Genome Wide Study of Micro RNAs and NBS-LRR Genes in Cucumis sativus and Exploring Their Role in Innate Immunity

Plants defense system comprises of various R-protein, which usually contain nucleotide binding site-Leucin rich repeat (NBS-LRR) domain. In addition, microRNAs (miRNAs) represents 2^nd layer of defense and play pivotal role behind the scene. In this study, genome wide analysis of miRNAs and NBS-LRR genes was conducted to explore their potential role, in provoking immune response of cucumber plants after challenging with cucumber mosaic virus (CMV). Nucleotide database was explored and the aminoacid sequence of NBS genes was retrieved by homology based search. Phylogenetic analysis and sequence alignment of 57 NBS genes, showed various classes of NBS- gene family. Screening of 21 genotypes of cucumber against CMV showed Ames-26917 the most susceptible and identified P1-618950 as the most resistant one. Transcript profiling of 17 CC domain containing genes along with non-NBS-LRR genes in the virus inoculated plants of selected resistant and susceptible genotypes at 8 DPI and 15DPI was carried out by real time RT-PCR. In resistant genotype, relatively higher transcript level of NBS-1, NBS-5 and NBS-7 was observed at 15 DPI in infected plants as compared to non-infected control. At 8DPI, the transcript accumulation was down-regulated upon infection. In susceptible genotype, same trend was observed but the transcripts were relatively lower in concentration as compared to resistant genotypes. To explore the potential role of miRNAs in attenuating these genes, bioinformatics prediction of miRNAs, targeting NBS-LRR genes, was conducted. Ten miRNAs showed their complementation with 7 NBS-LRR genes. To validate the role of these miRNAs in attenuating the target genes, co-expression analysis was carried out, which detected these miRNAs only in non-infected cucumber genotypes. This study concluded that cucumber plant’s immune system didn’t awake until virus obtained its threshold level at 15DPI. The detection of selected miRNAs (396c, 156a, 157b, 159a, 162a, 169a, 169c, 169e, 172d, 390b) only in the infected samples suggested their role in attenuating the expression of NBS-LRR genes but non NBS-LRRs are neutral to the abundance or absence of miRNA before or after infection.