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).