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Small RNAs are important regulators of plant development and resistance to viruses. To determine whether small RNAs mediate defense responses to herbivore attack, we silenced the expression of three RNA-directed RNA polymerases (RdRs) in the native tobacco Nicotiana attenuata by virus-induced gene silencing. Larvae of the leaf-chewing solanaceous specialist Manduca sexta grew faster on the RdR1-silenced plants than on empty vector (EV) controls; silencing RdR3 and 2 had little to no effect on larval performance. NaRdR1 transcripts were strongly elicited when puncture wounds were treated with M. sexta oral secretions (OS) to simulate herbivore attack, and with SA and JA, phytohormones that are elicited by herbivore attack. Stably silencing RdR1 by transforming N. attenuata with an inverted-repeat RdR1 construct produced plants (irRdR1) that grew normally but were highly susceptible to both M. sexta larvae and the cell-content-feeder Tupiocoris notatus. When irRdR1 lines were planted into N. attenuata’s native habitat in the Great Basin Desert (Utah, USA), they were highly susceptible to herbivore attack, due to deficiencies in direct rather than indirect defenses. Microarray analysis revealed the downregulation of ADC and ODC genes, which supply substrates for synthesizing the chemical defense compound nicotine; irRdR1 lines failed to accumulate nicotine after attack. We conclude that RdR1 mediates herbivore resistance, and infer that the small RNAs produced by RdR1 are probably involved in orchestrating some of the rapid metabolic adjustments required for plants to survive herbivore attack in their natural habitats. The experiment highlights the value of carrying out ’real-world’ tests of gene function early in the discovery process.