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

Abstract

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 βC1, 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 βC1. Transgenic plants over-expressing Nbrgs-CaM displayed developmental abnormities reminiscent of βC1-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 βC1 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 βC1-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.

Complexity of begomovirus and betasatellite populations associated with chilli leaf curl disease in India

“…Possible synergistic interactions, if any, between these monopartite and bipartite begomoviruses in chilli have not been studied so far. Cui Li et al, 2014;Saunders et al, 2004;Yang et al, 2011). At the time of sampling, only two betasatellite groups, ToLCBDB and ChiLCB, were known to be associated with ChiLCD in India (Chattopadhyay et al, 2008;Kumar et al, 2011).…”

“…Betasatellites typically contain a satellite conserved region (SCR), an adenine-rich region and a bC1 ORF. The bC1 ORF has been characterized as a pathogenicity determinant that suppresses host antiviral silencing and is involved in virus movement (Cui et al, 2005;Li et al, 2014;Saunders et al, 2004;Yang et al, 2011). A class of autonomously replicating satellite-like molecules identified as alphasatellites require helper begomoviruses for their intra-and intercellular movement and are reported to ameliorate symptoms in a few cases (Idris et al, 2011;Nawaz-ulRehman et al, 2010).…”

Regulation of Nicotine Biosynthesis by an Endogenous Target Mimicry of MicroRNA in Tobacco

“…The Agrobacterium tumefaciens-mediated tobacco leaf disc transformation method was used to generate transgenic tobacco plants. Selection (on 200 mg mL -1 kanamycin media) of transformants and positive transgenic plants was performed as described previously (Li et al, 2014a). Primers used in PCR and RT-qPCR analyses are listed in Supplemental Table S2.…”

rgs-CaM Detects and Counteracts Viral RNA Silencing Suppressors in Plant Immune Priming

“…In a previous study, rgs-CaM was shown to be an endogenous RSS that suppresses virus-induced gene silencing (VIGS) by the potato virus X (PVX) vector, which was developed from a member of the genus Potexvirus (28). We and other groups confirmed that rgs-CaM has RSS activity (32)(33)(34) and facilitates infection by viruses in the genus Begomovirus via its RSS activity (34,35). However, we also observed an antiviral function of rgs-CaM: it binds to and directs degradation of two viral RSSs, HC-Pro and CMV 2b, via autophagy, resulting in reinforcement of antiviral RNA silencing in virus-infected cells (32).…”

“…In that study, the overexpressed tobacco rgs-CaM gene interfered with VIGS of green fluorescent protein (GFP) by a PVX vector, resulting in increased fluorescence and accumulation of the GFP transgene and the PVX genomic RNA itself (28). Li et al (34) reported that infection by tomato yellow leaf curl China virus, a member of the genus Begomovirus, was facilitated or inhibited in transgenic N. benthamiana plants in which rgs-CaM was overexpressed or silenced, respectively. They also confirmed the RSS activity of rgs-CaM (34,70).…”