The Heterologous Expression of the p22 RNA Silencing Suppressor of the Crinivirus Tomato Chlorosis Virus from Tobacco Rattle Virus and Potato Virus X Enhances Disease Severity but Does Not Complement Suppressor-Defective Mutant Viruses.

To counteract host antiviral RNA silencing, plant viruses express suppressor proteins that function as pathogenicity enhancers. The genome of the Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae) encodes an RNA silencing suppressor, the protein p22, that has been described as having one of the longest lasting local suppressor activities when assayed in Nicotiana benthamiana. Since suppression of RNA silencing and the ability to enhance disease severity are closely associated, we analyzed the effect of expressing p22 in heterologous viral contexts. Thus, we studied the effect of the expression of ToCV p22 from viral vectors Tobacco rattle virus (TRV) and Potato virus X (PVX), and from attenuated suppressor mutants in N. benthamiana plants. Our results show that although an exacerbation of disease symptoms leading to plant death was observed in the heterologous expression of ToCV p22 from both viruses, only in the case of TRV did increased viral accumulation occur. The heterologous expression of ToCV p22 could not complement suppressor-defective mutant viruses.

The p22 RNA Silencing Suppressor of the Crinivirus Tomato chlorosis virus is Dispensable for Local Viral Replication but Important for Counteracting an Antiviral RDR6-Mediated Response during Systemic Infection.

Among the components of the RNA silencing pathway in plants, RNA-dependent RNA polymerases (RDRs) play fundamental roles in antiviral defence. Here, we demonstrate that the Nicotiana benthamiana RDR6 is involved in defence against the bipartite crinivirus (genus Crinivirus, family Closteroviridae) Tomato chlorosis virus (ToCV). Additionally, by producing a p22-deficient ToCV infectious mutant clone (ToCVΔp22), we studied the role of this viral suppressor of RNA silencing in viral infection in both wild-type and RDR6-silenced N. benthamiana (NbRDR6i) plants. We demonstrate that p22 is dispensable for the replication of ToCV, where RDR6 appears not to have any effect. Furthermore, the finding that ToCV∆p22 systemic accumulation was impaired in wild-type N. benthamiana but not in NbRDR6i plants suggests a role for p22 in counteracting an RDR6-mediated antiviral response of the plant during systemic infection.

The p22 RNA silencing suppressor of the crinivirus Tomato chlorosis virus preferentially binds long dsRNAs preventing them from cleavage.

Viruses encode silencing suppressor proteins to counteract RNA silencing. Because dsRNA plays a key role in silencing, a general silencing suppressor strategy is dsRNA binding. The p22 suppressor of the plant virus Tomato chlorosis virus (ToCV; genus Crinivirus, family Closteroviridae) has been described as having one of the longest lasting local suppressor activities. However, the mechanism of action of p22 has not been characterized. Here, we show that ToCV p22 binds long dsRNAs in vitro, thus interfering with their processing into small RNAs (sRNAs) by an RNase III-type Dicer homolog enzyme. Additionally, we have studied whether a putative zinc finger motif found in p22 has a role in dsRNA binding and suppressor function. The efficient ability of p22 to suppress RNA silencing, triggered by hairpin transcripts transiently expressed in planta, supports the relationship between its ability to bind dsRNA in vitro and its ability to inhibit RNA silencing in vivo.

Genetic diversity and silencing suppression activity of the p22 protein of Tomato chlorosis virus isolates from tomato and sweet pepper.

As for other bipartite criniviruses (genus Crinivirus, family Closteroviridae), the genome of Tomato chlorosis virus encodes an RNA silencing suppressor, the protein p22, in the 3'-proximal region of RNA1. This protein has been reported as having one of the longest lasting local suppressor activities when transiently expressed in Nicotiana benthamiana. Here, we examined the genetic diversity of the p22 gene in ToCV isolates from tomato and sweet pepper. The p22 gene sequences clearly grouped into two separated clades. However, functional analysis of both types of p22 proteins indicated no evident differences in suppressor activity. Our findings provide experimental evidence that the presence of a "strong" silencing suppressor is a conserved feature of ToCV isolates.