Analysis of gene content in sweet potato chlorotic stunt virus RNA1 reveals the presence of the p22 RNA silencing suppressor in only a few isolates: implications for viral evolution and synergism

Abstract

Sweet potato chlorotic stunt virus (genus Crinivirus) belongs to the family Closteroviridae, members of which have a conserved overall genomic organization but are variable in gene content. In the bipartite criniviruses, heterogeneity is pronounced in the 39-proximal region of RNA1, which in sweet potato chlorotic stuat virus (SPCSV) encodes two novel proteins, RNase3 (RNase III endonuclease) and p22 (RNA silencing suppressor). This study showed that two Ugandan SPCSV isolates contained the p22 gene, in contrast to three isolates of the East African strain from Tanzania and Peru and an isolate of the West African strain from Israel, which were missing a 767 nt fragment of RNA1 that included the p22 gene. Regardless of the presence of p22, all tested SPCSV isolates acted synergistically with potyvirus sweet potato feathery mottle virus (SPFMV; genus Potyvirus, family Potyviridae) in co-infected sweetpotato plants (Ipomoea batatas), which greatly enhanced SPFMV titres and caused severe sweetpotato virus disease (SPVD). Therefore, the results indicate that any efforts to engineer pathogen-derived RNA silencing-based resistance to SPCSV and SPVD in sweetpotato should not rely on p22 as the transgene. The data from this study demonstrate that isolates of this virus species can vary in the genes encoding RNA silencing suppressor proteins. This study also provides the first example of intraspecific variability in gene content of the family Closteroviridae and may be a new example of the recombination-mediated gene gain that is characteristic of virus evolution in this virus family. INTRODUCTIONViruses belonging to the family Closteroviridae have singlestranded, positive-sense RNA genomes that are the largest among plant viruses . These viruses share a conserved set of 'core' genes, but they also show a high level of variability in gene content at the 39 end of the monopartite genomes in the genera Closterovirus and Ampelovirus and at the 39 end of RNA1 in viruses of the genus Crinivirus, which have bipartite genomes . Gene content and organization of the variable 39-proximal genomic region have evolutionary implications (Karasev et al., 1996;Dolja et al., 2006), which are interesting not least because some of the genes in the 39 region encode RNA silencing suppression (RSS) proteins (Reed et al., 2003;Lu et al., 2004;Kreuze et al., 2005;Chiba et al., 2006). RSS proteins combat the fundamental antiviral resistance that is based on RNA silencing in plants (Lindbo & Dougherty, 2005). The functional similarities of the RSS proteins contrast with their very low or non-significant sequence homology with other viral and host proteins Mérai et al., 2006). It has been suggested that these characteristics might indicate a relatively recent acquisition of the genes for RSS proteins in viral genomes to counteract evolving eukaryotic host defence responses to viral pathogens (Voinnet, 2005).Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) is a phloem-limited, whitefly-transmitted, bipartite viru...

Elimination of antiviral defense by viral RNase III

“…The worst symptoms and yield losses develop in plants co-infected with SPCSV and an unrelated ssRNA virus, SPFMV (family Potyviridae). Consequently, titers of SPFMV increase by 2-3 orders of magnitude and severe symptoms develop (2,3,5). These results indicate a general loss of resistance to viruses in sweet potato plants infected with SPCSV.…”

“…The cloning strategy and vector plasmids used in the agroinfiltration assays in this study have been described (26). Sequences of the RNase3 genes of SPCSV-Ug, SPCSV-M2-47, and SPCSV-Is have been described and encode the most different RNase3 protein sequences currently known (amino acid sequence identity 80 -97%) (5). In the mutated RNase3 of SPCSV-Ug (designated as RNase3-Ala),2 substitutions (E37A and D44A) were made in the highly conserved RNase III signature motif required for the dsRNA endonuclease activity of RNase III enzymes (Fig.…”

“…We found previously that SPCSV encodes a unique class 1 RNase III enzyme (RNase3) whose endonuclease activity enhances RNAi suppression caused by another protein (p22) that also is encoded by SPCSV (26). Recent studies have revealed that many SPCSV isolates that lack p22 still synergize with unrelated viruses (4,5), indicating that p22 is dispensable for synergy between SPCSV and other viruses. Therefore, the aim of the current study was to elucidate the possible involvement of RNase3 in the synergism and development of SPVD.…”

“…The central part of HC-Pro that mediates viral synergism is involved in suppression of RNAi (24,25). However, the unidirectional synergy of SPCSV and SPFMV is unusual in that the concentration of the potyviral component increases by 2-3 orders of magnitude, whereas the titer of the non-potyviral component remains constant or slightly decreases (3,5). In addition, SPCSV synergizes several unrelated viruses, such as Sweet potato mild mottle virus (SPMMV, genus Ipomovirus) (3,4) and Sweet potato chlorotic fleck virus (SPCFV, genus Carlavirus) (4), which enhances the concentrations of these viruses and the severity of symptoms.…”

Evolution of Plant Viruses: Adaptation to Hosts and Vectors

“…No other RNA viruses are known to carry an RNase gene. It seems to be recently incorporated into the viral genome (Cuellar et al 2008). Tenui-and tospoviruses (family Bunyaviridae) snatch ca.…”