Abstract
Earlier work indicated that Tomato spotted wilt virus (TSWV) messenger transcripts, and not the (anti)genomic RNAs, are targeted by the RNA silencing machinery. Here, the predicted AU-rich hairpin (HP) structure encoded by the intergenic region (IGR) of the TSWV S RNA, and present at the 3′ end of viral mRNAs, was analyzed as a target and inducer for RNA silencing. Virus-derived siRNAs (vsiRNAs) purified from virus infected plants were found to derive from all three genomic RNA segments but predominantly the ambisense M and S RNAs. Further profiling on the S RNA sequence revealed that vsiRNAs were found from almost the entire S RNA sequence, except the IGR from where hardly any vsiRNAs were found. Similar profiles were observed with the distantly related Tomato yellow ring tospovirus (TYRV). Dicer cleavage assays using Drosophila melanogaster (Dm) embryo extracts showed that synthetic transcripts of the IGR-HP region were recognized as substrate for Dicer. Transient agroinfiltration assays of a GFP-sensor construct containing the IGR-HP sequence at its 3′ UTR (GFP-HP) did not show more rapid/strong silencing and profiling of the corresponding siRNAs, generated outside the context of a viral infection, still revealed relatively low levels of IGR-HP-derived siRNAs. These data support the idea that the IGR-HP is a weak inducer of RNA silencing and only plays a minor role in the amplification of a strong antiviral RNAi response.
Highlights
RNA silencing, named post transcriptional gene silencing (PTGS), is a conserved cellular mechanism in plants and animals in which double-strandedRNA, imperfect hairpin RNAs or highly structured single-strandedRNA trigger a chain of processes leading to sequence-specific RNA degradation [1,2]
To test for this, and analyse whether M and S RNA give rise to the production of higher levels of Virus-derived small interfering RNAs (siRNAs) (vsiRNAs), small RNA molecules were purified from Tomato spotted wilt virus (TSWV)-infected N. benthamiana leaf material and, after radiolabeling, probed on total RNA and genomic RNA purified from isolated viral RNPs (Fig. 3A)
While vsiRNAs were found hybridizing to the L, M and S RNA segments, strong hybridization signals were observed with the ambisense M and S RNA segments (Fig. 3A, lane 3)
Summary
RNA silencing, named post transcriptional gene silencing (PTGS), is a conserved cellular mechanism in plants and animals in which double-stranded (ds)RNA, imperfect hairpin RNAs or highly structured single-stranded (ss)RNA trigger a chain of processes leading to sequence-specific RNA degradation [1,2]. During this process, dsRNA is processed into small interfering RNAs (siRNAs) or microRNAs (miRNAs) of 21–26 nucleotides in length by RNase-III-type enzymes called Dicer or dicer-like (DCL) [3,4,5,6,7]. In all of these cases, the final outcome is similar, i.e. viral RNA target molecules are prevented from becoming degraded by the RISC complex
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