Project description:Arabidopsis thaliana plants were infested i) with sucking insect herbivores (the generalist aphid Myzus persicae and the specialist aphid Brevicoryne brassicae), ii) with chewing insect herbivores (generalist caterpillars of Spodoptera exigua and specialist caterpillars of Pieris rapae) or iii) were treated by wounding. For each treatment, rosette leaves were harvested at two time points (6h and 24h) after removal of insects. For chewing herbivores and wounding both local, i.e. immediately damaged leaves, and systemic, i.e. undamaged leaves from the same plant, were collected. Control plants were uninfested, but otherwise equally treated and harvested in parallel. We tested the hypothesis that Arabidopsis can recognize and respond differentially to insect species at the transcriptional level using a genome wide microarray. Transcriptional reprogramming was characterized using co-expression analysis in damaged and undamaged leaves at two times in response to mechanical wounding and four insect species. In all, 2778 (10.6%) of annotated genes on the array were differentially expressed in at least one treatment. Responses differed mainly between aphid and caterpillar and sampling times. Responses to aphids and caterpillars shared only 10% of up-regulated and 8% of down-regulated genes. Responses to two caterpillars shared 21% and 12% of up- and down-regulated genes, whereas responses to the two aphids shared only 7% and 4% of up-regulated and down-regulated genes. Overlap in genes expressed between 6h and 24h was 3-15%, and depended on the insect species. Responses in attacked and unattacked leaves differed at 6h but converged by 24h. Genes responding to the insects are also responsive to many stressors and included primary metabolism. Aphids down-regulated amino acid catabolism; caterpillars stimulated production of amino acids involved in glucosinolate synthesis. Co-expression analysis revealed 17 response networks. Transcription factors were a major portion of differentially expressed genes throughout and responsive genes shared most of the known or postulated binding sites.

Project description:We have implemented an integrated Systems Biology approach to analyze overall transcriptomic reprogramming and systems level defense responses in the model plant Arabidopsis thaliana during an insect (Brevicoryne brassicae) and a bacterial (Pseudomonas syringae pv. tomato strain DC3000) attack. The main aim of this study was to identify the attacker-specific and general defense response signatures in the model plant Arabidopsis thaliana while attacked by phloem feeding aphids or pathogenic bacteria. Defense responses and networks, unique and specific for aphid or Pseudomonas stresses were identified. Our analysis revealed a probable link between biotic stress and microRNAs in Arabidopsis and thus opened up a new direction to conduct large-scale targeted experiments to explore detailed regulatory links among them. The presented results provide a first comprehensive understanding of Arabidopsis - B. brassicae and Arabidopsis - P. syringae interactions at a systems biology level. Arabidopsis thaliana (ecotype Colombia-0) seeds were grown in 6-cm-diameter pots filled with a sterile soil mix (1.0 part soil and 0.5 part horticultural perlite), 3 plants per pot. Plants were kept in growth chambers VM-CM-6tsch VB 1514 (VM-CM-6tsch Industrietechnik GmbH, Germany) under the following conditions: a 8/16 h (light/dark) photoperiod at 22M-BM-0C/18M-BM-0C, 40%/70% relative humidity, and 70/0 M-NM-<mol m-2s-1 light intensity. After 32 days plants had 8 fully developed leaves. Each plant was infested with 32 wingless aphids [Brevicoryne Brassicae] (4 per leaf), which were transferred to leaves with a fine paintbrush. Infested plants and aphid-free controls were kept in plexiglass cylinders. Plants were harvested 72 h after infestation between the 6th and 8th hour of the light photoperiod. Four biological replicates were prepared from aphid infested and control plants, each sampled from 15 individual plants. Whole rosettes were cut at the hypocotyls and aphids were removed by washing with Milli-Q-filtered water. Differences in transcriptional responses were measured by comparing genes expression of aphid infested plants against non-infested control plants.

Project description:A pair of nearly isogenic tomato (Solanum lycopersicum) lines were used. These are: Motelle (Mi/Mi), root-knot nematode resistant and Moneymaker (mi/mi) root-knot nematode susceptible. A potato aphid (red biotype), imported from France, was used for infestation. An equal volume of sterile 1% carboxymethyl cellulose in water was added to the cleaned nematodes to keep them in suspension. The inoculum, consisting of 75-100 J2s, was dispensed in 10 µl aliquots using a repeat pipetter. The inoculum was pipetted 2 mm beneath the root tips. Control seedlings were inoculated with 0.5% carboxymethyl cellulose. After the desired incubation time (24 h or 36 h), seedlings were taken out of the boxes and a few set aside to check nematode infection rate. In our hand, this system results in 50% infection rate. The rest of the seedlings were placed on a water-saturated filter paper and the roots aligned. About 1 cm of the infected root tip was excised with a blade and quickly frozen on a polycarbonate membrane-lined 50 mm diameter Petri dish floating on liquid nitrogen and stored at -80C. Plants were grown for 6 weeks in a growth chamber under 16 h light/8 h dark at 25C in UCMix/sand, irrigated with water or MiracleGroR solution, then moved to an insect growth room (25C) 4-6 days before aphid infestation. Individual tomato leaflets were infested with 30-40 mixed stages of the potato aphids using plastic leaf cages. Four leaflets and two plants of each genotype were infested. Two plants of each genotype were used as controls. After the desired infestation time (6, 12, 24 or 48 h), leaflets were excised from the plants with a blade and sprayed with 1% SDS to force aphids to remove their stylets from the leaf tissue. Aphids were carefully removed from the leaflets and quickly frozen in liquid nitrogen. Control leaflets were excised and sprayed with 1% SDS before freezing. Samples were stored at -80C. RNA was isolated using the hot phenol method. Keywords: Direct comparison

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