Identification and characterisation of virus responsive miRNAs in banana musa (AAB) Nendran

Abstract

The study entitled “Identification and characterization of virus responsive miRNAs in banana Musa (AAB) ‘Nendran’” was carried out during 2017-2019, in the Department of Plant Biotechnology, College of Agriculture, Vellayani. The objective of the study was to identify the miRNAs associated with Banana Bract Mosaic Virus (BBrMV) infection in banana var. Nendran from the expression profile of selected miRNAs. Five miRNAs were selected from the 52 computationally predicted miRNAs in a previous study conducted in the Department of Plant Biotechnology. Selection was based on the function of their target genes i.e. their role in biotic stress conditions. The miRNAs selected were miR-3900-5p (targets: Plant viral response family protein and Putative disease resistance protein genes), miR-2172-5p (target: Putative ethylene responsive transcription factor1gene), miR-6928-5p (target: Flavin adenine dinucleotide dependent oxidoreductase gene), miR-5417 (target: Stress endoplasmic reticulum protein 2 gene) and miR-971-5p (targets: Argonaute protein and Transport inhibitor response-1 like protein genes). For studying the expression of miRNAs, three month old in vitro raised banana var. Nendran plants were infected with BBrMV through viruliferous aphids (Pentalonia nigronervosa) by acquisition feeding method. Fifteen to twenty aphids reared in healthy banana suckers were transferred to the BBrMV infected sucker for 2 hour acquisition feeding, after starving for 30 min. These aphids were released on to the leaf axils of the tissue culture plants for a period of 24 h. After this time period the aphids were killed using an insecticide. Infection was confirmed by PCR using replicase specific primers and the results showed the presence of BBrMV specific amplicons from 24h onwards in all the samples. For studying the expression of these miRNAs, leaf samples were collected from the infected plants 24 h, 48 h, 1 wk and 2 wk after infection. RNA was isolated using CTAB method, reverse transcribed to cDNA and PCR was done. PCR analysis confirmed the presence of all the five computationally predicted miRNAs in banana. The expression profiles of the miRNAs and their target genes were studied by RT-qPCR. The results showed upregulation of miR-3900-5p, miR-2172-5p, miR-6928-5p and miR-971-5p (1.2, 2.0, 1.27, 2.0 fold respectively) 24h after BBrMV infection. Among them miR-3900-5p, miR-2172-5p and miR-971-5p maintained higher expression upto one week compared to uninfected control. miR-6928-5p showed a 1.72 fold increase in expression 48 h after infection. Expression of miR-5417 was down regulated by BBrMV infection at 24 h of infection. The two targets of miR-3900-5p (Plant viral response family protein and Putative disease resistance protein genes) showed contrasting trends in their expression after BBrMV infection. While Putative disease resistance protein showed a drastic increase (13 fold) 24 h after infection, Plant viral response family protein expression showed a continuous reduction throughout the period of observation. miR-2172- 5p and its target Putative Ethylene-responsive transcription factor 1gene (3.39 fold) were found upregulated during the infection. While miR-6928-5p showed maximum expression at 48 h, its target FAD (Flavin adenine dinucleotide) dependent oxidoreductase gene showed maximum expression at 24 h after infection and maintained a higher level upto 48 h. Among the two targets of miR-971-5p, Transport inhibitor response-1like protein gene showed a quick response to virus infection with a 6.15 fold expression at 24 h, while Argonaute protein gene showed a lower expression upto 48h and a drastic increase upto 2 fold as the infection progressed. The study suggested that miR3900-5p, miR2172-5p, miR6928-5p and miR971-5p were associated with BBrMV infection in banana var. Nendran. Their targets viz., putative disease resistance protein gene, putative ethylene responsive transcription factor 1gene, FAD dependent oxidoreductase gene and transport inhibitor response-1 like protein gene showed significant increase immediately after the infection. Suppression of the targets viz., Plant viral response family protein and Argonaute protein genes during BBrMV infection suggested the possible role of miR-3900-5p and miR-971-5p in regulating the infection process.