Transcriptome Analysis and miRNA Target Profiling at Various Stages of Root-Knot Nematode Meloidogyne incognita Development for Identification of Potential Regulatory Networks

Abstract

Root-knot nematodes (RKNs) are a group of plant-parasitic nematodes that cause damage to various plant species and extensive economical losses. In this study, we performed integrated analysis of miRNA and mRNA expression data to explore the regulation of miRNA and mRNA in RKNs. In particular, we aimed to elucidate the mRNA targets of Meloidogyne incognita miRNAs and variations of the RKN transcriptome during five stages of its life cycle. Stage-wise RNA sequencing of M. incognita resulted in clean read numbers of 56,902,902, 50,762,456, 40,968,532, 47,309,223, and 51,730,234 for the egg, J2, J3, J4, and female stages, respectively. Overall, stage-dependent mRNA sequencing revealed that 17,423 genes were expressed in the transcriptome of M. incognita. The egg stage showed the maximum number of transcripts, and 12,803 gene transcripts were expressed in all stages. Functional Gene Ontology (GO) analysis resulted in three main GO classes: biological process, cellular components, and molecular function; the detected sequences were longer than sequences in the reference genome. Stage-wise selected fragments per kilobase of transcript per million mapped reads (FPKM) values of the top 10 stage-specific and common mRNAs were used to construct expression profiles, and 20 mRNAs were validated through quantitative real-time PCR (qRT-PCR). Next, we used three target prediction programs (miRanda, RNAhybrid, and PITA) to obtain 2431 potential target miRNA genes in RKNs, which regulate 8331 mRNAs. The predicted potential targets of miRNA were generally involved in cellular and metabolic processes, binding of molecules in the cell, membranes, and organelles. Stage-wise miRNA target analysis revealed that the egg stage contains heat shock proteins, transcriptional factors, and DNA repair proteins, whereas J2 includes DNA replication, heat shock, and ubiquitin-conjugating pathway-related proteins; the J3 and J4 stages are represented by the major sperm protein domain and translation-related proteins, respectively. In the female stage, we found proteins related to the maintenance of molybdopterin-binding domain-containing proteins and ubiquitin-mediated protein degradation. In total, 29 highly expressed stage-specific mRNA-targeting miRNAs were analyzed using qRT-PCR to validate the sequence analysis data. Overall, our findings provide new insights into the molecular mechanisms occurring at various developmental stages of the RKN life cycle, thus aiding in the identification of potential control strategies.