Interactions of Rice Tungro Bacilliform Pararetrovirus and Its Protein P4 with Plant RNA-Silencing Machinery

Abstract

Small interfering RNA (siRNA)-directed gene silencing plays a major role in antiviral defense. Virus-derived siRNAs inhibit viral replication in infected cells and potentially move to neighboring cells, immunizing them from incoming virus. Viruses have evolved various ways to evade and suppress siRNA production or action. Here, we show that 21-, 22-, and 24-nucleotide (nt) viral siRNAs together constitute up to 19% of total small RNA population of Oryza sativa plants infected with Rice tungro bacilliform virus (RTBV) and cover both strands of the RTBV DNA genome. However, viral siRNA hotspots are restricted to a short noncoding region between transcription and reverse-transcription start sites. This region generates double-stranded RNA (dsRNA) precursors of siRNAs and, in pregenomic RNA, forms a stable secondary structure likely inaccessible to siRNA-directed cleavage. In transient assays, RTBV protein P4 suppressed cell-to-cell spread of silencing but enhanced cell-autonomous silencing, which correlated with reduced 21-nt siRNA levels and increased 22-nt siRNA levels. Our findings imply that RTBV generates decoy dsRNA that restricts siRNA production to the structured noncoding region and thereby protects other regions of the viral genome from repressive action of siRNAs, while the viral protein P4 interferes with cell-to-cell spread of antiviral silencing.

MISIS-2: A bioinformatics tool for in-depth analysis of small RNAs and representation of consensus master genome in viral quasispecies

“…Using the script implemented in MISIS-2, analysis of the 5'-nucleotide identity and frequency was performed for the three major size-classes of plant and viral small from virus-infected rice, banana and tomato plants. The results confirmed and extended previous findings indicating that the plant and viral siRNAs are sorted by AGO proteins based on size and 5'nucleotide identity (Rajeswaran et al 2014a;Rajeswaran et al 2014b;Fuentes et al, 2016).…”

“…Interestingly, a novel class of abundant 20-nt small RNAs with 5'-terminal guanidine was identified in rice and banana plants (Rajeswaran et al 2014a;Rajeswaran et al 2014b).…”

Mal de Río Cuarto Virus Infection Triggers the Production of Distinctive Viral-Derived siRNA Profiles in Wheat and Its Planthopper Vector

“…In turn, control plants displayed a ∼0.6 21-/24-nt ratio. Along the same line, infections with a phloem-limited rice virus (Rajeswaran et al, 2014) and a wheat virus (Tatineni et al, 2014) displayed similar slight changes in 21- and 24-nt species abundance. These results contrast to what was reported in virus infections of dicotyledonous plants where 21-/24-nt ratios are much higher (Donaire et al, 2009; Herranz et al, 2015).…”

“…This result could be partially explained by the well-known dsRNAs panhandle structures formed by interactions between reovirus 5′ and 3′ terminal ends. Alternatively, these heavily targeted regions could be explained by a decoy mechanism such as the one observed upon the infection with rice tungro bacilliform virus, where decoy dsRNA restricts siRNA production to the upstream region to protect other regions of the viral genome from the repressive action of vsiRNAs (Rajeswaran et al, 2014). We evaluated possible associations between internal hotspots and RNA secondary structures within segments but this approach did not satisfactorily explain our results (data not shown).…”

RNA based viral silencing suppression in plant pararetroviruses

“…Since viral RNAs are targets rather than inhibitors of silencing they have not as yet been considered as silencing suppressors. However, recent data obtained with plant pararetroviruses, such as Cauliflower Mosaic Virus (CaMV), and Rice Tungro Bacilliform Virus (RTBV) allow the extension of the list of viral suppressors to ds viral suppressor RNAs ( Blevins et al, 2011 ; Rajeswaran et al, 2014a ).…”