PG Thesis

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Identification and characterisation of virus responsive miRNAs in banana musa (AAB) Nendran
(Department of Plant Pathology, College of Agriculture, Vellayani, 2019) Athira Subramanian; Soni, K B
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.
Computational prediction of mirnas in banana (musa spp.) and evaluation of their role in virus infection
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Kokila Sajeev, Anurag Mathew; Sony, K B
The study entitled "Computational prediction of miRNAs in banana (Musa spp.) and evaluation of their role in virus infection" was conducted at the Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram and Central Tuber Crops Research Institute (ICAR-CTCRI), Sreekariyam, Thiruvananthapuram during 2016–2018. The objective of the study was to predict miRNAs in banana using bioinformatics tools and to validate and analyze their expression during BBrMV infection. Computational miRNA prediction tool NovoMIR was used for the prediction of miRNAs in banana genome. Analysis was performed with all the gene coding or nucleotide sequences of banana and 85 pre-miRNAs were predicted from 11 chromosomes. Most of the pre-miRNAs ranged from 140–180 nt. G+C% content of pre-miRNAs ranged from 24-77% and A+U% content ranged from 23-76%. MFE of pre-miRNAs were found by using an online application RNAfold web server. MFE of pre-miRNAs ranged from -20 Kcal/mol to -194.4 Kcal/mol. AMFE of pre-miRNAs ranged from -18.9 Kcal/mol to -85.4 Kcal/mol. All the predicted pre-miRNAs by NOVOMIR had MFE ≤ -20 Kcal/mol. MFEI of pre-miRNAs ranged from 0.85 to 1.82. BLAST analysis of 85 pre-miRNAs against annotated mature miRNAs in miRBase resulted in 52 mature miRNAs. The targets for the 52 mature miRNAs were predicted using the web tool psRNATarget and were functionally annotated by Blast2Go analysis server. Targets were identified for 40 miRNAs in banana genome. A total of 124 targets were found, with each miRNA having more than one target. Validation of the predicted miRNAs were done in in vitro banana plants of variety Nendran. Three months old tissue culture plants were infected with BBrMV virus by using aphids. Twenty healthy aphids were released on BBrMV infected sucker for 30 min for acquisition feeding, after starving for 10 min. These aphids were further released on tissue culture plants for 12-14 h for infection. The infection process was repeated twice a day for 7 days. For experimental validation, five miRNAs and their target genes having role in biological process were selected and stem-loop/gene specific primers were designed. RNA was isolated from healthy and infected leaf samples and reverse transcribed to cDNA. Expression analysis using RT-qPCR showed the presence of all the five miRNAs in healthy and BBrMV infected leaf samples. Out of them, miR-3900-5p, miR-9112 and miR-5417 were found up-regulated, miR-6928-5p downregulated and miR-3900-5p remain unchanged in infected samples and there was a positive correlation with the expression of their corresponding target genes. In the present study, 52 mature miRNAs have been predicted using bioinformatics tools in banana genome. Targets for 40 miRNAs were identified in banana genome. The five miRNAs selected were validated along with their targets. Expression analysis showed regulation of four of them during virus infection, indicating the possibility of their role in stress response in banana. The remaining miRNAs predicted need validation.
Investigations on the possible relationship fields in Kerala State between the nutritional status of soils and the incidence of Bunchy top disease of Bananas (Musa species)
(Division of Agronomy, Agricultural College & Research Institute, Vellayani, 1963) Damodaran Nambiar, P K; Nair, C K N
Banana plays a very decisive role in the economy of the country. This is more so in the case of Kerala which commands the largest acreage under this crop. Such an economic crop is threatned with annihilation by the deadly disease "Bunchy Top" and the modest estimate of the annual loss is calculated to be about six crores of rupees.
Evaluation of siRNA mediated banana bract mosaic virus (BBrMV) resistance in banana plants with ihpRNA construct for replicase gene
(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2018) Harshitha, C K; Soni, K B

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