Artificial microRNA mediated RNA silencing to tomato yellow leaf curl virus infecting tomato in Oman
مؤلف
Al-Rushadiyah, Maha Rashid Musabah.
الملخص الإنجليزي
Tomato yellow leaf curl disease is the most devastating viral disease of tomato and other dicot species caused by Tomato yellow leaf curl virus (TYLCV). TYLCV is a whitefly (Bemisia tabaci) transmitted geminivirus belongs to the family Geminiviridae, genus Begomovirus. The management of the disease in Oman is mainly based on cultural practices and use of pesticides, which is not sufficient to reduce disease incidence. The deployment of host-plant resistance is the most practical mean to control this disease.
MicroRNAs (miRNAs) are short 21 to 23 nucleotide long RNA sequences homologous to target viral Rep sequences with important regulatory role. miRNAs control the expression of target transcripts by binding to complementary sequences leading to cleavage or translational inhibition of the target RNA. Plants carrying sense or antisense transgenes can exhibit spontaneous silencing.
To achieve this goal, sense and antisense transgenes derived from Rep (replication associated protein) ORF of TYLCV-OM driven by 350S CaMV promoter and Nos terminator were synthesized commercially. Susceptible tomato cultivar (Moneymaker) was used as explants and co-cultivated with Agrobacterium tumefaciens strain AGL1 harboring plant expression binary vector carrying miRNA gene. Transformed shoots were selected on kanamycin medium and the presence of the transgene in the primary transformants was confirmed by PCR using CaMV promoter specific primers. Seeds were collected and sown. Integration of amiRNA genes into F1 transgenic plants was confirmed by PCR. F1 tomato seedlings were challenged with TYLCV-OM.
Putative transgenic plants obtained through Agrobacterium mediated transformation have 70% transformation efficiency. Seeds from transgenic plants have been collected to test resistance against TYLCV-OM in F1 generation. Severe disease symptoms of TYLCV infection appeared on susceptible tomato plants whereas no symptoms developed on transgenic plants.
The findings of this study were consistent with previous studies demonstrating amiRNAmediated resistance is an effective silencing strategy to introduce anti-viral transgenes in host plants. Transgenic expression of Rep-specific amiRNAs was found to be an effective method to inhibit TYLCV infection in tomato plants.
