Identification and characterization of microRNA396 and its targets in Cucumis metuliferus infected with Meloidogyne incognita

MicroRNAs (miRNAs), a class of non-coding small RNAs, play an important role in biotic and abiotic stresses. Previously, great progress has been made in miRNA-guided gene regulation in plant-parasitic cyst nematodes interactions. However, little is known about miRNAs regulation of nematode stress response in Cucumis metuliferus, which is a relative of cultivated cucumber or melon and highly resistant to the root-knot nematode Meloidogyne incognita. On the basis of our investigation recently, five cmu-miR396 precursors were firstly identified and characterized from C. metuliferus in this study. Subquently, a total of 12 genes were predicted to be as the potential targets of cmu-miR396 and their function involved in transcription factor, metabolism, signal transduction and stress response. Moreover, three cmu-GRFs genes with significant differential expression in roots of C. metuliferus were successfully amplified and confirmed by RT-PCR experimently. Additionally, the analysis was performed for the phylogeny and subcellular localization of cmu-GRFs protein, and the three transcription factors cmu-GRFs were confirmed as the candidate targets of cmu-miR396 through 5′RLM-RACE assays. These data laid a foundation for further functional analyses to reveal the roles played by miR396 during the interaction between C. metuliferus and M. incognita, leading to a better understanding of the mechanisms and underlying regulatory networks of miR396-mediated resistance to M. incognita in C. metuliferus.