Project description:Characterization of Arabidopsis thaliana`s transcriptional response to Pieris brassicae eggs Overall design: Arabidopsis thaliana Col-0 wild type plants were grown under short day conditions at 20°C for seven weeks (6 weeks: 10 h/14 h light/dark + 1 week: 8 h/ 16 h light/dark). Seven week old plants were (i) deposited with eggs (one egg cluster with about 40 eggs was deposited onto the lower surface of a rosette leaf within position 14-17) or were (ii) left untreated. Six days after egg deposition, leaf tissue was harvested from egg-deposited and untreated plants. The tissue was harvested from a leaf region proximal to the egg deposition site.
Project description:Plants can prepare their defense of impending herbivory, when they previously perceived eggs deposited by herbivores insect which reliably indicates that larvae will soon hatch and feed on the plants. Using Arabidopsis thaliana and Pieris brassicae we addressed the question whether prior egg deposition affects transcriptional changes in feeding-damaged plants. We determined the transcriptomes of Arabidopsis leaves that (i) were for six days deposited with eggs and suffered larval feeding for two days, (ii) suffered larval feeding for two days without prior egg deposition or (iii) were left untreated. Overall design: Arabidopsis thaliana Col-0 wild type plants were grown under short day conditions at 20°C for seven weeks (6 weeks: 10 h/14 h light/dark + 1 week: 8 h/ 16 h light/dark). Seven week old plants were (i) deposited with eggs (one egg cluster with about 40 eggs was deposited onto the lower surface of a rosette leaf within position 14-17) or were (ii) left untreated. Six days after egg deposition a defined number of larvae was allowed to feed for two days on the leaf which carried the former egg cluster or on leaf with corresponding position of the untreated plants. Control plants were left untreated. From treated and untreated plants, tissue from a leaf region proximal to the egg deposition and/or feeding site was harvested for transcriptome analysis
Project description:Plant defence against insects is well known to be affected by previous exposure to cues warning of herbivory. Using Arabidopsis thaliana and the herbivore Pieris brassicae, we addressed the question whether the maintenance of the effects of the warning cue depends on its reliability. We determined the transcriptomes of Arabidopsis leaves that were treated by P. brassicae egg deposition (i) five days after oviposition, (ii) one day after removal of the eggs following the egg treatment, (iii) three days after removal of the eggs, (iv) after two days of herbivory that started one day after removal of the eggs; or that were treated by chilling (v) five days after transfer to 4°C, (vi) one day after transfering the plants to 20°C following the chilling treatment, (vii) three days after transfering the plants to 20°C, (viii) after two days of herbivory that started one day after transfering the plants to 20°C. Arabidopsis thaliana Col-0 wild type plants were grown under short day conditions (10 h/14 h light/dark) at 20°C for 7 weeks. Subsequently (i) the plants were transferred to 4°C for 5 days, or (ii) Pieris brassicae deposited ca. 40 eggs on leaf 17 where they remained for five days, or (iii) as controls plants grew untreated for five days. Next, the plants were transferred back to 20°C and the eggs were removed, respectively. Next, all plants rested for 1 day at 20°C. Next, P. brassicae larvae were allowed to feed for 2 days on leaf 17 adjacent to the former egg deposit site or at a respective leaf region of chilling-treated or untreated plants. Control plants were not exposed to larvae. From all treated and untreated plants material from a leaf region proximal to the egg deposition and/or feeding site was harvested for transcriptome analysis.
Project description:Plant resistance to the feeding by herbivorous insects has recently been found to be positively or negatively influenced by prior egg deposition. Here we show how crucial it is to conduct experiments on plant responses to herbivory under conditions that simulate natural insect behaviour. We used a well-studied plant--herbivore system, Arabidopsis thaliana and the cabbage white butterfly Pieris brassicae, testing the effects of naturally laid eggs (rather than egg extracts) and allowing larvae to feed gregariously as they do naturally (rather than placing single larvae on plants). Under natural conditions, newly hatched larvae start feeding on their egg shells before they consume leaf tissue, but access to egg shells had no effect on subsequent larval performance in our experiments. However, young larvae feeding gregariously on leaves previously laden with eggs caused less feeding damage, gained less weight during the first 2 days, and suffered twice as high a mortality until pupation compared to larvae feeding on plants that had never had eggs. The concentration of the major anti-herbivore defences of A. thaliana, the glucosinolates, was not significantly increased by oviposition, but the amount of the most abundant member of this class, 4-methylsulfinylbutyl glucosinolate was 1.8-fold lower in larval-damaged leaves with prior egg deposition compared to damaged leaves that had never had eggs. There were also few significant changes in the transcript levels of glucosinolate metabolic genes, except that egg deposition suppressed the feeding-induced up-regulation of FMOGS-OX2 , a gene encoding a flavin monooxygenase involved in the last step of 4-methylsulfinylbutyl glucosinolate biosynthesis. Hence, our study demonstrates that oviposition does increase A. thaliana resistance to feeding by subsequently hatching larvae, but this cannot be attributed simply to changes in glucosinolate content.
Project description:Insect egg deposition activates plant defence, but very little is known about signalling events that control this response. In Arabidopsis thaliana, oviposition by Pieris brassicae triggers salicylic acid (SA) accumulation and induces the expression of defence genes. This is similar to the recognition of pathogen-associated molecular patterns (PAMPs), which are involved in PAMP-triggered immunity (PTI). Here, the involvement of known signalling components of PTI in response to oviposition was studied. Treatment with P. brassicae egg extract caused a rapid induction of early PAMP-responsive genes. In addition, expression of the defence gene PR-1 required EDS1, SID2, and, partially, NPR1, thus implicating the SA pathway downstream of egg recognition. PR-1 expression was triggered by a non-polar fraction of egg extract and by an oxidative burst modulated through the antagonistic action of EDS1 and NUDT7, but which did not depend on the NADPH oxidases RBOHD and RBOHF. Searching for receptors of egg-derived elicitors, a receptor-like kinase mutant, lecRK-I.8, was identified which shows a much reduced induction of PR-1 in response to egg extract treatment. These results demonstrate the importance of the SA pathway in response to egg-derived elicitor(s) and unravel intriguing similarities between the detection of insect eggs and PTI in Arabidopsis.
Project description:Brassica nigra plants, a Brassicaceae close to Arabidopsis thaliana, was used for combined stresses experiments. In this study, we performed a whole-genome microarray analysis on five-week-old plants and compared untreated plants and plants treated different single or dual stresses: the larvae Pieris brassicae, egg extract of Pieris brassicae, the bacterial Xanthomonas campestris pv. raphani, the aphid Brevicoryne brassicae or by combined stresses eggs of P. brassicae / P. brassicae, X. campestris / P. brassicae, B. brassicae / P. brassicae.
Project description:Plants induce defense responses after insect egg deposition, but very little is known about the perception mechanisms. In Arabidopsis thaliana, eggs of the specialist insect Pieris brassicae trigger accumulation of reactive oxygen species (ROS) and salicylic acid (SA), followed by induction of defense genes and localized necrosis. Here, the involvement of the clade I L-type lectin receptor kinase LecRK-I.8 in these responses was studied. Expression of LecRK-I.8 was upregulated at the site of P. brassicae oviposition and egg extract (EE) treatment. ROS, SA, cell death, and expression of PR1 were substantially reduced in the Arabidopsis knock-out mutant lecrk-I.8 after EE treatment. In addition, EE-induced systemic resistance against Pseudomonas syringae was abolished in lecrk-I.8. Expression of ten clade I homologs of LecRK-I.8 was also induced by EE treatment, but single mutants displayed only weak alteration of EE-induced PR1 expression. These results demonstrate that LecRK-I.8 is an early component of egg perception.
Project description:Plants release volatiles induced by herbivore feeding that may affect the diversity and composition of plant-associated arthropod communities. However, the specificity and role of plant volatiles induced during the early phase of attack, i.e. egg deposition by herbivorous insects, and their consequences on insects of different trophic levels remain poorly explored. In olfactometer and wind tunnel set-ups, we investigated behavioural responses of a specialist cabbage butterfly (Pieris brassicae) and two of its parasitic wasps (Trichogramma brassicae and Cotesia glomerata) to volatiles of a wild crucifer (Brassica nigra) induced by oviposition of the specialist butterfly and an additional generalist moth (Mamestra brassicae). Gravid butterflies were repelled by volatiles from plants induced by cabbage white butterfly eggs, probably as a means of avoiding competition, whereas both parasitic wasp species were attracted. In contrast, volatiles from plants induced by eggs of the generalist moth did neither repel nor attract any of the tested community members. Analysis of the plant's volatile metabolomic profile by gas chromatography-mass spectrometry and the structure of the plant-egg interface by scanning electron microscopy confirmed that the plant responds differently to egg deposition by the two lepidopteran species. Our findings imply that prior to actual feeding damage, egg deposition can induce specific plant responses that significantly influence various members of higher trophic levels.
Project description:Expression profiling of the affect of eggs of the Pieris brassicae butterfly on Arabidopsis thaliana local leaf vs. the distal leaf using leaf disks the size of the egg batch.
Project description:Although a seemingly harmless developmental stage of herbivores, insect eggs trigger efficient plant defenses that include necrosis, callus formation, accumulation of ovicidal compounds and release of volatiles to attract egg predators. The large white butterfly Pieris brassicae deposits batches of 20-30 eggs onto Arabidopsis leaves, causing a large transcriptional reprograming that is drastically distinct from the expression profile triggered by larval feeding. Also, P. brassicae eggs induce localized cell death, accumulation of reactive oxygen species (ROS) and salicylic acid (SA), and expression of PTI-related genes, suggesting that egg-associated molecular patterns (EAMPs) activate a response that is similar to the response induced by microbial pathogens. We previously reported that a crude P. brassicae egg extract (EE, soluble fraction from crushed eggs) induced similar responses as oviposition, including ROS and SA accumulation, cell death and defense gene induction. In order to compare oviposition and EE treatment at the transcriptome level, we analyzed changes in transcipt abundance with P. brassicae EE or after natural oviposition. After 5 days, hundreds of genes were significantly upregulated by each treatment and their induction was highly similar between treatments. This conserved transcriptomic signature thus strongly supports our previous observations that oviposition and EE treatment trigger comparable responses in Arabidopsis. Overall design: For experiments with natural oviposition, 15 plants were placed in a 60 x 60 x 60 cm tent containing approximately 30 Pieris brassicae butterflies. After 24 h, four plants containing one egg batch on two leaves were placed in a growth chamber for 4 days. Just before hatching, eggs were gently removed with a forceps. For egg extract application, 2 x 2 µl of egg extract were spotted under the surface of each of two leaves of each treated plant. Four plants were treated with egg extract for 5 days. Treated or oviposited leaves were harvested and quickly stored in liquid N2. Untreated plants were used as controls. Total RNA from 3 biologically independent experiments was extracted using an RNeasy® plant mini kit (Qiagen). DNase treatment was added to the protocol. For cDNA synthesis, RNA samples were purified by NaAC 3M and EtOH precipitation. Library were synthetized from 500 ng of purified total RNA using the TrueSeq stranded mRNA kit (Illumina). RNA and library quality was assessed with a fragment analyzer from Advanced Analytical. Library were sequenced with the Illumina HiSeq 2500 sequencer at the Genomic Technologies Facility platform of the University of Lausanne (LGTF) (http://www.unil.ch/gtf/en/home.html).