Evaluation of Two Nontransformative Approaches in Triggering RNAi against Tomato Yellow Leaf Curl Virus

Tomato yellow leaf curl virus-Israel (TYLCV-IL) is a worldwide destructive monopartite begomovirus. Two nontransformative methods, namely the dsRNA (double-stranded RNA) vaccination and the hairpin construct expressing dsRNA were applied to induce RNA interference (RNAi) against TYLCV-IL infection. Analysis of TYLCV-IL-derived small interfering RNAs profile revealed two hot spots triggering post-transcriptional gene silencing, including a part of the coat protein (CP) and the replication-associated protein (Rep) genes. CP and Rep derived dsRNAs were produced in vitro and applied simultaneously or three days before agroinoculation of TYLCV-IL by rubbing on the leaf surface of Nicotiana benthamiana. All tested plants showed symptoms of typical infection at 21 days' post-inoculation (dpi), and viral genomic fragments were amplified in symptomatic plants by PCR as expected. Despite the systemic movement of applied dsRNAs in N. benthamiana plants, quantitative PCR showed no statistically significant difference between TYLCV-IL genome accumulation in virus infection alone or at the presence of dsRNA molecules. In contrast, the hairpin RNA construct (hpRep) triggered a high resistance against TYLCV-IL infection in tomato plants by targeting the same Rep sequence. HpRep construct suppressed expression of symptoms up to 21 dpi. Moreover, no virus genome was detected in symptomless plants indicating disruption of TYLCV-IL replication. These results indicate the distinct efficiency of two methods of RNAi induction against an important begomovirus species, TYLCV-IL. The role of different factors on the RNAi efficiency against TYLCV-IL has been discussed.