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

Abstract

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 3ʹ untranslated region with the virulence-modulating region (VMR) of PSTVd strain RG1, and was downregulated in PSTVd-infected potato plants. Analysis using 3ʹ RNA ligase-mediated rapid amplification of cDNA ends (3ʹ 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.

Effects of Host-Adaptive Mutations on Hop Stunt Viroid Pathogenicity and Small RNA Biogenesis

“…Although RNA silencing has been experimentally shown to be involved in the pathogenesis of both chloroplastic [ 27 , 28 ] and nuclear [ 29 , 30 , 31 , 32 ] viroids, this mechanism does not appear responsible for the differences in pathogenicity of HSVd-h and HSVd-g54. This conclusion is based on the observation that none of the HSVd-sRNA species which differ in sequence between HSVd-g54 and HSVd-h were able to pair with any of the genes differentially expressed between HSVd-g54 and HSVd-h infected cucumber plants ( Tables S5 and S6 ).…”

“…Also, a potato gene which encodes transcription factor StTCP23 was shown to be targeted by several sRNA species derived from the virulence-modulating region of PSTVd. The silencing of StTCP23 leads to the development of viroid-like symptoms in potato [ 32 ].…”

“…Only the predominant 21- and 22-nt HSVd-sRNA species [ 36 ] ( Figure 6 B) were analyzed. A maximum cutoff score = 2 was chosen, considering the following factors: (i) The duplexes between three PLMVd-sRNA species (PC-sRNA8a, PC-sRNA8b, and PYM-sRNA40) [ 27 , 28 ] and two PSTVd-sRNA species (PSTVd-sRNA45, 46) [ 32 ], and their verified targets showed scores from 1 to 2; (ii) a score of 2 is capable of predicting the targets of several cucumber miRNA molecules identified in previous studies [ 52 , 53 , 54 ] and conserved in other species (including miR156, miR159, miR167, miR164, miR172, miR319, and miR858). Next, we compared the expression levels of these genes in cucumbers infected with HSVd-g54 and HSVd-h using previously published transcriptome data [ 37 ].…”

“…Several studies have also implicated RNA silencing in pathogenicity of members of the family Pospiviroidae [ 29 , 30 , 31 ], but evidence for its involvement is less clear than for members of the family Avsunviroidae [ 26 , 28 ]. In this regard, the recent demonstration that silencing of transcription factor StTCP23 expression by several sRNA species derived from the virulence modulating region of potato spindle tuber viroid (PSTVd) is associated with symptom development in potato [ 32 ] provides solid support.…”

Identification and Molecular Mechanisms of Key Nucleotides Causing Attenuation in Pathogenicity of Dahlia Isolate of Potato Spindle Tuber Viroid

“…A vd-sRNA generated from the VMR of PSTVd was then shown to silence the callose synthase genes of tomato plants, and it was observed that suppression was more pronounced in plants infected with a severe strain than those infected with a mild strain [ 42 ]. More recently, vd-sRNAs derived from the VMR of PSTVd were reported to have silenced a transcription factor encoding gene StTCP23 in potato, leading to the development of disease symptoms [ 43 ]. Moreover, genes reported containing sequences that could be targeted by vd-sRNAs include tomato FRIGIDA- like protein 3 ( FRL3 ), chloride channel protein CLCb -like ( clcB ), ribosomal protein S3a - like ( RPS3A ), serine-threonine protein kinases , WD40-repeat protein ( WD40 ), and soluble inorganic pyrophosphatase of tobacco plants [ 44 , 45 , 46 , 47 , 48 , 49 , 50 ].…”