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Abstract

In Nicotiana attenuata, specific RNA directed RNA polymerase (RdR1) and the Dicer-like proteins (DCL3 and 4) are recruited during herbivore attack to mediate the regulation of defense responses. However, the identity and role(s) of Argonautes (AGOs) involved in herbivory remains unknown. Of the 11 AGOs in the N. attenuata genome, we silenced the expression of 10. Plants silenced in NaAGO8 expression grew normally but were highly susceptible to herbivore attack. Larvae of the Manduca sexta grew faster when consuming irAGO8 plants, but did not differ from WT when consuming plants silenced in AGO1 (a, b and c), AGO2, AGO4 (a and b), AGO7, or AGO10 expression. irAGO8 plants were significantly compromised in the herbivore-induced levels of defense metabolites such as nicotine, phenolamides and diterpenoid glycosides (DTGs). Time-course analyses revealed extensively altered miRNA profiles and the reduced accumulation of MYB8 transcripts and of the associated genes of the phenolamide and phenylpropanoid pathways as well as the nicotine biosynthetic pathway. A possible AGO8-modulated miRNA-mRNA target network was inferred. Further, comparative analysis of domains revealed the diversity of AGO conformations, particularly in the smRNA binding pocket, which may influence substrate recognition/binding and functional specificity. We infer that AGO8 plays a central role in the induction of direct defenses by modulating several regulatory nodes in defense signaling network during herbivore response. Thus, our study identifies the effector AGO of the herbivore-induced smRNA machinery, which in N. attenuata, now comprises of RdR1, DCL3/4 and AGO8.