Gene silencing for functional studies in plants has been largely facilitated by manipulating viral genomes with inserts from host genes to trigger virus-induced gene silencing (VIGS) against the corresponding mRNAs. However, viral genomes encode multiple proteins and can disrupt plant homeostasis by interfering with endogenous cell mechanisms. To try to circumvent this functional limitation, we have developed a silencing method based on the minimal autonomously-infectious nucleic acids currently known: viroids, which lack proven coding capability. The genome of Eggplant latent viroid, an asymptomatic viroid, was manipulated with insertions ranging between 21 and 42 nucleotides. Our results show that, although larger insertions might be tolerated, the maintenance of the secondary structure appears to be critical for viroid genome stability. Remarkably, these modified ELVd molecules are able to induce systemic infection promoting the silencing of target genes in eggplant. Inspired by the design of artificial microRNAs, we have developed a simple and standardized procedure to generate stable insertions into the ELVd genome capable of silencing a specific target gene. Analogously to VIGS, we have termed our approach viroid-induced gene silencing, and demonstrate that it is a promising tool for dissecting gene functions in eggplant.

Climate change has been associated with a higher incidence of combined adverse environmental conditions that can promote a significant decrease in crop productivity. However, knowledge on how a combination of stresses might affect plant development is still scarce. MicroRNAs (miRNAs) have been proposed as potential targets for improving crop-productivity. Here, we have combined deep-sequencing, computational characterization of responsive miRNAs and validation of their regulatory role in a comprehensive analysis of melon response to several combinations of four stresses (cold, salinity, short day, and infection with a fungus). Twenty-two miRNA families responding to double and/or triple stresses were identified. The regulatory role of the differentially expressed miRNAs was validated by quantitative measurements of the expression of the corresponding target genes. A high proportion (ca. 60%) of these families (mainly highly conserved miRNAs targeting transcription factors) showed a non-additive response to multiple stresses in comparison with that observed under each one of the stresses individually. Among those miRNAs showing non-additive response to stress-combinations, most interactions were negative suggesting the existence of functional convergence in the miRNA-mediated response to combined stresses. Taken together, our results provide compelling evidences that the response to combined stresses cannot be easily predicted from the study individual stresses

Gene silencing for functional studies in plants has been largely facilitated by manipulating viral genomes with inserts from host genes to trigger virus induced gene silencing (VIGS) against the corresponding mRNAs. However, viral genomes encode multiple proteins and disrupt plant homeostasis by interfering with endogenous cell mechanisms. To circumvent this issue, we have developed a silencing method based on the minimal autonomously-infectious nucleic acids currently known: viroids. In particular, Eggplant latent viroid (ELVd), an asymptomatic viroid, was manipulated with insertions between 21 to 42 nucleotides and our results show that larger insertions are tolerated but secondary structure is critical for their stability. Additionally, these ELVd constructs are able of local and systemic spread and can silence a target gene in eggplant. Inspired by the design of artificial microRNAs, we have developed a standardized procedure to generate stable insertions into the ELVd genome capable of silencing the desired target gene. Analogously to VIGS, we have termed our approach Viroid Induced Gene Silencing (VdIGS) and demonstrate that is a promising tool for dissecting gene functions in eggplant. Overall, this represents the use of minimal circular replicating RNAs able to spread systemically combined with the production of a tailored sRNA for targeted silencing.