Abstract
The tomato (Solanum lycopersicum) callose synthase genes CalS11-like and CalS12-like encode proteins that are essential for the formation of callose, a major component of pollen mother cell walls; these enzymes also function in callose formation during pathogen infection. This article describes the targeting of these callose synthase mRNAs by a small RNA derived from the virulence modulating region of two Potato spindle tuber viroid variants. More specifically, viroid infection of tomato plants resulted in the suppression of the target mRNAs up to 1.5-fold, depending on the viroid variant used and the gene targeted. The targeting of these mRNAs by RNA silencing was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Viroid mutants incapable of targeting callose synthase mRNAs failed to induce typical infection phenotypes, whereas a chimeric viroid obtained by swapping the virulence modulating regions of a mild and a severe variant of Potato spindle tuber viroid greatly affected the accumulation of viroids and the severity of disease symptoms. These data provide evidence of the silencing of multiple genes by a single small RNA derived from a viroid.
Highlights
In plant pathology, viroids are the most enigmatic pathogens, due to their small (246 to 401 nucleotides), single-stranded, highly structured RNA genome
To demonstrate RNA-induced silencing complex (RISC)-mediated targeting of the CalS11-like mRNA, we developed an artificial microRNA system for the transient expression of the viroid-derived small RNAs (vdsRNAs) derived from the VMR of both PSTVd variants in Nicotiana benthamiana leaves
For each set of experiments, the ratio of GFP to phosphoenolpyruvate carboxylase (PEPC) obtained with empty pBIN61 vector (EV) plus GFP:C11-vdXX was set at a value of 1
Summary
Viroids are the most enigmatic pathogens, due to their small (246 to 401 nucleotides), single-stranded, highly structured RNA genome. Viroid infection is often accompanied by a wide array of symptoms, including stunting, leaf epinasty, leaf distortion, flower distortion, and reduced numbers of flowers. How exactly these small, noncoding RNAs can invade plants and induce host responses has long been a subject of interest (Owens et al, 2012). The Avsunviroidae family, whose type species is the Avocado sunblotch viroid, includes four members that replicate in chloroplasts through a symmetric rolling-circle mechanism and exhibit selfcleavage activity. Members of the Pospiviroidae family, whose type species is the Potato spindle tuber viroid (PSTVd), replicate in the nucleus and contain five structural/functional domains: the terminal left, pathogenicity, central conserved region, variable, and terminal right regions. The importance of the viroid pathogenicity domain in symptom severity is well established
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