Argonaute 5 (AGO5) forms a component of the herbivore-induced small RNA pathway in Nicotiana attenuata

Argonautes (AGOs) are the effectors for the action of microRNAs (miRNAs). Plant genomes harbor large numbers of AGO genes whose functions remain to be fully understood. Here, we elucidated a function of AGO5 in the ecological model plant Nicotiana attenuata during its interactions with the specialist herbivore Manduca sexta. Plants silenced in NaAGO5 expression using inverted-repeat technology (irAGO5) were indistinguishable from the wild type (WT) in growth and development but were highly susceptible to M. sexta herbivory. M. sexta caterpillars grew faster and accumulated significantly more biomass on irAGO5 than on WT plants. Herbivory-elicited irAGO5 plants accumulated significantly lower amounts of auxin-dependent defense metabolites such as phenolamides, flavonoids, and diterpenoid glycosides, but not nicotine and trypsin protease inhibitors (TPI). Nicotine and TPI levels, which require intact jasmonate signaling, were attenuated in plants silenced in NaAGO8 expression (irAGO8). irAGO5 plants showed compromised herbivore-induced auxin levels and YUCCA gene expression but accumulated more salicylic acid; however, jasmonate accumulations were at WT levels. Exogenous auxin treatments restored resistance against M. sexta and auxin-dependent defense metabolites. Substantial temporal changes in the miRNome were observed in irAGO5 and were largely different from those in irAGO8. An AGO5-dependent miRNA-mRNA regulatory interaction network was inferred for defense-signaling components. Furthermore, double knockdowns of NaAGO5 and NaAGO8 revealed cooperative functions of the 2 genes during herbivory. We infer that AGO5 is a central component of the herbivore-induced smRNA pathway that modulates multiple nodes in the auxin-dependent metabolic space of the defense signaling network when N. attenuata plants interact with the specialist herbivore M. sexta.