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Title: REPLICATION OF POTATO SPINDLE TUBER VIROID IN TOBACCO PROTOPLASTS INOCULATED USING A TOMATO MOTTLE GEMINIVIRUS VECTOR

Author
item Watson, Michael
item Owens, Robert

Submitted to: Plant Cell Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/21/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary: Viroids are the smallest known agents of infectious disease -- small, highly structured RNA molecules which lack both a protein capsid and detectable mRNA activity yet are able to replicate independently and cause disease in many plant crops. As part of our efforts to create durable resistance to viroid infection, we are trying to understand how certain structural features of potato spindle tuber viroid (PSTVd) control its ability to replicate, move from cell to cell, and cause disease. Plant protoplasts (i.e., wall-less single cells) provide an ideal experimental system in which to carry out many such studies, and use of a virus vector to mediate the infection process should ensure a high initial rate of PSTVd RNA synthesis post inoculation. We have substituted an expression cassette containing a DNA copy of PSTVd flanked by spontaneously self-cleaving ribozyme sequences for the coat protein gene of tomato mottle geminivirus. Replication of the PSTVd cDNA-contai beginning 2-3 days post electroporation, but normal viroid replication appeared to be blocked by a failure of (+)PSTVd RNA to circularize in vivo. The fundamental information gathered during our studies will be of greatest interest to researchers studying host-pathogen interactions (especially interactions between plant viruses and their hosts) at the molecular level.

Technical Abstract: A coat protein replacement vector was constructed from the A component of tomato mottle geminivirus (ToMoV) by replacing the coat protein open reading frame with a potato spindle tuber viroid (PSTVd) cDNA cassette containing either wild-type or mutant PSTVd sequences flanked by hammerhead and paperclip ribozyme sequences derived from the satellite RNA of tobacco ringspot virus. Transfected Nicotiana tabacum (cv xanthi NC) protoplasts supported replication of the PSTVd cDNA-containing ToMoV beginning 2 days post-electroporation, and linear PSTVd RNAs of both polarities were also synthesized. Very little of the (+) PSTVd RNA was circularized in vivo.