Metabolomic analysis
Germinating seeds of OE, knockout, and NIP were placed in a plastic cup (10 cm in diameter), and 12 seedlings remained in each cup. They were then treated with six second to third instar nymphs for each 15-day-old seedling, and each cup was covered with a fine light-transmitting nylon bag. The outermost sheaths were quickly stripped and placed in liquid nitrogen 0, 48, and 72 h after BPH infestation. Six cups were used for each treatment group.
Metabolic extraction and detection were performed as described by Liu et al. (2017). To demonstrate the reliability and efficacy of the metabolite data, the retention time was confirmed with fine stability and repeatability, and some metabolites were verified using the retention index and standard substances. We annotated 216 metabolites by comparing the metabolite mass spectra with the NIST and Wiley Registry metabolic databases. Furthermore, PCA was performed using SIMCAP (v13.0). To fully illustrate the metabolite correlations and the regulation of metabolic pathways involved in the OE, knockout, and NIP lines, 216 of the identified metabolites were analyzed and mapped using PMN and KEGG (Zhang et al ., 2016).