Mutational analysis of eggplant latent viroid RNA processing in Chlamydomonas reinhardtii chloroplast.

Viroids of the family Avsunviroidae, such as eggplant latent viroid (ELVd), contain hammerhead ribozymes and replicate in the chloroplasts of the host plant through an RNA-based symmetrical rolling-circle mechanism in which oligomeric RNAs of both polarity are processed to monomeric linear RNAs (by cleavage) and to monomeric circular RNAs (by ligation). Using an experimental system consisting of transplastomic lines of the alga Chlamydomonas reinhardtii, a mutational analysis of sequence and structural elements in the ELVd molecule that are involved in transcript processing in vivo in a chloroplastic context was carried out. A collection of six insertion and three deletion ELVd mutants was created and expressed in C. reinhardtii chloroplast. All mutants cleaved efficiently except for the control with an insertion inside the hammerhead ribozyme domain, supporting the prediction that this domain is necessary and sufficient to mediate transcript cleavage in vivo. However, two deletion mutants that cleaved efficiently showed ligation defects, indicating that during RNA circularization, other parts of the molecule are involved in addition to the hammerhead ribozyme domain. This is probably a quasi double-stranded structure present in the central part of the molecule which contains the ligation site in an internal loop. However, the mutations prevented the viroid from infecting its natural host, eggplant, indicating that they affected other essential functions in ELVd infectious cycle. The insertion in the terminal loop of the right upper hairpin of ELVd did not have this effect; it was tolerated and partially maintained in the progeny.