Decoding insect feeding : identification of regulatory components mediating insect-specific defense responses in plants
Nicotiana attenuata has the capacity to respond specifically to herbivory by its natural herbivore, Manduca sexta, through the perception of elicitors in larval oral secretions (OS). Fatty acid-amino acid conjugates (FACs) in the OS of the M. sexta larvae elicit the herbivory-specific responses in N.attenuata, an annual wild tobacco species. How FACs are perceived and activate signal transduction mechanisms is unknown. To find transcriptional targets of the FAC signal transduction pathway with the goal of identifying regulatory components of insect mediated responses, I used the SuperSAGE technique. I generated two SuperSAGE libraries from FAC-elicited and wounded leaf for the identification of transcripts rapidly and differentially regulated by FACs. From 12,744 unique transcript sequences identified, 429 and 117 were significantly up- and down-regulated, respectively, after FAC elicitation compared to wounding. Based on gene ontology classification, 25% of the annotated Unitags corresponded to regulatory components. qPCR analysis confirmed the FAC regulation of 70% of a subset of 27 UniTags selected. Seven FAC-regulated genes were functionally characterized by VIGS and three, a lectin receptor-like kinase (LecRK1), a putative lipid phosphate phosphatase and a protein of unknown function, were identified as important mediators of the M. sexta-N. attenuata interaction. Silencing the expression of NaLecRK1 by VIGS or RNAi highlighted its importance in the regulation of the defense response to M. sexta herbivory. M. sexta larvae fed on ir-lecRK1 plants were larger than those fed on WT plants, and this corresponded to reductions in the accumulation of defense metabolites such as nicotine, 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) and tripsin protease inhibitors (TPI) or NaTD expression. The production of jasmonic acid (JA) and jasmonyl-isoleucine (JA-Ile) was unaffected during M. sexta herbivory in ir-lecRK1 plants; however, the production of salicylic acid (SA) was twice that in WT plants. Suppression of the SA burst by introgression of the nahG gene into ir-lecRK1 plants demonstrated that the increased OS-elicited SA formation was responsible for the inhibition of nicotine, HGL-DTG and TPIs accumulation and NaTD expression, as well as for the enhanced performance of M. sexta larvae on these plants. I also contributed to the understanding of the biogenesis and function of the C12 derivatives of the HPL pathway in N. attenuata plants after wounding and herbivory, as well as a functional understanding of the role of these C12 derivatives in plant responses to wounding and herbivory.