Abstract
A large amount of short interfering RNA (vsiRNA) is generated from plant viruses during infection, but the function, structure and biogenesis of these is not understood. We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs was the same in different plant species and in the absence of RDR6. We used the Terminator 5′-Phosphate-Dependent Exonuclease to study the 5′ end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5′ monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5′ end of short RNAs or after replacing any potential 5′ ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were complementary to non-abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double stranded RNA, and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double stranded RNA or by RNA dependent RNA polymerase.
Highlights
The RNA silencing based antiviral plant response is one of the best studied antiviral strategies in plants
We found that viral short RNAs are primarily produced from the positive strand of the virus and produced with very different frequency along the viral genome
The hybridisation approach showed that the profile of viral short RNAs is determined by the virus itself because the profiles were the same in different species and it showed that the process was RDR6 independent
Summary
The RNA silencing based antiviral plant response is one of the best studied antiviral strategies in plants. The key element of RNA silencing based antiviral strategies is the virus derived small interfering RNA (vsiRNA), which guides the RNA induced silencing complex (RISC) to target viral genomes in plants and invertebrates [1]. Plant RNA viruses are strong inducers as well as targets of RNA silencing and high levels of vsiRNAs accumulate during the viral infection. In plants two distinct classes of vsiRNAs have been identified: the primary siRNAs, which result from DCL mediated cleavage of an initial trigger RNA, and secondary siRNAs, whose biogenesis requires an RDR enzyme [5,6]. DCL4 and DCL2 are the most important plant DICERs involved in virus induced RNA silencing and they can process ds or hairpin viral RNAs into vsiRNAs of 21 and 22 nt, respectively. AGO1 is the major slicer in plants but other AGO paralogs are likely to be involved, potentially mediating translational repression [1,11]
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