Transgenic tomato plants expressing artificial microRNAs for silencing the pre-coat and coat proteins of a begomovirus, Tomato leaf curl New Delhi virus, show tolerance to virus infection

Designing artificial microRNAs (amiRs) targeting the genes responsible for viral replication, transmission and symptom development after viral infection offers a promising strategy to contain the multiplication and spread of geminiviruses in host plants. Here, we report the design of two amiRs targeting the middle region of the AV1 (coat protein) transcript (amiR-AV1-3) and the overlapping region of the AV1 and AV2 (pre-coat protein) transcripts (amiR-AV1-1) of a model geminivirus, Tomato leaf curl virus (ToLCV). Our analyses demonstrate that transgenic tomato plants expressing amiR-AV1-1, propagated until the T2 generation and were highly tolerant to Tomato leaf curl New Delhi virus (ToLCNDV), whereas those harboring amiR-AV1-3 exhibited only moderate tolerance. Biochemical analyses revealed that in these cases, the amiRs acted through the slicing mechanism, cleaving their respective targets. Although ToLCVs are generally difficult targets for manipulations related to virus resistance, our data reveal that an amiR strategy could be employed to protect plants in an effective manner.

► Design of artificial microRNAs (amiRs). ► Tomato transgenics expressing amiRs. ► Transgenics showing tolerance to infectious ToLCV clones. ► amiR cleavage of target sites. ► T2 level transgenics show high tolerance against virus challenges.