Combined Activity of DCL2 and DCL3 Is Crucial in the Defense against Potato Spindle Tuber Viroid

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.
Author Summary Viroids consist of a peculiar type of highly structured small circular RNAs, capable of infecting crop plants and ornamentals. They do not encode any protein, yet they manage to replicate, move through the plant and often cause severe symptoms. In order to achieve this, viroids hijack plant cellular machinery. In addition, they manage to overcome plant RNAi response, which is the major antiviral defense mechanism of plants. DCL proteins have a central role in the RNAi pathway. We have used Nicotiana benthamiana plants, an experimental host of some viroids, and produced plants suppressed for DCL proteins (individually or in combination). By infecting DCL knockdown plants with Potato spindle tuber viroid we were able to identify which DCL proteins are mainly involved in the antiviroid response. We found that it is the combined activity of DCL2 and DCL3 pathways which most potently suppress viroid infectivity. In contrast, DCL4, the main antiviral DCL, seemed to obscure the DCL2-DCL3 effect on viroid infectivity. This lead us to the hypothesis that viroids may have evolved to be primarily processed by DCL4, as it seems to be the DCL protein with less detrimental effects on viroid infectivity. Our findings may aid understanding the complex interaction of viroids with the plant defense machinery.