Genetics of insect resistance to plant defence

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