveyed Arabidopsis thaliana populations. We confirmed the presence in the isolates of the critical pathogenicity genes hrpS and hrpL. The pathogenicity of these isolates was verified by estimating in planta bacterial growth rates and by testing for disease symptoms and hypersensitive responses to A. thaliana. Infection of 21 A. thaliana ecotypes with six locally collected P. viridiflava isolates and with one P. syringae isolate showed both compatible (disease) and incompatible (resistance) responses. Significant variation in response to infection was evident among Arabidopsis ecotypes, both in terms of symptom development and in planta bacterial growth. The ability to grow and cause disease symptoms on particular ecotypes also varied for some P. viridiflava isolates. We believe that these pathogens will provide a powerful system for exploring coevolution in natural plant-pathogen interactions.     

Fitness consequences of infection of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> with its natural bacterial pathogen <Emphasis Type="Italic">Pseudomonas viridiflava</Emphasis>

Variation in plant resistance to pathogen infection is commonly observed in interactions between wild plants and their foliar pathogens. Models of host–pathogen interactions indicate that a large cost of infection is generally necessary to maintain this variation, yet there is limited evidence that foliar pathogens cause detectable fitness reductions in wild host plants. Most published work has focused on fungal pathogens. Pseudomonas viridiflava, a common bacterial pathogen of the annual weed Arabidopsis thaliana across its range, comprises two distinct genetic clades that cause disease symptoms of different severity. Here we measured the extent of infection of wild A. thaliana populations in the Midwest, USA, and examined the effect on seed production, in field and growth-chamber experiments, of experimental inoculation with isolates from the two clades. We found infection with P. viridiflava varied from 0 to 56% in Midwest A. thaliana populations, with the possibility of several leaves per plant infected later in the growing season. In the growth chambers, experimental inoculation reduced seed set by averages of 15 and 11% for clades A and B, respectively. In the field experiment, only clade A affected plant fitness significantly, reducing seed set by an average of 38%. Underlying these average effects we observed both negative and positive effects of infection, and variation in both fitness among plant genotypes and sensitivity to environmental conditions.     

Maladaptation in wild populations of the generalist plant pathogen Pseudomonas syringae

Multihost pathogens occur widely on both natural and agriculturally managed hosts. Despite the importance of such generalists, evolutionary studies of host-pathogen interactions have largely focused on tightly coupled interactions between species pairs. We characterized resistance in a collection of Arabidopsis thaliana hosts, including 24 accessions collected from the Midwest USA and 24 from around the world, and patterns of virulence in a collection of Pseudomonas syringae strains, including 24 strains collected from wild Midwest populations of A. thaliana (residents) and 18 from an array of cultivated species (nonresidents). All of the nonresident strains and half of the resident strains elicited a resistance response on one or more A. thaliana accessions. The resident strains that failed to elicit any resistance response possessed an alternative type III secretion system (T3SS) that is unable to deliver effectors into plant host cells; as a result, these seemingly nonpathogenic strains are incapable of engaging in gene for gene interactions with A. thaliana. The remaining resident strains triggered greater resistance compared to nonresident strains, consistent with maladaptation of the resident bacterial population. We weigh the plausibility of two explanations: general maladaptation of pathogen strains and a more novel hypothesis whereby community level epidemiological dynamics result in adaptive dynamics favoring ephemeral hosts like A. thaliana.     

Evolutionary diversification through hybridization in a wild host-pathogen interaction

Coevolutionary outcomes between interacting species are predicted to vary across landscapes, as environmental conditions, gene flow, and the strength of selection vary among populations. Using a combination of molecular, experimental, and field approaches, we describe how broad-scale patterns of environmental heterogeneity, genetic divergence, and regional adaptation have the potential to influence coevolutionary processes in the Linum marginale-Melampsora lini plant-pathogen interaction. We show that two genetically and geographically divergent pathogen lineages dominate interactions with the host across Australia, and demonstrate a hybrid origin for one of the lineages. We further demonstrate that the geographic divergence of the two lineages of M. lini in Australia is related to variation among lineages in virulence, life-history characteristics, and response to environmental conditions. When correlated with data describing regional patterns of variation in host resistance diversity and mating system these observations highlight the potential for gene flow and geographic selection mosaics to generate and maintain coevolutionary diversification in long-standing host-pathogen interactions.     

Coevolution at multiple spatial scales: Linum marginale–Melampsora lini – from the individual to the species

Coevolutionary processes are intrinsically spatial as well as temporal, and occur at many different scales. These range from single populations dominated by demographic and genetic stochasticity, to metapopulations in which colonisation/extinction dynamics have a large influence, and larger geographic regions where phylogenetic patterns and historical events become important. We present data for the genetically and demographically well-characterised plant host–pathogen interaction, the Linum marginale–Melampsora lini system, and use this to demonstrate the varying nature of resistance and virulence structure across these spatial scales. At the within population level, our results indicate considerable variability in resistance and virulence, but little evidence of coordinated changes in host and pathogen. Studies involving comparisons among multiple demes within a single metapopulation show that adjacent populations often have asynchronous disease dynamics and large differences in diversity and frequency of resistance and virulence phenotypes. Nevertheless, at this scale, there is also evidence of spatial structure in that more closely adjacent host populations are significantly more likely to have similar resistance phenotypes and mean levels of resistance. At larger scales, comparisons among adjacent metapopulations indicate that quantitative differences in host mating system and other life history features can have further major consequences for how host and pathogen variation is packaged. Finally, comparisons at continental and among host-species levels show variation consistent with specialisation and speciation in the pathogen. This revised version was published online in July 2006 with corrections to the Cover Date.     

Local adaptation and effect of host genotype on the rate of pathogen evolution: an experimental test in a plant pathosystem

Abstract Virulence is thought to be a driving force in host–pathogen coevolution. Theoretical models suggest that virulence is an unavoidable consequence of pathogens evolving towards a high rate of intrahost reproduction. These models predict a positive correlation between the reproductive fitness of a pathogen and its level of virulence. Theoretical models also suggest that the demography and genetic structure of a host population can influence the evolution of virulence. If evolution occurs faster in pathogen populations than in host populations, the predicted result is local adaptation of the pathogen population. In our studies, we used a combination of molecular and physiological markers to test these hypotheses in an agricultural system. We isolated five strains of the fungal pathogen Mycosphaerella graminicola from each of two wheat cultivars that differed in their level of resistance to this pathogen. Each of the 10 fungal strains had distinct genotypes as indicated by different DNA fingerprints. These fungal strains were re‐inoculated onto the same two host cultivars in a field experiment and their genotype frequencies were monitored over several generations of asexual reproduction. We also measured the virulence of these 10 fungal strains and correlated it to the reproductive fitness of each fungal strain. We found that host genotypes had a strong impact on the dynamics of the pathogen populations. The pathogen population collected from the moderately resistant cultivar Madsen showed greater stability, higher genotype diversity, and smaller selection coefficients than the pathogen populations collected from the susceptible cultivar Stephens or a mixture of the two host cultivars. The pathogen collection from the mixed host population was midway between the two pure lines for most parameters measured. Our results also revealed that the measures of reproductive fitness and virulence of a pathogen strain were not always correlated. The pathogen strains varied in their patterns of local adaptation, ranging from locally adapted to locally maladapted.     

Local adaptation in the Linum marginale-Melampsora lini host-pathogen interaction

The potential for local adaptation between pathogens and their hosts has generated strong theoretical and empirical interest with evidence both for and against local adaptation reported for a range of systems. We use the Linum marginale-Melampsora lini plant-pathogen system and a hierarchical spatial structure to investigate patterns of local adaptation within a metapopulation characterised by epidemic dynamics and frequent extinction of pathogen populations. Based on large sample sizes and comprehensive cross-inoculation trials, our analyses demonstrate strong local adaptation by Melampsora to its host populations, with this effect being greatest at regional scales, as predicted from the broader spatial scales at which M. lini disperses relative to L. marginale. However, there was no consistent trend for more distant pathogen populations to perform more poorly. Our results further show how the coevolutionary interaction between hosts and pathogens can be influenced by local structure such that resistant hosts select for generally virulent pathogens, while susceptible hosts select for more avirulent pathogens. Empirically, local adaptation has generally been tested in two contrasting ways: (1) pathogen performance on sympatric versus allopatric hosts; and (2) sympatric versus allopatric pathogens on a given host population. In situations where no host population is more resistant or susceptible than others when averaged across pathogen populations (and likewise, no pathogen population is more virulent or avirulent than others), results from these tests should generally be congruent. We argue that this is unlikely to be the case in the metapopulation situations that predominate in natural host-pathogen interactions, thus requiring tests that control simultaneously for variation in plant and pathogen populations.     

Spatial scale of local adaptation in a plant-pathogen metapopulation

The rate and scale of gene flow can strongly affect patterns of local adaptation in host-parasite interactions. I used data on regional pathogen occurrence to infer the scale of pathogen dispersal and to identify pathogen metapopulations in the interaction between Plantago lanceolata and its specialist phytopathogen, Podosphaera plantaginis. Frequent extinctions and colonizations were recorded in the metapopulations, suggesting substantial gene flow at this spatial scale. The level of pathogen local adaptation was assessed in a laboratory inoculation experiment at three different scales: in sympatric host populations, in sympatric host metapopulations and in allopatric host metapopulations. I found evidence for adaptation to sympatric host populations, as well as evidence indicating that local adaptation may extend to the scale of the sympatric host metapopulation. There was also variation among the metapopulations in the degree of pathogen local adaptation. This may be explained by regional differences in the rate of migration.     

The Pseudomonas syringae type III effector HopAM1 enhances virulence on water-stressed plants

Pseudomonas syringae strains deliver diverse type III effector proteins into host cells, where they can act as virulence factors. Although the functions of the majority of type III effectors are unknown, several have been shown to interfere with plant basal defense mechanisms. Type III effectors also could contribute to bacterial virulence by enhancing nutrient uptake and pathogen adaptation to the environment of the host plant. We demonstrate that the type III effector HopAM1 (formerly known as AvrPpiB) enhances the virulence of a weak pathogen in plants that are grown under drought stress. This is the first report of a type III effector that aids pathogen adaptation to water availability in the host plant. Expression of HopAM1 makes transgenic Ws-0 Arabidopsis hypersensitive to abscisic acid (ABA) for stomatal closure and germination arrest. Conditional expression of HopAM1 in Arabidopsis also suppresses basal defenses. ABA responses overlap with defense responses and ABA has been shown to suppress defense against P. syringae pathogens. We propose that HopAM1 aids P. syringae virulence by manipulation of ABA responses that suppress defense responses. In addition, host ABA responses enhanced by type III delivery of HopAM1 protect developing bacterial colonies inside leaves from osmotic stress.     

Host–Pathogen Coevolution: The Selective Advantage of Bacillus thuringiensis Virulence and Its Cry Toxin Genes

Reciprocal coevolution between host and pathogen is widely seen as a major driver of evolution and biological innovation. Yet, to date, the underlying genetic mechanisms and associated trait functions that are unique to rapid coevolutionary change are generally unknown. We here combined experimental evolution of the bacterial biocontrol agent Bacillus thuringiensis and its nematode host Caenorhabditis elegans with large-scale phenotyping, whole genome analysis, and functional genetics to demonstrate the selective benefit of pathogen virulence and the underlying toxin genes during the adaptation process. We show that: (i) high virulence was specifically favoured during pathogen–host coevolution rather than pathogen one-sided adaptation to a nonchanging host or to an environment without host; (ii) the pathogen genotype BT-679 with known nematocidal toxin genes and high virulence specifically swept to fixation in all of the independent replicate populations under coevolution but only some under one-sided adaptation; (iii) high virulence in the BT-679-dominated populations correlated with elevated copy numbers of the plasmid containing the nematocidal toxin genes; (iv) loss of virulence in a toxin-plasmid lacking BT-679 isolate was reconstituted by genetic reintroduction or external addition of the toxins. We conclude that sustained coevolution is distinct from unidirectional selection in shaping the pathogen''s genome and life history characteristics. To our knowledge, this study is the first to characterize the pathogen genes involved in coevolutionary adaptation in an animal host–pathogen interaction system.     

Host resistance and pathogen aggressiveness are key determinants of coinfection in the wild

Coinfection, whereby the same host is infected by more than one pathogen strain, may favor faster host exploitation rates as strains compete for the same limited resources. Hence, coinfection is expected to have major consequences for pathogen evolution, virulence, and epidemiology. Theory predicts genetic variation in host resistance and pathogen infectivity to play a key role in how coinfections are formed. The limited number of studies available has demonstrated coinfection to be a common phenomenon, but little is known about how coinfection varies in space, and what its determinants are. Our aim is to understand how variation in host resistance and pathogen infectivity and aggressiveness contribute to how coinfections are formed in the interaction between fungal pathogen Podosphaera plantaginis and Plantago lanceolata. Our phenotyping study reveals that more aggressive strains are more likely to form coinfections than less aggressive strains in the natural populations. In the natural populations most of the variation in coinfection is found at the individual plant level, and results from a common garden study confirm the prevalence of coinfection to vary significantly among host genotypes. These results show that genetic variation in both the host and pathogen populations are key determinants of coinfection in the wild.     

Detecting local adaptation in a natural plant-pathogen metapopulation: a laboratory vs. field transplant approach

Antagonistic coevolution between hosts and parasites in spatially structured populations can result in local adaptation of parasites. Traditionally parasite local adaptation has been investigated in field transplant experiments or in the laboratory under a constant environment. Despite the conceptual importance of local adaptation in studies of (co)evolution, to date no study has provided a comparative analysis of these two methods. Here, using information on pathogen population dynamics, I tested local adaptation of the specialist phytopathogen, Podosphaera plantaginis, to its host, Plantago lanceolata at three different spatial scales: sympatric host population, sympatric host metapopulation and allopatric host metapopulations. The experiment was carried out as a field transplant experiment with greenhouse-reared host plants from these three different origins introduced into four pathogen populations. In contrast to results of an earlier study performed with these same host and parasite populations under laboratory conditions, I did not find any evidence for parasite local adaptation. For interactions governed by strain-specific resistance, field studies may not be sensitive enough to detect mean parasite population virulence. Given that parasite transmission potential may be mediated by the abiotic environment and genotype-by-environment interactions, I suggest that relevant environmental variation should be incorporated into laboratory studies of parasite local adaptation.     

LOCAL MALADAPTATION IN THE ANTHER-SMUT FUNGUS MICROBOTRYUM VIOLACEUM TO ITS HOST PLANT SILENE LATIFOLIA: EVIDENCE FROM A CROSS-INOCULATION EXPERIMENT

Conventional wisdom holds that parasites evolve more rapidly than their hosts and are therefore locally adapted, that is, better at exploiting sympatric than allopatric hosts. We studied local adaptation in the insect-transmitted fungal pathogen Microbotryum violaceum and its host plant Silene latifolia. Infection success was tested in sympatric (local) and allopatric (foreign) combinations of pathogen and host from 14 natural populations from a metapopulation. Seedlings from up to 10 seed families from each population were exposed to sporidial suspensions from each of four fungal strains derived from the same population, from a near-by population (< 10 km distance), and from two populations at an intermediate (< 30 km) and remote (< 170 km) distance, respectively. We obtained significant pathogen X plant interactions in infection success (proportion of diseased plants) at both fungal population and strain level. There was an overall pattern of local maladaptation of this pathogen: average fungal infection success was significantly lower on sympatric hosts (mean proportion of diseased plants = 0.32 ± 0.03 SE) than on allopatric hosts (0.40 ± 0.02). Five of the 14 fungal populations showed no strong reduction in infection success on sympatric hosts, and three even tended to perform better on sympatric hosts. This pattern is consistent with models of time-lagged cycles predicting patterns of local adaptation in host-parasite systems to emerge only on average. Several factors may restrict the evolutionary potential of this pathogen relative to that of its host. First, a predominantly selfing breeding system may limit its ability to generate new virulence types by sexual recombination, whereas the obligately outcrossing host 5. latifolia may profit from rearrangement of resistance alleles by random mating. Second, populations often harbor only a few infected individuals, so virulence variation may be further reduced by drift. Third, migration rates among host plant populations are much higher than among pathogen populations, possibly because pollinators prefer healthy over diseased plants. Migration among partly isolated populations may therefore introduce novel host plant resistance variants more often than novel parasite virulence variants. That migration contributes to the coevolutionary dynamics in this system is supported by the geographic pattern of infectivity. Infection success increased over the first 10–km range of host-pathogen population distances, which is likely the natural range of gene exchange.     

The Pseudomonas syringae avrRpt2 gene product promotes pathogen virulence from inside plant cells

Several bacterial avr genes have been shown to contribute to virulence on susceptible plants lacking the corresponding resistance (R) gene. The mechanisms by which avr genes promote parasitism and disease, however, are not well understood. We investigated the role of the Pseudomonas syringae pv. tomato avrRpt2 gene in pathogenesis by studying the interaction of P. syringae pv. tomato strain PstDC3000 expressing avrRpt2 with several Arabidopsis thaliana lines lacking the corresponding R gene, RPS2. We found that PstDC3000 expressing avrRpt2 grew to significantly higher levels and often resulted in the formation of more severe disease symptoms in ecotype No-0 plants carrying a mutant RPS2 allele, as well as in two Col-0 mutant lines, cpr5 rps2 and coil rps2, that exhibit enhanced resistance. We also generated transgenic A. thaliana lines expressing avrRpt2 and demonstrated, by using several different assays, that expression of avrRpt2 within the plant also promotes virulence of PstDC3000. Thus, AvrRpt2 appears to promote pathogen virulence from within the plant cell.     

Global analysis of Arabidopsis/downy mildew interactions reveals prevalence of incomplete resistance and rapid evolution of pathogen recognition

Interactions between Arabidopsis thaliana and its native obligate oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) represent a model system to study evolution of natural variation in a host/pathogen interaction. Both Arabidopsis and Hpa genomes are sequenced and collections of different sub-species are available. We analyzed ～400 interactions between different Arabidopsis accessions and five strains of Hpa. We examined the pathogen's overall ability to reproduce on a given host, and performed detailed cytological staining to assay for pathogen growth and hypersensitive cell death response in the host. We demonstrate that intermediate levels of resistance are prevalent among Arabidopsis populations and correlate strongly with host developmental stage. In addition to looking at plant responses to challenge by whole pathogen inoculations, we investigated the Arabidopsis resistance attributed to recognition of the individual Hpa effectors, ATR1 and ATR13. Our results suggest that recognition of these effectors is evolutionarily dynamic and does not form a single clade in overall Arabidopsis phylogeny for either effector. Furthermore, we show that the ultimate outcome of the interactions can be modified by the pathogen, despite a defined gene-for-gene resistance in the host. These data indicate that the outcome of disease and disease resistance depends on genome-for-genome interactions between the host and its pathogen, rather than single gene pairs as thought previously.     

Natural variation in the Pto disease resistance gene within species of wild tomato (Lycopersicon). II. Population genetics of Pto

Disease resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) in the host species Lycopersicon esculentum, the cultivated tomato, and the closely related L. pimpinellifolium is triggered by the physical interaction between the protein products of the host resistance (R) gene Pto and the pathogen avirulence genes AvrPto and AvrPtoB. Sequence variation at the Pto locus was surveyed in natural populations of seven species of Lycopersicon to test hypotheses of host-parasite coevolution and functional adaptation of the Pto gene. Pto shows significantly higher nonsynonymous polymorphism than 14 other non-R-gene loci in the same samples of Lycopersicon species, while showing no difference in synonymous polymorphism, suggesting that the maintenance of amino acid polymorphism at this locus is mediated by pathogen selection. Also, a larger proportion of ancestral variation is maintained at Pto as compared to these non-R-gene loci. The frequency spectrum of amino acid polymorphisms known to negatively affect Pto function is skewed toward low frequency compared to amino acid polymorphisms that do not affect function or silent polymorphisms. Therefore, the evolution of Pto appears to be influenced by a mixture of both purifying and balancing selection.