Project description:In this study, Agrobacterium tumefaciens containing recombinant plasmids were used to agroinoculate cucumber seedlings with tandem repeats of DNA-A genome of Tomato leaf curl New Delhi virus (ToLCNDV). The inoculated seedlings, representing both resistant and susceptible accessions, were monitored for symptoms over a period of four weeks. Symptom development was observed in susceptible genotypes, while the resistant genotype remained symptomless. Leaf samples were collected from both agroinoculated and control plants for further analysis. This research sheds light on the pathogenicity of ToLCNDV in cucumber and highlights the potential resistance mechanisms in certain accessions.
Project description:RNA interference (RNAi) is a conserved, RNA-mediated, regulatory mechanism in eukaryotes. In plants, it plays an important role in growth, development and resistance against viral infections. As a counter-defence, plant viruses, e.g. geminiviruses, encode RNAi suppressors, such as AC2, AC4 and AV2. To obtain Nicotiana tabacum virus resistant plants against Tomato leaf curl New Delhi virus (ToLCNDV), we employ the biogenesis pathway of a class of endogenous siRNAs, the trans-acting siRNAs (ta-siRNAs), by engineering artificial ta-siRNAs (ata-siRNAs) targeting the AC2 (TRiV-AC2) and AC4 (TRiV-AC4) RNAi suppressors using miRNA390 dual target sites. The mode of action of ta-siRNAs comprises of the cleavage of the target (similar to the miRNA targeting). Using degradome approaches, the abundance of the resulting 3' fragment of the cleaved transcript can be quantified and the precise localization of the cleavage on the target mRNA can be identified. We sequenced degradome libraries of Nicotiana tabacum plants infected with ToLCNDV which were treated with the ata-siRNA-AC2 construct; mock-treated plants were used as controls. Following quality checks, the abundance distributions of the degradation fragments were normalized. The transcripts with different cleavage patterns was the AC2, supporting the conclusion that an efficient cleavage of the target occurred, without significant off-target effects.
Project description:RNA interference (RNAi) is a conserved, RNA-mediated, regulatory mechanism in eukaryotes. In plants, it plays an important role in growth, development and resistance against viral infections. As a counter-defence, plant viruses, e.g. geminiviruses, encode RNAi suppressors, such as AC2, AC4 and AV2. To obtain Nicotiana tabacum virus resistant plants against Tomato leaf curl New Delhi virus (ToLCNDV), we employ the biogenesis pathway of a class of endogenous siRNAs, the trans-acting siRNAs (ta-siRNAs), by engineering artificial ta-siRNAs (ata-siRNAs) targeting the AC2 (TRiV-AC2) and AC4 (TRiV-AC4) RNAi suppressors using miRNA390 dual target sites. The mode of action of ta-siRNAs comprises of the cleavage of the target (similar to the miRNA targeting). Using degradome approaches, the abundance of the resulting 3' fragment of the cleaved transcript can be quantified and the precise localization of the cleavage on the target mRNA can be identified. We sequenced degradome libraries of Nicotiana tabacum plants infected with ToLCNDV which were treated with the ata-siRNA-AC2 construct; mock-treated plants were used as controls. Following quality checks, the abundance distributions of the degradation fragments were normalized. The transcripts with different cleavage patterns was the AC2, supporting the conclusion that an efficient cleavage of the target occurred, without significant off-target effects.