Characterization of eIF4E gene in banana (Musa spp.) and analysis of its expression in relation to banana bract mosaic virus infection

Abstract

Banana and plantain (Musa spp.) are one of the most important food crops especially in the tropics. India is the leading producer of banana in the world with a share of around 25 per cent in total output. Despite its importance, banana cultivation in India is impeded by disease incidence and pest attack resulting in serious loss to farmers. Banana bract mosaic disease caused by banana bract mosaic virus (family Potyviridae) is a major virus disease affecting banana. Plant viruses need host factors for maintaining their life cycle. Eukaryotic translation initiation factors, particularly the eukaryotic translation initiation factor 4E (eIF4E) and its isoform are host factors essential for infection by plant viruses in the genus Potyvirus. In many crop species, natural variation in eIF4E protein confers resistance to potyviruses while in others, which lack natural eIF4E alleles conferring resistance, gene editing can be applied to transfer genetic resistance. Therefore, gene editing approaches for mutation of eIF4E gene have potential in conferring potyvirus resistance to plants. Sequence characterization of eIF4E gene in Musa spp. is necessary for future studies on gene editing for resistance breeding. Moreover, the expression studies of eIF4E gene in relation to BBrMV infection will provide an insight into its probable role in disease development. In this background, the present study entitled “Characterization of eIF4E gene in banana (Musa spp.) and analysis of its expression in relation to banana bract mosaic virus infection” was undertaken during the period 2019 to 2022 at the Department of Plant Biotechnology, College of Agriculture, Vellanikkara, Thrissur, with an objective to characterize the eIF4E gene in banana cultivar Nendran and to analyse its expression in relation to BBrMV infection. Initially, leaf samples were collected from Nendran banana plants from field at Banana Research Station, Kannara, Kerala. Purification of total RNA was attempted using TRI reagent (Sigma), Purelink Plant RNA reagent (Invitrogen) and RNeasy plant mini kit (Qiagen). Out of the three methods, good quality RNA was obtained using RNeasy plant mini kit (Qiagen). The total RNA was converted to cDNA using Oligo(dT)18 primer and reverse transcriptase enzyme. This was followed by PCR amplification of the eIF4E gene using two pairs of primers targeting overlapping regions of the gene. The PCR amplicons were sequenced and the overlapping sequences were aligned to obtain the final contig of 853 bp using Cap3 software. The BLASTN analysis revealed that the sequence showed 99.06 % identity with 99 % query coverage with predicted sequence of Musa acuminata subsp. malaccensis eukaryotic translation initiation factor 4E-1 (LOC103981303) (Accession no. XM_009397982.2). The BLASTX search against NCBI protein database revealed that the sequence showed 99.58 % identity with predicted protein sequence of eIF4E-1 in Musa acuminata subsp. malaccensis (Accession no. XP_009396257.1). Five exons were identified in the coding sequence. The amino acid sequence of the eIF4E gene was deduced from the nucleotide sequence using ExPASy translate tool. Eight open reading frames were identified using ORF finder. The multiple sequence alignment and phylogenetic analysis showed that the Nendran eIF4E gene was more related to sequence of eIF4E gene of Musa acuminata subsp. malaccensis. Quantitative real-time PCR analysis was performed to study the differential expression of eIF4E gene in banana cultivar Nendran under healthy and diseased conditions. Five banana plants showing typical symptoms of BBrMV and a symptomless banana plant were initially identified from the field and later confirmed for the presence and absence of virus respectively based on diagnostic RT-PCR with BBrMV coat protein gene specific primers. For gene expression study, two samples each were collected from the identified diseased and healthy plants. One pair of primer each for banana eIF4E gene (test gene) and actin gene (endogenous control) was designed using Primer3 software for the qRT-PCR study. The real time PCR reaction based on SYBR Green chemistry was carried out for quantification of expression of eIF4E gene in diseased and healthy Nendran banana. The relative expression levels of the gene was normalised with the expression of endogenous control actin gene following 2-∆∆Ct method. The diseased banana plants showed 1.48 to 16.92 fold increase in the expression of eIF4E gene when compared to the healthy plants. Moreover, the plants showing severe symptoms of the disease had higher expression of eIF4E gene. Hence, in the present study, up regulation of eIF4E gene in relation to BBrMV infection was observed which indicates its role in disease development.