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Coevolutionary interactions are thought to have spurred the evolution of key innovations and driven the diversification of much of life on Earth. However, the genetic and evolutionary basis of the innovations that facilitate such interactions remains poorly understood. We examined the coevolutionary interactions between plants (Brassicales) and butterflies (Pieridae), and uncovered evidence for an escalating evolutionary arms-race. Although gradual changes in trait complexity appear to have been facilitated by allelic turnover, key innovations are associated with gene and genome duplications. Furthermore, we show that the origins of both chemical defenses and of molecular counter adaptations were associated with shifts in diversification rates during the arms-race. These findings provide an important connection between the origins of biodiversity, coevolution, and the role of gene and genome duplications as a substrate for novel traits.

Saccharomyces cells occasionally carry cytoplasmic ds-RNA “killer” viruses coding for low-mass proteins, which upon secretion to the environment can kill related cells that do not carry the viral particles. Such killer viruses are not infectious, and can spread only through cell division and during mating. Three principal classes of Saccharomyces viruses (ScV-M1, ScV-M2 and ScV-M28) belonging to the Totiviridae family have been characterised, each capable of forming a specific anti-competitor toxin and corresponding antidote. Presumably, toxic killing provides competitive benefits to the yeast host. However, the ecological and evolutionary significance of toxin production remains poorly understood. For example, it is unknown where yeast killers occur and at what frequency, how evolvable killing ability is, whether it is constrained by possible trade-offs with resource competitive ability and how it is shaped by interactions with toxin-sensitive competitors. Also unknown is how stable yeast-virus symbioses are, and how coevolution between host and virus may affect this stability and the killing phenotype itself. It is believed that killer yeasts are common based on the fact that they have been found among yeasts isolated from different sources over several decades. In chapter 2, we assay two large yeast collections from diverse habitats, including nature and man-made habitats (in total 136 strains with known genome sequences), for killer phenotype and toxin resistance. We find that ~10.3% carry a killer virus, while about 25% are resistant to at least one of the three known killer toxins (12.5% to different combinations of two and ~9% to all three), most likely due to chromosomal mutations. Analyses of their evolutionary relationship indicate that host-virus associations are relatively short lived, whereas the relatively high frequency of resistance suggests that toxins have a substantial impact on yeast evolution. In order to understand the ecological and evolutionary role of toxin production, it is essential to reliably assess the killing rate of toxin producers by measuring how many toxin-sensitive individuals are killed by a single toxin producer during a given time interval. To identify a convenient method with high sensitivity and reproducibility, in chapter 3 we perform a systematic comparative analysis of four methods, including the conventional “Halo method” and three more quantitative liquid assays. We apply these methods to a set of three known yeast killer strains (K1, K2 and K28) and find that the easy applicable Halo method provides the most sensitive and reproducible killing rate estimates (with best discrimination between killer strains). Understanding the evolution of the yeast-virus association is crucial for a full understanding of the ecological and evolutionary role of killer strains. In chapter 4, we present experimental tests of the strength of the dependence of yeast host strains on their killer viruses. We cross-infect several viruses among killer strains of the genus Saccharomyces – all expressing the K1-type toxin, and test native and new combinations for the strength of host-virus co-adaptation. We find explicit host-virus co-adaptation, because native yeasts hosts display the highest toxicity and highest stability of killer viruses relative to hosts carrying non-native viruses. Even stronger, we find that curing these wild killer yeasts from their virus reduces their competitive fitness, despite initial fitness costs of viral carriage reported for constructed killer strains. These results demonstrate co-adaptation of host and virus in the natural killer strains resulting in their dependence on the killer virus. To explore the evolutionary costs and benefits of virus carriage and toxin production, and understand whether they are shaped by the coevolution between host and virus and the presence of toxin-sensitive competitors in the environment, we conduct a series of laboratory experiments where we manipulate the opportunity for coevolution (chapter 5). Analyses of killing ability, toxin sensitivity and fitness (i.e. resource competitive ability), show rapid reciprocal changes in killer and sensitive strain when coevolution is allowed, modulated by the rapid invasion of toxin-resistant mutants and subsequent reduction of killing ability. Remarkably, we find that the rapid invasion of toxin-resistant mutants involves two mutational steps, the first being a mutation showing a meiotic drive phenotype as well as a strong fitness benefit in heterozygotes, the second the resistance mutation. Shifts in the competitive fitness of evolved killer isolates with increased killing ability show a clear trade-off between killing rate and resource competitive ability, indicating that resource and interference competitive ability are alternative competitive strategies. Moreover, by cross-infecting the killer virus between the ancestral and an evolved strain, we are able to demonstrate the rapid co-adaptation between host and killer virus, supporting our previous findings of co-adaptive responses in wild yeast killers (chapter 4). Our analyses are based on screens of natural isolates, laboratory evolution experiments and phenotypic analyses, complemented by classical genetics. To more fully understand the reciprocal nature and molecular mechanisms of adaptive responses, genome analyses are required. The motivation for such analyses and other follow-up studies are proposed in chapter 6. My studies show the usefulness of the killer yeast system to address questions related to interference competition and coevolution, which may proof valuable also given potential applications of killer yeasts in the fermentation industry.

Saccharomyces cells occasionally carry cytoplasmic ds-RNA “killer” viruses coding for low-mass proteins, which upon secretion to the environment can kill related cells that do not carry the viral particles. Such killer viruses are not infectious, and can spread only through cell division and during mating. Three principal classes of Saccharomyces viruses (ScV-M1, ScV-M2 and ScV-M28) belonging to the Totiviridae family have been characterised, each capable of forming a specific anti-competitor toxin and corresponding antidote. Presumably, toxic killing provides competitive benefits to the yeast host. However, the ecological and evolutionary significance of toxin production remains poorly understood. For example, it is unknown where yeast killers occur and at what frequency, how evolvable killing ability is, whether it is constrained by possible trade-offs with resource competitive ability and how it is shaped by interactions with toxin-sensitive competitors. Also unknown is how stable yeast-virus symbioses are, and how coevolution between host and virus may affect this stability and the killing phenotype itself. It is believed that killer yeasts are common based on the fact that they have been found among yeasts isolated from different sources over several decades. In chapter 2, we assay two large yeast collections from diverse habitats, including nature and man-made habitats (in total 136 strains with known genome sequences), for killer phenotype and toxin resistance. We find that ~10.3% carry a killer virus, while about 25% are resistant to at least one of the three known killer toxins (12.5% to different combinations of two and ~9% to all three), most likely due to chromosomal mutations. Analyses of their evolutionary relationship indicate that host-virus associations are relatively short lived, whereas the relatively high frequency of resistance suggests that toxins have a substantial impact on yeast evolution. In order to understand the ecological and evolutionary role of toxin production, it is essential to reliably assess the killing rate of toxin producers by measuring how many toxin-sensitive individuals are killed by a single toxin producer during a given time interval. To identify a convenient method with high sensitivity and reproducibility, in chapter 3 we perform a systematic comparative analysis of four methods, including the conventional “Halo method” and three more quantitative liquid assays. We apply these methods to a set of three known yeast killer strains (K1, K2 and K28) and find that the easy applicable Halo method provides the most sensitive and reproducible killing rate estimates (with best discrimination between killer strains). Understanding the evolution of the yeast-virus association is crucial for a full understanding of the ecological and evolutionary role of killer strains. In chapter 4, we present experimental tests of the strength of the dependence of yeast host strains on their killer viruses. We cross-infect several viruses among killer strains of the genus Saccharomyces – all expressing the K1-type toxin, and test native and new combinations for the strength of host-virus co-adaptation. We find explicit host-virus co-adaptation, because native yeasts hosts display the highest toxicity and highest stability of killer viruses relative to hosts carrying non-native viruses. Even stronger, we find that curing these wild killer yeasts from their virus reduces their competitive fitness, despite initial fitness costs of viral carriage reported for constructed killer strains. These results demonstrate co-adaptation of host and virus in the natural killer strains resulting in their dependence on the killer virus. To explore the evolutionary costs and benefits of virus carriage and toxin production, and understand whether they are shaped by the coevolution between host and virus and the presence of toxin-sensitive competitors in the environment, we conduct a series of laboratory experiments where we manipulate the opportunity for coevolution (chapter 5). Analyses of killing ability, toxin sensitivity and fitness (i.e. resource competitive ability), show rapid reciprocal changes in killer and sensitive strain when coevolution is allowed, modulated by the rapid invasion of toxin-resistant mutants and subsequent reduction of killing ability. Remarkably, we find that the rapid invasion of toxin-resistant mutants involves two mutational steps, the first being a mutation showing a meiotic drive phenotype as well as a strong fitness benefit in heterozygotes, the second the resistance mutation. Shifts in the competitive fitness of evolved killer isolates with increased killing ability show a clear trade-off between killing rate and resource competitive ability, indicating that resource and interference competitive ability are alternative competitive strategies. Moreover, by cross-infecting the killer virus between the ancestral and an evolved strain, we are able to demonstrate the rapid co-adaptation between host and killer virus, supporting our previous findings of co-adaptive responses in wild yeast killers (chapter 4). Our analyses are based on screens of natural isolates, laboratory evolution experiments and phenotypic analyses, complemented by classical genetics. To more fully understand the reciprocal nature and molecular mechanisms of adaptive responses, genome analyses are required. The motivation for such analyses and other follow-up studies are proposed in chapter 6. My studies show the usefulness of the killer yeast system to address questions related to interference competition and coevolution, which may proof valuable also given potential applications of killer yeasts in the fermentation industry.

Plants are chemically defended against insect herbivory in various ways. They produce a broad range of secondary metabolites that may be toxic or deterrent to insects. Specialist insects, however, are often capable of overcoming these defences. The yellow striped flea beetle (Phyllotreta nemorumL.) is a specialist that feeds on crucifers (Brassicaceae) such as Sinapis arvensisand Barbarea vulgaris. In Denmark, two types of Barbarea vulgarisvar. arcuataare distinguished: one with pubescent leaves (P-type) and one with glabrous leaves (G-type). All individuals of P. nemorumcan feed on B. vulgarisP-type. Barbarea vulgarisG-type, on the other hand, is chemically defended against most P. nemorumindividuals during the flea beetle reproductive season. The defence compounds are hypothesized to be saponins, a class of compounds with various biological effects and insecticidal properties. Despite high levels of these saponins during summer, some flea beetles can and do feed on B. vulgaris G-type. The ability of P. nemorumto feed on B. vulgarisG-type is heritable; resistance against the defence of B. vulgarisG-type is controlled by dominant major resistance genes (R-genes). One dominant R-allele of an R-gene is enough to convert a susceptible beetle into a resistant one. Despite knowledge of the inheritance patterns of resistance in the flea beetles, which have been demonstrated to be variable, the underlying mechanism of flea beetle resistance has, so far, remained unclear. This prompted me to investigate, as an initial part of my thesis, the genetic basis of the flea beetle adaptation to the defence of B. vulgarisG-type. The interaction between B. vulgarisand the flea beetle is a unique natural model system to study chemical defences in plants and counter-adaptations in insects. Plant and insect are both polymorphic with respect to the trait involved in resistance and hereby provide an excellent opportunity to study the geographic aspects of the evolution of the resistance trait in both interacting species. In this thesis, I focus on the resistance of the flea beetle, and take the presence of different genotypes of the plant as a given. Phyllotreta nemorumis a major pest, for example in oil seed rape. Understanding how resistance evolves in P. nemorumwill not only benefit flea beetle control, but also control of other pest insects. Understanding insect resistance includes knowledge of seasonal, geographic and genetic variation in both plant defense and herbivore adaptation.The R-gene has a remarkable distribution. Flea beetle populations living on B. vulgarisG-type consist solely of resistant individuals, but on host plant patches nearby B. vulgarisG-type lower frequencies of resistant beetles are found than one would expect with the amount of gene flow found at the neutral level between these subpopulations. The aim of this thesis was to find the gene that is held responsible for the resistance of P. nemorumto the defences of B. vulgaris, investigate the distribution of this resistance trait and explain the distribution of this trait in natural populations. The following questions were addressed: (1) what is the genetic basis of the adaptation under study? (2) how is the resistance distributed across flea beetle populations in Denmark? and (3) which factors underlie this distribution? In order to answer these questions, I used an integrated approach. I have combined a candidate gene approach (CHAPTER3) with an empirical approach via the study of variation in resistance in flea beetle populations (CHAPTER4), and a population genomics approach by using molecular markers to gain insight in the genomic make-up of the population and its connection with the resistance trait (CHAPTERS5 and 6). The population genomics approach is a recent advance in methods to detect the involvement of selection in the distribution of alleles at presumably adaptive loci. Using this approach one can distinguish locus-specific effects, like directional selection, from genome-wide effects, on the distribution of alleles at loci of interest. The population genomics approach is introduced in CHAPTER2 together with the Geographic Mosaic Theory of Coevolution. I illustrate how processes underlying this theory of coevolution can be investigated with the population genomics approach. According to the geographic mosaic theory of coevolution, reciprocal selection between interacting species only happens in so-called hot-spots. Hot spots can be identified using population genomics and genetic variation found at specific loci can be attributed to locus-specific processes such as directional selection. For the B. vulgaris- flea beetle system this means that with a population genomics approach we can examine whether the distribution of resistant flea beetles on alternative host plants is only influenced by migration, or also by selection (CHAPTER5). Another valuable utility of the population genomics approach is to investigate whether a candidate gene for the R-gene is under selection, by looking whether a candidate gene is experiencing locus-specific effects beside genome-wide effects when comparing flea beetle populations living on B. vulgarisG-type with populations living on alternative host plants (CHAPTER6). However, before using a population genomics approach to compare the resistance trait or a candidate gene with parts of the genome that only experience genome-wide effects, I have tried to identify the genetic basis of the flea beetle adaptation to the defence ofB. vulgarisG-type. In CHAPTER3, I have addressed this question by using a candidate gene approach to examine the involvement of a possible detoxifying enzyme in P. nemorum. Genes coding for β-glucosidase were a candidate for genes underlying the difference between resistant and susceptible beetles, because β-glucosidase is used as detoxifying enzyme by other organisms resistant to saponin defence. Three different β-glucosidase cDNA sequences were cloned from Danish flea beetle lines. We named them β-glucosidase A, B and C. β-glucosidase C was only found in resistant lines and not in the susceptible line. We then tested if recombinant β-glucosidase C breaks down the most abundant and most effective defence compound in B. vulgarisG-type, hederagenin cellobioside. β-glucosidase C was able to deglycosylate one glucose unit of hederagenin cellobioside, when expressed in an insect cell line. This suggests that expressed β-glucosidase C can deglycosylate antifeedant saponins and may play a role in the resistant flea beetle’s ability to overcome the defence of B. vulgaris. Next, a segregating family was created in which offspring differed in resistance genotype. Again β-glucosidase cDNA sequences were cloned to find a difference in the presence of these β-glucosidases between resistant and susceptible individuals. This time cDNA sequences of β-glucosidases A, B and C were present in both resistant and susceptible individuals although significantly fewer β-glucosidase C cDNA sequence variants were found in susceptible individuals than in resistant individuals. Thus, the genetic basis of flea beetle resistance remains unclear. Further investigation is needed to explore if the β-glucosidase C protein is also capable of inactivating hederagenin cellobioside by hydrolysizing the second glucose unit from the saponin and if there is a difference in enzyme activity of β-glucosidase C between resistant and susceptible beetles. Subsequently, in CHAPTER4 I have investigated whether the frequency of resistant beetles decreased in populations living on other host plant patches than B. vulgarisG-type and whether the change in frequency was significant within the flea beetle season. I found that the frequency of resistant beetles varied significantly among years, but there was no evidence for a decrease in the frequency of resistant beetles, the latter being expected if selection acts against the resistance on other host plants than B. vulgarisG-type. Furthermore, I found that the frequency of resistant beetles varied significantly within a flea beetle season. This study demonstrates that relative frequencies of different resistance phenotypes of P. nemorumon other host plants than B. vulgarisG-type are highly dynamic, both within and across years. It is, therefore, important to sample season-wide when one wants to monitor the changes in frequencies of insect resistance in natural systems. In CHAPTERS5 and 6 I took a population genomics approach to investigate if the observed geographicaldistribution of resistance of P. nemorumto chemically defendedB. vulgarisin flea beetle populations could be explained by factors that are solely associated with genome-wide effects, such as migration, or also by locus-specific factors like selection at the resistance locus. First, neutral microsatellites were used to reveal the genetic differentiation at parts of the genome that are only influenced by genome-wide processes. Next, the level of neutral genetic differentiation was compared with the genetic differentiation found for the resistance trait. The resistance trait was an outlier in pairwise comparisons between flea beetle populations on B. vulgarisand S. arvensis, meaning that the level of genetic differentiation was significantly higher than expected if the resistance trait experiences only genome-wide effects. The resistance trait was also an outlier in the pairwise comparison between populations on S. arvensis, which suggests that the resistance trait is also under directional selection on other host plants than B. vulgarisG-type. Additionally, I examined in CHAPTER6 if the homologous β-glucosidases B and C sequences found in CHAPTER3 correspond to two alleles of the major resistance gene, because of their similarity and their presence in flea beetle lines. The sequence of β-glucosidases C had so far only been found in resistant individuals, so we hypothesized it to be the dominant resistance allele and the sequence of β-glucosidases B would then correspond to the susceptible allele. In order to find out if this hypothesized PneR-gene (Phyllotreta nemorum R-gene) is the resistance gene, we first directly compared resistance phenotypes of beetles collected from populations on B. vulgarisG-type and S. arvensiswith genotypes derived with primers developed for β-glucosidase B and C. The phenotype of the flea beetles did not match the genotype derived with the β-glucosidase primers. Additionally, the frequency of heterozygotes and homozygotes of the PneR-gene genotype was not significantly deviating from Hardy-Weinberg Equilibrium which implies that there are no locus-specific effects involved when both sequences are seen as one gene with two alleles. A population approach was taken like in CHAPTER5, this time including the genetic differentiation estimated for the candidate gene as well. The candidate gene behaved similar to the neutral loci while the resistance trait was an outlier in most pairwise comparisons between flea beetle populations. If both sequences are alleles of the same gene, then the candidate gene is not directly responsible for the flea beetle resistance to B. vulgarisG-type defence. The results presented in this thesis show the complexity of genetic processes (either genome-wide or locus specific) affecting local adaptation and the distribution of a resistance trait in insects in natural populations. Furthermore, the present study shows that when studying coevolution between insect and host plant by means of adaptive traits, also geographical and seasonal variation in allele frequencies should be considered. A multidisciplinary approach to study adaptation in plant-insect interactions such as used in this thesis, will benefit research on plant-insect interactions, including applied research such as studying the potential of host plants as dead-end traps for pest insects and preventing/diminishing the development of resistance by pest insects to crop defences.

Plants are chemically defended against insect herbivory in various ways. They produce a broad range of secondary metabolites that may be toxic or deterrent to insects. Specialist insects, however, are often capable of overcoming these defences. The yellow striped flea beetle (Phyllotreta nemorumL.) is a specialist that feeds on crucifers (Brassicaceae) such as Sinapis arvensisand Barbarea vulgaris. In Denmark, two types of Barbarea vulgarisvar. arcuataare distinguished: one with pubescent leaves (P-type) and one with glabrous leaves (G-type). All individuals of P. nemorumcan feed on B. vulgarisP-type. Barbarea vulgarisG-type, on the other hand, is chemically defended against most P. nemorumindividuals during the flea beetle reproductive season. The defence compounds are hypothesized to be saponins, a class of compounds with various biological effects and insecticidal properties. Despite high levels of these saponins during summer, some flea beetles can and do feed on B. vulgaris G-type. The ability of P. nemorumto feed on B. vulgarisG-type is heritable; resistance against the defence of B. vulgarisG-type is controlled by dominant major resistance genes (R-genes). One dominant R-allele of an R-gene is enough to convert a susceptible beetle into a resistant one. Despite knowledge of the inheritance patterns of resistance in the flea beetles, which have been demonstrated to be variable, the underlying mechanism of flea beetle resistance has, so far, remained unclear. This prompted me to investigate, as an initial part of my thesis, the genetic basis of the flea beetle adaptation to the defence of B. vulgarisG-type. The interaction between B. vulgarisand the flea beetle is a unique natural model system to study chemical defences in plants and counter-adaptations in insects. Plant and insect are both polymorphic with respect to the trait involved in resistance and hereby provide an excellent opportunity to study the geographic aspects of the evolution of the resistance trait in both interacting species. In this thesis, I focus on the resistance of the flea beetle, and take the presence of different genotypes of the plant as a given. Phyllotreta nemorumis a major pest, for example in oil seed rape. Understanding how resistance evolves in P. nemorumwill not only benefit flea beetle control, but also control of other pest insects. Understanding insect resistance includes knowledge of seasonal, geographic and genetic variation in both plant defense and herbivore adaptation.The R-gene has a remarkable distribution. Flea beetle populations living on B. vulgarisG-type consist solely of resistant individuals, but on host plant patches nearby B. vulgarisG-type lower frequencies of resistant beetles are found than one would expect with the amount of gene flow found at the neutral level between these subpopulations. The aim of this thesis was to find the gene that is held responsible for the resistance of P. nemorumto the defences of B. vulgaris, investigate the distribution of this resistance trait and explain the distribution of this trait in natural populations. The following questions were addressed: (1) what is the genetic basis of the adaptation under study? (2) how is the resistance distributed across flea beetle populations in Denmark? and (3) which factors underlie this distribution? In order to answer these questions, I used an integrated approach. I have combined a candidate gene approach (CHAPTER3) with an empirical approach via the study of variation in resistance in flea beetle populations (CHAPTER4), and a population genomics approach by using molecular markers to gain insight in the genomic make-up of the population and its connection with the resistance trait (CHAPTERS5 and 6). The population genomics approach is a recent advance in methods to detect the involvement of selection in the distribution of alleles at presumably adaptive loci. Using this approach one can distinguish locus-specific effects, like directional selection, from genome-wide effects, on the distribution of alleles at loci of interest. The population genomics approach is introduced in CHAPTER2 together with the Geographic Mosaic Theory of Coevolution. I illustrate how processes underlying this theory of coevolution can be investigated with the population genomics approach. According to the geographic mosaic theory of coevolution, reciprocal selection between interacting species only happens in so-called hot-spots. Hot spots can be identified using population genomics and genetic variation found at specific loci can be attributed to locus-specific processes such as directional selection. For the B. vulgaris- flea beetle system this means that with a population genomics approach we can examine whether the distribution of resistant flea beetles on alternative host plants is only influenced by migration, or also by selection (CHAPTER5). Another valuable utility of the population genomics approach is to investigate whether a candidate gene for the R-gene is under selection, by looking whether a candidate gene is experiencing locus-specific effects beside genome-wide effects when comparing flea beetle populations living on B. vulgarisG-type with populations living on alternative host plants (CHAPTER6). However, before using a population genomics approach to compare the resistance trait or a candidate gene with parts of the genome that only experience genome-wide effects, I have tried to identify the genetic basis of the flea beetle adaptation to the defence ofB. vulgarisG-type. In CHAPTER3, I have addressed this question by using a candidate gene approach to examine the involvement of a possible detoxifying enzyme in P. nemorum. Genes coding for β-glucosidase were a candidate for genes underlying the difference between resistant and susceptible beetles, because β-glucosidase is used as detoxifying enzyme by other organisms resistant to saponin defence. Three different β-glucosidase cDNA sequences were cloned from Danish flea beetle lines. We named them β-glucosidase A, B and C. β-glucosidase C was only found in resistant lines and not in the susceptible line. We then tested if recombinant β-glucosidase C breaks down the most abundant and most effective defence compound in B. vulgarisG-type, hederagenin cellobioside. β-glucosidase C was able to deglycosylate one glucose unit of hederagenin cellobioside, when expressed in an insect cell line. This suggests that expressed β-glucosidase C can deglycosylate antifeedant saponins and may play a role in the resistant flea beetle’s ability to overcome the defence of B. vulgaris. Next, a segregating family was created in which offspring differed in resistance genotype. Again β-glucosidase cDNA sequences were cloned to find a difference in the presence of these β-glucosidases between resistant and susceptible individuals. This time cDNA sequences of β-glucosidases A, B and C were present in both resistant and susceptible individuals although significantly fewer β-glucosidase C cDNA sequence variants were found in susceptible individuals than in resistant individuals. Thus, the genetic basis of flea beetle resistance remains unclear. Further investigation is needed to explore if the β-glucosidase C protein is also capable of inactivating hederagenin cellobioside by hydrolysizing the second glucose unit from the saponin and if there is a difference in enzyme activity of β-glucosidase C between resistant and susceptible beetles. Subsequently, in CHAPTER4 I have investigated whether the frequency of resistant beetles decreased in populations living on other host plant patches than B. vulgarisG-type and whether the change in frequency was significant within the flea beetle season. I found that the frequency of resistant beetles varied significantly among years, but there was no evidence for a decrease in the frequency of resistant beetles, the latter being expected if selection acts against the resistance on other host plants than B. vulgarisG-type. Furthermore, I found that the frequency of resistant beetles varied significantly within a flea beetle season. This study demonstrates that relative frequencies of different resistance phenotypes of P. nemorumon other host plants than B. vulgarisG-type are highly dynamic, both within and across years. It is, therefore, important to sample season-wide when one wants to monitor the changes in frequencies of insect resistance in natural systems. In CHAPTERS5 and 6 I took a population genomics approach to investigate if the observed geographicaldistribution of resistance of P. nemorumto chemically defendedB. vulgarisin flea beetle populations could be explained by factors that are solely associated with genome-wide effects, such as migration, or also by locus-specific factors like selection at the resistance locus. First, neutral microsatellites were used to reveal the genetic differentiation at parts of the genome that are only influenced by genome-wide processes. Next, the level of neutral genetic differentiation was compared with the genetic differentiation found for the resistance trait. The resistance trait was an outlier in pairwise comparisons between flea beetle populations on B. vulgarisand S. arvensis, meaning that the level of genetic differentiation was significantly higher than expected if the resistance trait experiences only genome-wide effects. The resistance trait was also an outlier in the pairwise comparison between populations on S. arvensis, which suggests that the resistance trait is also under directional selection on other host plants than B. vulgarisG-type. Additionally, I examined in CHAPTER6 if the homologous β-glucosidases B and C sequences found in CHAPTER3 correspond to two alleles of the major resistance gene, because of their similarity and their presence in flea beetle lines. The sequence of β-glucosidases C had so far only been found in resistant individuals, so we hypothesized it to be the dominant resistance allele and the sequence of β-glucosidases B would then correspond to the susceptible allele. In order to find out if this hypothesized PneR-gene (Phyllotreta nemorum R-gene) is the resistance gene, we first directly compared resistance phenotypes of beetles collected from populations on B. vulgarisG-type and S. arvensiswith genotypes derived with primers developed for β-glucosidase B and C. The phenotype of the flea beetles did not match the genotype derived with the β-glucosidase primers. Additionally, the frequency of heterozygotes and homozygotes of the PneR-gene genotype was not significantly deviating from Hardy-Weinberg Equilibrium which implies that there are no locus-specific effects involved when both sequences are seen as one gene with two alleles. A population approach was taken like in CHAPTER5, this time including the genetic differentiation estimated for the candidate gene as well. The candidate gene behaved similar to the neutral loci while the resistance trait was an outlier in most pairwise comparisons between flea beetle populations. If both sequences are alleles of the same gene, then the candidate gene is not directly responsible for the flea beetle resistance to B. vulgarisG-type defence. The results presented in this thesis show the complexity of genetic processes (either genome-wide or locus specific) affecting local adaptation and the distribution of a resistance trait in insects in natural populations. Furthermore, the present study shows that when studying coevolution between insect and host plant by means of adaptive traits, also geographical and seasonal variation in allele frequencies should be considered. A multidisciplinary approach to study adaptation in plant-insect interactions such as used in this thesis, will benefit research on plant-insect interactions, including applied research such as studying the potential of host plants as dead-end traps for pest insects and preventing/diminishing the development of resistance by pest insects to crop defences.

This thesis aimed to elucidate the evolutionary history of the associations between Cymothoeforest butterflies (Nymphalidae, Limenitidinae) and their Rinoreahost plants (Violaceae) in tropical Africa. Insects are by far the most diverse group of multicellular organisms on earth. Because most insect species are herbivores, understanding the evolution of interactions between herbivorous insects and their host plants is therefore crucial to comprehend global patterns in terrestrial biodiversity. The Cymothoe-Rinoreasystem is especially suitable for untangling processes shaping patterns of insect-host plant associations because of its high level of specificity (mostly monophagous) and the large number of related species involved (33 insect herbivores and 32 hosts). Obviously, any evolutionary study relies on a solid classification and taxonomy of the organisms under study. Unfortunately, however, in Cymothoeas well as Rinorea, taxonomy and classification is still partly unresolved. To improve taxonomy of Cymothoeand facilitate efficient identification of immature specimens found on Rinoreahost plants, we generated an extensive dataset of 1204 DNA barcode sequences (Chapter 2). Application of a novel taxonomic decision pipeline for integrating DNA barcodes with morphology and biogeography proved instrumental for solving taxonomic problems in Cymothoeand five taxa within Cymothoecould be confidently raised to species level. In addition, our DNA barcode data set allowed for the identification of 42 immature specimens from six different countries, significantly increasing the data on Cymothoehost plant associations. Nevertheless, our results also demonstrated that not all species of Cymothoecan be confidently delimited or identified. We hypothesize that this is probably due to incomplete lineage sorting and introgression (the latter possibly mediated through Wolbachiaendosymbionts) between recently diverged Cymothoespecies. In order to assess what are the best methods for matching DNA barcodes from recently diverged species, we compared six methods in their ability to correctly match DNA barcodes from selected published empirical data sets as well as simulated data (Chapter 3). Our results showed that, even though recently diverged species pose a significant problem for effective DNA barcoding, sensitive similarity-based and diagnostic methods can significantly improve identification performance compared with the commonly used tree-based methods. To improve classification and clarify the biogeographic history of Rinorea, we presented an updated phylogenetic tree of Rinoreawith increased taxonomic sampling, using plastid as well as nuclear DNA sequences (Chapter 5). Phylogenetic relationships inferred from nuclear DNA data were generally congruent with those based on evidence from plastid haplotypes from earlier studies of Rinoreaand helped resolve additional clades, some of which warrant further taxonomic study. Divergence time estimations indicated that Rinoreaoriginated in the Neotropics and reached Africa in the Eocene through trans-Atlantic dispersal. From Africa, Rinoreasubsequently dispersed into Asia in the Oligocene or early Miocene, and colonized Madagascar multiple times independently within a relatively recent time scale (Pliocene), suggesting that factors governing the independent colonizations of Rinoreato Madagascar may have been similar. In Chapter 4 we assessed whether differential rates of net species diversification in the African butterfly sister genera Harma(1 species) and Cymothoe(approximately 82 species) could best be explained by shifts to novel host plants (from Achariaceae to Rinorea) or by environmental factors such as changing climate. We generated the first time-calibrated species-level molecular phylogenetic tree of Harmaand Cymothoeand found that, after their divergence in the Miocene (15 Mya), net species diversification was low during the first 7 Myr. Coinciding with the onset of diversification of Cymothoein the late Miocene (around 7.5 Mya) there was a sharp and significant increase in diversification rate, suggesting a rapid radiation. This increased rate did not correlate with host plant transition from Achariaceae to Rinoreahost plants, but rather with a period of global cooling and desiccation, indicating that tropical forest fragmentation may well have driven the elevated diversification rates in Cymothoe. Finally, in Chapter 6 we integrated the time-calibrated phylogenetic evidence from Cymothoeand Rinoreapresented in chapters 4 and 5 with updated host association records from the field, with the aim to distinguish between alternative scenarios for the evolution of insect-host plant associations. Our results showed that: (i) divergences among extant Cymothoeare more recent than those among their associated Rinoreahosts, suggesting asynchronous diversification of Cymothoeherbivores onto already diversified clades within African Rinorea; (ii) phylogenetic trees of Cymothoeand their associated Rinoreahost plants are discordant and current associations between Cymothoeherbivores and their Rinoreahosts have developed primarily through a process of host shifting rather than by cospeciation; and (iii) related Cymothoetend to feed on related Rinoreahosts. Based on the available data, we propose a recent origin of Rinorea{}-feeding by Cymothoebutterflies with a single colonization of pre-existing lineages in the late Miocene. Current associations are best explained by a predominance of shifts among related plants, probably due to constraints in larval physiology and oviposition behaviour. Overall, these findings are in agreement with a growing body of substantial evidence to suggest that divergences of herbivorous insects and their host plants are asynchronous, and that evolutionary dynamics of hosts and parasites do not favour cospeciation. Insect-plant interactions are receiving increasing attention because of their importance in crop production and protection. At the same time, an increasing number of insects and plants that have evolved in separation are currently coming into contact due to human activities and climatic changes. It is therefore tempting to find implications of our findings for insect-host plant associations for agricultural systems (Chapter 7). Based on our results, one might predict that insects will only become pests of crops that are closely related to their natural host. Extrapolating our findings to an agricultural setting is difficult, however, because of the difference in selective pressures between natural and agricultural ecosystems.

This thesis aimed to elucidate the evolutionary history of the associations between Cymothoeforest butterflies (Nymphalidae, Limenitidinae) and their Rinoreahost plants (Violaceae) in tropical Africa. Insects are by far the most diverse group of multicellular organisms on earth. Because most insect species are herbivores, understanding the evolution of interactions between herbivorous insects and their host plants is therefore crucial to comprehend global patterns in terrestrial biodiversity. The Cymothoe-Rinoreasystem is especially suitable for untangling processes shaping patterns of insect-host plant associations because of its high level of specificity (mostly monophagous) and the large number of related species involved (33 insect herbivores and 32 hosts). Obviously, any evolutionary study relies on a solid classification and taxonomy of the organisms under study. Unfortunately, however, in Cymothoeas well as Rinorea, taxonomy and classification is still partly unresolved. To improve taxonomy of Cymothoeand facilitate efficient identification of immature specimens found on Rinoreahost plants, we generated an extensive dataset of 1204 DNA barcode sequences (Chapter 2). Application of a novel taxonomic decision pipeline for integrating DNA barcodes with morphology and biogeography proved instrumental for solving taxonomic problems in Cymothoeand five taxa within Cymothoecould be confidently raised to species level. In addition, our DNA barcode data set allowed for the identification of 42 immature specimens from six different countries, significantly increasing the data on Cymothoehost plant associations. Nevertheless, our results also demonstrated that not all species of Cymothoecan be confidently delimited or identified. We hypothesize that this is probably due to incomplete lineage sorting and introgression (the latter possibly mediated through Wolbachiaendosymbionts) between recently diverged Cymothoespecies. In order to assess what are the best methods for matching DNA barcodes from recently diverged species, we compared six methods in their ability to correctly match DNA barcodes from selected published empirical data sets as well as simulated data (Chapter 3). Our results showed that, even though recently diverged species pose a significant problem for effective DNA barcoding, sensitive similarity-based and diagnostic methods can significantly improve identification performance compared with the commonly used tree-based methods. To improve classification and clarify the biogeographic history of Rinorea, we presented an updated phylogenetic tree of Rinoreawith increased taxonomic sampling, using plastid as well as nuclear DNA sequences (Chapter 5). Phylogenetic relationships inferred from nuclear DNA data were generally congruent with those based on evidence from plastid haplotypes from earlier studies of Rinoreaand helped resolve additional clades, some of which warrant further taxonomic study. Divergence time estimations indicated that Rinoreaoriginated in the Neotropics and reached Africa in the Eocene through trans-Atlantic dispersal. From Africa, Rinoreasubsequently dispersed into Asia in the Oligocene or early Miocene, and colonized Madagascar multiple times independently within a relatively recent time scale (Pliocene), suggesting that factors governing the independent colonizations of Rinoreato Madagascar may have been similar. In Chapter 4 we assessed whether differential rates of net species diversification in the African butterfly sister genera Harma(1 species) and Cymothoe(approximately 82 species) could best be explained by shifts to novel host plants (from Achariaceae to Rinorea) or by environmental factors such as changing climate. We generated the first time-calibrated species-level molecular phylogenetic tree of Harmaand Cymothoeand found that, after their divergence in the Miocene (15 Mya), net species diversification was low during the first 7 Myr. Coinciding with the onset of diversification of Cymothoein the late Miocene (around 7.5 Mya) there was a sharp and significant increase in diversification rate, suggesting a rapid radiation. This increased rate did not correlate with host plant transition from Achariaceae to Rinoreahost plants, but rather with a period of global cooling and desiccation, indicating that tropical forest fragmentation may well have driven the elevated diversification rates in Cymothoe. Finally, in Chapter 6 we integrated the time-calibrated phylogenetic evidence from Cymothoeand Rinoreapresented in chapters 4 and 5 with updated host association records from the field, with the aim to distinguish between alternative scenarios for the evolution of insect-host plant associations. Our results showed that: (i) divergences among extant Cymothoeare more recent than those among their associated Rinoreahosts, suggesting asynchronous diversification of Cymothoeherbivores onto already diversified clades within African Rinorea; (ii) phylogenetic trees of Cymothoeand their associated Rinoreahost plants are discordant and current associations between Cymothoeherbivores and their Rinoreahosts have developed primarily through a process of host shifting rather than by cospeciation; and (iii) related Cymothoetend to feed on related Rinoreahosts. Based on the available data, we propose a recent origin of Rinorea{}-feeding by Cymothoebutterflies with a single colonization of pre-existing lineages in the late Miocene. Current associations are best explained by a predominance of shifts among related plants, probably due to constraints in larval physiology and oviposition behaviour. Overall, these findings are in agreement with a growing body of substantial evidence to suggest that divergences of herbivorous insects and their host plants are asynchronous, and that evolutionary dynamics of hosts and parasites do not favour cospeciation. Insect-plant interactions are receiving increasing attention because of their importance in crop production and protection. At the same time, an increasing number of insects and plants that have evolved in separation are currently coming into contact due to human activities and climatic changes. It is therefore tempting to find implications of our findings for insect-host plant associations for agricultural systems (Chapter 7). Based on our results, one might predict that insects will only become pests of crops that are closely related to their natural host. Extrapolating our findings to an agricultural setting is difficult, however, because of the difference in selective pressures between natural and agricultural ecosystems.

Sedentary plant parasitic nematodes have evolved sophisticated strategies that allow them to transform host cells in the roots of host plants into feeding structures. These complex structures enable the nematodes to complete their life cycle inside a single host plant. Feeding structure initiation and maintenance are thought to be determined by the concerted action of effectors produced by the esophageal glands of the nematodes. However, the molecular mechanisms underlying the transformation of host cells into feeding structures and the role of the effectors in this process are poorly understood. For example, it is generally thought that virulent nematodes also use effectors to protect their complex feeding structures from plant innate immune responses. However, nematode effectors suppressing plant innate immunity have not been identified to date. The host ranges of sedentary plant parasitic nematodes can vary from very wide (e.g. root-knot nematodes) to relatively narrow, limited to single plant families (e.g. cyst nematodes). Potato cyst nematodes (PCN) are able to parasitize only Solanaceous plants. Every year, they cause substantial yield losses in potato production areas. PCN are very difficult to control by the use of cultivation methods or the application of pesticides alone. The introduction of novel nematode resistant potato cultivars to the market is therefore of great importance for potato growers all over the world. Resistances, however, can be overcome by the emergence of virulent nematode populations. The aim of this thesis is to study incompatible interactions between the potato cyst nematode Globodera rostochiensis and potato (Solanum tuberosum), by analyzing resistance loci that make the plant immune to potato cyst nematodes as well as nematode effectors that suppress plant innate immunity. This thesis begins with an extensive review of the literature on molecular and cellular aspects of plant resistance to sedentary endoparasitic nematodes, including pre-infectional, non-host and host resistance (Chapter 2). Most research in this field has focused on host resistance to nematodes, which is determined by single (e.g.H1 and Gpa2) or multiple (e.g. GpaVSsplandGpaXISspl) resistance gene loci. Two potato cyst nematode resistance loci were studied in this thesis (i.e. Grp1 in Chapter 3 and H1 in Chapter 4) and the results of these studies are summarized below. Grp1 locus confers broad-spectrum quantitative resistance to the potato cyst nematode species Globodera pallida and G. rostochiensis in potato. It was previously mapped to a 3 cM interval on the short arm of potato chromosome V between the markers GP21 and GP179 in a hot spot for resistance (Rouppe Van Der Voort et al., 1998). The aim of the work described in chapter 3 was to fine map the Grp1 locus. First, a diploid mapping population RHAM026, comprising 1536 genotypes was screened with the flanking markers GP21 and GP179, resulting in the identification of 61 recombinants in this region. Next, thirteen new markers were developed using the genomic sequence information available from the same region of Solanum demissum. Together with markers available from the literature, these thirteen markers were used to screen a subset of 54 recombinants. Finally, these recombinants were tested for resistance to G. pallida Pa2 and G. rostochiensis Ro5. This mapping of both resistance specificities resulted in two nearly identical LOD graphs with the highest score just north of marker TG432. We conclude that the resistances to both G. pallida and G. rostochiensis map to the same 1.08 cM interval between the markers SPUD838 and TG432. Other studies have revealed that this locus in potato harbors several gene clusters encoding classical NB-ARC-LRR resistance proteins. This finding led us to the hypothesis that the Grp1 resistance depends on one, or perhaps several tightly linked major genes. Near-absolute resistance to G. rostochiensis pathotypes 1 and 4 is conferred by the H1 resistance locus at the distal end of chromosome V of the diploid S. tuberosum ssp. andigena genotype SH83-92-488 (SH). The H1 resistance involves a hypersensitive response in the cells surrounding the nematode feeding structure, so that it becomes isolated from the vascular tissues in the host. A high-resolution map of H1 locus was generated previously using SHxRH mapping population (Bakker et al., 2004). In chapter 4, we used markers from thismap to screen a BAC library of SH. The BAC inserts identified with the markers were used to construct a physical map covering this region in the resistant haplotype. Further sequencing of the BAC inserts, included in the physical map, revealed a genomic fragment of 341 Kb harboring a large cluster of CC-NB-ARC-LRR genes. We compared this cluster of resistance gene homologs with the sequences of the corresponding regions in the two susceptible haplotypes from the diploid genotype RH89-039-16 (S. tuberosum ssp. tuberosum/ S. phureja), spanning 700 and 319 Kb respectively. The genomic regions in all three haplotypes harbor from 17 up to 23 resistance gene homologs interspersed with numerous transposable elements, genes coding for extensin-like proteins, and an amino acid transporter. Strikingly, the three haplotypes do not reveal gene order conservation and the overall sequence homology is only confined to the coding sequences of the resistance gene homologs. These findings suggest that extensive rearrangements have shaped the H1 locus. Sequence data and marker information gained from this study will benefit future efforts to clone the H1 nematode resistance gene. At the start of the research described in this thesis no suppressor of plant immune response had been found in plant parasitic nematodes. In chapter 5, we report the first identification and functional characterization of a G. rostochiensis effector suppressing plant innate immune responses. The Nematode Suppressors of Immunity 1 (NSI-1) are specifically expressed in the dorsal esophageal gland of the nematodes, and their expression is upregulated in stages that feed on host cells. We identified many variants of NSI-1 in the Ro1-Mierenbos field population, and showed that this gene family is under diversifying selection. Knocking-down NSI-1 transcription by RNA interference strongly reduced the number of nematodes developing into full-grown cysts. Overexpression of four NSI-1 variants in susceptible potato plants resulted in enhanced susceptibility to nematodes. Moreover, overexpression of three other variants enhanced the susceptibility of potato plants to the fungus Verticillium dahliae. Down-regulation of the potato homologs of the Arabidopsis thaliana transcription factors WRKY22 and WRKY53 in these plants indicated that NSI-1 target immune signaling in plants. In an agroinfiltration assay in leaves of Nicotiana benthamiana several NSI-1 variants suppressed the hypersensitive response caused by the co-expression of specific resistance proteins and matching pathogen effectors (i.e. RBP-1/Gpa2 and AvrBlb2/Rpi-blb2) and by autoactive mutants of the resistance protein Mi-1.2 and an H1 resistance gene homolog RGH10-H1. Interestingly, other NSI-1 effector variant suppressed the hypersensitivity response induced by an autoactive mutant of the immune signaling protein NRC1. These findings altogether lead to the conclusion that potato cyst nematodes secrete suppressors of plant immunity, most likely to protect their feeding structures. In the final chapter of this thesis, we discuss our most important findings within the broader context of recent developments in the field of molecular plant-microbe interactions. First, we argue that quantitative nematode resistance Grp1 is encoded by one or more NB-ARC-LRR genes located in one of the resistance gene clusters mapped to the GP21-GP179 interval on potato chromosome V. We further examine obstacles and offer possible solutions with regard to future cloning of the H1 nematode resistance gene. Lastly, we elaborate on possible functions, activities, and evolution of NSI-1 effectors as suppressors of plant innate immunity.

Sedentary plant parasitic nematodes have evolved sophisticated strategies that allow them to transform host cells in the roots of host plants into feeding structures. These complex structures enable the nematodes to complete their life cycle inside a single host plant. Feeding structure initiation and maintenance are thought to be determined by the concerted action of effectors produced by the esophageal glands of the nematodes. However, the molecular mechanisms underlying the transformation of host cells into feeding structures and the role of the effectors in this process are poorly understood. For example, it is generally thought that virulent nematodes also use effectors to protect their complex feeding structures from plant innate immune responses. However, nematode effectors suppressing plant innate immunity have not been identified to date. The host ranges of sedentary plant parasitic nematodes can vary from very wide (e.g. root-knot nematodes) to relatively narrow, limited to single plant families (e.g. cyst nematodes). Potato cyst nematodes (PCN) are able to parasitize only Solanaceous plants. Every year, they cause substantial yield losses in potato production areas. PCN are very difficult to control by the use of cultivation methods or the application of pesticides alone. The introduction of novel nematode resistant potato cultivars to the market is therefore of great importance for potato growers all over the world. Resistances, however, can be overcome by the emergence of virulent nematode populations. The aim of this thesis is to study incompatible interactions between the potato cyst nematode Globodera rostochiensis and potato (Solanum tuberosum), by analyzing resistance loci that make the plant immune to potato cyst nematodes as well as nematode effectors that suppress plant innate immunity. This thesis begins with an extensive review of the literature on molecular and cellular aspects of plant resistance to sedentary endoparasitic nematodes, including pre-infectional, non-host and host resistance (Chapter 2). Most research in this field has focused on host resistance to nematodes, which is determined by single (e.g.H1 and Gpa2) or multiple (e.g. GpaVSsplandGpaXISspl) resistance gene loci. Two potato cyst nematode resistance loci were studied in this thesis (i.e. Grp1 in Chapter 3 and H1 in Chapter 4) and the results of these studies are summarized below. Grp1 locus confers broad-spectrum quantitative resistance to the potato cyst nematode species Globodera pallida and G. rostochiensis in potato. It was previously mapped to a 3 cM interval on the short arm of potato chromosome V between the markers GP21 and GP179 in a hot spot for resistance (Rouppe Van Der Voort et al., 1998). The aim of the work described in chapter 3 was to fine map the Grp1 locus. First, a diploid mapping population RHAM026, comprising 1536 genotypes was screened with the flanking markers GP21 and GP179, resulting in the identification of 61 recombinants in this region. Next, thirteen new markers were developed using the genomic sequence information available from the same region of Solanum demissum. Together with markers available from the literature, these thirteen markers were used to screen a subset of 54 recombinants. Finally, these recombinants were tested for resistance to G. pallida Pa2 and G. rostochiensis Ro5. This mapping of both resistance specificities resulted in two nearly identical LOD graphs with the highest score just north of marker TG432. We conclude that the resistances to both G. pallida and G. rostochiensis map to the same 1.08 cM interval between the markers SPUD838 and TG432. Other studies have revealed that this locus in potato harbors several gene clusters encoding classical NB-ARC-LRR resistance proteins. This finding led us to the hypothesis that the Grp1 resistance depends on one, or perhaps several tightly linked major genes. Near-absolute resistance to G. rostochiensis pathotypes 1 and 4 is conferred by the H1 resistance locus at the distal end of chromosome V of the diploid S. tuberosum ssp. andigena genotype SH83-92-488 (SH). The H1 resistance involves a hypersensitive response in the cells surrounding the nematode feeding structure, so that it becomes isolated from the vascular tissues in the host. A high-resolution map of H1 locus was generated previously using SHxRH mapping population (Bakker et al., 2004). In chapter 4, we used markers from thismap to screen a BAC library of SH. The BAC inserts identified with the markers were used to construct a physical map covering this region in the resistant haplotype. Further sequencing of the BAC inserts, included in the physical map, revealed a genomic fragment of 341 Kb harboring a large cluster of CC-NB-ARC-LRR genes. We compared this cluster of resistance gene homologs with the sequences of the corresponding regions in the two susceptible haplotypes from the diploid genotype RH89-039-16 (S. tuberosum ssp. tuberosum/ S. phureja), spanning 700 and 319 Kb respectively. The genomic regions in all three haplotypes harbor from 17 up to 23 resistance gene homologs interspersed with numerous transposable elements, genes coding for extensin-like proteins, and an amino acid transporter. Strikingly, the three haplotypes do not reveal gene order conservation and the overall sequence homology is only confined to the coding sequences of the resistance gene homologs. These findings suggest that extensive rearrangements have shaped the H1 locus. Sequence data and marker information gained from this study will benefit future efforts to clone the H1 nematode resistance gene. At the start of the research described in this thesis no suppressor of plant immune response had been found in plant parasitic nematodes. In chapter 5, we report the first identification and functional characterization of a G. rostochiensis effector suppressing plant innate immune responses. The Nematode Suppressors of Immunity 1 (NSI-1) are specifically expressed in the dorsal esophageal gland of the nematodes, and their expression is upregulated in stages that feed on host cells. We identified many variants of NSI-1 in the Ro1-Mierenbos field population, and showed that this gene family is under diversifying selection. Knocking-down NSI-1 transcription by RNA interference strongly reduced the number of nematodes developing into full-grown cysts. Overexpression of four NSI-1 variants in susceptible potato plants resulted in enhanced susceptibility to nematodes. Moreover, overexpression of three other variants enhanced the susceptibility of potato plants to the fungus Verticillium dahliae. Down-regulation of the potato homologs of the Arabidopsis thaliana transcription factors WRKY22 and WRKY53 in these plants indicated that NSI-1 target immune signaling in plants. In an agroinfiltration assay in leaves of Nicotiana benthamiana several NSI-1 variants suppressed the hypersensitive response caused by the co-expression of specific resistance proteins and matching pathogen effectors (i.e. RBP-1/Gpa2 and AvrBlb2/Rpi-blb2) and by autoactive mutants of the resistance protein Mi-1.2 and an H1 resistance gene homolog RGH10-H1. Interestingly, other NSI-1 effector variant suppressed the hypersensitivity response induced by an autoactive mutant of the immune signaling protein NRC1. These findings altogether lead to the conclusion that potato cyst nematodes secrete suppressors of plant immunity, most likely to protect their feeding structures. In the final chapter of this thesis, we discuss our most important findings within the broader context of recent developments in the field of molecular plant-microbe interactions. First, we argue that quantitative nematode resistance Grp1 is encoded by one or more NB-ARC-LRR genes located in one of the resistance gene clusters mapped to the GP21-GP179 interval on potato chromosome V. We further examine obstacles and offer possible solutions with regard to future cloning of the H1 nematode resistance gene. Lastly, we elaborate on possible functions, activities, and evolution of NSI-1 effectors as suppressors of plant innate immunity.

Potato is consumed worldwide and represents the fourth most important staple food crop after rice and wheat. Potato cultivars display a large variety of color, shape, taste, cooking properties and starch content but are all derived from the same species; Solanum tuberosum. Potato breeding is an economic important activity for international breeding companies, but also plays an important role in breaking the circle of poverty for small farmers. In the Andean region, most farmers use many different potato genotypes combined with farming practices transmitted orally over thousands of years. The most prominent menace to potato production is Late Blight caused by the oomycete Phytophthora infestans which destroys leaves, stems and tubers. Differences of breeding methods between the potato grown in South America and in the rest of the world is related to differences in the consequences of Late Blight infection. In the 19th, century, entire potato fields in Ireland were devastated while in South America P. infestans proliferation was readily inhibited. This difference is found in the biodiversity reserve such as that of the Chiloé archipelago in Chile where local people cultivate about 200 varieties of native potato. Obviously, the genetic diversity of cultivated native potato acts as a shield against this versatile pathogen. Inspired by this model to solve the problems raised by the extensive use of potato monoculture, growers and breeders need to maintain genetic diversity in the European staple food crops. In exploring the South American native potato collection, Solanum demissum and later on Solanum bulbocastanum appeared to be a source of resistance genes (Rpi) to P. infestans. The S. demissum Rpi genes were transmitted to potato breeding clones by traditional introgression breeding. However the fading of their ability in providing effective resistance against Late Blight infection was witnessed within a decade. In the pursuit to provide a hopefully more durable protection in existing potato cultivars, plant breeding scientists proposed to directly introduce South American native potato Rpi genes in modern potato varieties by using a so-called cisgenic approach. This in contrast to transgenic plants which can contain genes which have originated from non related genera or even different kingdoms. Breeding of cisgenic plants is on its way to public acceptance because of its inherent resemblance to natural crossing and because efforts are made by the scientific community to explain the principles of cisgenesis. Lessons were learned from the flexibility of P. infestans to overcome the effect of newly introduced Rpi genes and, therefore, efforts are still ongoing to discover and clone new Rpi genes from native potatoes. With this in mind, a new family of Rpi genes represented by Rpi-blb3, Rpi-abpt, R2, R2-like and Rpi-mcd1.1 were characterized in clones derived from S. bulbocastanum, S. demissum, S. edinense and S. microdontum. We accomplished in this research the physical isolation of these genes, the molecular characterization of their functionality and the allelic distribution in the Petota collection. Rpi-blb3, Rpi-abpt, R2, R2-like and Rpi-mcd1.1 belong to the potato linkage group IV and all contain signature sequences characteristic of LZ-NBS-LRR proteins. The closest known R gene so far is RPP13 from Arabidopsis thaliana which shares an amino-acid sequence similarity of 35%. The LRR domains of Rpi-blb3, Rpi-abpt, R2 and R2-like proteins are highly homologous, whilst LZ and NBS domains are more polymorphic with those of R2 being the most divergent. All four Rpi genes recognize the recently identified RXLR effector protein PiAVR2 which is secreted by P.infestans in the cytoplasm of plant cells during the infection process. Unlike Rpi-blb3, Rpi-abpt, R2 and R2-like , the S. microdontum resistance gene Rpi-mcd1.1 does not interact with PiAVR2 and provides a different resistance spectrum. Rpi-mcd1.1 shares 90% nucleotide identity with Rpi-blb3 and polymorphic nucleotides are mainly located in the LRR region. The S. bulbocastanum haplotypes of Rpi-blb1, Rpi-blb2 and Rpi-blb3 were discovered in several Mexican diploid as well as polyploid species closely related to S. bulbocastanum. These three resistance genes occurred in different combinations and frequencies in S. bulbocastanum accessions and their distribution is confined to Central America. A selected set of genotypes was tested for their response to the avirulence effectors IPIO-2, Avr-blb2 and Pi-Avr2, which interact with Rpi-blb1, Rpi-blb2 and Rpi-blb3, respectively, as well as by disease assays with a diverse set of isolates. Using this approach some accessions could be identified that contain novel, yet unknown, Late Blight resistance factors in addition to the Rpi-blb1, Rpi-blb2 and Rpi-blb3 genes Analysis of the sequences obtained in different allele mining strategies suggests an evolution of the major late blight locus on linkage group IV through recombination and point mutations. By making use of the sequence information provided by the alleles, we identified the repeats and amino acids in the LRR domain which are specific for PiAVR2 recognition. Finally, we discussed the results described in this thesis in a potato/ P. infestans co-evolution context.

Potato is consumed worldwide and represents the fourth most important staple food crop after rice and wheat. Potato cultivars display a large variety of color, shape, taste, cooking properties and starch content but are all derived from the same species; Solanum tuberosum. Potato breeding is an economic important activity for international breeding companies, but also plays an important role in breaking the circle of poverty for small farmers. In the Andean region, most farmers use many different potato genotypes combined with farming practices transmitted orally over thousands of years. The most prominent menace to potato production is Late Blight caused by the oomycete Phytophthora infestans which destroys leaves, stems and tubers. Differences of breeding methods between the potato grown in South America and in the rest of the world is related to differences in the consequences of Late Blight infection. In the 19th, century, entire potato fields in Ireland were devastated while in South America P. infestans proliferation was readily inhibited. This difference is found in the biodiversity reserve such as that of the Chiloé archipelago in Chile where local people cultivate about 200 varieties of native potato. Obviously, the genetic diversity of cultivated native potato acts as a shield against this versatile pathogen. Inspired by this model to solve the problems raised by the extensive use of potato monoculture, growers and breeders need to maintain genetic diversity in the European staple food crops. In exploring the South American native potato collection, Solanum demissum and later on Solanum bulbocastanum appeared to be a source of resistance genes (Rpi) to P. infestans. The S. demissum Rpi genes were transmitted to potato breeding clones by traditional introgression breeding. However the fading of their ability in providing effective resistance against Late Blight infection was witnessed within a decade. In the pursuit to provide a hopefully more durable protection in existing potato cultivars, plant breeding scientists proposed to directly introduce South American native potato Rpi genes in modern potato varieties by using a so-called cisgenic approach. This in contrast to transgenic plants which can contain genes which have originated from non related genera or even different kingdoms. Breeding of cisgenic plants is on its way to public acceptance because of its inherent resemblance to natural crossing and because efforts are made by the scientific community to explain the principles of cisgenesis. Lessons were learned from the flexibility of P. infestans to overcome the effect of newly introduced Rpi genes and, therefore, efforts are still ongoing to discover and clone new Rpi genes from native potatoes. With this in mind, a new family of Rpi genes represented by Rpi-blb3, Rpi-abpt, R2, R2-like and Rpi-mcd1.1 were characterized in clones derived from S. bulbocastanum, S. demissum, S. edinense and S. microdontum. We accomplished in this research the physical isolation of these genes, the molecular characterization of their functionality and the allelic distribution in the Petota collection. Rpi-blb3, Rpi-abpt, R2, R2-like and Rpi-mcd1.1 belong to the potato linkage group IV and all contain signature sequences characteristic of LZ-NBS-LRR proteins. The closest known R gene so far is RPP13 from Arabidopsis thaliana which shares an amino-acid sequence similarity of 35%. The LRR domains of Rpi-blb3, Rpi-abpt, R2 and R2-like proteins are highly homologous, whilst LZ and NBS domains are more polymorphic with those of R2 being the most divergent. All four Rpi genes recognize the recently identified RXLR effector protein PiAVR2 which is secreted by P.infestans in the cytoplasm of plant cells during the infection process. Unlike Rpi-blb3, Rpi-abpt, R2 and R2-like , the S. microdontum resistance gene Rpi-mcd1.1 does not interact with PiAVR2 and provides a different resistance spectrum. Rpi-mcd1.1 shares 90% nucleotide identity with Rpi-blb3 and polymorphic nucleotides are mainly located in the LRR region. The S. bulbocastanum haplotypes of Rpi-blb1, Rpi-blb2 and Rpi-blb3 were discovered in several Mexican diploid as well as polyploid species closely related to S. bulbocastanum. These three resistance genes occurred in different combinations and frequencies in S. bulbocastanum accessions and their distribution is confined to Central America. A selected set of genotypes was tested for their response to the avirulence effectors IPIO-2, Avr-blb2 and Pi-Avr2, which interact with Rpi-blb1, Rpi-blb2 and Rpi-blb3, respectively, as well as by disease assays with a diverse set of isolates. Using this approach some accessions could be identified that contain novel, yet unknown, Late Blight resistance factors in addition to the Rpi-blb1, Rpi-blb2 and Rpi-blb3 genes Analysis of the sequences obtained in different allele mining strategies suggests an evolution of the major late blight locus on linkage group IV through recombination and point mutations. By making use of the sequence information provided by the alleles, we identified the repeats and amino acids in the LRR domain which are specific for PiAVR2 recognition. Finally, we discussed the results described in this thesis in a potato/ P. infestans co-evolution context.

Like many other plants, chemical defence compounds are involved in the defense of Barbarea vulgaris against natural enemies. Barbarea vulgaris produces glucosinolates, which are present in most crucifers such as cabbage, mustard, and the scientific model species Arabidopsis thaliana. Glucosinolates form, together with an enzyme (myrosinase), a two-component system: the enzyme and glucosinolates are stored in spatially separated compartments. Upon cell disruption they come into contact, and the glucosinolates are catabolised by the enzyme. For generalist herbivores the glucosinolates, and especially their breakdown products, are often toxic and protect the plants against herbivory. However, specialist herbivores often use these same compounds to recognize suitable host plants. The breakdown products are also responsible for the specific taste to many cabbage and mustard species There are over 100 different glucosinolates, each with a different chemical structure. The structure determines, amongst other factors, which breakdown product will be formed upon damage. Every plant species has its own typical composition of glucosinolates. I studied how these glucosinolate profiles may affect plant resistance against herbivores. In a screening of several Dutch populations it was found that Barbarea vulgaris plants differed in glucosinolate profile. The majority of the plants contained mainly glucobarbarin, a glucosinolate typical for this species and named after the genus Barbarea. Also populations sampled in Germany, Belgium, France, and Switserland, consisted completely of plants with mainly glucobarbarin. In half of the Dutch populations I found that a minority of the plants (2-22%) produced another glucosinolate, named gluconasturtiin. The difference in the chemical structure between these two glucosinolates is very small; glucobarbarin has only one hydroxyl group more than gluconasturtiin. However, this small structural difference may be of large biological relevance. When gluconasturtiin reacts with myrosinase a toxic and unpalatable isothiocyanate is formed, whereas glucobarbarin, due to the position of the hydroxyl group, produces oxazolidinethions. It is unknown whether these oxazolidinethiones are toxic, but in mammals they can inhibit the iodine intake, thereby causing thyroid problems. The Barbarea vulgaris glucosinolate polymorphism thus has two chemotypes. I characterized these chemotypes and used them to study the effects of different glucosinolates on herbivores. The difference in glucosinolate profile is consistently present in all plant organs of B. vulgaris, but it is larger in the aboveground organs than in the roots. The glucosinolate profile does not change upon induction by insects nor upon artificial induction by addition of jasmonic acid. I crossed plants, analysed the chemotype of the offspring and showed that the ability to produce glucobarbarin is heritable and regulated by a dominant gene. Based on the assumption that there is a specific enzyme that converts gluconasturtiin to glucobarbarin by a single hydroxylation step, I identified some candidate genes. Further research is needed to determine whether one of these candidate genes is indeed responsible the difference between the chemotypes. Subsequently, I studied the effect of chemotype on leaf and root herbivores. Plants with mainly glucobarbarin turned out to be very resistant to the generalist leaf-eating larvae of the Mamestra brassicae moth. Almost none of the larvae survived on plants with mainly glucobarbarin. If the larvae were given the choice, they strongly preferred to feed on plants with mainly gluconasturtiin. However, female moths deposited approximately the same number of eggs on each chemotype. Larvae of the specialist small cabbage white grew equally well on each chemotype and did not distinguish between the chemotypes in choice experiments. Larvae of the specialist cabbage root fly, however, performed worse on roots of the gluconasturtiin type than on roots of the glucobarbarin type. Plants responded to the root fly infection by a reduction of the root and shoot biomass with 50%, and decreased levels of nutrients such as sugars and amino acids. In order to translate the results obtained in the greenhouse to the natural situation, I planted plants of both chemotypes in an experimental garden. Over a period of two years, the numbers of several herbivores were counted on each chemotype every week during the growth season. This revealed that some aboveground insects had a preference for a certain chemotype, and others did not. Butterflies of the small cabbage white preferred to oviposit on plants with gluconasturtiin, but flea beetles and gall midges were more abundant on plants with glucobarbarin. The cabbage aphid and the green peach aphid did not show any preference and were equally abundant on both chemotypes. Three to four times a year, I dug up a subset of the plants to analyse the root herbivores and the soil nematode community. The numbers and community structure of these belowground organisms did not differ between the chemotypes. Additionally, I investigated whether there are other chemical differences between the chemotypes, apart from glucosinolate profile, that could explain the preference of the above insect species. Therefore, I performed extensive metabolomics analyses, using LC-TOF-MS. Multivariate analyses showed that the major chemical differences between the chemotypes was due to differences in glucosinolates. These differences were larger in shoots than in roots. Apart from glucosinolates only eight unidentified compounds differed between the chemotypes. Known defense compounds such as flavonoids and saponins were identified but did not differ between the chemotypes. Therefore, it is very likely that the differences in herbivore performance and preference are predominantly caused by the differences in glucosinolate profile. Based on my results, I conclude that the structure of glucosinolates can cause significant differences in resistance against several herbivores. However, in the case of B. vulgaris there is no supreme chemotype that is more resistant in all cases. Which of the two chemotypes accrues the largest benefit in a natural environment thus will depend on the herbivore community in their population. In this way the B. vulgaris glucosinolate polymorphism can be maintained in natural populations.

Plant pathogens inject so-called effector molecules into the cells of a host plant to promote their growth and reproduction in these hosts. In plant parasitic nematodes, these effector molecules are produced in the salivary glands. The objective of this thesis was to identify and characterize effector molecules produced in the salivary glands of the potato cyst nematode Globodera rostochiensis. A combination of cDNA-AFLP and mining of EST-databases resulted in the identification of a large family of effectors named the SPRYSECs. The SPRYSECs essentially consist of a conserved SPRY domain preceded by a signal peptide for secretion. The SPRYSECs are injected into host cells through the oral stylet. A protein structure model of the SPRYSECs indicated that one particular surface of the proteins in the SPRYSEC family was hypervariable and seemed to undergo diversifying selection. This led us to believe that the SPRYSECs are important players in the co-evolution between plant and nematode. Transgenic potato plants overexpressing SPRYSEC-19 appeared to be two- to five-fold more susceptible to infections of nematodes, the fungus Verticillium dahliae, and tomato spotted wilt virus (TSWV). This hypersusceptibility to a range of unrelated plant-pathogens suggests that SPRYSECs somehow suppress the basal defense responses that are controlled by the plant’s innate immunity. SPRYSEC-19 was found to engage in a specific physical interaction with the Leucine Rich Repeat domain of a protein from the CC-NBS-LRR class of resistance genes. Many immune receptors in the plant’s innate immunity belong to the same class of NB-LRR proteins. The host interactor of SPRYSEC19 is most similar to members of SW5 R gene cluster that confers resistance to tospoviruses. Remarkably, plants harboring the CC-NB-LRR interactor of SPRYSEC19 are not resistant to nematodes. Therefore, we hypothesize that the nematode effector SPRYSEC-19 promotes its virulence in susceptible host plants by suppressing basal defense through its interaction with an NB-LRR immune receptor.

Like many other plants, chemical defence compounds are involved in the defense of Barbarea vulgaris against natural enemies. Barbarea vulgaris produces glucosinolates, which are present in most crucifers such as cabbage, mustard, and the scientific model species Arabidopsis thaliana. Glucosinolates form, together with an enzyme (myrosinase), a two-component system: the enzyme and glucosinolates are stored in spatially separated compartments. Upon cell disruption they come into contact, and the glucosinolates are catabolised by the enzyme. For generalist herbivores the glucosinolates, and especially their breakdown products, are often toxic and protect the plants against herbivory. However, specialist herbivores often use these same compounds to recognize suitable host plants. The breakdown products are also responsible for the specific taste to many cabbage and mustard species There are over 100 different glucosinolates, each with a different chemical structure. The structure determines, amongst other factors, which breakdown product will be formed upon damage. Every plant species has its own typical composition of glucosinolates. I studied how these glucosinolate profiles may affect plant resistance against herbivores. In a screening of several Dutch populations it was found that Barbarea vulgaris plants differed in glucosinolate profile. The majority of the plants contained mainly glucobarbarin, a glucosinolate typical for this species and named after the genus Barbarea. Also populations sampled in Germany, Belgium, France, and Switserland, consisted completely of plants with mainly glucobarbarin. In half of the Dutch populations I found that a minority of the plants (2-22%) produced another glucosinolate, named gluconasturtiin. The difference in the chemical structure between these two glucosinolates is very small; glucobarbarin has only one hydroxyl group more than gluconasturtiin. However, this small structural difference may be of large biological relevance. When gluconasturtiin reacts with myrosinase a toxic and unpalatable isothiocyanate is formed, whereas glucobarbarin, due to the position of the hydroxyl group, produces oxazolidinethions. It is unknown whether these oxazolidinethiones are toxic, but in mammals they can inhibit the iodine intake, thereby causing thyroid problems. The Barbarea vulgaris glucosinolate polymorphism thus has two chemotypes. I characterized these chemotypes and used them to study the effects of different glucosinolates on herbivores. The difference in glucosinolate profile is consistently present in all plant organs of B. vulgaris, but it is larger in the aboveground organs than in the roots. The glucosinolate profile does not change upon induction by insects nor upon artificial induction by addition of jasmonic acid. I crossed plants, analysed the chemotype of the offspring and showed that the ability to produce glucobarbarin is heritable and regulated by a dominant gene. Based on the assumption that there is a specific enzyme that converts gluconasturtiin to glucobarbarin by a single hydroxylation step, I identified some candidate genes. Further research is needed to determine whether one of these candidate genes is indeed responsible the difference between the chemotypes. Subsequently, I studied the effect of chemotype on leaf and root herbivores. Plants with mainly glucobarbarin turned out to be very resistant to the generalist leaf-eating larvae of the Mamestra brassicae moth. Almost none of the larvae survived on plants with mainly glucobarbarin. If the larvae were given the choice, they strongly preferred to feed on plants with mainly gluconasturtiin. However, female moths deposited approximately the same number of eggs on each chemotype. Larvae of the specialist small cabbage white grew equally well on each chemotype and did not distinguish between the chemotypes in choice experiments. Larvae of the specialist cabbage root fly, however, performed worse on roots of the gluconasturtiin type than on roots of the glucobarbarin type. Plants responded to the root fly infection by a reduction of the root and shoot biomass with 50%, and decreased levels of nutrients such as sugars and amino acids. In order to translate the results obtained in the greenhouse to the natural situation, I planted plants of both chemotypes in an experimental garden. Over a period of two years, the numbers of several herbivores were counted on each chemotype every week during the growth season. This revealed that some aboveground insects had a preference for a certain chemotype, and others did not. Butterflies of the small cabbage white preferred to oviposit on plants with gluconasturtiin, but flea beetles and gall midges were more abundant on plants with glucobarbarin. The cabbage aphid and the green peach aphid did not show any preference and were equally abundant on both chemotypes. Three to four times a year, I dug up a subset of the plants to analyse the root herbivores and the soil nematode community. The numbers and community structure of these belowground organisms did not differ between the chemotypes. Additionally, I investigated whether there are other chemical differences between the chemotypes, apart from glucosinolate profile, that could explain the preference of the above insect species. Therefore, I performed extensive metabolomics analyses, using LC-TOF-MS. Multivariate analyses showed that the major chemical differences between the chemotypes was due to differences in glucosinolates. These differences were larger in shoots than in roots. Apart from glucosinolates only eight unidentified compounds differed between the chemotypes. Known defense compounds such as flavonoids and saponins were identified but did not differ between the chemotypes. Therefore, it is very likely that the differences in herbivore performance and preference are predominantly caused by the differences in glucosinolate profile. Based on my results, I conclude that the structure of glucosinolates can cause significant differences in resistance against several herbivores. However, in the case of B. vulgaris there is no supreme chemotype that is more resistant in all cases. Which of the two chemotypes accrues the largest benefit in a natural environment thus will depend on the herbivore community in their population. In this way the B. vulgaris glucosinolate polymorphism can be maintained in natural populations.

Plant pathogens inject so-called effector molecules into the cells of a host plant to promote their growth and reproduction in these hosts. In plant parasitic nematodes, these effector molecules are produced in the salivary glands. The objective of this thesis was to identify and characterize effector molecules produced in the salivary glands of the potato cyst nematode Globodera rostochiensis. A combination of cDNA-AFLP and mining of EST-databases resulted in the identification of a large family of effectors named the SPRYSECs. The SPRYSECs essentially consist of a conserved SPRY domain preceded by a signal peptide for secretion. The SPRYSECs are injected into host cells through the oral stylet. A protein structure model of the SPRYSECs indicated that one particular surface of the proteins in the SPRYSEC family was hypervariable and seemed to undergo diversifying selection. This led us to believe that the SPRYSECs are important players in the co-evolution between plant and nematode. Transgenic potato plants overexpressing SPRYSEC-19 appeared to be two- to five-fold more susceptible to infections of nematodes, the fungus Verticillium dahliae, and tomato spotted wilt virus (TSWV). This hypersusceptibility to a range of unrelated plant-pathogens suggests that SPRYSECs somehow suppress the basal defense responses that are controlled by the plant’s innate immunity. SPRYSEC-19 was found to engage in a specific physical interaction with the Leucine Rich Repeat domain of a protein from the CC-NBS-LRR class of resistance genes. Many immune receptors in the plant’s innate immunity belong to the same class of NB-LRR proteins. The host interactor of SPRYSEC19 is most similar to members of SW5 R gene cluster that confers resistance to tospoviruses. Remarkably, plants harboring the CC-NB-LRR interactor of SPRYSEC19 are not resistant to nematodes. Therefore, we hypothesize that the nematode effector SPRYSEC-19 promotes its virulence in susceptible host plants by suppressing basal defense through its interaction with an NB-LRR immune receptor.

Phytophthora is a genus comprised of over 65 destructive plant pathogenic species that cause severe damages in agriculture, forestry and natural habitats. Economically important pathogens are Phytophthora infestans (causing potato late blight) and Phytophthora sojae (causing soybean root and stem rot). A newly discovered species, Phytophthora ramorum is destroying oak trees along the west-coast of the USA by causing the Sudden Oak Death syndrome. Phytophthora belongs to the oomycetes, which together with phyopathogenic fungi constitute the major groups of plant pathogens. Oomycetes and fungi resemble each other morphologically but belong to different kingdoms, Stramenopila and Fungi, respectively. Convergent evolution has shaped a similar set of weaponry for attacking plants in oomycetes and fungi.Phytophthora secretes a variety of molecules into the plant apoplast presumably to promote infection. These molecules with a potential role in virulence or pathogenicity are named virulence factors or effectors. Despite their intrinsic virulence functions, effectors may cause failure of infection if plants recognize them and initiate defense responses. Effectors that trigger plant defense responses are called avirulence factors or elicitors. This thesis describes the drastic genome rearrangements at an avirulence locus in P. infestans, the characterization of effector gene reservoirs in the fully sequenced P. sojae and P. ramorum genomes, and evolutionary patterns of effector genes.Potato and P. infestans interact accordmg to the gene-for-gene model. As a first step towards unraveling the molecular interaction mechanisms, P. infestans avriulence (Avr) genes have to be isolated. Chapter 2 describes a transcriptional profiling slrategy to identify avirulence associated transcripts. cDNA-AFLP was used for comparing transcripts in P. infestans strains with different virulence phenotypes. A large number of avirulence-associated TDFs (Transcript Derived Fragments) were cloned and sequenced, and EST and genome databases were mined to generate more sequence data. To identify promising candidates, bioinformatic predictions such as the presence of signal peptides, number of cysteine residues and putative virulence functions were used as important selection criteria. Chapter 3 describes how a combination of transcriptional profiling, and genetic and physical mapping resulted in the characterisation of a complex avirulence locus. Four avirulence associated TDFs were shown to be derived from one gene, named p't3.4. Genetic mapping and physical mapping placed pi3.4 at the Avr3b-Avr10-Avr11 locus on linkage group VIIl. Comparative genomic hybridization (CGH) revealed that this A^rlocus belongs to one of the six loci in the genome that show copy number variation (CNV)- An amplified pi3.4 gene cluster is present in the avirulent haplotype but absent in the virulent haplotype. OnIy the 3' half of the pi3.4 gene is amplified, and this amplification was found to provide diverse modules for assembly of novel full length genes. Among the proteins secreted by Phytophthora. elicitins are produced most abundantly. Elicitins show elicitor activity by causing a hypersensitive response in tobacco. In Chapter 4 and Chapter 5, the diversity and genome organization of elicitin genes in four Phytophthora species are described. Elicitins were found to be encoded by a large complex gene family, and they belong to one of the most highly conserved groups of proteins in the Phytophthora genus. Many elicitin (ELI) and elicitin-like (ELL) genes are clustered in the genome. Phylogeny construction indicated that the complex elicitin gene family existed before the ancestral Phytophthora species gave rise to the current Phytophthora species. Molecular phylogeny classified elicitin family members into 17 different clades, namely 4 ELI clades and 13 ELL clades. Based on expression patterns and bioinformatic predictions, different clades are proposed to possess distinct functions.The two fully sequenced Phytophthora species, P. sojae and P. ramorum, differ in their genome sizes, sexual behavior and host specificity. Comparative genomics was carried out to gain insight into the evolution of effector genes. In Chapter 6 the genome organization of potential effector genes is described. Overall co-linearity was found between large genomic regions in P. sojae and P. ramorum. However, insertions, deletions and expansions revealed some hotspots for genome rearrangements, and such rearrangement hotspots often harbor genes associated with virulence. Contrasting evolutionary patterns were found for neighboring gene families, for example, families encoding extracellular enzymes showed more rearrangements than those encoding intracellular enzymes. Also genes encoding host specific elicitors showed more rearrangements than those encoding general elicitors.In Chapter 7 the whole reservoir of secreted proteins present in the proteome was revealed by bioinformatic-predictions. A large secretome comprised of overthousand proteins was found in both P. sojae and P. ramorum. The majority of secreted protein encoding genes form fami!ies and many of them are clustered in the genome. Comparison between secretomes of P. sojae and P. ramorum showed that different families are evolving at a different pace. The most rapidly evolving families include the surface anchored proteins, mating associated factors and 'RXLR-DEER' proteins. They may piay important roles in either host-pathogen interactions or in reproduction.In Chapter 8 the base compositions of P. sojae and P. ramorum were calculated and compared. This information is usefui for analyzing basic genome features. The coding regions of Phytophthora clearly show high GC3 (3rd position codon usage) and this preference causes codon bias in Phytophthora genes. Evolutionary forces such as selective pressure and mutation bias were found to drive codon bias in Phytophthora. The higher GC3 value of highly expressed genes in different Phytophthora species is indicative for selection pressure, whereas lineage specific GC increase of non-coding regions is reminiscent of whole genome mutation bias. The most widespread groups of mobile elements were retrieved from the genomes and they show a codon bias that is similar to the genes of the host Phytophthora.Finally, the evolutionary implications of the findings presented in this thesis are discussed in Chapter 9. For pathogenic organisms, genes encoding effectors are instrumental for interaction with their hosts. As a result, the evolutionary pace of effector genes is in general faster than that of average genes. The results presented in this thesis demonstrate that comparative genomics is a powerful toot to discover these genes and to point out promising candidates responsible for the process of pathogenesis. The ongoing P. infestans genome sequencing project will provide new resources for fundamental and applied research, and with the b!ueprint of P. infestans in hand, late bight research will gain momentum.

Phytophthora is a genus comprised of over 65 destructive plant pathogenic species that cause severe damages in agriculture, forestry and natural habitats. Economically important pathogens are Phytophthora infestans (causing potato late blight) and Phytophthora sojae (causing soybean root and stem rot). A newly discovered species, Phytophthora ramorum is destroying oak trees along the west-coast of the USA by causing the Sudden Oak Death syndrome. Phytophthora belongs to the oomycetes, which together with phyopathogenic fungi constitute the major groups of plant pathogens. Oomycetes and fungi resemble each other morphologically but belong to different kingdoms, Stramenopila and Fungi, respectively. Convergent evolution has shaped a similar set of weaponry for attacking plants in oomycetes and fungi.Phytophthora secretes a variety of molecules into the plant apoplast presumably to promote infection. These molecules with a potential role in virulence or pathogenicity are named virulence factors or effectors. Despite their intrinsic virulence functions, effectors may cause failure of infection if plants recognize them and initiate defense responses. Effectors that trigger plant defense responses are called avirulence factors or elicitors. This thesis describes the drastic genome rearrangements at an avirulence locus in P. infestans, the characterization of effector gene reservoirs in the fully sequenced P. sojae and P. ramorum genomes, and evolutionary patterns of effector genes.Potato and P. infestans interact accordmg to the gene-for-gene model. As a first step towards unraveling the molecular interaction mechanisms, P. infestans avriulence (Avr) genes have to be isolated. Chapter 2 describes a transcriptional profiling slrategy to identify avirulence associated transcripts. cDNA-AFLP was used for comparing transcripts in P. infestans strains with different virulence phenotypes. A large number of avirulence-associated TDFs (Transcript Derived Fragments) were cloned and sequenced, and EST and genome databases were mined to generate more sequence data. To identify promising candidates, bioinformatic predictions such as the presence of signal peptides, number of cysteine residues and putative virulence functions were used as important selection criteria. Chapter 3 describes how a combination of transcriptional profiling, and genetic and physical mapping resulted in the characterisation of a complex avirulence locus. Four avirulence associated TDFs were shown to be derived from one gene, named p't3.4. Genetic mapping and physical mapping placed pi3.4 at the Avr3b-Avr10-Avr11 locus on linkage group VIIl. Comparative genomic hybridization (CGH) revealed that this A^rlocus belongs to one of the six loci in the genome that show copy number variation (CNV)- An amplified pi3.4 gene cluster is present in the avirulent haplotype but absent in the virulent haplotype. OnIy the 3' half of the pi3.4 gene is amplified, and this amplification was found to provide diverse modules for assembly of novel full length genes. Among the proteins secreted by Phytophthora. elicitins are produced most abundantly. Elicitins show elicitor activity by causing a hypersensitive response in tobacco. In Chapter 4 and Chapter 5, the diversity and genome organization of elicitin genes in four Phytophthora species are described. Elicitins were found to be encoded by a large complex gene family, and they belong to one of the most highly conserved groups of proteins in the Phytophthora genus. Many elicitin (ELI) and elicitin-like (ELL) genes are clustered in the genome. Phylogeny construction indicated that the complex elicitin gene family existed before the ancestral Phytophthora species gave rise to the current Phytophthora species. Molecular phylogeny classified elicitin family members into 17 different clades, namely 4 ELI clades and 13 ELL clades. Based on expression patterns and bioinformatic predictions, different clades are proposed to possess distinct functions.The two fully sequenced Phytophthora species, P. sojae and P. ramorum, differ in their genome sizes, sexual behavior and host specificity. Comparative genomics was carried out to gain insight into the evolution of effector genes. In Chapter 6 the genome organization of potential effector genes is described. Overall co-linearity was found between large genomic regions in P. sojae and P. ramorum. However, insertions, deletions and expansions revealed some hotspots for genome rearrangements, and such rearrangement hotspots often harbor genes associated with virulence. Contrasting evolutionary patterns were found for neighboring gene families, for example, families encoding extracellular enzymes showed more rearrangements than those encoding intracellular enzymes. Also genes encoding host specific elicitors showed more rearrangements than those encoding general elicitors.In Chapter 7 the whole reservoir of secreted proteins present in the proteome was revealed by bioinformatic-predictions. A large secretome comprised of overthousand proteins was found in both P. sojae and P. ramorum. The majority of secreted protein encoding genes form fami!ies and many of them are clustered in the genome. Comparison between secretomes of P. sojae and P. ramorum showed that different families are evolving at a different pace. The most rapidly evolving families include the surface anchored proteins, mating associated factors and 'RXLR-DEER' proteins. They may piay important roles in either host-pathogen interactions or in reproduction.In Chapter 8 the base compositions of P. sojae and P. ramorum were calculated and compared. This information is usefui for analyzing basic genome features. The coding regions of Phytophthora clearly show high GC3 (3rd position codon usage) and this preference causes codon bias in Phytophthora genes. Evolutionary forces such as selective pressure and mutation bias were found to drive codon bias in Phytophthora. The higher GC3 value of highly expressed genes in different Phytophthora species is indicative for selection pressure, whereas lineage specific GC increase of non-coding regions is reminiscent of whole genome mutation bias. The most widespread groups of mobile elements were retrieved from the genomes and they show a codon bias that is similar to the genes of the host Phytophthora.Finally, the evolutionary implications of the findings presented in this thesis are discussed in Chapter 9. For pathogenic organisms, genes encoding effectors are instrumental for interaction with their hosts. As a result, the evolutionary pace of effector genes is in general faster than that of average genes. The results presented in this thesis demonstrate that comparative genomics is a powerful toot to discover these genes and to point out promising candidates responsible for the process of pathogenesis. The ongoing P. infestans genome sequencing project will provide new resources for fundamental and applied research, and with the b!ueprint of P. infestans in hand, late bight research will gain momentum.