Proteomic analysis was performed using a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap instrument (Thermo Fisher Scientific Inc., Waltham, MA) with a Dionex U 3000 RSLC nano high performance liquid chromatography (HPLC) system. An ESI source fitted with a fused silica emitter tip (New Objective, Woburn, MA) was employed with a mobile phase consisting of the water:acetonitrile (98:2 [v/v]) containing 0.1% formic acid. The trypsin-treated samples were trapped on an Acclaim PepMap 100 trap column (100 μm × 2 cm, nanoViper C18, 5 μm, 100 Å) and washed for 6 min at a flow rate of 4 μL/min, and then separated on an Acclaim PepMap 100 capillary column (75 μm × 15 cm, nanoViper C18, 3 μm, 100 Å) at a flow rate of 300 μL/min. The resulting peptides were electrosprayed through a coated silica tip with ion spray voltage of 2,000 eV. Mass data were collected and analyzed using Proteome Discoverer 1.4, MaxQuant 1.6, and Scaffold 4.8.4 against the protein databases of S. oryzae and T. castaneum. Detailed information including analysis conditions and data processing can be found in Supplementary Methods.
The expression of 18 genes expected to be associated with PH3 resistance from proteome analysis were validated by qRT-PCR. The method of qRT-PCR was in Supplementary Methods.
The expression of 18 genes expected to be associated with PH3 resistance from proteome analysis were validated by qRT-PCR. The method of qRT-PCR was in Supplementary Methods.