#METABOLOMICS WORKBENCH shijuan_20200313_023939 DATATRACK_ID:1942 STUDY_ID:ST001331 ANALYSIS_ID:AN002219 PROJECT_ID:PR000910 VERSION 1 CREATED_ON March 24, 2020, 2:05 pm #PROJECT PR:PROJECT_TITLE Rice panicle blast resistence PR:PROJECT_SUMMARY Lipidomics studies of OsGF14b-mediated innate immunity against panicle blast in PR:PROJECT_SUMMARY rice PR:INSTITUTE Guangdong Academy of Agricultural Sciences PR:DEPARTMENT Agro-biological Gene Research Center PR:LAST_NAME Yan PR:FIRST_NAME Shijuan PR:ADDRESS No. 20 Jinying Road, Tianhe District, Guangzhou City, Guangdong Province, PR:ADDRESS 510640, China. PR:EMAIL shijuan@agrogene.ac.cn PR:PHONE +86-020-38213643 #STUDY ST:STUDY_TITLE Multi-omics of OsGF14b-mediated innate immunity against panicle blast in rice ST:STUDY_TITLE (part-II) ST:STUDY_SUMMARY In the present study, we used a multi-omics approach to decipher the molecular ST:STUDY_SUMMARY mechanisms of OsGF14b in governing panicle resistance to Magnaporthe ST:STUDY_SUMMARY oryzae.Results revealed OsGF14b mediated panicle blast resistance was involved ST:STUDY_SUMMARY in the activation of auxin and JA signaling pathways, resulting in reprogramming ST:STUDY_SUMMARY of the phenylpropanoid and diterpenoid pathway. ST:INSTITUTE Agro-biological Gene Research Center , Guangdong Academy of Agricultural ST:INSTITUTE Sciences ST:LAST_NAME Yan ST:FIRST_NAME Shijuan ST:ADDRESS No. 20 Jinying Road, Tianhe District, Guangzhou City, Guangdong Province, ST:ADDRESS 510640, China. ST:EMAIL shijuan@agrogene.ac.cn ST:PHONE +86-020-38213643 #SUBJECT SU:SUBJECT_TYPE Plant SU:SUBJECT_SPECIES Oryza sativa Japonica Group SU:TAXONOMY_ID 39947 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - OXGF14b-2-0h-1 Genotype:OsGF14b-overexpression line 2 | Treatment:0h RAW_FILE_NAME=GF14b-2-0h-1 SUBJECT_SAMPLE_FACTORS - OXGF14b-2-0h-2 Genotype:OsGF14b-overexpression line 2 | Treatment:0h RAW_FILE_NAME=GF14b-2-0h-2 SUBJECT_SAMPLE_FACTORS - OXGF14b-2-0h-3 Genotype:OsGF14b-overexpression line 2 | Treatment:0h RAW_FILE_NAME=GF14b-2-0h-3 SUBJECT_SAMPLE_FACTORS - OXGF14b-2-0h-4 Genotype:OsGF14b-overexpression line 2 | Treatment:0h RAW_FILE_NAME=GF14b-2-0h-4 SUBJECT_SAMPLE_FACTORS - OXGF14b-2-0h-5 Genotype:OsGF14b-overexpression line 2 | Treatment:0h RAW_FILE_NAME=GF14b-2-0h-5 SUBJECT_SAMPLE_FACTORS - OXGF14b-4-0h-1 Genotype:OsGF14b-overexpression line 4 | Treatment:0h RAW_FILE_NAME=GF14b-4-0h-1 SUBJECT_SAMPLE_FACTORS - OXGF14b-4-0h-2 Genotype:OsGF14b-overexpression line 4 | Treatment:0h RAW_FILE_NAME=GF14b-4-0h-2 SUBJECT_SAMPLE_FACTORS - OXGF14b-4-0h-3 Genotype:OsGF14b-overexpression line 4 | Treatment:0h RAW_FILE_NAME=GF14b-4-0h-3 SUBJECT_SAMPLE_FACTORS - OXGF14b-4-0h-4 Genotype:OsGF14b-overexpression line 4 | Treatment:0h RAW_FILE_NAME=GF14b-4-0h-4 SUBJECT_SAMPLE_FACTORS - OXGF14b-4-0h-5 Genotype:OsGF14b-overexpression line 4 | Treatment:0h RAW_FILE_NAME=GF14b-4-0h-5 SUBJECT_SAMPLE_FACTORS - OXGF14b-6-0h-1 Genotype:OsGF14b-overexpression line 6 | Treatment:0h RAW_FILE_NAME=GF14b-6-0h-1 SUBJECT_SAMPLE_FACTORS - OXGF14b-6-0h-2 Genotype:OsGF14b-overexpression line 6 | Treatment:0h RAW_FILE_NAME=GF14b-6-0h-2 SUBJECT_SAMPLE_FACTORS - OXGF14b-6-0h-3 Genotype:OsGF14b-overexpression line 6 | Treatment:0h RAW_FILE_NAME=GF14b-6-0h-3 SUBJECT_SAMPLE_FACTORS - OXGF14b-6-0h-4 Genotype:OsGF14b-overexpression line 6 | Treatment:0h RAW_FILE_NAME=GF14b-6-0h-4 SUBJECT_SAMPLE_FACTORS - OXGF14b-6-0h-5 Genotype:OsGF14b-overexpression line 6 | Treatment:0h RAW_FILE_NAME=GF14b-6-0h-5 SUBJECT_SAMPLE_FACTORS - Nip-0h-1 Genotype:Wild-type | Treatment:0h RAW_FILE_NAME=Nip-0h-1 SUBJECT_SAMPLE_FACTORS - Nip-0h-2 Genotype:Wild-type | Treatment:0h RAW_FILE_NAME=Nip-0h-2 SUBJECT_SAMPLE_FACTORS - Nip-0h-3 Genotype:Wild-type | Treatment:0h RAW_FILE_NAME=Nip-0h-3 SUBJECT_SAMPLE_FACTORS - Nip-0h-4 Genotype:Wild-type | Treatment:0h RAW_FILE_NAME=Nip-0h-4 SUBJECT_SAMPLE_FACTORS - Nip-0h-5 Genotype:Wild-type | Treatment:0h RAW_FILE_NAME=Nip-0h-5 SUBJECT_SAMPLE_FACTORS - OXGF14b-2-24h-1 Genotype:OsGF14b-overexpression line 2 | Treatment:24h RAW_FILE_NAME=GF14b-2-24h-1 SUBJECT_SAMPLE_FACTORS - OXGF14b-2-24h-2 Genotype:OsGF14b-overexpression line 2 | Treatment:24h RAW_FILE_NAME=GF14b-2-24h-2 SUBJECT_SAMPLE_FACTORS - OXGF14b-2-24h-3 Genotype:OsGF14b-overexpression line 2 | Treatment:24h RAW_FILE_NAME=GF14b-2-24h-3 SUBJECT_SAMPLE_FACTORS - OXGF14b-2-24h-4 Genotype:OsGF14b-overexpression line 2 | Treatment:24h RAW_FILE_NAME=GF14b-2-24h-4 SUBJECT_SAMPLE_FACTORS - OXGF14b-2-24h-5 Genotype:OsGF14b-overexpression line 2 | Treatment:24h RAW_FILE_NAME=GF14b-2-24h-5 SUBJECT_SAMPLE_FACTORS - OXGF14b-4-24h-1 Genotype:OsGF14b-overexpression line 4 | Treatment:24h RAW_FILE_NAME=GF14b-4-24h-1 SUBJECT_SAMPLE_FACTORS - OXGF14b-4-24h-5 Genotype:OsGF14b-overexpression line 4 | Treatment:24h RAW_FILE_NAME=GF14b-4-24h-5 SUBJECT_SAMPLE_FACTORS - OXGF14b-4-24h-2 Genotype:OsGF14b-overexpression line 4 | Treatment:24h RAW_FILE_NAME=GF14b-4-24h-2 SUBJECT_SAMPLE_FACTORS - OXGF14b-4-24h-3 Genotype:OsGF14b-overexpression line 4 | Treatment:24h RAW_FILE_NAME=GF14b-4-24h-3 SUBJECT_SAMPLE_FACTORS - OXGF14b-4-24h-4 Genotype:OsGF14b-overexpression line 4 | Treatment:24h RAW_FILE_NAME=GF14b-4-24h-4 SUBJECT_SAMPLE_FACTORS - OXGF14b-6-24h-1 Genotype:OsGF14b-overexpression line 6 | Treatment:24h RAW_FILE_NAME=GF14b-6-24h-1 SUBJECT_SAMPLE_FACTORS - OXGF14b-6-24h-2 Genotype:OsGF14b-overexpression line 6 | Treatment:24h RAW_FILE_NAME=GF14b-6-24h-2 SUBJECT_SAMPLE_FACTORS - OXGF14b-6-24h-3 Genotype:OsGF14b-overexpression line 6 | Treatment:24h RAW_FILE_NAME=GF14b-6-24h-3 SUBJECT_SAMPLE_FACTORS - OXGF14b-6-24h-4 Genotype:OsGF14b-overexpression line 6 | Treatment:24h RAW_FILE_NAME=GF14b-6-24h-4 SUBJECT_SAMPLE_FACTORS - OXGF14b-6-24h-5 Genotype:OsGF14b-overexpression line 6 | Treatment:24h RAW_FILE_NAME=GF14b-6-24h-5 SUBJECT_SAMPLE_FACTORS - Nip-24h-1 Genotype:Wild-type | Treatment:24h RAW_FILE_NAME=Nip-24h-1 SUBJECT_SAMPLE_FACTORS - Nip-24h-2 Genotype:Wild-type | Treatment:24h RAW_FILE_NAME=Nip-24h-2 SUBJECT_SAMPLE_FACTORS - Nip-24h-3 Genotype:Wild-type | Treatment:24h RAW_FILE_NAME=Nip-24h-3 SUBJECT_SAMPLE_FACTORS - Nip-24h-4 Genotype:Wild-type | Treatment:24h RAW_FILE_NAME=Nip-24h-4 SUBJECT_SAMPLE_FACTORS - Nip-24h-5 Genotype:Wild-type | Treatment:24h RAW_FILE_NAME=Nip-24h-5 #COLLECTION CO:COLLECTION_SUMMARY The panicles at the initial heading stage of the wild-type Nipponbare (Nip) and CO:COLLECTION_SUMMARY OsGF14b-overexpressing plants were harvested before (Nip-0h; OXGF14b-2-0h; CO:COLLECTION_SUMMARY OXGF14b-4-0h; OXGF14b-6-0h) and after M. oryzae 24-hour inoculation (Nip-24h; CO:COLLECTION_SUMMARY OXGF14b-2-24h; OXGF14b-4-24h; OXGF14b-6-24h) respectively. They were immediately CO:COLLECTION_SUMMARY frozen in liquid nitrogen, with each biological replicate containing panicle CO:COLLECTION_SUMMARY pooled from 10 individual plants. CO:SAMPLE_TYPE Seeds #TREATMENT TR:TREATMENT_SUMMARY Wild-type japonica rice (Oryzae sativa cv. Nipponbare) and three OsGF14b gene TR:TREATMENT_SUMMARY overexpressing lines, including transgenic line 2 (OXGF14b-2), transgenic line 4 TR:TREATMENT_SUMMARY (OXGF14b-4), transgenic line 6 (OXGF14b-6) were used in this study. Rice seeds TR:TREATMENT_SUMMARY were surface-sterilized and transferred to 1/2 MS medium and incubated in a TR:TREATMENT_SUMMARY growth chamber for germination under light of 200 μmol/m2/s with a 12-h TR:TREATMENT_SUMMARY photoperiod at 26℃. Subsequently, rice seedlings were transplanted into soil TR:TREATMENT_SUMMARY and kept in a greenhouse. M. oryzae GD08-T13 was used for rice blast TR:TREATMENT_SUMMARY inoculation. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The rice panicle pre-cooled in liquid nitrogen were ground using a Mixer/mill SP:SAMPLEPREP_SUMMARY (MM400; Retsch) with steel ball for 30 seconds at 30 HZ. Fifty milligram of rice SP:SAMPLEPREP_SUMMARY panicle powder of each sample was extracted with a fixed volume (1 ml) of SP:SAMPLEPREP_SUMMARY pre-cooled (−20 °C) extraction solvent (methanol: chloroform: water = 5: 2: SP:SAMPLEPREP_SUMMARY 2) was added to homogenized tissues. After adding the extraction solvent, the SP:SAMPLEPREP_SUMMARY vials/tubes were thoroughly vortexed for 1 min and then incubated on an orbital SP:SAMPLEPREP_SUMMARY shaker (200 rpm) for 10 min at 4 °C followed by a 15 min sonication step. SP:SAMPLEPREP_SUMMARY For phase separation, a volume of 500 µl of solvent (methanol: water = 1: 3), SP:SAMPLEPREP_SUMMARY was added to each vial/tube and the samples were again thoroughly vortexed for SP:SAMPLEPREP_SUMMARY 1 min. After that, the samples are centrifuged at a speed of 14000rpm for SP:SAMPLEPREP_SUMMARY 10 min at 4 °C. one fixed volume 500 μL of the unpolar phase (the upper SP:SAMPLEPREP_SUMMARY phase) was transferred into pre-labeled 1.5 ml microcentrifuge tube. Then the SP:SAMPLEPREP_SUMMARY samples were dried in a SpeedVac concentrator without heating. The dried 500 μL SP:SAMPLEPREP_SUMMARY aliquot from the upper phase for lipids profiling was re-suspended in 200 μL SP:SAMPLEPREP_SUMMARY UPLC-grade ACN: 2-propanol: dichloromethane (1:1:1, v/v/v) and analyzed using SP:SAMPLEPREP_SUMMARY LC-MS for lipidomics study. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Five μL of each sample was eluted using a CORTECS® C18 column (150 mm × 2.1 CH:CHROMATOGRAPHY_SUMMARY mm, 2.7μm, Waters) with 0.4 mL/min flow rate. The mobile phase A was water: ACN CH:CHROMATOGRAPHY_SUMMARY (40: 60, v/v) with 0.1% formic acid and 10mM ammonium formate, and the mobile CH:CHROMATOGRAPHY_SUMMARY phase B was 2-propanol: ACN (90:10, v/v) with 0.1% formic acid and 10mM ammonium CH:CHROMATOGRAPHY_SUMMARY formate. The compounds were separated by a elution gradient: 20% B was firstly CH:CHROMATOGRAPHY_SUMMARY maintained for 0.2 min, then linearly decreased to 60% B from 0.2 to 2min, to CH:CHROMATOGRAPHY_SUMMARY 100% B from 2 to 9min, and maintained at 100% B from 9 to 10 min, then linearly CH:CHROMATOGRAPHY_SUMMARY increased to 20% B from 10 to 10.5 min followed by equilibration at 20% B for CH:CHROMATOGRAPHY_SUMMARY 3.5 min. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Waters CORTECS C18 (150 mm×2.1 mm, 2.7µm) CH:FLOW_RATE 0.4 mL/min CH:SOLVENT_A Water: Acetonitrile(40: 60, v/v) with0.1% formic acid and 10mM ammonium formate CH:SOLVENT_B 2-propanol: Acetonitrile (90:10, v/v) with 0.1% formic acid and 10mM ammonium CH:SOLVENT_B formate #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Fusion Tribrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The spray voltage was set to 3500 V in the positive-ion modes, with the MS:MS_COMMENTS following ion-source properties: sheath gas, 45 Arbs; auxiliary gas, 10 Arbs; MS:MS_COMMENTS sweep gas, 0 Arbs; ion-transfer tube temperature, 320 °C; vaporizer temp, 350 MS:MS_COMMENTS °C. All FTMS data were acquired using the following conditions: detector type, MS:MS_COMMENTS orbitrap; orbitrap resolution, 120000; scan range was set to m/z 200-1200. The MS:MS_COMMENTS AGC was set at 5.0 e5 and the maximum injection time was set to 100 ms. RF lens MS:MS_COMMENTS was set to 60%, and the microscans was 1. data type, profile. All FTMS2 data MS:MS_COMMENTS were acquired using the following conditions: isolation mode, quadrupole; MS:MS_COMMENTS isolation window, 1 m/z; detector type, orbitrap; scan range, auto; AGC target, MS:MS_COMMENTS 5.0 e4; maximum injection time, 100 ms; microscans, 1; orbitrap resolution, MS:MS_COMMENTS 30000; first mass, 50 m/z; data type, profile. The HCD collision energy was set MS:MS_COMMENTS to 25% with ± HCD collision energy was set 5% in positive mode, and the HCD MS:MS_COMMENTS collision energy was set to 30% with ± HCD collision energy was set 5% in MS:MS_COMMENTS negative mode. All data was acquired by the Xcalibur 4.1 software (Thermo Fisher MS:MS_COMMENTS Scientific, USA). Thermo Scientific™ LipidSearch™ 5.0 software was used for MS:MS_COMMENTS lipidome data processing. MS:MS_RESULTS_FILE ST001331_AN002219_Results.txt UNITS:Relative content Has m/z:Yes Has RT:Yes RT units:Minutes #END