Abstract
Infectious viroid clones consist of dimeric cDNAs used to generate transcripts which mimic the longer-than-unit replication intermediates. These transcripts can be either generated in vitro or produced in vivo by agro-inoculation. We have designed a new plasmid, which allows both inoculation methods, and we have compared them by infecting Solanum lycopersicum and Solanum melongena with clones of Citrus exocortis virod (CEVd), Tomato chlorotic dwarf viroid (TCDVd), and Potato spindle tuber viroid (PSTVd). Our results showed more uniform and severe symptoms in agro-inoculated plants. Viroid accumulation and the proportion of circular and linear forms were different depending on the host and the inoculation method and did not correlate with the symptoms, which correlated with an increase in PR1 induction, accumulation of the defensive signal molecules salicylic (SA) and gentisic (GA) acids, and ribosomal stress in tomato plants. The alteration in ribosome biogenesis was evidenced by both the upregulation of the tomato ribosomal stress marker SlNAC082 and the impairment in 18S rRNA processing, pointing out ribosomal stress as a novel signature of the pathogenesis of nuclear-replicating viroids. In conclusion, this updated binary vector has turned out to be an efficient and reproducible method that will facilitate the studies of viroid–host interactions.
Highlights
Viroids are the simplest pathogens with autonomous replication currently known, and have only been found naturally in higher plants [1]
To evaluate the influence of the inoculation method on the viroid infection progress in Solanum hosts, we have developed infectious clones of these three pospiviroids in an updated binary vector which allows both agro-infiltration, and a more efficient in vitro transcription of the dimeric viroid cDNA
These results indicate that agro-inoculation with Citrus exocortis virod (CEVd), Tomato chlorotic dwarf viroid (TCDVd), and Potato spindle tuber viroid (PSTVd) impairs ribosome processing in tomato plants, and that this impairment is correlated with disease severity and the activation of the plant defense response (Figures 3 and 4)
Summary
Viroids are the simplest pathogens with autonomous replication currently known, and have only been found naturally in higher plants [1]. Due to the extreme simplicity of their circular RNA genome, viroids extensively rely on host factors for their movement and replication [7,8] They can exert wide transcriptional changes in host plants, interfering with the silencing machinery of the cell [9,10], or producing epigenetic modifications [11,12,13], alternative splicing [14,15] or, as described recently, ribosomal stress [16,17]. This latter phenomenon refers to the impaired processing of the 18S rRNA provoked by the viroid, which causes defects in ribosome biogenesis, and important alterations in the host translation machinery [16,17,18]
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