Conserved Arabidopsis response to oviposition and crude egg extract treatment

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. 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).