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Previous attempts to develop RNAi-mediated viroid-resistant transgenic plants using nearly full-length  Potato spindle tuber viroid  (PSTVd) hairpin RNA (hpRNA) were successful; however unusual phenotypes resembling viroid infection occurred. Therefore, in the present work, transgenic  Nicotiana benthamiana  lines expressing both partial and truncated versions of PSTVd hpRNA were developed. Specifically, seven partial or truncated versions of PSTVd sequences were selected according to the hotspots of both PSTVd-sRNAs and functional domains of the PSTVd. A total of 21 transgenic lines  Nicotiana benthamiana  were developed under the control of either the CaMV-35S or the CoYMV promoters. All of the transgenic lines established here were monitored for the induction of phenotypic changes, for PSTVd-sRNA expression and for the resistance against PSTVd infection. Additionally, this study demonstrates the use of inverted repeat construct sequences as short as 26- to -49 nucleotides for both the efficient expression of the PSTVd-sRNA and the inhibition of PSTVd infection.

RNAi mediated inhibition of viroid infection in transgenic plants expressing viroid-specific small RNAs derived from various functional domains