P0 of Beet Western Yellows Virus Is a Suppressor of Posttranscriptional Gene Silencing

Abstract

Higher plants employ a homology-dependent RNA-degradation system known as posttranscriptional gene silencing (PTGS) as a defense against virus infection. Several plant viruses are known to encode proteins that can suppress PTGS. Here we show that P0 of beet western yellows virus (BWYV) displays strong silencing suppressor activity in a transient expression assay based upon its ability to inhibit PTGS of green fluorescent protein (GFP) when expressed in agro-infiltrated leaves of Nicotiana benthamiana containing a GFP transgene. PTGS suppressor activity was also observed for the P0s of two other poleroviruses, cucurbit aphid-borne yellows virus and potato leafroll virus. P0 is encoded by the 5-proximal gene in BWYV RNA but does not accumulate to detectable levels when expressed from the genome-length RNA during infection. The low accumulation of P0 and the resulting low PTGS suppressor activity are in part a consequence of the suboptimal translation initiation context of the P0 start codon in viral RNA, although other factors, probably related to the viral replication process, also play a role. A mutation to optimize the P0 translation initiation efficiency in BWYV RNA was not stable during virus multiplication in planta. Instead, the P0 initiation codon in the progeny was frequently replaced by a less efficient initiation codon such as ACG, GTG, or ATA, indicating that there is selection against overexpression of P0 from the viral genome.Most if not all plants possess a defense mechanism against virus infection known as posttranscriptional gene silencing (PTGS; see reference 44 for a recent review). PTGS is a homology-dependent RNA degradation system to recognize and degrade viral RNA (and any homologous transgene mRNA) in the cytoplasm. Mechanistically similar phenomena exist in animals and fungi (reviewed in references 4, 13, and 35). The mechanism by which a virus infection triggers PTGS in plants is not fully understood, but double-stranded RNA is a strong inducer of PTGS (43) and such a form is produced during replication of an RNA virus. Production of small RNAs of ϳ21 to 23 nucleotides, called small interfering RNAs (siRNAs), corresponding to both the plus and minus strands of the target RNA is associated with PTGS (12,13,45). In plants, gene silencing at one site can trigger PTGS in distant tissues and across a graft union (29, 39a). The mobile signal, which must consist at least in part of homologous RNA, is thought to move from cell to cell through plasmodesmata and systemically via the vasculature. Some plant viruses have been shown to encode proteins which can counteract PTGS (8,22,39,40,42). These silencing suppressor proteins may act at different steps in the PTGS pathway. Thus, (i) the potyvirus helper component-proteinase (HCPro) interferes with initiation and maintenance of silencing at a step coincident with or upstream of siRNA production (1,7,16,23,24), (ii) the 2b protein of cucumber mosaic virus (CMV) prevents initiation of PTGS in new growth by inhibiting the long-range PTGS signaling acti...

P0 proteins of European beet-infecting poleroviruses display variable RNA silencing suppression activity

“…Tu (Pfeffer et al, 2002). In contrast, for all P0 BC studied, as well as for two P0 BM (P0 BM-N32 and P0 ), no mRNA GFP overaccumulation was observed ( Fig.…”

“…To determine whether the P0 proteins of the poleroviruses BChV and BMYV could suppress GFP silencing in GFPexpressing transgenic N. benthamiana line 16c, binary vectors expressing P0 of BChV (P0 BC ) or BMYV (P0 BM ) were constructed, and those expressing P0 of TuYV (pBin-P0 Tu ) and P14 of BNYVV (pBin-P14) were used as described previously (Pfeffer et al, 2002). Expression of GFP in the leaves and stems of N. benthamiana line 16c resulted in a green fluorescence that could be visualized under UV light.…”

“…So far, polerovirus P0 has been shown to be the RSS of TuYV and CABYV (Pfeffer et al, 2002), acting as an F-box protein and targeting a key step in the RNA-dependent RNA degradation pathway (Pazhouhandeh et al, 2006). The aim of this work was to evaluate and compare P0 RSS activity from a range of BMYV and BChV isolates, using both agro-infiltration of fluorescent Nicotiana benthamiana plants and inoculation of Chenopodium quinoa via a BNYVV replicon.…”

“…The RSSs characterized for sugar beet viruses are: L2 of beet curly top virus (genus Curtovirus; Yang et al, 2007), P14 of beet necrotic yellow vein virus (BNYVV, genus Benyvirus; Dunoyer et al, 2002), P21 of beet yellows virus (genus Closterovirus; Reed et al, 2003) and P0 of turnip yellows virus (TuYV, genus Polerovirus; Pfeffer et al, 2002). TuYV [syn.…”

Detecção e análise filogenética de vírus em melancia e construção de novos vetores virais para expressão de proteínas em células de inseto

“…O tamanho do genoma é bastante uniforme variando de 5,6 kb a 6,0 kb (King et al, 2011). Os RNAs genômicos possuem cinco ou seis ORFs diferenciadas entre os seus três generos são: ORF 1 e 2 (sobrepostas), codificam proteínas relacionadas à replicação (RdRp, RNA polimerases dependentes de RNA); ORF 3, responsável pela codificação da CP viral e a ORF 4, envolvida na codificação da proteína para o movimento a longa distância, à qual é transcrita a partir de uma mudança da fase da leitura da ORF 3; ORF 5, codifica a proteína de transmissão, que só é expressa quando não ocorre o reconhecimento da extremidade 3'da ORF 3 (Pfeffer et al, 2002;King et al, 2011). A ORF 0, possivelmente codifica uma proteína envolvida na supressão de silenciamento gênico e os luteovírus produzem um ou dois sgRNAs adicionais, o maior contém ORF 6 (Terradot et al, 2001;King et al, 2011).…”