The Dicer-like Protein 4 and RNA-dependent RNA Polymerase 6 are Involved in Tomato Torrado Virus Pathogenesis in Nicotiana benthamiana.

Tomato torrado virus (ToTV) is a type member of the Torradovirus genus in the Secoviridae family known to cause severe necrosis in susceptible tomato varieties. ToTV also infects other Solanaceae plants, including Nicotiana benthamiana, where it induces distinctive disease symptoms: plant growth drop with the emergence of spoon-like malformed systemic leaves. Virus-induced post-transcriptional gene silencing (PTGS) is significant among plant defense mechanisms activated upon virus invasion. The PTGS, however, can be counteracted by suppressors of RNA silencing commonly found in viruses, which efficiently disrupt the antiviral defense of their host. Here we addressed a question of PTGS antiviral activity and its suppression in N. benthamiana during ToTV infection - a phenomenon not described for any representative from the Torradovirus genus so far. First, we showed that neither the Vp26 - necrosis-inducing pathogenicity determinant of ToTV nor other structural viral proteins - limited the locally induced PTGS similarly to the p19, a well-characterized potent suppressor of RNA silencing of tombusviruses. Moreover, by employing wild-type and transgenic lines of N. benthamiana with suppressed Dicer-like 2, Dicer-like 4 (DCL2 and DCL4), Argonaute2 (AGO2) and RNA-dependent RNA6 (RDR6) proteins, we proved their involvement in anti-ToTV defense. Additionally, we identified the DCL4 as the major processor of ToTV-derived siRNA. More importantly, our results indicate the essential role of the SGS3/RDR6 pathway in anti-ToTV defense. Finally, we conclude that ToTV might not require a potent RNA silencing suppressor during infection of the model plant N. benthamiana.