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Abstract

DICER‐like (DCL) proteins produce small RNAs that silence genes involved in development and defenses against viruses and pathogens. Which DCLs participate in plant‐herbivore interactions remains unstudied. We identified and stably silenced four distinct DCL genes by RNAi in Nicotiana attenuata (Torrey ex. Watson), a model for the study of plant‐herbivore interactions. Silencing DCL1 expression was lethal. Manduca sexta larvae performed significantly better on ir‐dcl3 and ir‐dcl4 plants, but not on ir‐dcl2 plants compared to wild type plants. Phytohormones, defense metabolites and microarray analyses revealed that when DCL3 and DCL4 were silenced separately, herbivore resistance traits were regulated in distinctly different ways. Crossing of the lines revealed complex interactions in the patterns of regulation. Single ir‐dcl4 and double ir‐dcl2 ir‐dcl3 plants were impaired in JA accumulation, while JA‐Ile was increased in ir‐dcl3 plants. Ir‐dcl3 and ir‐dcl4 plants were impaired in nicotine accumulation; silencing DCL2 in combination with either DCL3 or DCL4 restored nicotine levels to those of WT. Trypsin proteinase inhibitor activity and transcripts were only silenced in ir‐dcl3 plants. We conclude that DCL2/3/4 interact in a complex manner to regulate anti‐herbivore defenses and that these interactions significantly complicate the already challenging task of understanding smRNA function in the regulation of biotic interactions.