From a2a1cbb346cf5bbe9447f35eb2ff89c18c773c3b Mon Sep 17 00:00:00 2001 From: Viktoria Petrova <vipet103@hhu.de> Date: Tue, 26 Nov 2024 17:34:28 +0100 Subject: [PATCH 01/23] create accession selection study and protocol --- studies/AccessionSelection/README.md | 0 studies/AccessionSelection/isa.study.xlsx | Bin 0 -> 7495 bytes studies/AccessionSelection/protocols/.gitkeep | 0 .../protocols/AccessionSelectionProtocol.md | 3 +++ studies/AccessionSelection/resources/.gitkeep | 0 5 files changed, 3 insertions(+) create mode 100644 studies/AccessionSelection/README.md create mode 100644 studies/AccessionSelection/isa.study.xlsx create mode 100644 studies/AccessionSelection/protocols/.gitkeep create mode 100644 studies/AccessionSelection/protocols/AccessionSelectionProtocol.md create mode 100644 studies/AccessionSelection/resources/.gitkeep diff --git a/studies/AccessionSelection/README.md b/studies/AccessionSelection/README.md new file mode 100644 index 0000000..e69de29 diff --git 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HcmV?d00001 diff --git a/studies/AccessionSelection/protocols/.gitkeep b/studies/AccessionSelection/protocols/.gitkeep new file mode 100644 index 0000000..e69de29 diff --git a/studies/AccessionSelection/protocols/AccessionSelectionProtocol.md b/studies/AccessionSelection/protocols/AccessionSelectionProtocol.md new file mode 100644 index 0000000..1f646b8 --- /dev/null +++ b/studies/AccessionSelection/protocols/AccessionSelectionProtocol.md @@ -0,0 +1,3 @@ +## Accession selection + +In order to establish clusters of *A. thaliana* accessions with high versus low S content in their leaves, we re-analyzed ionomics data from Baxter et al. (2007) and Campos et al. (2021), with 174 accessions overlapping in the two datasets (Baxter et al., 2007; Campos et al., 2021). The two datasets of Baxter et al. (2007) and Campos et al. (2021) have different ranges of S concentrations, and individual accessions differ in absolute value but are usually in a similar percentile or relative range, with some exceptions. Therefore, we aimed to use only the overlapping accessions and to use both datasets to decide which accessions to choose. From these 174 overlapping accessions, a principal component analysis (PCA) was performed on the total of 37 ion data (Supplementary Table S1) to establish groupings of accessions with similar leaf S content. Based on the availability of these accessions, we selected 20 accessions with low, medium, and high sulfate levels for further investigation (Supplementary Fig. S1; Supplementary Table S1). \ No newline at end of file diff --git a/studies/AccessionSelection/resources/.gitkeep b/studies/AccessionSelection/resources/.gitkeep new file mode 100644 index 0000000..e69de29 -- GitLab From e057001a3650b9986d072f157781abc6e6f8ff8c Mon Sep 17 00:00:00 2001 From: Viktoria Petrova <vipet103@hhu.de> Date: Tue, 26 Nov 2024 17:40:03 +0100 Subject: [PATCH 02/23] create plant material and growth conditions study and protocol --- .../PlantMaterialAndGrowthConditions/README.md | 0 .../isa.study.xlsx | Bin 0 -> 7515 bytes .../protocols/.gitkeep | 0 .../PlantMaterialAndGrowthConditionsProtocol.md | 5 +++++ .../resources/.gitkeep | 0 5 files changed, 5 insertions(+) create mode 100644 studies/PlantMaterialAndGrowthConditions/README.md create mode 100644 studies/PlantMaterialAndGrowthConditions/isa.study.xlsx create mode 100644 studies/PlantMaterialAndGrowthConditions/protocols/.gitkeep create mode 100644 studies/PlantMaterialAndGrowthConditions/protocols/PlantMaterialAndGrowthConditionsProtocol.md create mode 100644 studies/PlantMaterialAndGrowthConditions/resources/.gitkeep diff --git a/studies/PlantMaterialAndGrowthConditions/README.md b/studies/PlantMaterialAndGrowthConditions/README.md new file mode 100644 index 0000000..e69de29 diff --git a/studies/PlantMaterialAndGrowthConditions/isa.study.xlsx b/studies/PlantMaterialAndGrowthConditions/isa.study.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..501926fc794d91e3ebfba9687fc9ca5d5634675a GIT binary patch literal 7515 zcmai3bzBr$(_Xq+K)O4ZZeamIq`SLQ=|)N#q*+1fMjAmH1O$|BB$g6sM5G(PRbKU8 z@BQBQ{INgIGS8Xwo0&5+`;4jr0wOv906+y;hWhFyLOam(-~fQHhycJH*tZ5!PLA$Y zj_xL!KF(HdMjYM_k18Naj&Hee!cM``tL*ZaMw(jEZ}VOc5SAPPCcGs<<j{lX?NBc~ z=2BMbQqwzZF9i1<XLXaP)W^zwAeI$Lf2t;ck2W{X;y~1NmeF1mPn(OX{ltaP?UfW> z@`|JAl21WsI{R8)+?cqWoK-BA>bj_Yr6va-&ba`nAi@<519IB+F$9zuYimJj091=9 z*6r-?UbPyCKfMP}ir05&JX7New@79rx!P=Ka2%0DiAv(-Nm^Q042KaaDrd{K0T=Pc zD@$<{J((o*3>m9z^+Rtg#sKF=%B)rL9Tp?4Exc!JI#>;ss|dk)q}&3GJ0Hs798j*W zloe|X9tdK)9_{tvp8J2g@%0ZKE<%xYu}N?QpA5f_H<Xe%#zmltNk=)`_5YY`WFohA zV;6*_G3)>-tX&q}$HhAPW^|L$Sub+@D3oS<ARml3d$Ik>T!bxb8_~d1ynKaB^*^2f z{O|<K_`eo1>@O2nt4D5}KV@#cb&xGLw*~v30z3eK@k@r|yJSUDr*an;ro^6~^y+1r zqbzD}VGDJ^0wbST2fZU%U2J}L$d|pS1PRPoJCrz=&l@%aJ42$e+p+2=<d~o`QhcO= z;fUdmfg3v)AS(uP1RV;Ga{*@hy7jdpoqS1OTf82DY%j8J;#rPIJy_{ETO5AB%7C3D z%gE$bq-RImQEBC#`$b>SdT2@gu*6(5!^>;#asEe+22NXvyJmURHu_in%$AB2QBQm( z_cFqD)^k}^M>mYLr(3ST#aXhWxjFS2wzwK4$}0o<?H=S%Beac?7zN3OZ%yQBVZn4> zdT16`JmXA)qke#&7Wt>u5MUPZe)P*^{}e#G75F#HhSI;%{%+YOm}N=-D(2<n`q13T z>EZX6@YB%tU0gW#_pZ@jo$7n)%d;f9u{<#}I7JA3U9b2^&Q{Rj`c#WdmK-$~CA+(P z=EZy2ouN~qNEO9Nl&Tz7d7Mq9>b5<6KR)QC4Nmu*0T?kJosoWK(rZVMoNt4|)t^35 zNtDHTa9>)U2U|u-I&$++iER9(@W5o>%OPVgW*w_@`iXgjZ48xHwdmu++SF+*D}k+2 z&=X{n5aF23bNNvEtnfij(#*S-NHRv2R0-3cbQIpXE%vxwWQ$^#8DIjRYA?G>UJkQ} ze{GFAKO-Fcl*DC1v)e~=K(fkrAwBfIgv4k6(>B%rZ)ogW1ARA*o2`|V`=4R@Nl)3m z7v_ynLI8mHAF@9n+~4i_Y-TJjT?ij}`BDsNB>>s)QL-GQz+R+O-%h8r*=V;e;(0f# zsbz+uO?Ci<HPhWK=gF+6vNPdSA=gM>FLvWAhm-|khELNC=Tm&FFX#3Q+m}`Y124VZ zQf(~wu7_X0X-L27i#vzzrXS}6m)?ieaVx-GH}(O!vCHy^cS3dty?0Qc#VPM1hPyQ} zFU`q=`CGPfD%o>DWqG`nBiVPfKYH=_O*GCeUS7=1iit%aLeri%i&M^Xy(#cG-s|>Y z^b^MxtlJC6-(S`z$}Soe-PxYn@ZSsZv7NB`hSNXEIjiwe%Wq?QS!Sslihmj?V`6ex zqf5wQ*Rt5;j<nH|g#>+c;AMXNhV{_ACCl?h$m2kv*R!7cy+FWme0DjM%AAxg)8>ZQ zU>Yj^xv##&E;)7DgUU~2N(pSVoAqT^xA?NIukKC8%HsW6Bb0B?3q?4`nStw~apnxx zacoZaij}7~{Fe$T)Tf^J7dtd@C__EeicO-MSK<0YOxED~@1(Yt?1CLb&fVmcml*n! 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The seeds were placed onto 0.8% agarose plates containing a modified Long-Ashton medium (Dietzen et al., 2020). The medium consisted of 1.5 mM Ca(NO3)2·4H2O, 1 mM KNO3, 0.75 mM KH2PO4, 0.75 mM MgSO4·7H2O, and 0.1 mM Fe-EDTA in terms of macroelements. Microelements consisted of 10 µM MnCl2·4H2O, 50 µM H3BO3, 1.75 µM ZnCl2, 0.5 µM CuCl2, 0.8 µM Na2MoO4, 1 µM KI, and 0.1 µM CoCl2·6H2O. In the S-deficient medium, the 0.75 mM MgSO4·7H2O was replaced with a mixture of 0.7125 mM MgCl2·6H2O and 0.000225 mM MgSO4·7H2O, supplemented with 0.8 gl–1 MES salts, and 0.5% sucrose, and pH adjusted to 5.7 with KOH. The plates were placed at 4 °C for 2 d for optimum germination and then incubated vertically for 18 d in Sanyo light chambers with a photoperiod consisting of a 16 h:8 h light and dark cycle, with humidity at 60% and 21 °C. Alternatively, for determination of sulfate uptake, the plants were grown in 12-well plates as described in Koprivova et al. (2023). Sterile seeds were distributed onto square sterile nylon membranes and placed in 12-well plates on top of 1 ml of the modified Long-Ashton medium with 0.5% sucrose. After stratification for 2 d in the dark and cold, the plates were incubated for 3 d in the dark at 22 °C to promote etiolation, and further for 2 weeks in the growth cabinets under the long-day conditions as described above. + +The bacterial pathogen *Burkholderia glumae* PG1 (Gao et al., 2015) was obtained from K.-E. Jäger, Heinrich Heine Universität Düsseldorf, Germany. 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For uptake measurement, the medium was exchanged with 1 ml of the Long-Ashton medium with 0.2 mM sulfate supplemented with 12 mCi of [35S]sulfuric acid and incubated for 30 min in the light. Whole seedlings still on the mesh were washed thoroughly, blotted dry, and shoot and root samples were cut, weighed, and stored separately in liquid nitrogen until further processing on the same day. Samples were extracted in a 10-fold volume of 0.1 M HCl. 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/dev/null +++ b/assays/RNAIsolationAndExpressionAnalysis/protocols/RNAIsolationAndExpressionAnalysisProtocol.md @@ -0,0 +1,3 @@ +## RNA isolation and expression analysis + +Total RNA was extracted from the roots of 18-day-old plants by standard phenol/chloroform extraction and LiCl precipitation. Thereafter, DNase treatment was performed and cDNA was synthesized from 600 ng of total RNA using the QuantiTect Reverse Transcription kit (QIAGEN) according to the manufacturer’s protocol. The product was diluted with autoclaved water to a final volume of 200 μl. Using quantitative real-time PCR (qPCR), 11 genes were examined using the gene-specific primers indicated in Supplementary Table S2. The qPCR was performed using the SYBR green as per the manufacturer’s instructions using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). All quantifications were normalized to the *TIP41* (AT4G34270) and *UBC21* (AT5G25760) genes using the 2−ΔΔCt method. 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The samples were centrifuged at maximum speed for 15 min at 4 °C, and 200 μl of the supernatants were transferred to an ion chromatography vial. Standard curves were generated using 0.5, 1, and 2 mM KH2PO4, KNO3, and K2SO4. The inorganic anions were measured with the Dionex ICS-1100 chromatography system and separated using a Dionex IonPac AS22 RFIC 43 250 mm analytic column (Thermo Scientific). The running buffer was made up of 4.5 mM NaCO3 and 1.4 mM NaHCO3 as described in Dietzen et al. (2020). \ No newline at end of file diff --git a/assays/MetaboliteAnalysis/protocols/GlucosinolatesAnalysis.md b/assays/MetaboliteAnalysis/protocols/GlucosinolatesAnalysis.md new file mode 100644 index 0000000..2ab2162 --- /dev/null +++ b/assays/MetaboliteAnalysis/protocols/GlucosinolatesAnalysis.md @@ -0,0 +1,3 @@ +## Glucosinolates + +GSLs were extracted from ~20 mg of homogenized plant material using 500 ml of hot 70% (v/v) methanol, and 10 μl of sinigrin was added as internal standard. The extract was vortexed and incubated at 70 °C for 45 min, vortexing twice during the incubation. The samples were left to cool and centrifuged at maximum speed for 5 min at room temperature. The supernatant was transferred to prepared columns containing 0.5 ml of DEAE Sephadex A-25, washed twice with 0.5 ml of sterile H2O, and subsequently twice again with 0.5 ml of 0.02 M sodium acetate buffer. With a new tube placed underneath each column, a layer of 75 μl of sulfatase was placed onto the column. The samples were left at room temperature overnight, and the resulting desulfo-GSLs were eluted twice with 0.5 ml of sterile H2O, followed by a final elution by 0.25 ml. The eluates were combined, vortexed, centrifuged for 5 min, and 200 μl of the supernatant were transferred to HPLC vials. The desulfo-GSLs were resolved by HPLC (Spherisorb ODS2, 250 3 4.6 mm, 5 μm; Waters) using a gradient of acetonitrile in water and detected by UV absorption at 229 nm. The GSLs were quantified using the internal standard and response factors as described in Dietzen et al. (2020). \ No newline at end of file diff --git a/assays/MetaboliteAnalysis/protocols/ThiolsAnalysis.md b/assays/MetaboliteAnalysis/protocols/ThiolsAnalysis.md new file mode 100644 index 0000000..6e3433e --- /dev/null +++ b/assays/MetaboliteAnalysis/protocols/ThiolsAnalysis.md @@ -0,0 +1,3 @@ +## Thiols + +To analyze low molecular weight thiols (Cys and GSH), ~20 mg of homogenized plant material was extracted with 0.1 M HCl at a 1:10 ratio (w/v) and subsequently centrifuged at maximum speed at 4 °C. To reduce the thiols in the samples, 60 μl of the supernatant was transferred to a new tube and 100 μl of 0.25 M CHES-NaOH (pH 9.4) was added. Thereafter, 35 μl of 10 mM DTT was added, and the tubes were vortexed and incubated for 40 min at room temperature. A 5 μl aliquot of 25 mM monobromobimane was added to the reduced extracts, and the samples were vortexed and incubated in darkness for 15 min at room temperature. The reaction was stopped by adding 110 μl of 100 mM methansulfonic acid and vortexing. After centrifugation at 4 °C for 20 min, 200 μl of supernatant was transferred into HPLC vials. Standards, ranging from 0 mM to 100 mM, were prepared using 2 mM l-Cys and GSH stocks. The conjugated thiols were resolved using reverse phase (RP)-HPLC (Eurospher 100-3 C18, 150 × 4 mm; Knauer) and a gradient of 90% (v/v) methanol and 0.25% (v/v) acetic acid, pH 4.1 in 10% (v/v) methanol and 0.25% (v/v) acetic acid, pH 4.1, and detected fluorimetrically with a 474 detector with an excitation wavelength at 380 nm and emission wavelength at 470 nm. 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z#8Z|e;+a5&Q$!N}XPw)9_19wGm5{={1ONBZ2Myi>dPOsa4f32G!U*BzrM><Gl7GYB -- GitLab From 1514c8d774066007741ac810267aa84dbe8fd927 Mon Sep 17 00:00:00 2001 From: Viktoria Petrova <vipet103@hhu.de> Date: Sat, 11 Jan 2025 14:35:33 +0100 Subject: [PATCH 12/23] add camalexin analysis protocol --- assays/MetaboliteAnalysis/protocols/CamalexinAnalysis.md | 3 +++ 1 file changed, 3 insertions(+) create mode 100644 assays/MetaboliteAnalysis/protocols/CamalexinAnalysis.md diff --git a/assays/MetaboliteAnalysis/protocols/CamalexinAnalysis.md b/assays/MetaboliteAnalysis/protocols/CamalexinAnalysis.md new file mode 100644 index 0000000..aac8b39 --- /dev/null +++ b/assays/MetaboliteAnalysis/protocols/CamalexinAnalysis.md @@ -0,0 +1,3 @@ +## Camalexin + +Camalexin was extracted from ~50 mg of leaves in 200 µl of dimethlysulfoxide (DMSO) as described in Koprivova et al. (2019). After centrifugation at room temperature for 20 min, 20 µl aliquots of the supernatant were injected into a Thermo Scientific Dionex UltiMate 3000 HPLC system with a Waters Spherisorb ODS-2 column (250 mm×4.6 mm, 5 µm). The samples were resolved using a gradient of acetonitrile in 0.01% (v/v) formic acid. Camalexin was detected by a fluorescence detector with an excitation at 318 nm and emission at 368 nm. 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b/assays/AssessmentOfPlantResistanceToPseudomonasSyringae/protocols/AssessmentOfPlantResistanceToPseudomonasSyringaeProtocol.md @@ -0,0 +1,3 @@ +## Assessment of plant resistance to *Pseudomonas syringae* + +To assess bacterial growth in naive 5-week-old plants, overnight log phase cultures of *Pseudomonas syringae* pathovar *maculicola* were washed three times with 10 mM MgCl2 and diluted to a final optical density at 600nm (OD600)=0.001 before infiltrating the resulting bacterial suspensions from the abaxial side into three fully grown leaves using a 1 ml syringe without a needle. The infiltration was performed between 10.00 h and 11.00 h, as previously described (DOI: 10.1016/j.cell.2018.02.049). Approximately 60 h later, the bacterial growth was quantified by measuring the bacterial bioluminescence in leaf discs (10 mm in diameter) of infiltrated leaves (one disc per leaf, three discs per plant) using a Sirius FB12 luminometer (Berthold Detection Systems). 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a/assays/Co-cultivationWithBurkholderiaGlumae/protocols/.gitkeep b/assays/Co-cultivationWithBurkholderiaGlumae/protocols/.gitkeep new file mode 100644 index 0000000..e69de29 diff --git a/assays/Co-cultivationWithBurkholderiaGlumae/protocols/Co-cultivationWithBurkholderiaGlumaeProtocol.md b/assays/Co-cultivationWithBurkholderiaGlumae/protocols/Co-cultivationWithBurkholderiaGlumaeProtocol.md new file mode 100644 index 0000000..95d0f55 --- /dev/null +++ b/assays/Co-cultivationWithBurkholderiaGlumae/protocols/Co-cultivationWithBurkholderiaGlumaeProtocol.md @@ -0,0 +1,3 @@ +## Co-cultivation with *Burkholderia glumae* + +Plants were grown in the 12-well plates as described above for 14 d. For inoculation, overnight bacterial cultures were washed twice with sterile 10 mM MgCl2 and the final OD600 was measured. The *B. glumae* PG1 was diluted stepwise to OD600=0.0005 in 10 mM MgCl2. An 8 µl aliquot of these suspensions was used for inoculation into each well and 8 µl of 10 mM MgCl2 was used as mock treatment. 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described in Koprivova et al. (2023). Genomic DNA was extracted by a buffer containing 0.025 M EDTA, 0.2 M Tris pH 8.0, 0.25 M NaCl, and 0.5% SDS. After 10 min incubation at 65 °C and centrifugation, the supernatant was precipitated with isopropanol, washed with 70% ethanol, and resuspended in 100 µl of sterile water. For the qPCR, 13 ng of corresponding DNA samples were used with *Arabidopsis*- (At primer AT4G26410) and *B. glumae* PG1-specific primer (Burk1 for NR042931). The qPCR conditions were the same as for expression analysis. The qPCRs were performed in duplicate for each of the four independent samples. The qPCR results were related to the bacterial titer as established in Koprivova et al. 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assays/StatisticalAnalysis/isa.assay.xlsx | Bin 0 -> 6900 bytes assays/StatisticalAnalysis/protocols/.gitkeep | 0 .../protocols/StatisticalAnalysisProtocol.md | 5 +++++ 5 files changed, 5 insertions(+) create mode 100644 assays/StatisticalAnalysis/README.md create mode 100644 assays/StatisticalAnalysis/dataset/.gitkeep create mode 100644 assays/StatisticalAnalysis/isa.assay.xlsx create mode 100644 assays/StatisticalAnalysis/protocols/.gitkeep create mode 100644 assays/StatisticalAnalysis/protocols/StatisticalAnalysisProtocol.md diff --git a/assays/StatisticalAnalysis/README.md b/assays/StatisticalAnalysis/README.md new file mode 100644 index 0000000..e69de29 diff --git a/assays/StatisticalAnalysis/dataset/.gitkeep b/assays/StatisticalAnalysis/dataset/.gitkeep new file mode 100644 index 0000000..e69de29 diff --git a/assays/StatisticalAnalysis/isa.assay.xlsx b/assays/StatisticalAnalysis/isa.assay.xlsx new file mode 100644 index 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These processed data were used for statistical analysis. All experiments made use of 3–4 biological replicates for each accession. The accessions were grouped into low, medium, and high sulfate groups, which were then used in a one-way ANOVA. Furthermore, a Tukey honest significant difference (HSD) post-hoc between significantly different groups was performed. The level of significance was set at P≤0.05. + +For clustering analysis, we extracted ~400 genes, annotated in S, N, and phosphorus (P) homeostasis and metabolism (www.arabidopsis.org) from a previously published dataset (Kawakatsu et al., 2016), and transformed the counts into z-scores on a gene-by-gene basis in the seven accessions, three from our low S content group, three from our high S content group, and the reference accession, Col-0. Multiple Experiment Viewer software (TIGR; http://mev.tm4.org) was used to create heat maps and perform cluster analysis using QTC with Pearson correlation, hierarchical clustering: average linkage method, and diameter 0.1. The pair-wise correlation analysis between the traits was performed using the ‘Hmisc’ and ‘corrplot’ packages in R (https://www.Rproject.org). 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