evaluation of two nontransformative approaches in triggering rnai against tomato yellow leaf curl virus

Tomato yellow leaf curl virusisrael (tylcvil) is a worldwide destructive monopartite begomovirus. two nontransformative methods, namely the dsrna (doublestranded rna) vaccination and the hairpin construct expressing dsrna were applied to induce rna interference (rnai) against tylcvil infection. analysis of tylcvilderived small interfering rnas profile revealed two hot spots triggering posttranscriptional gene silencing, including a part of the coat protein (cp) and the replicationassociated protein (rep) genes. cp and rep derived dsrnas were produced in vitro and applied simultaneously or three days before agroinoculation of tylcvil by rubbing on the leaf surface of nicotiana benthamiana. all tested plants showed symptoms of typical infection at 21 days’ postinoculation (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 tylcvil 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 tylcvil 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 tylcvil replication. these results indicate the distinct efficiency of two methods of rnai induction against an important begomovirus species, tylcvil. the role of different factors on the rnai efficiency against tylcvil has been discussed.

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

Tomato yellow leaf curl virusIsrael (TYLCVIL) is a worldwide destructive monopartite begomovirus. Two nontransformative methods, namely the dsRNA (doublestranded RNA) vaccination and the hairpin construct expressing dsRNA were applied to induce RNA interference (RNAi) against TYLCVIL infection. Analysis of TYLCVILderived small interfering RNAs profile revealed two hot spots triggering posttranscriptional gene silencing, including a part of the coat protein (CP) and the replicationassociated protein (Rep) genes. CP and Rep derived dsRNAs were produced in vitro and applied simultaneously or three days before agroinoculation of TYLCVIL by rubbing on the leaf surface of Nicotiana benthamiana. All tested plants showed symptoms of typical infection at 21 days’ postinoculation (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 TYLCVIL 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 TYLCVIL 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 TYLCVIL replication. These results indicate the distinct efficiency of two methods of RNAi induction against an important begomovirus species, TYLCVIL. The role of different factors on the RNAi efficiency against TYLCVIL has been discussed.