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 3MODIFIER LETTER PRIME untranslated region with the virulence-modulating region (VMR) of PSTVd strain RG1, and was downregulated in PSTVd-infected potato plants. Analysis using 3MODIFIER LETTER PRIME RNA ligase-mediated rapid amplification of cDNA ends (3MODIFIER LETTER PRIME 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. Author summary Potato spindle tuber viroid (PSTVd) is a small RNA pathogen that causes severe pandemic diseases in potato. How this non-protein-coding RNA induces disease symptom development in potato is unknown, thereby hindering the development of effective control measures. Here we report the first evidence that PSTVd disease is caused by the silencing of StTCP23, a potato transcription factor encoding gene, by PSTVd-derived small-interfering RNA (siRNAs). Specifically, we demonstrate that 3MODIFIER LETTER PRIME untranslated region (UTR) region of StTCP23 mRNA contains a 21-nt sequence that is complementary to the virulence-modulating region (VMR) of PSTVd. Furthermore, we show that StTCP23 expression is repressed in PSTVd-infected potato, and this repression is accompanied by StTCP23 transcript cleavage within the identified region of complementary. In planta expression of VMR sequences as 21-nt artificial microRNAs (amiRNAs) or infection of potato plants with a virus-induced gene silencing vector containing a portion the StTCP23 coding sequence, results in reduced StTCP23 transcript abundance and the expression of PSTVd-like disease symptoms. Consistent with the predicted functional role of StTCP23 in regulating the gibberellic acid (GA) biosynthesis and signaling pathways, GA levels were reduced both in PSTVd-infected and amiRNA-expressing plants. Our results provide compelling evidence that StTCP23 positively regulates potato sprouting and tuber development via a GA-related mechanism, and that the disease symptoms that develop upon PSTVd infection result from silencing of StTCP23 by VMR-derived siRNAs.