#METABOLOMICS WORKBENCH shijuan_20200313_021343 DATATRACK_ID:1941 STUDY_ID:ST001330 ANALYSIS_ID:AN002218 PROJECT_ID:PR000909 VERSION 1 CREATED_ON March 22, 2020, 8:59 pm #PROJECT PR:PROJECT_TITLE Rice panicle blast resistence PR:PROJECT_SUMMARY Metabolomics studies of OsGF14b-mediated innate immunity against panicle blast PR:PROJECT_SUMMARY in 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_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. Two fixed volume of 200 μL of the polar phase (the lower SP:SAMPLEPREP_SUMMARY phase) were transferred into pre-labeled 1.5 ml microcentrifuge tube SP:SAMPLEPREP_SUMMARY respectively. Then the samples were dried in a SpeedVac concentrator without SP:SAMPLEPREP_SUMMARY heating. Two dried 200 μL aliquots of the polar phase in each sample were SP:SAMPLEPREP_SUMMARY analyzed using gas chromatography tandem mass spectrometry (GC-MS) and liquid SP:SAMPLEPREP_SUMMARY chromatography tandem mass spectrometry (LC-MS) for metabolomics study. The SP:SAMPLEPREP_SUMMARY dried 200 µl aliquots of the polar phase for GC-MS analysis were re-suspended SP:SAMPLEPREP_SUMMARY in methoxyamine-hydrochloride/pyridine solution for methoxymization of carbonyl SP:SAMPLEPREP_SUMMARY groups followed by heating at 37 °C for 2 h. The samples were further SP:SAMPLEPREP_SUMMARY derivatized with N-methyl-N-trimethylsilyltrifloracetamide (MSTFA) for 30 min SP:SAMPLEPREP_SUMMARY at 37 °C. then one µl of the derivatized sample mixture was injected onto the SP:SAMPLEPREP_SUMMARY GC-column and measured. Another dried 200 µl aliquots of the polar phase were SP:SAMPLEPREP_SUMMARY re-suspended in 150 µl UPLC-grade methanol: water (1:1, vol/vol) and subjected SP:SAMPLEPREP_SUMMARY to LC-MS analysis. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Firstly, 10 μL of each sample was eluted using a TSS T3 column (100 mm × 2.1 CH:CHROMATOGRAPHY_SUMMARY mm containing 1.8 μm diameter particles, Waters) with 0.4 mL/min flow rate. The CH:CHROMATOGRAPHY_SUMMARY mobile phase A was water with 0.1% formic acid, and the mobile phase B was ACN CH:CHROMATOGRAPHY_SUMMARY with 0.1% formic acid. The compounds were separated by a elution gradient: 1% B CH:CHROMATOGRAPHY_SUMMARY was initially firstly maintained for 1 min, then linearly increased to 40% B CH:CHROMATOGRAPHY_SUMMARY from 1 to 11 min, to 70% B from 11 to 13 min, then to 99% B from 13 to 15 min, CH:CHROMATOGRAPHY_SUMMARY and maintained at 99% B from 15 to 16 min, then linearly decreased to 1% B from CH:CHROMATOGRAPHY_SUMMARY 16 to 17 min followed by equilibration at 1% B for 3 min. and the column CH:CHROMATOGRAPHY_SUMMARY temperature was set at 40°C. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Waters Acquity BEH HSS T3 (100 x 2.1mm, 1.8um) CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 40°C CH:SOLVENT_A Water with 0.1% formic acid CH:SOLVENT_B Acetonitrile with 0.1% formic acid #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Fusion Tribrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The spray voltage was set to -3000 V in the negative-ion modes, with the MS:MS_COMMENTS following ion-source properties: ion source type, H-ESI; Sheath gas, 45 Arb; Aux MS:MS_COMMENTS gas, 10 Arb; sweep gas, 0 Arbs; Ion transfer tube temperature, 320 °C; MS:MS_COMMENTS Vaporizer temperature, 350 °C. Full scan data was acquired with a scan range of MS:MS_COMMENTS m/z 100-1000, which was acquired with orbitrap resolution of 120000. The MS:MS_COMMENTS automatic gain control (AGC) was set at 2E5 and the maximum injection time was MS:MS_COMMENTS set to 100 ms. RF lens was set to 60%, and the micro scans was 1. data type, MS:MS_COMMENTS profile. All FTMS2 data were acquired using the following conditions: isolation MS:MS_COMMENTS mode, quadrupole; isolation window, 1.6 m/z; detector type, Orbitrap; scan MS:MS_COMMENTS range, auto; AGC target, 5.0e4; maximum injection time, 35 ms; microscans, 1; MS:MS_COMMENTS orbitrap resolution, 15000; first mass, 100 m/z; data type, profile. Both HCD MS:MS_COMMENTS and CID were used for FTMS2 as the activation type. The HCD collision energy was MS:MS_COMMENTS set to 30%, 40%, and 50%, respectively, which ± HCD collision energy was set MS:MS_COMMENTS 10%. The CID collision energy was set to 30% and 40%, and the activation Q was MS:MS_COMMENTS set to 0.25. The Xcalibur v4.1 software (Thermo Fisher Scientific, USA) were MS:MS_COMMENTS used for HPLC-MS control. Compound Discovery (Thermo Fisher Scientific, San MS:MS_COMMENTS Jose, CA, USA) and Trace Finder 3.3 (Thermo Fisher Scientific, San Jose, CA, MS:MS_COMMENTS USA) were used for the LC-MS-based secondary metabolome data analysis, combining MS:MS_COMMENTS qualitative and quantitative analysis. MS:MS_RESULTS_FILE ST001330_AN002218_Results.txt UNITS:Relative content Has m/z:Yes Has RT:Yes RT units:Minutes #END