Prediction and characterization of Tomato leaf curl New Delhi virus (ToLCNDV) responsive novel microRNAs in Solanum lycopersicum

Abstract

Tomato leaf curl New Delhi virus (ToLCNDV) infects tomato (Solanum lycopersicum) plants and causes severe crop losses. As the microRNAs (miRNAs) are deregulated during stressful events, such as biotic stress, we wanted to study the effect of ToLCNDV infection on tomato miRNAs. We constructed two libraries, isolating small RNAs (sRNAs) from healthy (HT) and ToLCNDV infected (IT) tomato leaves, and sequenced the library-specific sRNAs using the next generation sequencing (NGS) approach. These data helped predict 112 mature miRNA sequences employing the miRDeep-P program. A substantial number (58) of the sequences were 24-mer in size, which was a bit surprising. Based on the calculation of precision values, 53 novel miRNAs were screened from the predicted sequences. Nineteen of these were chosen for expression analysis; a northern blot analysis showed 15 to be positive. Many of the predicted miRNAs were up-regulated following viral infection. The target genes of the miRNAs were also predicted and the expression analysis of selected transcripts showed a typical inverse relation between the accumulation of target transcripts and the abundance of corresponding miRNAs. Furthermore, the cleavage sites of the target transcripts for three novel miRNAs were mapped, confirming the correct annotation of the miRNA-targets. The sRNA deep sequencing clearly revealed that the virus modulated global miRNA expression in the host. The validated miRNAs (Tom_4; Tom_14; Tom_17; Tom_21; Tom_29; Tom_43) could be valuable tools for understanding the ToLCNDV-tomato interaction, ultimately leading to the development of a virus-resistant tomato plant.