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

While the potato spindle tuber viroid (PSTVd) variant, PSTVd-Dahlia (PSTVd-D or PSTVd-D_wt) induces very mild symptoms in tomato cultivar 'Rutgers', PSTVd-Intermediate (PSTVd-I or PSTVd-I_wt) induces severe symptoms. These two variants differ by nine nucleotides, of which six mutations are located in the terminal left (TL) to the pathogenicity (P) domains. To evaluate the importance of mutations located in the TL to the P domains, ten types of point mutants were created by swapping the nucleotides between the two viroid variants. Bioassay in tomato plants demonstrated that two mutants created on PSTVd-I_wt at positions 42 and 64 resulted in symptom attenuation. Phenotypic and RT-qPCR analysis revealed that mutation at position 42 of PSTVd-I_wt significantly reduced disease severity and accumulation of the viroid, whereas mutation at position 64 showed a significant reduction in stunting when compared to the PSTVd-I_wt infected plant. RT-qPCR analysis on pathogenesis-related protein 1b1 and chalcone synthase genes showed a direct correlation with symptom severity whereas the expansin genes were down-regulated irrespective of the symptom severity. These results indicate that the nucleotides at positions 42 and 64 are in concert with the ones at positions 43, 310, and 311/312, which determines the slower and stable accumulation of PSTVd-D without eliciting excessive host defense responses thus contributing in the attenuation of disease symptom.