From left to right: Gustavo Gómez, Joan Márquez, Jose Antonio Navarro, Vicente Pallás

This doctoral thesis, directed by Gustavo Gómez, Vicente Pallás and José Antonio Navarro Bohigues, provides new data on the nature and mode of interaction of viroids with their hosts. Part of the results have been published in the journals RNA Biology, Molecular Plant Pathology and Plant Methods. The thesis was defended on May 16, 2022.

Viroids are the simplest pathogens with autonomous replication and have only been found naturally infecting plants. A deeper knowledge of the nature and mode of action of viroids have been the main objectives of the thesis "New knowledge about the biology of viroids and host responses to their infection." A new efficient method for constructing infectious viroid clones was developed and tested with one viroid from each family: eggplant latent viroid (ELVd, Avsunviroidae) and hop dwarf viroid (HSVd, Pospiviroidae). This procedure was based on type IIS restriction enzymes that cut outside the recognition site and represents a universal procedure to obtain infectious clones of a viroid regardless of its sequence, with high efficiency. Although viroids have been considered since their discovery as phytopathogenic non-coding RNAs, the computational analysis performed in this research predicted small open reading frames (ORFs) in each of the HSVd and ELVd genomes. No significant similarities were found with proteins from the plant database, but some of these predicted peptides were highly conserved among all HSVd and ELVd variants. Interestingly, fusion of these conserved sequences with a fluorescent protein revealed a specific subcellular localization in the corresponding organelle where replication and accumulation takes place for each viroid: nucleolus and chloroplast for HSVd and ELVd, respectively. Mutations truncating the nucleolar domain of HSVd were deleterious to the viroid, while truncation of either of the two ELVd ORFs containing a transit signal to the chloroplast also decreased (but to a lesser extent) the biological efficiency of the viroid, perhaps due to functional redundancy. Circular forms of HSVd and ELVd RNAs were found in polysomal fractions, revealing their physical interaction with the plant cell translation machinery. Taken together, these experimental observations indicate that the coding capacity of viroids cannot be ruled out, although definitive testing is a technological challenge that will have to be addressed in the future. Finally, to study the host changes that occur during symptomatic viroid infection, an integrative analysis of the timing and intensity of genome-wide alterations in HSVd-infected cucumber plants was performed. Various omics data were integrated to determine the temporal response to viroid infection. The results support that HSVd promotes the redesign of cucumber regulatory pathways predominantly affecting specific regulatory layers in different phases of infection. This research contributes to the first comprehensive map of plant responses to viroid infection.

Joan Márquez Molins conducted her doctoral research under the supervision of Gustavo Gómez (Institute of Integrative Systems Biology I2SysBio, UV-CSIC), Vicente Pallás (Institute of Molecular and Cellular Plant Biology IBMCP, CSIC-UPV) and José Antonio Navarro Bohigues (IBMCP, CSIC-UPV). During the development of his thesis he has enjoyed a predoctoral contract from the ACIF program of the Generalitat Valenciana, as well as a short-term scholarship from the BEFPI program of the GV and has carried out a research stay in the laboratory of German Martinez (Plant Biology department of the Swedish University of Agricultural Sciences SLU). The qualifying panel was made up of Miguel Aranda Regules (Center for Soil Science and Applied Biology of Segura CEBAS, CSIC), Marco de la Peña Del Rivero (IBMCP, CSIC-UPV), and Selma Gago Zachert (Institute of Biochemistry and Biotechnology, Martín Lutero University), who rated the thesis as outstanding.