Silencing of transcription factor encoding gene StTCP23 by small RNAs derived from the virulence modulating region of potato spindle tuber viroid is associated with symptom development in potato.

Viroids are small, non-protein-coding RNAs which can induce disease symptoms in a variety of plant species. Potato (Solanum tuberosum L.) is the natural host of Potato spindle tuber viroid (PSTVd) where infection results in stunting, distortion of leaves and tubers and yield loss. Replication of PSTVd is accompanied by the accumulation of viroid-derived small RNAs (sRNAs) proposed to play a central role in disease symptom development. Here we report that PSTVd sRNAs direct RNA silencing in potato against StTCP23, a member of the TCP (teosinte branched1/Cycloidea/Proliferating cell factor) transcription factor family genes that play an important role in plant growth and development as well as hormonal regulation, especially in responses to gibberellic acid (GA). The StTCP23 transcript has 21-nucleotide sequence complementarity in its 3' untranslated region with the virulence-modulating region (VMR) of PSTVd strain RG1, and was downregulated in PSTVd-infected potato plants. Analysis using 3' RNA ligase-mediated rapid amplification of cDNA ends (3' RLM RACE) confirmed cleavage of StTCP23 transcript at the expected sites within the complementarity with VMR-derived sRNAs. Expression of these VMR sRNA sequences as artificial miRNAs (amiRNAs) in transgenic potato plants resulted in phenotypes reminiscent of PSTVd-RG1-infected plants. Furthermore, the severity of the phenotypes displayed was correlated with the level of amiRNA accumulation and the degree of amiRNA-directed down-regulation of StTCP23. In addition, virus-induced gene silencing (VIGS) of StTCP23 in potato also resulted in PSTVd-like phenotypes. Consistent with the function of TCP family genes, amiRNA lines in which StTCP23 expression was silenced showed a decrease in GA levels as well as alterations to the expression of GA biosynthesis and signaling genes previously implicated in tuber development. Application of GA to the amiRNA plants minimized the PSTVd-like phenotypes. Taken together, our results indicate that sRNAs derived from the VMR of PSTVd-RG1 direct silencing of StTCP23 expression, thereby disrupting the signaling pathways regulating GA metabolism and leading to plant stunting and formation of small and spindle-shaped tubers.

The phenotypic and molecular assessment of the non-conserved Arabidopsis MICRORNA163/S-ADENOSYL-METHYLTRANSFERASE regulatory module during biotic stress.

In plants, microRNAs (miRNAs) have evolved in parallel to the protein-coding genes that they target for expression regulation, and miRNA-directed gene expression regulation is central to almost every cellular process. MicroRNA, miR163, is unique to the Arabidopsis genus and is processed into a 24-nucleotide (nt) mature small regulatory RNA (sRNA) from a single precursor transcript transcribed from a single locus, the MIR163 gene. The MIR163 locus is a result of a recent inverted duplication event of one of the five closely related S-ADENOSYL-METHYLTRANSFERASE genes that the mature miR163 sRNA targets for expression regulation. Currently, however, little is known about the role of the miR163/S-ADENOSYL-METHYLTRANSFERASE regulatory module in response to biotic stress. Here, we document the expression domains of MIR163 and the S-ADENOSYL-METHYLTRANSFERASE target genes following fusion of their putative promoter sequences to the ß-glucuronidase (GUS) reporter gene and subsequent in planta expression. Further, we report on our phenotypic and molecular assessment of Arabidopsis thaliana plants with altered miR163 accumulation, namely the mir163-1 and mir163-2 insertion knockout mutants and the miR163 overexpression line, the MIR163-OE plant. Finally, we reveal miR163 accumulation and S-ADENOSYL-METHYLTRANSFERASE target gene expression post treatment with the defence elicitors, salicylic acid and jasmonic acid, and following Fusarium oxysporum infection, wounding, and herbivory attack. Together, the work presented here provides a comprehensive new biological insight into the role played by the Arabidopsis genus-specific miR163/S-ADENOSYL-METHYLTRANSFERASE regulatory module in normal A. thaliana development and during the exposure of A. thaliana plants to biotic stress.