Arabidopsis thaliana Responses to Aphid Infestation
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- Institutt for biologi 
Interactions between plants and herbivorous insects, as they are shaped today, result from a long process of evolutionary adaptation. A whole arsenal of defence mechanisms has been developed to protect plants from a variety of enemies. Some of them are general and provide protection from a wide range of insects, whereas others are more specific against particular types of attackers. Plants are equipped with systems for perception of attack which trigger activation of signalling pathways and initiation of defence-related responses. Coordinated regulation of gene expression results in biosynthesis of proteins and secondary metabolites involved in plant defence. In this study plant responses to insect attack and insect responses to plant defence has been investigated. Plants with different genetic backgrounds were infested with aphids and transcriptional and metabolic changes which were triggered under infestation were examined. Following experiments measuring fitness of aphids aimed to assess susceptibility of the studied plants to infestation. Arabidopsis thaliana , similarly to all Brassicaceae, produces a variety of glucose-derived, sulphur containing secondary metabolites, commonly named glucosinolates. Glucosinolates and their hydrolysis products, which are formed during destruction of plant tissue, are a part of the plants defence system. The ability to produce a particular glucosinolate type is genetically controlled and naturally varies between A. thaliana ecotypes. Because bioactive properties are not the same for different glucosinolates or their hydrolysis products, glucosinolate profile may influence susceptibility of plant to attack by certain species of insects. Three A. thaliana ecotypes differing with respect to glucosinolate profiles, Wassilewskija (Ws), Cape Verde Islands (Cvi) and Landsberg erecta (Ler), were chosen to study their interactions with two species of aphids. One of the insects, Brevicoryne brassicae, is a specialist adapted to feed on plants containing glucosinolates, whereas the other one Myzus persicae , is a generalist feeding on a wide range of plant species. Transcriptionaler profiling of plants infested for 72 h revealed that aphid attack introduced similar responses in the three ecotypes studied. However, intensities of the observed changes differed between plants. The glucosinolate biosynthesis pathway was induced most strongly in Ler, while jasmonate-related responses were most pronounced in Cvi. Most of the genes responded similarly to specialist and generalist attack, and only some transcripts showed aphid-specific regulation or aphid-specific spatial induction pattern. These results indicate that, regardless of their specialisation, both attackers trigger similar types of defensive mechanisms. Insect bioassays conducted to assess susceptibility of the three ecotypes revealed that the fitness of B. brassicae was lower on Cvi than on Ler and Ws, whereas M. persicae performed equallywell on all three ecotypes. While the above study provided extensive information regarding plant transcriptional responses at a fixed time point after insect attack, our understanding of plant molecular defences during the early phase of aphid infestation was limited. Therefore, a time series experiment was designed to assess gene expression changes and metabolite levels at four different time points after infestation: 6, 12, 24 and 48 h. To obtain a comprehensive picture of the plant immune system in action, full genome transcriptional profiling was followed by visualisation of callose depositions, examination of hydrogen peroxide accumulation, monitoring of changes in glucosinolate profiles, and assessment of camalexin accumulation. This study describes for the first time dynamics of the B. brassicae-induced changes captured at four time points during the first 48 h of infestation. Based on the experimental data and knowledge provided by previously published research, a model of plant-aphid interactions on molecular and cellular level was proposed. In this model, calcium signalling and reactive oxygen species signalling, together with several WRKY transcription factors, play the crucial role in activation of transcriptional changes. Jasmonic acid and salicylic acid were also found to be important for regulation of defence responses. Induction of genes involved in biosynthesis of secondary metabolites led to accumulation of 4-methoxyindol-3-ylmethyl glucosinolate and camalexin. The putative role of camalexin in the defence against aphids was further evaluated in insect fitness experiments. The involvement of jasmonate-mediated signalling in regulation of plant response to phloem feeding insects has been well documented. However, the impact jasmonates have on modulation of transcriptional changes upon aphid infestation is not fully understood. In the third article included in this thesis we aimed to investigate the role of endogenous jasmonate levels on induction of plant defence responses. Two mutants were used in this study: aos and fou2 in which jasmonic acid biosynthesis pathway is blocked or constitutively activated, respectively. Transcriptional responses induced after Brevicoryne brassicae attack in aos, fou2 and wild type plants were compared. Several defence-related transcripts, genes whose products are involved in signal transduction and a number of transcription factors showed differential regulation in the mutants compared to the wt. Genes that were already induced in resting fou2 plants, as a result of enhanced jasmonate signalling, were less responsive to infestation than corresponding transcripts in wt. Physiological changes introduced by fou2 mutation resulted, however, in improved resistance to B. brassicae infestation as assessed by insect fitness experiments.