Date Published: October 12, 2016
Publisher: Public Library of Science
Author(s): Konstantina Katsarou, Eleni Mavrothalassiti, Wannes Dermauw, Thomas Van Leeuwen, Kriton Kalantidis, Shou-Wei Ding.
Viroids are self replicating non-coding RNAs capable of infecting a wide range of plant hosts. They do not encode any proteins, thus the mechanism by which they escape plant defenses remains unclear. RNAi silencing is a major defense mechanism against virus infections, with the four DCL proteins being principal components of the pathway. We have used Nicotiana benthamiana as a model to study Potato spindle tuber viroid infection. This viroid is a member of the Pospiviroidae family and replicates in the nucleus via an asymmetric rolling circle mechanism. We have created knock-down plants for all four DCL genes and their combinations. Previously, we showed that DCL4 has a positive effect on PSTVd infectivity since viroid levels drop when DCL4 is suppressed. Here, we show that PSTVd levels remain decreased throughout infection in DCL4 knockdown plants, and that simultaneous knockdown of DCL1, DCL2 or DCL3 together with DCL4 cannot reverse this effect. Through infection of plants suppressed for multiple DCLs we further show that a combined suppression of DCL2 and DCL3 has a major effect in succumbing plant antiviral defense. Based on our results, we further suggest that Pospoviroids may have evolved to be primarily processed by DCL4 as it seems to be a DCL protein with less detrimental effects on viroid infectivity. These findings pave the way to delineate the complexity of the relationship between viroids and plant RNA silencing response.
Viroids are infectious, naked circular RNAs sized from 246 to 401 nucleotides (nt), capable of infecting a wide range of hosts, causing important economic loses . They are divided into two families, Pospiviroidae and Avsunviroidae [1–3]. Potato spindle tuber viroid (PSTVd), a type species of the Pospiviroidae family, has a rod-like secondary structure with five distinct domains, and replicates in the nucleus through an asymmetric rolling circle mechanism using RNA polymerase II (RNAPII) [1,4–6]. It is known to infect crop plants of the Solanaceae family such as tomato and potato, as well as some ornamental plants of the Scrophulariaceae and Asteracae family, but does not infect the plant model Arabidopsis thaliana systemically.
The complex relationship of viroids with the RNAi pathways has been puzzling researchers since the discovery of RNAi in 1998. The partially dsRNA nature of their genome, in combination with the lack of any silencing suppressor proteins, suggested that viroids could be a good target for RNAi resistance. This was supported a few years later by the finding that indeed vd-siRNAs are abundant in infected plants [4,36,37]. However, even though viroids seem to have evolved in such way that they can escape this fate leading to infection, the way they manage to do so remains unclear [39–42]. Nevertheless, different studies have implicated RNAi components in viroid infection [45–47]. In a previous study, we have shown that, at least, DCL2, DCL3 and DCL4 of N. benthamiana are involved in the production of cognate vd-siRNAs of specific size classes. In addition, we identified the importance of DCL4, since when DCL4 is suppressed, PSTVd titer and symptoms follow . This effect is in contrast to what is usually observed during viral infections, where DCL4 acts as a major antiviral protein [13,19,20].