Abstract
Antiviral RNAi is the main protective measure employed by plants in the fight against viruses. The main steps of this process have been clarified in recent years, primarily relying on the extensive genetic resources of Arabidopsis thaliana. Our knowledge of viral diseases of crops, however, is still limited, mainly due to the fact that A. thaliana is a non-host for many agriculturally important viruses. In contrast, Nicotiana benthamiana has an unparalleled susceptibility to viruses and, since it belongs to the Solanaceae family, it is considered an adequate system for modeling infectious diseases of crops such as tomatoes. We used a series of N. benthamiana mutants created by genome editing to analyze the RNAi response elicited by the emerging tomato pathogen, pepino mosaic virus (PepMV). We uncovered hierarchical roles of several Argonaute proteins (AGOs) in anti-PepMV defense, with the predominant contribution of AGO2. Interestingly, the anti-PepMV activities of AGO1A, AGO5, and AGO10 only become apparent when AGO2 is mutated. Taken together, our results prove that hierarchical actions of several AGOs are needed for the plant to build effective anti-PepMV resistance. The genetic resources created here will be valuable assets for analyzing RNAi responses triggered by other agriculturally important pathogenic viruses.