Silencing 7 herbivory-regulated proteins in Nicotiana attenuata to understand their function in plant-herbivore interactions

1. Large-scale transcriptomic and proteomic analyses reveal that a plant's primary metabolism is reconfigured when herbivores attack. However, unbiased means of evaluating the function of this reconfiguration for the plant-herbivore interaction are lacking. 2. The production of secondary metabolites can be genetically silenced for in planta tests of defensive function without dramatically altering growth. This is not possible with primary metabolites, which may also function defensively. 3. We test this hypothesis by silencing the expression of genes coding for seven herbivore-induced proteins not known to play obvious defensive roles in the Nicotiana attenuata-Manduca sexta interaction and analysed the performance of plants and insects and traits known to mediate resistance and performance. Silencing genes of up-regulated proteins in N. attenuata tended to benefit larval performance, while silencing genes of down-regulated proteins tended to decrease it: these results are consistent with expectations that herbivory-induced regulation reflects the role of proteins in attacked plants. 4. Surprisingly, plant trypsin proteinase inhibitors (TPIs) and larval midgut protease activity were not associated with larval performance and neither was the variation in established defence metabolites (nicotine), suggesting greater importance of other metabolites, such as diterpene glycosides. 5. We conclude that silencing the genes of proteins differentially regulated during a plant-herbivore interaction and querying each player in the interaction reveals novel functions of genes at an organismic level of analysis and the direct-indirect influence of non-defence proteins in plant-herbivore interactions.