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Many plant species are hosts of powdery mildew fungi, including Arabidopsis and economically important crops such as wheat, barley and tomato. Resistance has been explored using induced mutagenesis and natural variation in the plant species. The isolated genes encompass loss-of-function susceptibility genes and dominantly inherited genes encoding NB-LRR proteins, receptor-like kinases or proteins that do not have typical resistance protein domains. Cultivated tomato is susceptible to powdery mildew species Oidium neolycopersici, and exploiting the resistance genes present in wild tomato species is a favourable strategy to control the disease. In chapter 2, we give an overview of all the identified resistance genes in wild tomato species and their resistance mechanisms inferred from cytological and molecular data. Furthermore, resistance genes and their mechanisms are compared between tomato and other plant species, such as dicot Arabidopsis and monocots barley and wheat. This comparison illustrates that both common and species-specific mechanisms are involved with respect to resistance to powdery mildews in different plant species. Resistance gene Ol-1 originates from wild tomato species S. habrochaites. It confers race-non-specific resistance to tomato powdery mildew. To elucidate the resistance signalling pathway, we adopted a virus induced gene silencing (VIGS) approach to suppress genes which are differentially expressed when comparing genotypes with and without the Ol-1 introgression. In chapter 3, we showed that ALS (acetolactate synthase) activity is important for Ol-1-mediated resistance, as simultaneous silencing of two ALS genes attenuated the resistance level of NIL-Ol-1. ALS is a key enzyme in the biosynthesis of branched-chain amino acids, and a target of commercial herbicides. Reducing ALS activity via herbicidal treatment did not result in altered responses to powdery mildew infection in susceptible cultivar Moneymaker and resistant line NIL-Ol-4, indicating that ALS is not involved in basal defense nor in NB-LRR gene-mediated resistance. Whether the role of ALS in Ol-1-mediated resistance is associated with amino acid homeostasis is unknown and needs further investigation. Besides tomato, Arabidopsis is a host of powdery mildew O. neolycopersici. The large collection of Arabidopsis accessions and several mutant collections are valuable resources to identify novel resistance genes. In chapter 4, we first screened 123 Arabidopsis accessions for O. neolycopersici resistance and then studied the genetic basis of theresistance by segregation analysis in 19 F2 populations. The results showed that polygenic resistance is the main form of resistance. Accession C24 displays complete resistance with polygenic nature, as shown by QTL analysis of the F2 population derived from the cross between C24 and susceptible accession Sha. The recessively inherited locus on chromosome 1 was fine-mapped by recombinant screening, and analysis of candidate genes resulted in the isolation of the gene conferring resistance. It proved to be a mutant allele of EDR1, harbouring a deletion upstream of the kinase domain resulting in a truncated protein. Previously, an induced edr1mutationin Col-0 background was obtained. However, the edr1 mutation in our C24 source (referred to as C24-W) occurred in a different position. The resistance conferred by edr1 in C24-W was not associated with constitutively expressed pathogenesis-related genes. Remarkably, we observed that although C24-W carried the edr1 mutation this mutation was absent in other C24 sources. In addition, C24-W was smaller in size than C24 from other sources. Since the edr1 mutation confers resistance to tomato powdery mildew in Arabidopsis, we investigated whether this resistance system is conserved in tomato. The results showed that individual silencing of two tomato EDR1 candidate genes in susceptible cultivar Moneymaker did not result in decreased sporulation of tomato powdery mildew. In chapter 5, we screened an activation tag Arabidopsis mutant collection. In these mutants, tagged genes are overexpressed by the strong 35S enhancers resulting in a dominant gain-of-function phenotype. One mutant line, 3221, was identified due to its resistance to powdery mildew O. neolycopersici. Additional disease tests showed that 3221 displayed resistance to the downy mildew Hyaloperonospora arabidopsidis and the aphid Myzus persicae, but susceptibility to the bacterial pathogen Pseudomonas syringae pv tomato DC3000. The mutant line 3221 also showed reduced size and serrated leaves, and the altered morphology was associated with resistance. Inverse PCR and expression analysis revealed that the gene underlying the resistance was ATHB13, a HD-Zip transcription factor. Suppression ofATHB13 in 3221 by RNAi transformation resulted in the loss of resistance and altered morphology, while overexpression of ATHB13 in wild-type plants induced resistance and altered morphology. Microarray analysis of 3221 and the parental line Ws resulted in the identification of a large number of genes showing differential expression. Analysis of these results did not give a clear indication that the resistance phenotype in 3221 is due to the activation of classical hormone pathway genes involved in resistance. The possibility of utilizing ATHB13 for engineering pathogen resistance in tomato needs to be investigated in the future. Finally, in chapter 6 the results from the previous chapters are discussed in a broader context.

Tomato (Solanum lycopersicum) is affected by a wide range of biotic stresses, of which Bemisia tabaci is one of the most important.Bemisia tabaci affects tomato directly through phloem sap feeding, and indirectly through its ability to be the vector of a large number of viruses. Different methods are available for whitefly control, and although several biological control agents are used against whiteflies in greenhouse cultivation, chemical control still is an essential component in open field tomato production. Breeding for host plant resistance is considered as one of the most promising methods in insect pest control in crop plants, and especially it is a promising alternative in whitefly control. Resistance to whiteflies was found in several wild relatives of tomato like Solanum peruvianum, S. pennellii, S. habrochaites, S. lycopersicum var. cerasiforme, S. pimpinellifolium andS. galapagense. In spite of previous breeding efforts, whiteflies are still a problem in tomato cultivation. The aim of my research was to identify and understand resistance mechanisms targeting specific stages of the whitefly life cycle in order to provide breeders with tools for developing whitefly resistant varieties. I assessed the natural variation and whitefly resistance in Solanum galapagense and S. cheesmaniae, two wild tomato species endemic to the Galapagos Islands. Previously, Solanum galapagense and S. cheesmaniae were classified as two species based on a morphological species concept, but with molecular markers no clear separation could be made. So far, only a limited number of accessions/populations of S. galapagense and S. cheesmaniae have been evaluated for insect resistance and therefore it was unknown if the insect resistance coincides with the morphological species boundaries. Neither was there any knowledge about the relation between geographical and climatic conditions today on the Galapagos and the occurrence of the two species. We characterized twelve accessions of S. galapagense, 22 of S. cheesmaniae, and as reference one of S. lycopersicum for whitefly resistance using no-choice experiments. Whitefly resistance was found in S. galapagense only and was associated with the presence of relatively high levels of acyl sugars and the presence of glandular trichomes of type I and IV.It is likely that a minimum level of acyl sugars and the presence of glandular trichomes type IV are needed to achieve an effective level of resistance. Genetic fingerprinting using 3316 polymorphic SNP markers did not show a clear differentiation between the two species endemic to the Galapagos. Acyl sugar accumulation as well as the climatic and geographical conditions at the collection sites of the accessions did not follow the morphological species boundaries. Altogether, our results suggest that S. galapagense and S. cheesmaniae might be considered as morphotypes rather than two species and that their co-existence is likely the result of selective pressure. Plants possess several resistance mechanisms acting at different time points during the interaction with herbivorous insect. Before any contact with the insects, plants emit an array of volatile organic compounds that can act as attractant or repellent of insects.Bemisia tabaci use a set of plant-derived cues in the process of host plant selection. It recognizes mainly monoterpenes (p-cymene, γ-terpinene and β-myrcene, α-phellandrene) and sesquiterpenes (7-epizingiberene and R-curcumene). Previously the line FCN93-6-2, which was derived from a cross between a susceptible tomato cultivar (Uco Plata INTA) and S. habrochaites (FCN3-5) was proved to be non-preferred by the greenhouse whitefly Trialeurodes vaporariorum. We identified chemical cues produced by FCN93-6-2 and S. habrochaites that can affect the preference of the whitefly B. tabaci as well as the potential chromosomal region(s) of S. habrochaites harbouring the genes involved in the preference. Two S. habrochaites accessions (CGN1.1561 and in FCN3-5) and the line FCN93-6-2 were non-preferred by B. tabaci when the whiteflies could get in direct contact with the plant and also when the whiteflies were offered olfactory cues only. The non-preference was independent of trichome type IV and of the presence of methyl-ketones but associated to the presence of monoterpenes in lower concentrations. Functional validation of the candidate metabolites and of the different introgressions is still needed. Once the insect has landed on a plant, another set of resistance mechanisms enter into action. We have described a 3.06 Mbp introgression on top of Chromosome 5 (OR-5) from the wild tomato species S. habrochaites (CGN1.1561). For the identification of OR-5, we went from the selection of specific F2 plants to the development of F2BC4S1 and F2BC4S2 families. This introgression was sufficient to reduce whitefly fecundity without an evident effect on whitefly survival. The identification of mechanisms exclusively affecting whitefly fecundity and independent of trichomes type IV opens new doors for resistance breeding to whiteflies that may be especially interesting in greenhouse cultivation combination with natural enemies of the whitefly. As an additional layer of defences, plants can perceive stress signals and respond to them in a specific way through induction of their immune system. This induction can also be triggered by exposing the plants to priming agents like hormones, some xenobiotic chemicals, like benzothiadiazole (BTH), β-aminobutyric acid (BABA), and sugars. Although the effect of priming agents was shown in laboratory and field studies, little is known about the effect of the genetic background of tomato on the extent of the priming, e.g. do genotypes varying in their level of resistance to insects and pathogens respond in the same way to a priming agent. We assessed the effect of selected priming agents on the effectiveness of natural defence in tomato. A set of no-choice and choice bioassays was conducted using tomato genotypes varying in their level of basal resistance to Bemisia tabaci and pathogens. We observed that whitefly survival and oviposition were not affected by the priming treatment in no-choice assays. Overall, in choice assays, fructose treated plants were more preferred by whiteflies than control plants. A genotype specific effect of priming was seen for the line FCN93-6-2. On this tomato line, JA and BABA applications decreased the number of whiteflies, e.g. making them less preferred. In this thesis, I have gone from the screening of wild relatives of tomatoes to in depth characterization of resistance mechanisms. I have identified resistance mechanisms targeting specific stages of the whitefly life cycle, thus providing new tools for breeding durable whitefly resistance in tomato.

Many plant species are hosts of powdery mildew fungi, including Arabidopsis and economically important crops such as wheat, barley and tomato. Resistance has been explored using induced mutagenesis and natural variation in the plant species. The isolated genes encompass loss-of-function susceptibility genes and dominantly inherited genes encoding NB-LRR proteins, receptor-like kinases or proteins that do not have typical resistance protein domains. Cultivated tomato is susceptible to powdery mildew species Oidium neolycopersici, and exploiting the resistance genes present in wild tomato species is a favourable strategy to control the disease. In chapter 2, we give an overview of all the identified resistance genes in wild tomato species and their resistance mechanisms inferred from cytological and molecular data. Furthermore, resistance genes and their mechanisms are compared between tomato and other plant species, such as dicot Arabidopsis and monocots barley and wheat. This comparison illustrates that both common and species-specific mechanisms are involved with respect to resistance to powdery mildews in different plant species. Resistance gene Ol-1 originates from wild tomato species S. habrochaites. It confers race-non-specific resistance to tomato powdery mildew. To elucidate the resistance signalling pathway, we adopted a virus induced gene silencing (VIGS) approach to suppress genes which are differentially expressed when comparing genotypes with and without the Ol-1 introgression. In chapter 3, we showed that ALS (acetolactate synthase) activity is important for Ol-1-mediated resistance, as simultaneous silencing of two ALS genes attenuated the resistance level of NIL-Ol-1. ALS is a key enzyme in the biosynthesis of branched-chain amino acids, and a target of commercial herbicides. Reducing ALS activity via herbicidal treatment did not result in altered responses to powdery mildew infection in susceptible cultivar Moneymaker and resistant line NIL-Ol-4, indicating that ALS is not involved in basal defense nor in NB-LRR gene-mediated resistance. Whether the role of ALS in Ol-1-mediated resistance is associated with amino acid homeostasis is unknown and needs further investigation. Besides tomato, Arabidopsis is a host of powdery mildew O. neolycopersici. The large collection of Arabidopsis accessions and several mutant collections are valuable resources to identify novel resistance genes. In chapter 4, we first screened 123 Arabidopsis accessions for O. neolycopersici resistance and then studied the genetic basis of theresistance by segregation analysis in 19 F2 populations. The results showed that polygenic resistance is the main form of resistance. Accession C24 displays complete resistance with polygenic nature, as shown by QTL analysis of the F2 population derived from the cross between C24 and susceptible accession Sha. The recessively inherited locus on chromosome 1 was fine-mapped by recombinant screening, and analysis of candidate genes resulted in the isolation of the gene conferring resistance. It proved to be a mutant allele of EDR1, harbouring a deletion upstream of the kinase domain resulting in a truncated protein. Previously, an induced edr1mutationin Col-0 background was obtained. However, the edr1 mutation in our C24 source (referred to as C24-W) occurred in a different position. The resistance conferred by edr1 in C24-W was not associated with constitutively expressed pathogenesis-related genes. Remarkably, we observed that although C24-W carried the edr1 mutation this mutation was absent in other C24 sources. In addition, C24-W was smaller in size than C24 from other sources. Since the edr1 mutation confers resistance to tomato powdery mildew in Arabidopsis, we investigated whether this resistance system is conserved in tomato. The results showed that individual silencing of two tomato EDR1 candidate genes in susceptible cultivar Moneymaker did not result in decreased sporulation of tomato powdery mildew. In chapter 5, we screened an activation tag Arabidopsis mutant collection. In these mutants, tagged genes are overexpressed by the strong 35S enhancers resulting in a dominant gain-of-function phenotype. One mutant line, 3221, was identified due to its resistance to powdery mildew O. neolycopersici. Additional disease tests showed that 3221 displayed resistance to the downy mildew Hyaloperonospora arabidopsidis and the aphid Myzus persicae, but susceptibility to the bacterial pathogen Pseudomonas syringae pv tomato DC3000. The mutant line 3221 also showed reduced size and serrated leaves, and the altered morphology was associated with resistance. Inverse PCR and expression analysis revealed that the gene underlying the resistance was ATHB13, a HD-Zip transcription factor. Suppression ofATHB13 in 3221 by RNAi transformation resulted in the loss of resistance and altered morphology, while overexpression of ATHB13 in wild-type plants induced resistance and altered morphology. Microarray analysis of 3221 and the parental line Ws resulted in the identification of a large number of genes showing differential expression. Analysis of these results did not give a clear indication that the resistance phenotype in 3221 is due to the activation of classical hormone pathway genes involved in resistance. The possibility of utilizing ATHB13 for engineering pathogen resistance in tomato needs to be investigated in the future. Finally, in chapter 6 the results from the previous chapters are discussed in a broader context.

Tomato (Solanum lycopersicum) is affected by a wide range of biotic stresses, of which Bemisia tabaci is one of the most important.Bemisia tabaci affects tomato directly through phloem sap feeding, and indirectly through its ability to be the vector of a large number of viruses. Different methods are available for whitefly control, and although several biological control agents are used against whiteflies in greenhouse cultivation, chemical control still is an essential component in open field tomato production. Breeding for host plant resistance is considered as one of the most promising methods in insect pest control in crop plants, and especially it is a promising alternative in whitefly control. Resistance to whiteflies was found in several wild relatives of tomato like Solanum peruvianum, S. pennellii, S. habrochaites, S. lycopersicum var. cerasiforme, S. pimpinellifolium andS. galapagense. In spite of previous breeding efforts, whiteflies are still a problem in tomato cultivation. The aim of my research was to identify and understand resistance mechanisms targeting specific stages of the whitefly life cycle in order to provide breeders with tools for developing whitefly resistant varieties. I assessed the natural variation and whitefly resistance in Solanum galapagense and S. cheesmaniae, two wild tomato species endemic to the Galapagos Islands. Previously, Solanum galapagense and S. cheesmaniae were classified as two species based on a morphological species concept, but with molecular markers no clear separation could be made. So far, only a limited number of accessions/populations of S. galapagense and S. cheesmaniae have been evaluated for insect resistance and therefore it was unknown if the insect resistance coincides with the morphological species boundaries. Neither was there any knowledge about the relation between geographical and climatic conditions today on the Galapagos and the occurrence of the two species. We characterized twelve accessions of S. galapagense, 22 of S. cheesmaniae, and as reference one of S. lycopersicum for whitefly resistance using no-choice experiments. Whitefly resistance was found in S. galapagense only and was associated with the presence of relatively high levels of acyl sugars and the presence of glandular trichomes of type I and IV.It is likely that a minimum level of acyl sugars and the presence of glandular trichomes type IV are needed to achieve an effective level of resistance. Genetic fingerprinting using 3316 polymorphic SNP markers did not show a clear differentiation between the two species endemic to the Galapagos. Acyl sugar accumulation as well as the climatic and geographical conditions at the collection sites of the accessions did not follow the morphological species boundaries. Altogether, our results suggest that S. galapagense and S. cheesmaniae might be considered as morphotypes rather than two species and that their co-existence is likely the result of selective pressure. Plants possess several resistance mechanisms acting at different time points during the interaction with herbivorous insect. Before any contact with the insects, plants emit an array of volatile organic compounds that can act as attractant or repellent of insects.Bemisia tabaci use a set of plant-derived cues in the process of host plant selection. It recognizes mainly monoterpenes (p-cymene, γ-terpinene and β-myrcene, α-phellandrene) and sesquiterpenes (7-epizingiberene and R-curcumene). Previously the line FCN93-6-2, which was derived from a cross between a susceptible tomato cultivar (Uco Plata INTA) and S. habrochaites (FCN3-5) was proved to be non-preferred by the greenhouse whitefly Trialeurodes vaporariorum. We identified chemical cues produced by FCN93-6-2 and S. habrochaites that can affect the preference of the whitefly B. tabaci as well as the potential chromosomal region(s) of S. habrochaites harbouring the genes involved in the preference. Two S. habrochaites accessions (CGN1.1561 and in FCN3-5) and the line FCN93-6-2 were non-preferred by B. tabaci when the whiteflies could get in direct contact with the plant and also when the whiteflies were offered olfactory cues only. The non-preference was independent of trichome type IV and of the presence of methyl-ketones but associated to the presence of monoterpenes in lower concentrations. Functional validation of the candidate metabolites and of the different introgressions is still needed. Once the insect has landed on a plant, another set of resistance mechanisms enter into action. We have described a 3.06 Mbp introgression on top of Chromosome 5 (OR-5) from the wild tomato species S. habrochaites (CGN1.1561). For the identification of OR-5, we went from the selection of specific F2 plants to the development of F2BC4S1 and F2BC4S2 families. This introgression was sufficient to reduce whitefly fecundity without an evident effect on whitefly survival. The identification of mechanisms exclusively affecting whitefly fecundity and independent of trichomes type IV opens new doors for resistance breeding to whiteflies that may be especially interesting in greenhouse cultivation combination with natural enemies of the whitefly. As an additional layer of defences, plants can perceive stress signals and respond to them in a specific way through induction of their immune system. This induction can also be triggered by exposing the plants to priming agents like hormones, some xenobiotic chemicals, like benzothiadiazole (BTH), β-aminobutyric acid (BABA), and sugars. Although the effect of priming agents was shown in laboratory and field studies, little is known about the effect of the genetic background of tomato on the extent of the priming, e.g. do genotypes varying in their level of resistance to insects and pathogens respond in the same way to a priming agent. We assessed the effect of selected priming agents on the effectiveness of natural defence in tomato. A set of no-choice and choice bioassays was conducted using tomato genotypes varying in their level of basal resistance to Bemisia tabaci and pathogens. We observed that whitefly survival and oviposition were not affected by the priming treatment in no-choice assays. Overall, in choice assays, fructose treated plants were more preferred by whiteflies than control plants. A genotype specific effect of priming was seen for the line FCN93-6-2. On this tomato line, JA and BABA applications decreased the number of whiteflies, e.g. making them less preferred. In this thesis, I have gone from the screening of wild relatives of tomatoes to in depth characterization of resistance mechanisms. I have identified resistance mechanisms targeting specific stages of the whitefly life cycle, thus providing new tools for breeding durable whitefly resistance in tomato.

The silverleaf whitefly (Bemisia tabaciGenn.) poses a serious threat to tomato cultivation. A large part of the damage is done directly through heavy host plant colonization. Colonization has a negative impact on the plant, as the whitefly takes up nutrients from the phloem and induces phytotoxic responses, which result in irregular ripening of the fruits. However, most damage is done indirectly as the silverleaf whitefly vectors a broad range of plant pathogenic viruses. The silverleaf whitefly can successfully be controlled biologically in greenhouse cultivations, but control of the whitefly in the field is mainly based on the application of pesticides. The use of pesticides can have a negative effect on non-harmful or beneficial organisms in the field. Moreover, the effectiveness of pesticides can decline or even completely disappear through adaptation of the whitefly. An effective alternative for the use of pesticides could be the deployment of resistant cultivars. Nowadays, genetic factors responsible for whitefly resistance can be transferred faster and more efficiently into tomato cultivars through marker-assisted backcross breeding programs. Complete resistance against the whitefly is present in some crossable wild relatives of the cultivated tomato and the literature reports extensively about accessions with a high level of resistance against the whitefly. In this work, I have studied different populations that were developed by interspecific crosses between cultivated tomato and the tomato wild relativesS. habrochaitesLYC4 and S. pennelliiLA3791. By integrating datasets from different research disciplines, I have studied the background of whitefly resistance in these populations. Furthermore, these data were used to identify the chromosomal loci in the wild tomato relatives that harbor genes responsible for the resistance and that can be bred into cultivated tomato. The mechanisms underlying the resistance in S. pennelliiLA3791 were studied through phenotypic resistance assays that demonstrated that survival and oviposition of the whitefly were not possible on this wild relative. Through removal of glandular cells, present on the leaf trichomes, the resistance was almost completely lost and only adult survival was still significantly different from the wild type. This result led to the hypothesis that glandular trichomes play an important role in the resistance. This was confirmed in a segregating population based on a cross between S. pennelliiLA3791 and a susceptible cultivated tomato. Plants that lacked glandular trichomes type I and IV, had the same resistance level as the susceptible parent. Further analyses of the segregating population showed that the presence of glandular trichomes was not the only factor determining resistance, but that the composition and quantity of the metabolites in the glandular trichomes also played an important role. To gain more knowledge on the role of individual metabolites on whitefly resistance and susceptibility, we analyzed the total metabolite content of extreme phenotypes of the F2 population. Gas Chromatography-Mass Spectrometry (GC-MS) and Liquid Chromatography Time-Of-Flight Mass Spectrometry (LC-TOF-MS) were employed for the analyses of the total metabolite content. Analyses revealed that on basis of the total metabolite profiles the extreme phenotypes (susceptible versus resistant for the silverleaf whitefly) could be discriminated into two groups that were correlated with resistance or susceptibility. A number of these metabolites could be annotated, but for the majority of the components this was not possible on the basis of available literature and databases. Subsequently, I have studied the genetic basis of the phenotypic resistance parameters as well as the genetic basis of the metabolites from the GC-MS and LC-TOF-MS analyses. A genetic linkage map of the F2 mapping population was developed using DNA markers (Amplification Fragment Length Polymorphisms,AFLPs and Single Nucleotide Polymorphisms, SNPs). QTLs (Quantitative Trait Loci) were identified between the majority of the metabolites and the genetic markers (>90%) and also we found genetic linkages between whitefly resistance parameters and markers. The QTLs for metabolites and phenotypic parameters partly co-localized at the same positions on the genetic map. Several metabolite QTLs (mQTLs) co-localized with each other in so-called ‘hotspots’. Remarkably, the results of the individual phenotypic QTLs (phQTLs) for adult survival and oviposition as well as the mQTLs for the individual components did not give high explained variances (

The silverleaf whitefly (Bemisia tabaciGenn.) poses a serious threat to tomato cultivation. A large part of the damage is done directly through heavy host plant colonization. Colonization has a negative impact on the plant, as the whitefly takes up nutrients from the phloem and induces phytotoxic responses, which result in irregular ripening of the fruits. However, most damage is done indirectly as the silverleaf whitefly vectors a broad range of plant pathogenic viruses. The silverleaf whitefly can successfully be controlled biologically in greenhouse cultivations, but control of the whitefly in the field is mainly based on the application of pesticides. The use of pesticides can have a negative effect on non-harmful or beneficial organisms in the field. Moreover, the effectiveness of pesticides can decline or even completely disappear through adaptation of the whitefly. An effective alternative for the use of pesticides could be the deployment of resistant cultivars. Nowadays, genetic factors responsible for whitefly resistance can be transferred faster and more efficiently into tomato cultivars through marker-assisted backcross breeding programs. Complete resistance against the whitefly is present in some crossable wild relatives of the cultivated tomato and the literature reports extensively about accessions with a high level of resistance against the whitefly. In this work, I have studied different populations that were developed by interspecific crosses between cultivated tomato and the tomato wild relativesS. habrochaitesLYC4 and S. pennelliiLA3791. By integrating datasets from different research disciplines, I have studied the background of whitefly resistance in these populations. Furthermore, these data were used to identify the chromosomal loci in the wild tomato relatives that harbor genes responsible for the resistance and that can be bred into cultivated tomato. The mechanisms underlying the resistance in S. pennelliiLA3791 were studied through phenotypic resistance assays that demonstrated that survival and oviposition of the whitefly were not possible on this wild relative. Through removal of glandular cells, present on the leaf trichomes, the resistance was almost completely lost and only adult survival was still significantly different from the wild type. This result led to the hypothesis that glandular trichomes play an important role in the resistance. This was confirmed in a segregating population based on a cross between S. pennelliiLA3791 and a susceptible cultivated tomato. Plants that lacked glandular trichomes type I and IV, had the same resistance level as the susceptible parent. Further analyses of the segregating population showed that the presence of glandular trichomes was not the only factor determining resistance, but that the composition and quantity of the metabolites in the glandular trichomes also played an important role. To gain more knowledge on the role of individual metabolites on whitefly resistance and susceptibility, we analyzed the total metabolite content of extreme phenotypes of the F2 population. Gas Chromatography-Mass Spectrometry (GC-MS) and Liquid Chromatography Time-Of-Flight Mass Spectrometry (LC-TOF-MS) were employed for the analyses of the total metabolite content. Analyses revealed that on basis of the total metabolite profiles the extreme phenotypes (susceptible versus resistant for the silverleaf whitefly) could be discriminated into two groups that were correlated with resistance or susceptibility. A number of these metabolites could be annotated, but for the majority of the components this was not possible on the basis of available literature and databases. Subsequently, I have studied the genetic basis of the phenotypic resistance parameters as well as the genetic basis of the metabolites from the GC-MS and LC-TOF-MS analyses. A genetic linkage map of the F2 mapping population was developed using DNA markers (Amplification Fragment Length Polymorphisms,AFLPs and Single Nucleotide Polymorphisms, SNPs). QTLs (Quantitative Trait Loci) were identified between the majority of the metabolites and the genetic markers (>90%) and also we found genetic linkages between whitefly resistance parameters and markers. The QTLs for metabolites and phenotypic parameters partly co-localized at the same positions on the genetic map. Several metabolite QTLs (mQTLs) co-localized with each other in so-called ‘hotspots’. Remarkably, the results of the individual phenotypic QTLs (phQTLs) for adult survival and oviposition as well as the mQTLs for the individual components did not give high explained variances (

This PhD project started with the composition of a diverse panel of genotypes that represented, (i) roughly the flavor variation in the commercial Capsicum annuum breeding program of Rijk Zwaan, (ii) parents of available mapping populations and (iii) some genotypes that were expected to have extraordinary flavors. The complete set consisted of 35 genotypes of which 24 genotypes were non-pungent. Volatile and non-volatile compounds as well as some breeding parameters were measured in mature fruits of all genotypes throughout the growing season. In addition, from three harvests the non-pungent genotypes were evaluated for taste by a trained descriptive sensory panel. The biochemical profiling with use of SPME-GC-MS allowed visualization of between- and within-species volatile compound variation. Principal components analysis (PCA) on the intensity patterns of 391 putative volatile compounds revealed individual grouping of C. chinense, C. baccatum var. pendulum and C. annuum, indicating potentially interesting volatile variation present in the former two groups. A large group of saturated and unsaturated esters were mainly responsible for the individual grouping of the C. chinense accessions. Due to the elevated acid concentrations and aberrant volatile profiles of the C. baccatum var. pendulum accessions PEN45 and PEN79, the two BIL populations derived from these accessions were identified as interesting candidates for further study. Compared to e.g. Mazurka the citrate concentration of the C. baccatum accessions was 2.5-3 times higher and the malate concentrations were even up to 12 times higher (Chapter 2). Based on the non-pungent genotypes, we found highly correlated clusters of volatiles and non-volatiles, which could be related to metabolic pathways and common biochemical precursors (Chapter 3). Contrasts between genotypes were caused by both qualitative and quantitative differences in these metabolic clusters, with the phenolic derivatives, higher alkanes, sesquiterpenes and lipid derived volatiles forming the major determinants. For the description of the non-pungent genotypes the panelists used fourteen attributes to describe the flavor sensation in the mouth/throat, which were the texture attributes crunchiness, stickiness of the skin, toughness and juiciness, the basic taste attributes sweetness and sourness and the retronasal flavor attributes aroma intensity, grassiness, green bean, carrot, fruity/apple, perfume, petrochemical and musty. The variation in flavor could be reduced into two major sensory contrasts, which were a texture related contrast and the basic sweet-sour contrast. The structure of the PCA plots resulting from the analysis with one harvest (Chapter 3) and the analysis with the combined three harvests (Chapter 4) remained almost identical, indicating the stability of these contrasts. To relate the sensory attributes to the metabolite data and to determine the importance of the individual compounds we used Random Forest regression on the individual harvests and on the three harvests together. Several predictors for the attributes aroma, fruity/apple, sourness and sweetness were found in common between harvests, which we proposed as key-metabolites involved in flavor determination of sweet pepper (Chapter 4). This list contains compounds with known relations to attributes, like sweetness and sugars, but also several compounds with new relations. In this respect we have demonstrated for the first time, that the metabolites p-menth-1-en-9-al, (E)-β-ocimene, (Z)-2-penten-1-ol, and 1-methyl-1,4-cyclohexadiene are related to fruity/apple taste and/or sweetness of pepper. For sourness the only compound with a consistent significant contribution was an unknown C6H8O2 compound. We postulated therefore the hypothesis that in pepper the effect of sourness related metabolites is masked by other volatile and non-volatile compounds or texture differences (Chapter 3). Subsequently in Chapter 4 we described a clear sweetness-sourness interaction and demonstrated that the masking effect of fructose and other sugars explained why we did not find organic acids contributing to the prediction of sourness. The major sensory attributes were also predicted between harvests. The Random Forest predictions of the texture related attributes (juiciness, toughness, crunchiness and stickiness of the skin) and sweetness were very good. The predictions of the attributes aroma intensity, sourness and fruity/apple were somewhat lower and more variable between harvests, especially in the second harvest. In general, we concluded that prediction of attributes with higher heritabilities works better and is more consistent over harvests (Chapter 4). Based on the results of the initial experiments (Chapter 2) the species C. baccatum was chosen for further study. To exploit the potential flavor wealth of C. baccatum PEN45 we combined interspecific crossing with embryo rescue, resulting in a multi-parent BC2S1 population, that was characterized for sensory and biochemical variation (Chapter 5). We developed a population specific genetic linkage map for QTL mapping of characterized traits. Because of the complex structure of our BC2S1 mapping population we encountered several limitations, such as accidental co-segregation, underrepresentation of color linked markers and pre-selection leading to skewness, which might have resulted in false positive or missed QTLs. Despite these limitations, we were still fairly well able to map several biochemical, physical and sensory traits, as demonstrated at first for the (monogenic) control traits red color and pungency in the BC2S1 mapping population and in second instance by validation of genetic effects via an experiment with near-isogenic lines (NILs).This two-step approach turned out to be very powerful, since it led to the identification of the main results from this thesis: (i) Asmall C. baccatum LG3 introgression causing an extraordinary effect on flavor, which resulted in significantly higher scores for the attributes aroma, flowers, spices, celery and chives. In an attempt to identify the responsible biochemical compounds few consistently up- and down-regulated metabolites were detected, including the well-known pepper compound 2-isobutyl-3-methoxypyrazine (down) and 6-methyl-4-oxo-5-heptenal (up); (ii) Two introgressions (LG10.1 and LG1) had major effects on terpenoid content of mature fruits, affecting at least fifteen different monoterpenes; (iii) A second LG3 fragment resulted in a strong increase in Brix (total soluble solids) without negative effects on fruit size (Chapter 5). In Chapter 6 some extra sensory results of the pungent genotypes are given and a comparison between the two C. baccatum pendulum BILs (PEN45 and PEN79 derived) is made in light of the overall results. Finally the perspectives for breeding are discussed and presented in the form of a flowchart for flavor improvement.

Many plant species can hybridise and produce fertile offspring. Hybridization between cultivated species and their wild relatives has raised concerns with regard to GM crops, as it constitutes a possible route along which the transgene could disperse from crops into related wild species, establish itself in the natural population, and persist under natural conditions. This may cause unintended ecological consequences such as the formation of more invasive weeds and genetic erosion. After crop-wild hybridization, the persistence of the hybrids and of the crop genes (including the transgenes) in later generations depends on their genetic make-up, which consists of specific combinations of wild and crop genomic segments, and on the environmental conditions. Therefore, knowledge on the dynamics of crop-wild hybridization and introgression using conventional crop varieties is needed as it constitutes the baseline for putting into perspective the effects of transgene introgression under natural conditions. This study focused on understanding the genetic process of hybridization and introgression from cultivated to wild relative species using cultivated lettuce (Lactuca sativa L.) and its closest wild relative, prickly lettuce (L. serriola L.) as a crop-wild complex model. Natural populations of prickly lettuce in Europe were tested for the occurrence of crop-wild hybrids using Bayesian-based programmes (Structure, InStruct and NewHybrids) which returned a crop-wild hybrid occurrence of 7%. However, the geographical location of the hybrids led to the conclusion that crop-wild hybridization is not the cause of the recent northward spread of L. serriola in Europe. To test for the importance of the crop genomic segments to the performance of lettuce crop-wild hybrids, three hybrid generations were created (F2 as a selfing generation, and BC1 and BC2, backcrossed to the wild genotype to mimick the introgression process) by crossing L. sativa (cv. Dynamite) with L. serriola collected from Eys (the Netherlands). The three populations were genotyped with Single Nucleotide Polymorphism markers for genetic analysis. Because of the importance of the abiotic stresses as selection factors under natural conditions and the prospective of generating GM crop varieties with enhanced abiotic stress tolerance, the three populations were evaluated for vigour at the rosette stage under greenhouse conditions of non-stress, drought, salinity and nutrient deficiency. The BC1 population was also evaluated under field conditions at two locations (Wageningen and Sijbekarspel, the Netherlands) for hybrid germination, vigour, survival and reproduction. Based on the location and allelic effect of the QTLs for germination, vigour, survival and reproductive traits in the current lettuce crop-wild cross, genomic regions were suggested where transgenes could be or could not be located in order to mitigate their persistence in crop-wild hybrids through genetic hitchhiking and background selection.

Tomato, Solanum lycopersicum, is a host for Oidium neolycopersici, the cause of powdery mildew (PM). Though cultivated tomatoes are susceptible to PM, resistance is reported in wild Solanum species. By screening wild tomato species, nine loci conferring resistance to PM have been identified, namely Ol-1, ol-2, Ol-3, Ol-4, Ol-5, Ol-6, Ol-qtl1, Ol-qtl2, andOl-qtl3. These genes are located on different chromosomes and mediate different levels of resistance by different mechanisms. In this thesis we mainly focused on the Ol genes located on chromosome 6 (Ol-1, Ol-4, Ol-5 and Ol-6) with the aim to fine-map and eventually clone these genes. In addition, we studied the contribution of different phytohormone pathways to the resistance mediated by Ol-1, ol-2, Ol-4 and Ol-qtls. We first focused on the Ol genes on the short arm of tomato chromosome 6, Ol-4 originating from S. peruvianum LA2172 and Ol-6 with unknown origin (Chapter 2). We showed that Ol-4 and Ol-6 are homologues of the Mi-1 gene. On the short arm of tomato chromosome 6, the Mi-1gene cluster is about 400 Kb in size and consists of several other genes besides the Mi-1 homologues. There are transport inhibitor responses-like (TIR-like) genes embedded in this cluster. Interestingly, the copy number of these TIR-like genes in the nematode-resistant tomatoes is less than that in nematode-susceptible ones (Chapter 3). Furthermore, lower expression of these TIR-like genes was observed in roots, but not in leaves, of nematode-resistant plants compared to nematode-susceptible plants. These observations prompted us to suggest and to discuss two different scenarios explaining how TIR-like genes could play a role in the plant response to root-knot nematodes. Then, we studied the Ol-1 and Ol-5, which are located on the long arm of chromosome 6, and originated from different S. habrochaites accessions. Ol-1 is closely linked to Ol-5. With fine-mapping, we narrowed down this locus to a 73 Kb interval which contains at least 10 putative genes. Interestingly we observed an interaction between chromosome regions harboring Ol-1 and Ol-5, indicating that the interaction between Ol-1 and Ol-5 is needed to confer PM resistance. Both Ol-1 and Ol-5 trigger delayed cell death that is distinguishable from hypersensitive response (HR), the hallmark of R gene response to biotrophic pathogens. The delayed cell death associated with Ol-1 and Ol-5 resembles the autophagic PCD. We observed that Ol-1 and Ol-5 were both required for on-time and effective cell death to stop PM. If one of these two genes was not present, cell deathcould not happen or not be effective enough to stop pathogen growth. Finally, we investigated the involvement of phytohormone pathways in PM resistance conferred by the Olgenes, including Ol-1, ol-2, Ol-4 and Ol-qtls (Chapter 5). There is overwhelming evidence implicating plant hormones in plant responses to pathogens. In this experiment we, in addition to Ol-1 and Ol-4, included other resistance loci for PM resistance in tomato. The first one is ol-2, a homologue of the barley mlo gene and derived from S. lycopersicum var cerasiforme LA1230. This gene confers resistance to PM by triggering callose deposition and, thereby, cell wall fortification. The other is Ol-qtls, a combination of three QTLs for PM resistance associated with both delayed cell deathand callose deposition. NILs carrying Ol-1, ol-2, Ol-4 and Ol-qtls, plus the background of these NILs (S. lycopersicum cv Moneymaker, MM), provided us the possibility to compare the involvement of hormonal pathways in different kinds of tomato responses. These responses include basal defense, cell wall fortification, delayed cell death,and HR. We quantified the expression of marker genes for the pathways of salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), and ethylene (ET) over a time-course after inoculation with PM. As a complementary approach, we crossed our NILs with tomato mutants for JA, ET and ABA. Our results suggested that Ol-4-mediated resistance probably relies on the SA pathway. Ol-1 and Ol-qtls require ET to promote the delayed cell deathfor PM resistance. JA deficiency can compromise resistance mediated by ol-2. Our results also suggested that ABA is required for those interactions demanding callose deposition, resistance associated with ol-2 and Ol-qtls. These results present a nice example of the involvement of different phytohormones in different phases of resistance against PM in tomato. Altogether, this thesis describes different tomato resistance mechanisms triggered by different resistance genes in the same pathosystem, underscoring the plant ability to adopt diverse molecular mechanisms to defense itself against intruders.

The powdery mildew disease, caused by obligate biotrophic fungi belonging to the Ascomycete order of Erysiphales, is common among higher plants and represents one of the most important threats for the cultivation of many crop species. Although powdery mildew resistance is usually a dominant trait, recessively inherited resistance has been reported to occur in Arabidopsis, barley, tomato and pea. In Chapter 1 of this thesis, we provide a state of the art on the understanding of mechanisms underlying plant immunity and review monogenic dominant and recessive sources used in breeding in order to develop resistant cultivars. In Chapter 2 and 3, we describe a successful homology-based cloning approach for the isolation of the recessive ol-2 gene, naturally occurring in a tomato accession collected in Ecuador and conferring broad-spectrum resistance to the powdery mildew fungus Oidium neolycopersici. We realized that ol-2 resistance shares striking similarities with well-known barley and Arabidopsis mlo powdery mildew resistance, originating from loss-of-function mutations of genes encoding for specific seven transmembrane domains MLO isoforms. The following chain of evidence was provided demonstrating that ol-2 resistance is due to the loss of the tomato MLO homolog SlMLO1: a) Ol-2 and SlMLO1 loci share the same genetic and cytogenetic position; b) resistant ol-2/ol-2 lines are homozygous for a loss-of-function deletion in the sequence of SlMLO1; c) a molecular marker developed on the mutation site co-segregates with resistant individuals in an F2population; d) SlMLO1 transgenic expression in ol-2/ol-2 individuals results in disease susceptibility; e) SlMLO1 virus-induced silencing in Ol-2/Ol-2 individuals is associated to increased powdery mildew resistance. In Chapter 4, we first illustrate a chemical mutagenesis program allowing the identification of a pea line showing recessive resistance towards the powdery mildew fungus Erysiphe pisi. Histological and genetic analyses revealed that the mutated gene is allelic to er1, commonly used in pea breeding for the development of resistant cultivars. As defense mechanisms associated to er1 resistance are reminiscent of mlo immunity, we sequenced the pea MLO homolog PsMLO1 and found a loss-of-function point mutation characterizing the resistant line. A polymorphic CAPS marker was developed on the mutation site and found to be fully co-segregating with resistance in a large F2population. Finally, PsMLO1 sequencing in three er1 resistant cultivars also resulted in the identification of aberrant alleles, further substantiating the identification of another case of mlo-based immunity. In Chapter 5, we report the identification (in vitro and in silico) of a series of MLO homolog sequences in five cultivated Solanaceae species affected by the powdery mildew disease. Comparative analyses using a dataset of several dicot MLO proteins allowed the identification of candidate isoforms for disease susceptibility and the detection of cluster-specific transmembrane amino acid motifs. In Chapter 6, we look at disease resistance as a condition due to the lack of susceptibility genes like MLO. We review several susceptibility genes isolated in crop species and in Arabidopsis, with respect to their molecular characterization, their role in plant-pathogen interactions and the resistant phenotype deriving from their loss-of-function mutations/silencing. A breeding strategy based on the lack of plant susceptibility genes is discussed.

Tomato, Solanum lycopersicum, is a host for Oidium neolycopersici, the cause of powdery mildew (PM). Though cultivated tomatoes are susceptible to PM, resistance is reported in wild Solanum species. By screening wild tomato species, nine loci conferring resistance to PM have been identified, namely Ol-1, ol-2, Ol-3, Ol-4, Ol-5, Ol-6, Ol-qtl1, Ol-qtl2, andOl-qtl3. These genes are located on different chromosomes and mediate different levels of resistance by different mechanisms. In this thesis we mainly focused on the Ol genes located on chromosome 6 (Ol-1, Ol-4, Ol-5 and Ol-6) with the aim to fine-map and eventually clone these genes. In addition, we studied the contribution of different phytohormone pathways to the resistance mediated by Ol-1, ol-2, Ol-4 and Ol-qtls. We first focused on the Ol genes on the short arm of tomato chromosome 6, Ol-4 originating from S. peruvianum LA2172 and Ol-6 with unknown origin (Chapter 2). We showed that Ol-4 and Ol-6 are homologues of the Mi-1 gene. On the short arm of tomato chromosome 6, the Mi-1gene cluster is about 400 Kb in size and consists of several other genes besides the Mi-1 homologues. There are transport inhibitor responses-like (TIR-like) genes embedded in this cluster. Interestingly, the copy number of these TIR-like genes in the nematode-resistant tomatoes is less than that in nematode-susceptible ones (Chapter 3). Furthermore, lower expression of these TIR-like genes was observed in roots, but not in leaves, of nematode-resistant plants compared to nematode-susceptible plants. These observations prompted us to suggest and to discuss two different scenarios explaining how TIR-like genes could play a role in the plant response to root-knot nematodes. Then, we studied the Ol-1 and Ol-5, which are located on the long arm of chromosome 6, and originated from different S. habrochaites accessions. Ol-1 is closely linked to Ol-5. With fine-mapping, we narrowed down this locus to a 73 Kb interval which contains at least 10 putative genes. Interestingly we observed an interaction between chromosome regions harboring Ol-1 and Ol-5, indicating that the interaction between Ol-1 and Ol-5 is needed to confer PM resistance. Both Ol-1 and Ol-5 trigger delayed cell death that is distinguishable from hypersensitive response (HR), the hallmark of R gene response to biotrophic pathogens. The delayed cell death associated with Ol-1 and Ol-5 resembles the autophagic PCD. We observed that Ol-1 and Ol-5 were both required for on-time and effective cell death to stop PM. If one of these two genes was not present, cell deathcould not happen or not be effective enough to stop pathogen growth. Finally, we investigated the involvement of phytohormone pathways in PM resistance conferred by the Olgenes, including Ol-1, ol-2, Ol-4 and Ol-qtls (Chapter 5). There is overwhelming evidence implicating plant hormones in plant responses to pathogens. In this experiment we, in addition to Ol-1 and Ol-4, included other resistance loci for PM resistance in tomato. The first one is ol-2, a homologue of the barley mlo gene and derived from S. lycopersicum var cerasiforme LA1230. This gene confers resistance to PM by triggering callose deposition and, thereby, cell wall fortification. The other is Ol-qtls, a combination of three QTLs for PM resistance associated with both delayed cell deathand callose deposition. NILs carrying Ol-1, ol-2, Ol-4 and Ol-qtls, plus the background of these NILs (S. lycopersicum cv Moneymaker, MM), provided us the possibility to compare the involvement of hormonal pathways in different kinds of tomato responses. These responses include basal defense, cell wall fortification, delayed cell death,and HR. We quantified the expression of marker genes for the pathways of salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), and ethylene (ET) over a time-course after inoculation with PM. As a complementary approach, we crossed our NILs with tomato mutants for JA, ET and ABA. Our results suggested that Ol-4-mediated resistance probably relies on the SA pathway. Ol-1 and Ol-qtls require ET to promote the delayed cell deathfor PM resistance. JA deficiency can compromise resistance mediated by ol-2. Our results also suggested that ABA is required for those interactions demanding callose deposition, resistance associated with ol-2 and Ol-qtls. These results present a nice example of the involvement of different phytohormones in different phases of resistance against PM in tomato. Altogether, this thesis describes different tomato resistance mechanisms triggered by different resistance genes in the same pathosystem, underscoring the plant ability to adopt diverse molecular mechanisms to defense itself against intruders.

The powdery mildew disease, caused by obligate biotrophic fungi belonging to the Ascomycete order of Erysiphales, is common among higher plants and represents one of the most important threats for the cultivation of many crop species. Although powdery mildew resistance is usually a dominant trait, recessively inherited resistance has been reported to occur in Arabidopsis, barley, tomato and pea. In Chapter 1 of this thesis, we provide a state of the art on the understanding of mechanisms underlying plant immunity and review monogenic dominant and recessive sources used in breeding in order to develop resistant cultivars. In Chapter 2 and 3, we describe a successful homology-based cloning approach for the isolation of the recessive ol-2 gene, naturally occurring in a tomato accession collected in Ecuador and conferring broad-spectrum resistance to the powdery mildew fungus Oidium neolycopersici. We realized that ol-2 resistance shares striking similarities with well-known barley and Arabidopsis mlo powdery mildew resistance, originating from loss-of-function mutations of genes encoding for specific seven transmembrane domains MLO isoforms. The following chain of evidence was provided demonstrating that ol-2 resistance is due to the loss of the tomato MLO homolog SlMLO1: a) Ol-2 and SlMLO1 loci share the same genetic and cytogenetic position; b) resistant ol-2/ol-2 lines are homozygous for a loss-of-function deletion in the sequence of SlMLO1; c) a molecular marker developed on the mutation site co-segregates with resistant individuals in an F2population; d) SlMLO1 transgenic expression in ol-2/ol-2 individuals results in disease susceptibility; e) SlMLO1 virus-induced silencing in Ol-2/Ol-2 individuals is associated to increased powdery mildew resistance. In Chapter 4, we first illustrate a chemical mutagenesis program allowing the identification of a pea line showing recessive resistance towards the powdery mildew fungus Erysiphe pisi. Histological and genetic analyses revealed that the mutated gene is allelic to er1, commonly used in pea breeding for the development of resistant cultivars. As defense mechanisms associated to er1 resistance are reminiscent of mlo immunity, we sequenced the pea MLO homolog PsMLO1 and found a loss-of-function point mutation characterizing the resistant line. A polymorphic CAPS marker was developed on the mutation site and found to be fully co-segregating with resistance in a large F2population. Finally, PsMLO1 sequencing in three er1 resistant cultivars also resulted in the identification of aberrant alleles, further substantiating the identification of another case of mlo-based immunity. In Chapter 5, we report the identification (in vitro and in silico) of a series of MLO homolog sequences in five cultivated Solanaceae species affected by the powdery mildew disease. Comparative analyses using a dataset of several dicot MLO proteins allowed the identification of candidate isoforms for disease susceptibility and the detection of cluster-specific transmembrane amino acid motifs. In Chapter 6, we look at disease resistance as a condition due to the lack of susceptibility genes like MLO. We review several susceptibility genes isolated in crop species and in Arabidopsis, with respect to their molecular characterization, their role in plant-pathogen interactions and the resistant phenotype deriving from their loss-of-function mutations/silencing. A breeding strategy based on the lack of plant susceptibility genes is discussed.

Many plant species can hybridise and produce fertile offspring. Hybridization between cultivated species and their wild relatives has raised concerns with regard to GM crops, as it constitutes a possible route along which the transgene could disperse from crops into related wild species, establish itself in the natural population, and persist under natural conditions. This may cause unintended ecological consequences such as the formation of more invasive weeds and genetic erosion. After crop-wild hybridization, the persistence of the hybrids and of the crop genes (including the transgenes) in later generations depends on their genetic make-up, which consists of specific combinations of wild and crop genomic segments, and on the environmental conditions. Therefore, knowledge on the dynamics of crop-wild hybridization and introgression using conventional crop varieties is needed as it constitutes the baseline for putting into perspective the effects of transgene introgression under natural conditions. This study focused on understanding the genetic process of hybridization and introgression from cultivated to wild relative species using cultivated lettuce (Lactuca sativa L.) and its closest wild relative, prickly lettuce (L. serriola L.) as a crop-wild complex model. Natural populations of prickly lettuce in Europe were tested for the occurrence of crop-wild hybrids using Bayesian-based programmes (Structure, InStruct and NewHybrids) which returned a crop-wild hybrid occurrence of 7%. However, the geographical location of the hybrids led to the conclusion that crop-wild hybridization is not the cause of the recent northward spread of L. serriola in Europe. To test for the importance of the crop genomic segments to the performance of lettuce crop-wild hybrids, three hybrid generations were created (F2 as a selfing generation, and BC1 and BC2, backcrossed to the wild genotype to mimick the introgression process) by crossing L. sativa (cv. Dynamite) with L. serriola collected from Eys (the Netherlands). The three populations were genotyped with Single Nucleotide Polymorphism markers for genetic analysis. Because of the importance of the abiotic stresses as selection factors under natural conditions and the prospective of generating GM crop varieties with enhanced abiotic stress tolerance, the three populations were evaluated for vigour at the rosette stage under greenhouse conditions of non-stress, drought, salinity and nutrient deficiency. The BC1 population was also evaluated under field conditions at two locations (Wageningen and Sijbekarspel, the Netherlands) for hybrid germination, vigour, survival and reproduction. Based on the location and allelic effect of the QTLs for germination, vigour, survival and reproductive traits in the current lettuce crop-wild cross, genomic regions were suggested where transgenes could be or could not be located in order to mitigate their persistence in crop-wild hybrids through genetic hitchhiking and background selection.

Potato (Solanum tuberosum L.) is nowadays the most important non-cereal food crop in the world. It is prone to huge annual losses due to late blight, the disease caused by the oomycete pathogen Phytophthora infestans. Modern management of late blight necessitates the use of multiple resistance (R) genes, which requires efficient pipelines for identification, isolation and characterization of R genes. This thesis employs effectoromics, i.e. the use of effectors (pathogenic secreted protein) to probe corresponding R gene(s) in a host plant and sort out their functional redundancy and specificity. Using cytoplasmic RXLR effectors of P. infestans to probe resistant Solanum germplasm for late blight R genes, we were able to: (i) assess the biodiversity of Avr-blb1, characterize the genomic structure of virulent P. infestans isolates on Rpi-blb1 plants and thus provide a technical solution for long-term disease management; (ii) identify the centre of origin of R3a, characterize R3a gene homologues and a functional R gene (Rpi-sto2), and (iii) uncover the potential co-evolution at both R and Avr side for the R2/PiAvr2-PexRD11 interactions, providing more diversity and specificity of R2 homologues, which may be valuable for potato breeding

The potato late blight disease, caused by the oomycete Phytophthora infestans, is a major threat for potato production worldwide. To breed potato varieties with durable resistance against P. infestans, it is necessary to combine two or more resistance (R) genes. Single R genes are easily overcome by the rapidly evolving pathogen, whereas the presence of several R genes could probably prevent gain of virulence from a single mutation in the pathogen. The large gene pool available within wild potato species offers sufficient possibilities to identify new and diverse R genes conferring resistance to P. infestans (Rpi). Map-based cloning is the most suitable strategy to isolate such new Rpi genes. The objective of this research was mapping, if possible, followed by cloning of Rpi genes from wild Solanum species. Resistance to P. infestans occurring in four different wild Solanum species were mapped in a major R gene cluster on chromosome 11. Natural stacking of three R genes located on different chromosomes was identified in a natural hybrid. In addition, we initiated studies on another type of defense system that is not based on the typical R genes, namely the response of Solanum to INF1 elicitin.

The potato late blight disease, caused by the oomycete Phytophthora infestans, is a major threat for potato production worldwide. To breed potato varieties with durable resistance against P. infestans, it is necessary to combine two or more resistance (R) genes. Single R genes are easily overcome by the rapidly evolving pathogen, whereas the presence of several R genes could probably prevent gain of virulence from a single mutation in the pathogen. The large gene pool available within wild potato species offers sufficient possibilities to identify new and diverse R genes conferring resistance to P. infestans (Rpi). Map-based cloning is the most suitable strategy to isolate such new Rpi genes. The objective of this research was mapping, if possible, followed by cloning of Rpi genes from wild Solanum species. Resistance to P. infestans occurring in four different wild Solanum species were mapped in a major R gene cluster on chromosome 11. Natural stacking of three R genes located on different chromosomes was identified in a natural hybrid. In addition, we initiated studies on another type of defense system that is not based on the typical R genes, namely the response of Solanum to INF1 elicitin.

Potato (Solanum tuberosum L.) is nowadays the most important non-cereal food crop in the world. It is prone to huge annual losses due to late blight, the disease caused by the oomycete pathogen Phytophthora infestans. Modern management of late blight necessitates the use of multiple resistance (R) genes, which requires efficient pipelines for identification, isolation and characterization of R genes. This thesis employs effectoromics, i.e. the use of effectors (pathogenic secreted protein) to probe corresponding R gene(s) in a host plant and sort out their functional redundancy and specificity. Using cytoplasmic RXLR effectors of P. infestans to probe resistant Solanum germplasm for late blight R genes, we were able to: (i) assess the biodiversity of Avr-blb1, characterize the genomic structure of virulent P. infestans isolates on Rpi-blb1 plants and thus provide a technical solution for long-term disease management; (ii) identify the centre of origin of R3a, characterize R3a gene homologues and a functional R gene (Rpi-sto2), and (iii) uncover the potential co-evolution at both R and Avr side for the R2/PiAvr2-PexRD11 interactions, providing more diversity and specificity of R2 homologues, which may be valuable for potato breeding

This book text presents methodologies and case studies that provide recommendations for the conservation and use of crop wild relatives. In a national, regional or global context, the status of crop wild relatives, that are closely related to crop plants, is examined. Conservation of crop wild relatives is important to enable these species to be included in plant breeding activities for beneficial traits such as pest or disease resistance and yield improvement.

The aphidMyzuspersicae(Sulzer) constitute a threat to potato crops because of their efficiency to transmit viruses. Many wildSolanumspecies have been reported to have resistance to M.persicae . These species represent an important potential source of resistance, which can be used to enhance resistance in crops. The aim of this thesis was to find and study in tuber bearingSolanumspecies different mechanisms of resistance to M.persicae that also would reduced the spread of viruses. Resistance and susceptibility of wildSolanumgenotypes and cultivated potatoes were characterized and linked to plant-aphid interactions with respect to aphid behaviour and performance and plant responses to aphid attack. A combination of aphid colony-development assay with the study of probing activities was used to localize resistance factors in tissues ofSolanumgenotypes. Genotypes with different degrees of resistance were found. S.stoloniferum showed pre-phloem resistance to M.persicae, and hence it was used as a model plant to unravel and characterisephenotypicallyand at molecular level the interactions of a single plant with different aphid species. Aphid performance, settling behaviour, and probing studies of M.persicae andMacrosiphumeuphorbiae (Thomas) on S.stoloniferum showed that while S.stoloniferum could be a host plant for M.euphorbiae , it is a poor host for M.persicae .The resistance found in S.stoloniferum against M.persicae seems to rely on constitutively expressed physical traits with some age effects.The transcriptional response of S.stoloniferum to the attack of M.euphorbiae (compatible interactions) was stronger than in response to M.persicae (incompatible). This stronger response to M.euphorbiae involves up-regulation of genes related to pathogenesis (PR), regulatory, and protein metabolism and down-regulation of regulatory genes, general metabolism and photosynthesis related genes.Infestation of S.stoloniferum leaves with M.persicae aphids leads to the development of pustules. Microscopic analysis of the pustules showed a burst of tissue on the surrounding of the vascular bundle. In contrast, the infestation with M.euphorbiae did not induce any visible cellular changes. The formation of pustules and the induction of some genes in S.stoloniferum suggest that a similar situation might exist between plants-pathogenic bacteria interactions, and plants-aphids interactions.Response of S.tuberosumcv.Kardalto the attack of M.persicae was evaluated by studying gene expression. The plant responses depend on foliage maturity. Young leaves ofcv.Kardalare resistant to M.persicae whereas mature to senescent leaves are susceptible. In old leaves M.persicae attack elicits higher number of differentially regulated genes than in young leaves.The transcriptional results obtained with the two systems: (1) S.stoloniferum after the attack of M.persicae and M.euphorbiae , and (2) S.tuberosumcv.Kardalat different maturity leaf stages after the attack of M.persicae were compared. The gene-expression studies provide evidence that S.tuberosum and S.stoloniferum respond by activating the salicylic acid (SA) and ethylene (ET) pathways. Genes responsive tojasmonicacid were differentially regulated in low number in the S.stoloniferum - M.euphorbiae but not in thecv.Kardal- M.persicae interaction.At the local level a compatible plant-aphid interaction resulted in a broader gene expression response than that a systemic level. Genes were identified related to changes in sink-source relationship at the feeding site which may indicate a plant manipulation by the aphids related to the process of changing the physiological status of the tissue towards a local metabolic sink; also genes related to signal-transduction pathways, regulation and signalling, protein metabolism, maintenance of cell homeostasis, transport, secondary metabolism, and structural features were found to be differentially regulated. On the contrary, in incompatible interactions the transcriptional response of the plant seems to be more restricted.PLRV-infection in potato plants ofcv.Kardalwas found to affect aphid behaviour. M.persicae responses to volatiles emitted from PLRV-infected and non-infected plants depend on the age of the leaf. PLRV infection affects also the probing behaviour of M.persicae . On virus-infected plantsstyletpenetration into the plant tissue is enhanced. The transmission efficiency of PLRV is also expected to be affected in PLRV-infected plants because these plants attract more aphids than non-infected plants.The results obtained on this research (e.g., aphid performance, probing behaviour, colony development, settling behaviour, and gene expression analysis ofSolanumspp. attacked by aphids) contribute to the understanding of plant responses towards aphid attack as a basis for further unravelling the resistance mechanisms at the metabolic, molecular, and genetic levels.

The aphidMyzuspersicae(Sulzer) constitute a threat to potato crops because of their efficiency to transmit viruses. Many wildSolanumspecies have been reported to have resistance to M.persicae . These species represent an important potential source of resistance, which can be used to enhance resistance in crops. The aim of this thesis was to find and study in tuber bearingSolanumspecies different mechanisms of resistance to M.persicae that also would reduced the spread of viruses. Resistance and susceptibility of wildSolanumgenotypes and cultivated potatoes were characterized and linked to plant-aphid interactions with respect to aphid behaviour and performance and plant responses to aphid attack. A combination of aphid colony-development assay with the study of probing activities was used to localize resistance factors in tissues ofSolanumgenotypes. Genotypes with different degrees of resistance were found. S.stoloniferum showed pre-phloem resistance to M.persicae, and hence it was used as a model plant to unravel and characterisephenotypicallyand at molecular level the interactions of a single plant with different aphid species. Aphid performance, settling behaviour, and probing studies of M.persicae andMacrosiphumeuphorbiae (Thomas) on S.stoloniferum showed that while S.stoloniferum could be a host plant for M.euphorbiae , it is a poor host for M.persicae .The resistance found in S.stoloniferum against M.persicae seems to rely on constitutively expressed physical traits with some age effects.The transcriptional response of S.stoloniferum to the attack of M.euphorbiae (compatible interactions) was stronger than in response to M.persicae (incompatible). This stronger response to M.euphorbiae involves up-regulation of genes related to pathogenesis (PR), regulatory, and protein metabolism and down-regulation of regulatory genes, general metabolism and photosynthesis related genes.Infestation of S.stoloniferum leaves with M.persicae aphids leads to the development of pustules. Microscopic analysis of the pustules showed a burst of tissue on the surrounding of the vascular bundle. In contrast, the infestation with M.euphorbiae did not induce any visible cellular changes. The formation of pustules and the induction of some genes in S.stoloniferum suggest that a similar situation might exist between plants-pathogenic bacteria interactions, and plants-aphids interactions.Response of S.tuberosumcv.Kardalto the attack of M.persicae was evaluated by studying gene expression. The plant responses depend on foliage maturity. Young leaves ofcv.Kardalare resistant to M.persicae whereas mature to senescent leaves are susceptible. In old leaves M.persicae attack elicits higher number of differentially regulated genes than in young leaves.The transcriptional results obtained with the two systems: (1) S.stoloniferum after the attack of M.persicae and M.euphorbiae , and (2) S.tuberosumcv.Kardalat different maturity leaf stages after the attack of M.persicae were compared. The gene-expression studies provide evidence that S.tuberosum and S.stoloniferum respond by activating the salicylic acid (SA) and ethylene (ET) pathways. Genes responsive tojasmonicacid were differentially regulated in low number in the S.stoloniferum - M.euphorbiae but not in thecv.Kardal- M.persicae interaction.At the local level a compatible plant-aphid interaction resulted in a broader gene expression response than that a systemic level. Genes were identified related to changes in sink-source relationship at the feeding site which may indicate a plant manipulation by the aphids related to the process of changing the physiological status of the tissue towards a local metabolic sink; also genes related to signal-transduction pathways, regulation and signalling, protein metabolism, maintenance of cell homeostasis, transport, secondary metabolism, and structural features were found to be differentially regulated. On the contrary, in incompatible interactions the transcriptional response of the plant seems to be more restricted.PLRV-infection in potato plants ofcv.Kardalwas found to affect aphid behaviour. M.persicae responses to volatiles emitted from PLRV-infected and non-infected plants depend on the age of the leaf. PLRV infection affects also the probing behaviour of M.persicae . On virus-infected plantsstyletpenetration into the plant tissue is enhanced. The transmission efficiency of PLRV is also expected to be affected in PLRV-infected plants because these plants attract more aphids than non-infected plants.The results obtained on this research (e.g., aphid performance, probing behaviour, colony development, settling behaviour, and gene expression analysis ofSolanumspp. attacked by aphids) contribute to the understanding of plant responses towards aphid attack as a basis for further unravelling the resistance mechanisms at the metabolic, molecular, and genetic levels.

Oidium neolycopersiciis the causal agent of the tomato powdery mildew disease. Besides a few recently released resistant cultivars, all tomato cultivars are susceptible to O. neolycopersici. Therefore, resistance to O. neolycopersici is an important trait in tomato breeding.The aim of this research is to study theinteraction between tomato ( Lycopersicon esculentum ) and tomato powdery mildew ( O. neolycopersici ). Firstly, we characterized the genetic basis for several resistances against O. neolycopersicioriginating from wild Lycopersicon species and mapped the correspondingresistance genes on the tomato genome. Remarkably, all the mapped dominant Ol -genes are located in three loci on tomato Chromosome 6. On the long arm, one locus contains the resistance genes Ol-1 and Ol-3 , originating from L. hirsutum G1.1560 and G1.1290, respectively, while another closely linked locus harbors Ol-5 , a resistance gene originating from L.hirsutum PI247087. On the short arm of Chromosome 6, one locus contains the two resistance genes Ol-4 (originating from L. peruvianum LA2172) and Ol-6 (unknown origin). In addition to these Ol -loci, three quantitative trait loci (QTLs) have been identified governing the O. neolycopersici resistance in L. parviflorum G1. 1601. One QTL ( Ol-qtl1 ) interval overlaps with Ol-1 , Ol-3 and Ol-5 . Two other QTLs ( Ol-qtl2 and Ol-qtl3 ) are linked on Chromosome 12 with Ol-qtl2 in the vicinity of the Lv locus that confers resistance to another powdery mildew species, Leveillula taurica . Secondly, we generated a set of near isogenic lines (NILs), each of which contains an individual Ol -gene in a genetic background of L.esculentum cv. Moneymaker. These NILs represent well-defined plant material and are a very useful tool to study the interaction between Ol -genes and O. neolycopersici isolates. Thirdly, we determined the race-specificity of the resistance conferred by the Ol -genes by testing the NILs with local isolates of O.neolycopersici in different geographic places. In addition to the dominant Ol -genes, the recessive gene ol-2 that originates from L. esculentum var cerasiforme and is located on Chromosome 4 (Ciccarese et al., 1998; De Giovanni et al., 2004) was also included in our study. Our data suggested that the resistance to O. neolycopersici conferred by different Ol -genes ( Ol-1, ol-2, Ol-3, Ol-4, Ol-5 and Ol-6 ) can be race-specific. Finally, we studied the resistance mechanisms associated with different Ol -genes by using the NILs. Microscopically, the resistance conferred by the dominant Ol -genes is associated with a hypersensitive reaction (HR), while, the resistance governed by the recessive gene ol-2 is related to papilla formation.

Oidium neolycopersiciis the causal agent of the tomato powdery mildew disease. Besides a few recently released resistant cultivars, all tomato cultivars are susceptible to O. neolycopersici. Therefore, resistance to O. neolycopersici is an important trait in tomato breeding.The aim of this research is to study theinteraction between tomato ( Lycopersicon esculentum ) and tomato powdery mildew ( O. neolycopersici ). Firstly, we characterized the genetic basis for several resistances against O. neolycopersicioriginating from wild Lycopersicon species and mapped the correspondingresistance genes on the tomato genome. Remarkably, all the mapped dominant Ol -genes are located in three loci on tomato Chromosome 6. On the long arm, one locus contains the resistance genes Ol-1 and Ol-3 , originating from L. hirsutum G1.1560 and G1.1290, respectively, while another closely linked locus harbors Ol-5 , a resistance gene originating from L.hirsutum PI247087. On the short arm of Chromosome 6, one locus contains the two resistance genes Ol-4 (originating from L. peruvianum LA2172) and Ol-6 (unknown origin). In addition to these Ol -loci, three quantitative trait loci (QTLs) have been identified governing the O. neolycopersici resistance in L. parviflorum G1. 1601. One QTL ( Ol-qtl1 ) interval overlaps with Ol-1 , Ol-3 and Ol-5 . Two other QTLs ( Ol-qtl2 and Ol-qtl3 ) are linked on Chromosome 12 with Ol-qtl2 in the vicinity of the Lv locus that confers resistance to another powdery mildew species, Leveillula taurica . Secondly, we generated a set of near isogenic lines (NILs), each of which contains an individual Ol -gene in a genetic background of L.esculentum cv. Moneymaker. These NILs represent well-defined plant material and are a very useful tool to study the interaction between Ol -genes and O. neolycopersici isolates. Thirdly, we determined the race-specificity of the resistance conferred by the Ol -genes by testing the NILs with local isolates of O.neolycopersici in different geographic places. In addition to the dominant Ol -genes, the recessive gene ol-2 that originates from L. esculentum var cerasiforme and is located on Chromosome 4 (Ciccarese et al., 1998; De Giovanni et al., 2004) was also included in our study. Our data suggested that the resistance to O. neolycopersici conferred by different Ol -genes ( Ol-1, ol-2, Ol-3, Ol-4, Ol-5 and Ol-6 ) can be race-specific. Finally, we studied the resistance mechanisms associated with different Ol -genes by using the NILs. Microscopically, the resistance conferred by the dominant Ol -genes is associated with a hypersensitive reaction (HR), while, the resistance governed by the recessive gene ol-2 is related to papilla formation.

Cladosporium fulvumis a biotrophic fungal leaf pathogen of tomato. Numerous C. fulvum resistance genes ( Cf genes) are present in wild tomato. In this thesis, a molecular study on the evolution of Cf genes is presented. Cf-9 originates from the wild tomato species Lycopersicon pimpinellifolium , and mediates recognition of the Avr9 elicitor of C. fulvum . Cf-9 (also known as 9C ) is a member of a large gene family and is located in a cluster of five homologues. In the L. pimpinellifolium population a second gene was identified that also mediates recognition of Avr9. The first half of this 9DC gene is nearly identical to the Cf-9 homologue 9D , whereas the second half is nearly identical to Cf-9 . Isolation of the 9DC cluster of L. pimpinellifolium allowed comparison of the Cf-9 and 9DC gene clusters. 9DC was shown to be the result of an intragenic recombination between Cf-9 and 9D . Of the three 9DC genes in the 9DC cluster, 9DC2 is probably the main determinant of Avr9 recognition. To further study the evolution of Cf genes, all eight wild tomato species were screened for recognition of the Avr4 and Avr9 elicitors. In all species recognition of one or both elicitors was found, and several corresponding Cf genes were isolated. Sequence analysis showed that both the Cf-4 and Cf-9 genes are highly conserved throughout the genus Lycopersicon , suggesting that C. fulvum is an ancient pathogen of wild tomato. Finally, from L. peruvianum two Cf gene homologues were isolated, which induce an elicitor-independent hypersensitive response (cell death) upon overexpression in several tobacco species. More of these so-called auto-activators were generated in vitro by gene shuffling of a number of Cf gene homologues. The auto-activators function in the same signalling pathway as Cf-9 . Several models that explain the elicitor-independency of the auto-activators are discussed.

Cladosporium fulvumis a biotrophic fungal leaf pathogen of tomato. Numerous C. fulvum resistance genes ( Cf genes) are present in wild tomato. In this thesis, a molecular study on the evolution of Cf genes is presented. Cf-9 originates from the wild tomato species Lycopersicon pimpinellifolium , and mediates recognition of the Avr9 elicitor of C. fulvum . Cf-9 (also known as 9C ) is a member of a large gene family and is located in a cluster of five homologues. In the L. pimpinellifolium population a second gene was identified that also mediates recognition of Avr9. The first half of this 9DC gene is nearly identical to the Cf-9 homologue 9D , whereas the second half is nearly identical to Cf-9 . Isolation of the 9DC cluster of L. pimpinellifolium allowed comparison of the Cf-9 and 9DC gene clusters. 9DC was shown to be the result of an intragenic recombination between Cf-9 and 9D . Of the three 9DC genes in the 9DC cluster, 9DC2 is probably the main determinant of Avr9 recognition. To further study the evolution of Cf genes, all eight wild tomato species were screened for recognition of the Avr4 and Avr9 elicitors. In all species recognition of one or both elicitors was found, and several corresponding Cf genes were isolated. Sequence analysis showed that both the Cf-4 and Cf-9 genes are highly conserved throughout the genus Lycopersicon , suggesting that C. fulvum is an ancient pathogen of wild tomato. Finally, from L. peruvianum two Cf gene homologues were isolated, which induce an elicitor-independent hypersensitive response (cell death) upon overexpression in several tobacco species. More of these so-called auto-activators were generated in vitro by gene shuffling of a number of Cf gene homologues. The auto-activators function in the same signalling pathway as Cf-9 . Several models that explain the elicitor-independency of the auto-activators are discussed.

This report was assigned by COGEM. It is meant to uncover possible lacunas in scientific knowledge in the field of crop-wild relative hybridisation and consecutive introgression of crop genes into wild relatives, as well as its ecological and evolutionary consequences. The motivation for this enquiry comes from the impending release of GM crops; the assessment and the a posteriori monitoring of the possible effects of such releases should be based on the best available scientific knowledge. The report reviews the current data and identifies the main research priorities to be implemented for further progress in this field

This report was assigned by COGEM. It is meant to uncover possible lacunas in scientific knowledge in the field of crop-wild relative hybridisation and consecutive introgression of crop genes into wild relatives, as well as its ecological and evolutionary consequences. The motivation for this enquiry comes from the impending release of GM crops; the assessment and the a posteriori monitoring of the possible effects of such releases should be based on the best available scientific knowledge. The report reviews the current data and identifies the main research priorities to be implemented for further progress in this field

Ever since the late blight epidemics of the mid-nineteenth century, man has endeavored to protect his potato crop. Every year, extensive chemical protection is applied, which is expensive and may be harmful to the natural environment. The use of resistant potato cultivars would provide an elegant alternative, however breeding for late blight resistance has not yet resulted in adequately resistant cultivars. In addition to the demand for a high level of resistance, the resistance should be durable. The causal agent of the late blight disease is Phytophthora infestans , a biotrophic oomycete pathogen. Oomycetes are often incorrectly referred to as fungi, but they evolved the ability to infect plants independently from fungi, and may therefore have distinct mechanisms for interacting with plants. Insight in the mechanisms of resistance to P.infestans may assist the breeders in their operation to achieve durable resistance.Genetic resistance can be determined at the subspecies or variety level (race-specific resistance), or at the species or genus level (nonhost resistance). Nonhost resistance is full resistance, and is present in most plant species. In addition, resistance may be a quantitative trait (partial resistance). A rich pool of diverse resistances to P.infestans has been found in wild Solanum species, with levels ranging from full resistance to various levels of partial resistance. Some old potato cultivars also exhibit partial resistance, which proved to be durable. To achieve a durable late blight resistance, a better understanding of the molecular basis of the various types of resistances is essential. To this end, we compiled a set of Solanum species with various types and levels of resistance to P.infestans , and studied the cellular and molecular aspects of the resistance mechanisms present in these plants.Laboratory studies at the cellular and molecular level require an experimental assay, which is both comparable to the natural situation, and assures a high percentage of successful infections. To this end, we developed a resistance assay with detached leaves under controlled conditions in the laboratory and compared this assay with a field trial. The tested growing conditions of the plants did not affect the resistance to P.infestans. Leaves on intact plants however, were more resistant than detached leaves. The incubation conditions of the detached leaves in the laboratory assay rather than the detachment itself appeared to affect the resistance expression. However, on intact plants the infection frequency was too low for molecular studies. Since the ranking of resistance levels within a set of twenty plant clones was similar under laboratory and field conditions, the laboratory assay proved adequate to study the Solanum - P.infestans interaction.A cytological survey of the interaction between twenty Solanum clones and three P.infestans isolates provided the first impression of the nature of the resistance responses. Potato cultivars with race-specific resistance ( R ) genes displayed the hypersensitive response (HR), a programmed cell death of plant cells, upon inoculation with P.infestans . Through this rapid cell death, the biotrophic pathogen became localized between dead cells, and was prevented from further growth. Also durably resistant potato cultivars without known R genes, wild Solanum species, and nonhosts displayed the HR. Interestingly, in highly resistant Solanum species such as S. berthaultii and Scircaeifolium, and nonhosts such as Arabidopsis thaliana and S.nigrum (black nightshade), the HR was extremely fast and effective, resulting in very localized cell death. In partially resistant plants, the HR was delayed, and resulted in larger HR lesions. Occasionally, hyphae were able to escape from these lesions and established a biotrophic interaction with the host. The effectiveness of the HR in restricting growth of the pathogen differed considerably between clones, and correlated with resistance levels. In addition to the HR, local depositions of callose and phenolic compounds occurred, which may function as physical barriers. Although these responses did not correlate with resistance levels, they may influence the balance between growth of the pathogen and induction of the HR. Ultimately, this fine balance may determine resistance at the cellular level, and illustrates the quantitative nature of the resistance to P.infestans at the plant or field level .The HR is initiated upon recognition of pathogen elicitors by plant cell receptors or R gene products as suggested by the gene-for-gene hypothesis. Several types of R genes are recognized in plants, including the nucleotide binding site leucine-rich repeat (NBS-LRR) type, and the Pto-like serine/threonine protein kinase type. The Pto gene was originally identified from wild relatives of tomato. We exploited our Solanum collection to identify Pto -like sequences, and studied evolutionary scenarios for Pto -like genes. Polymerase chain reaction (PCR) amplifications using primers based on conserved and variable regions of Pto yielded 32 intact Pto -like sequences from six Solanum species, and revealed an extensive Pto family. Pto -like transcripts were also detected in leaf tissue of all tested plants. The kinase consensus and autophosphorylation residues were highly conserved, in contrast to the kinase activation domain which is involved in ligand recognition in Pto. Phylogenetic analyses distinguished nine classes of Pto -like genes, and revealed that orthologues (homologues separated by a speciation process) were more similar than paralogues (homologues generated by a gene duplication event). This suggests that the Pto gene family evolved through a series of ancient gene duplication events prior to speciation in Solanum . The phylogenetic data are in line with recent results on the NBS-LRR class of R genes, and suggest that Pto- like genes are ancient, and highly diverse.Various levels of nonspecific resistance were revealed in Solanum species after inoculation with five P.infestans isolates. In partially resistant plants where hyphal escape occurred, the lesions expanded often slower than in susceptible plants. Here, defense mechanisms other than HR are thought to operate; this might for example involve systemic acquired resistance (SAR). SAR can be induced by various signals, but also basal levels of SAR may vary between plants. When we monitored basal expression levels of SAR marker genes in healthy leaves , we found variation between the Solanum clones in constitutive mRNAs levels of the pathogenesis-related ( PR ) genes PR-1 , PR-2 , and PR-5 . At the genus level, there was no correlation between basal PR mRNA levels and nonspecific resistance to P.infestans . In contrast, a positive correlation was found at the species level in S.arnezii x hondelmannii , S.microdontum , S.sucrense and S.tuberosum . In S.tuberosum cultivars, the levels of PR gene expression were the highest in resistant 'Robijn', intermediate in partially resistant 'Première', 'Estima' and 'Ehud', and the lowest in susceptible 'Bintje'. These results suggest that constitutive expression of PR genes may contribute to nonspecific resistance to P.infestans in Solanum . Therefore, PR mRNA levels could serve as molecular markers in potato breeding programs.In conclusion, diverse resistance reactions to P.infestans operate at various levels in Solanum species, including specific and nonspecific mechanisms. The ubiquitous association of the HR in all types of resistance suggests that numerous R genes are present in Solanum against the oomycete P.infestans . A remaining challenge is the identification and transfer of these R genes into commercially grown potato cultivars.

Ever since the late blight epidemics of the mid-nineteenth century, man has endeavored to protect his potato crop. Every year, extensive chemical protection is applied, which is expensive and may be harmful to the natural environment. The use of resistant potato cultivars would provide an elegant alternative, however breeding for late blight resistance has not yet resulted in adequately resistant cultivars. In addition to the demand for a high level of resistance, the resistance should be durable. The causal agent of the late blight disease is Phytophthora infestans , a biotrophic oomycete pathogen. Oomycetes are often incorrectly referred to as fungi, but they evolved the ability to infect plants independently from fungi, and may therefore have distinct mechanisms for interacting with plants. Insight in the mechanisms of resistance to P.infestans may assist the breeders in their operation to achieve durable resistance.Genetic resistance can be determined at the subspecies or variety level (race-specific resistance), or at the species or genus level (nonhost resistance). Nonhost resistance is full resistance, and is present in most plant species. In addition, resistance may be a quantitative trait (partial resistance). A rich pool of diverse resistances to P.infestans has been found in wild Solanum species, with levels ranging from full resistance to various levels of partial resistance. Some old potato cultivars also exhibit partial resistance, which proved to be durable. To achieve a durable late blight resistance, a better understanding of the molecular basis of the various types of resistances is essential. To this end, we compiled a set of Solanum species with various types and levels of resistance to P.infestans , and studied the cellular and molecular aspects of the resistance mechanisms present in these plants.Laboratory studies at the cellular and molecular level require an experimental assay, which is both comparable to the natural situation, and assures a high percentage of successful infections. To this end, we developed a resistance assay with detached leaves under controlled conditions in the laboratory and compared this assay with a field trial. The tested growing conditions of the plants did not affect the resistance to P.infestans. Leaves on intact plants however, were more resistant than detached leaves. The incubation conditions of the detached leaves in the laboratory assay rather than the detachment itself appeared to affect the resistance expression. However, on intact plants the infection frequency was too low for molecular studies. Since the ranking of resistance levels within a set of twenty plant clones was similar under laboratory and field conditions, the laboratory assay proved adequate to study the Solanum - P.infestans interaction.A cytological survey of the interaction between twenty Solanum clones and three P.infestans isolates provided the first impression of the nature of the resistance responses. Potato cultivars with race-specific resistance ( R ) genes displayed the hypersensitive response (HR), a programmed cell death of plant cells, upon inoculation with P.infestans . Through this rapid cell death, the biotrophic pathogen became localized between dead cells, and was prevented from further growth. Also durably resistant potato cultivars without known R genes, wild Solanum species, and nonhosts displayed the HR. Interestingly, in highly resistant Solanum species such as S. berthaultii and Scircaeifolium, and nonhosts such as Arabidopsis thaliana and S.nigrum (black nightshade), the HR was extremely fast and effective, resulting in very localized cell death. In partially resistant plants, the HR was delayed, and resulted in larger HR lesions. Occasionally, hyphae were able to escape from these lesions and established a biotrophic interaction with the host. The effectiveness of the HR in restricting growth of the pathogen differed considerably between clones, and correlated with resistance levels. In addition to the HR, local depositions of callose and phenolic compounds occurred, which may function as physical barriers. Although these responses did not correlate with resistance levels, they may influence the balance between growth of the pathogen and induction of the HR. Ultimately, this fine balance may determine resistance at the cellular level, and illustrates the quantitative nature of the resistance to P.infestans at the plant or field level .The HR is initiated upon recognition of pathogen elicitors by plant cell receptors or R gene products as suggested by the gene-for-gene hypothesis. Several types of R genes are recognized in plants, including the nucleotide binding site leucine-rich repeat (NBS-LRR) type, and the Pto-like serine/threonine protein kinase type. The Pto gene was originally identified from wild relatives of tomato. We exploited our Solanum collection to identify Pto -like sequences, and studied evolutionary scenarios for Pto -like genes. Polymerase chain reaction (PCR) amplifications using primers based on conserved and variable regions of Pto yielded 32 intact Pto -like sequences from six Solanum species, and revealed an extensive Pto family. Pto -like transcripts were also detected in leaf tissue of all tested plants. The kinase consensus and autophosphorylation residues were highly conserved, in contrast to the kinase activation domain which is involved in ligand recognition in Pto. Phylogenetic analyses distinguished nine classes of Pto -like genes, and revealed that orthologues (homologues separated by a speciation process) were more similar than paralogues (homologues generated by a gene duplication event). This suggests that the Pto gene family evolved through a series of ancient gene duplication events prior to speciation in Solanum . The phylogenetic data are in line with recent results on the NBS-LRR class of R genes, and suggest that Pto- like genes are ancient, and highly diverse.Various levels of nonspecific resistance were revealed in Solanum species after inoculation with five P.infestans isolates. In partially resistant plants where hyphal escape occurred, the lesions expanded often slower than in susceptible plants. Here, defense mechanisms other than HR are thought to operate; this might for example involve systemic acquired resistance (SAR). SAR can be induced by various signals, but also basal levels of SAR may vary between plants. When we monitored basal expression levels of SAR marker genes in healthy leaves , we found variation between the Solanum clones in constitutive mRNAs levels of the pathogenesis-related ( PR ) genes PR-1 , PR-2 , and PR-5 . At the genus level, there was no correlation between basal PR mRNA levels and nonspecific resistance to P.infestans . In contrast, a positive correlation was found at the species level in S.arnezii x hondelmannii , S.microdontum , S.sucrense and S.tuberosum . In S.tuberosum cultivars, the levels of PR gene expression were the highest in resistant 'Robijn', intermediate in partially resistant 'Première', 'Estima' and 'Ehud', and the lowest in susceptible 'Bintje'. These results suggest that constitutive expression of PR genes may contribute to nonspecific resistance to P.infestans in Solanum . Therefore, PR mRNA levels could serve as molecular markers in potato breeding programs.In conclusion, diverse resistance reactions to P.infestans operate at various levels in Solanum species, including specific and nonspecific mechanisms. The ubiquitous association of the HR in all types of resistance suggests that numerous R genes are present in Solanum against the oomycete P.infestans . A remaining challenge is the identification and transfer of these R genes into commercially grown potato cultivars.