Suppression of RNA Silencing by a Plant DNA Virus Satellite Requires a Host Calmodulin-Like Protein to Repress RDR6 Expression

In plants, RNA silencing plays a key role in antiviral defense. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that target different effector molecules in the RNA silencing pathway. Evidence has shown that plants also encode endogenous suppressors of RNA silencing (ESRs) that function in proper regulation of RNA silencing. The possibility that these cellular proteins can be subverted by viruses to thwart host defense is intriguing but has not been fully explored. Here we report that the Nicotiana benthamiana calmodulin-like protein Nbrgs-CaM is required for the functions of the VSR C1, the sole protein encoded by the DNA satellite associated with the geminivirus Tomato yellow leaf curl China virus (TYLCCNV). Nbrgs-CaM expression is up-regulated by the C1. Transgenic plants over-expressing Nbrgs-CaM displayed developmental abnormities reminiscent of C1-associated morphological alterations. Nbrgs-CaM suppressed RNA silencing in an Agrobacterium infiltration assay and, when over-expressed, blocked TYLCCNV-induced gene silencing. Genetic evidence showed that Nbrgs-CaM mediated the C1 functions in silencing suppression and symptom modulation, and was required for efficient virus infection. Moreover, the tobacco and tomato orthologs of Nbrgs-CaM also possessed ESR activity, and were induced by betasatellite to promote virus infection in these Solanaceae hosts. We further demonstrated that C1-induced Nbrgs-CaM suppressed the production of secondary siRNAs, likely through repressing RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) expression. RDR6-deficient N. benthamiana plants were defective in antiviral response and were hypersensitive to TYLCCNV infection. More significantly, TYLCCNV could overcome host range restrictions to infect Arabidopsis thaliana when the plants carried a RDR6 mutation. These findings demonstrate a distinct mechanism of VSR for suppressing PTGS through usurpation of a host ESR, and highlight an essential role for RDR6 in RNA silencing defense response against geminivirus infection. Author Summary In plants, RNA silencing plays a key role in developmental regulation and antiviral defense. To successfully infect their hosts, plant viruses encode silencing suppressors (VSRs) as counter-defense measures. These VSRs function to disable host antiviral RNA silencing defenses through various mechanisms that are not well understood. Here we report that a host calmodulin-like protein called Nbrgs-CaM, which appears to be an endogenous suppressor of RNA silencing, plays essential roles in suppression of RNA silencing and induction of symptoms by the VSR C1, the sole protein encoded by a geminivirus-associated DNA satellite. The Nbrgs-CaM was up-regulated by Tomato yellow leaf curl China geminivirus (TYLCCNV)-encoded VSR C1 upon virus infection or stable expression via a transgene. Further analyses revealed that up-regulation of Nbrgs-CaM by C1 suppressed RNA silencing likely through repressing the expression of RNA-DEPENDENT RNA POLYMERASE 6 (RDR6). We have demonstrated that RDR6-mediated RNA silencing plays an important role in antiviral defense in Nicotiana benthamiana and confers host range restriction against TYLCCNV infection on Arabidopsis thaliana. Our study suggests that exploiting a cellular suppressor can be an efficient mechanism for viruses to counteract host RNA silencing defense response.