RESUMO
Tomato yellow leaf curl disease (TYLCD) has become the key limiting factor for the production of tomato in many areas because of the continuous infection and recombination of several tomato yellow leaf curl virus (TYLCV)-like species (TYLCLV) which produce novel and destructive viruses. Artificial microRNA (AMIR) is a recent and effective technology used to create viral resistance in major crops. This study applies AMIR technology in two ways, i.e., amiRNA in introns (AMINs) and amiRNA in exons (AMIEs), to express 14 amiRNAs targeting conserved regions in seven TYLCLV genes and their satellite DNA. The resulting pAMIN14 and pAMIE14 vectors can encode large AMIR clusters and their function in silencing reporter genes was validated with transient assays and stable transgenic N. tabacum plants. To assess the efficacy of conferring resistance against TYLCLV, pAMIE14 and pAMIN14 were transformed into tomato cultivar A57 and the resulting transgenic tomato plants were evaluated for their level of resistance to mixed TYLCLV infection. The results suggest that pAMIN14 transgenic lines have a more effective resistance than pAMIE14 transgenic lines, reaching a resistance level comparable to plants carrying the TY1 resistance gene.
RESUMO
Plant viruses impose serious threats on crop production. Artificial miRNAs can mediate specific and effective gene silencing in plants and are widely used in plant gene function studies and to engineer plant viral resistance. To facilitate the design of artificial miRNA genes, we developed a webserver, AMIRdesigner, which can be used to design oligos for artificial miRNA synthesis using wild-type and permutated MIR171 and MIR164 backbones. The artificial miRNA genes designed by AMIRdesigner can be easily assembled into miRNA clusters for multiple target sites. To validate the server functionality, we designed four artificial miRNA genes targeting four conserved regions in the potato leafroll virus genome using AMIRdesigner. These genes were synthesized with the server-designed oligos and further assembled into a quadruple miRNA cluster, which was cloned into an overexpression vector and transformed into potato plants. Small RNA Northern blot and virus inoculation analyses showed that a high level of artificial miRNA expression and good viral resistance were achieved in some of the transgenic lines. These results demonstrate the utility of our webserver AMIRdesigner for engineering crop viral resistance.
RESUMO
Small RNAs regulate a large set of gene expression in all plants and constitute a natural immunity against viruses. Small RNA based genetic engineering (SRGE) technology had been explored for crop protection against viruses for nearly 30 years. Viral resistance has been developed in diverse crops with SRGE technology and a few viral resistant crops have been approved for commercial release. In this review we summarized the efforts generating viral resistance with SRGE in different crops, analyzed the evolution of the technology, its efficacy in different crops for different viruses and its application status in different crops. The challenge and potential solution for application of SRGE in crop protection are also discussed.
