NIK1 (NSP-Interacting Kinase 1) is a protein located in the plasm membrane involved in plant antiviral immunity. In Arabidopsis, NIK1 belongs to the RLK family which has an N-Terminal domain component by Leucine-rich repeat receptor-like kinase (LRR), and C-terminal by Threonine/ Serine kinase domain. NIK1 activation is triggered by begomovirus-derived nucleic acids which promote phosphorylation on Thr-474 leading in turn to phosphorylation of the ribosomal protein RPL10. Phosphorylated RPL10 is then targeted to the nucleus and interacts with the LIMYB (L10- interacting Myb domain-containing protein) transcription factor, mediating host translation suppression by down-regulating genes from the translational machinery. The immune mechanism initiated by the NIK1 activation seems to work also against RNA viruses such as the Tobacco rattle virus (TRV), which is a member of the genus Trobavirus. TRV contains a bipartite, single-stranded positive-sense RNA genome formed by TRV RNA1 and TRV RNA2 genomic components. To verify whether RNA viruses can activate NIK1 and induce an antiviral response, an infection assay was conducted in leaf disc from Arabidopsis Col-0, nik1 knockout line, and NIK1-5 complemented line using plant infected RNA from TRV. The accumulation of viral transcripts and the TRV genomic copy number were evaluated by RT-qPCR. The absolute and relative levels of TRV genomic RNA were higher in the nik-1 knockout line than in Col-0 and NIK1-5 complemented line, indicating that nik1 was more susceptible to TRV and supporting the hypothesis that TRV may activate the NIK1 antiviral defense. This interpretation was confirmed by treating the different genotypes nik1, nik2, nik1nik2 and NIK1-5 lines with RNA from infected plants and analyzing the readouts of the NIK1-mediated antiviral immunity activation. The expression of ribosomal genes in plants infected with TRV was lower compared to plants treated with uninfected RNA and water. Collectively, these results indicate that NIK1 is activated by RNA from TRV-infected plants, which stimulate the same mechanisms as the begomovirus-activated NIK1 antiviral signaling.