EMBO/SourceData huggingface dataset:
Figure 1(A) Immunoblot with anti-TG2 or anti-β-actin as loading control in in whole lysates from small intestine homogenates of CftrF508del/F508del and wild-type (CftrWT) mice (n=5 per group) and densitometric analysis of immunoblots. Mean ± SD of triplicates of independent pooled samples. **p<0.01 (Student's t test). Data information: The blots are representative of one experiment for group of treatment.(B) Detection of NLRP3 expression (top) and caspase-1 cleavage (bottom) by immunoblot of whole lysates from small intestine homogenates of CftrWT and CftrF508del/F508del mice (n= 5) and densitometric analysis of immunoblots. Mean ± SD of triplicates of independent pooled samples. **p<0.01 CftrWT vs CftrF508del/F508del (Student's t test). Data information: The blots are representative of one experiment for group of treatment.(C) Protein levels of IL-17A and IFN-γ from small intestine homogenates of CftrWT and CftrF508del/F508del (n= 10). Mean ± SD of triplicates of independent pooled samples. ***p<0.001 CftrWT vs CftrF508del/F508del (Student's t test).(D) IL-15 mRNA (left) and protein (right) levels in small intestine homogenates from CftrF508del/F508del, Cftr-/- or their CftrWT littermates (n= 10 per group). Mean ± SD of triplicates of independent pooled samples. **p<0.01 CftrF508del/F508del vs CftrWT(FVB/129), or °°p<0.01 Cftr-/- vs CftrWT(B6.129P2) (ANOVA, Bonferroni post hoc test).(E) IL-15 mRNA levels in small intestine homogenates from CftrWT mice or CftrF508del/F508del or TG-/-/CftrF508del/F508del or TG-/- mice (n= 10 per group). Mean ± SD of triplicates of independent pooled samples. ***p<0.001 vs CftrWT, °°p<0.01, ##p<0.01 vs CftrF508del/F508del (ANOVA, Bonferroni post hoc test).(F) Effects of 4 weeks of oral administration of gliadin on IL-15, IL-17A and IFN-γ protein levels in small intestine homogenates from CftrF508del/F508del and CftrWT mice (n =10 mice per group of treatment). Mean ± SD of triplicates of independent pooled samples. **p<0.01, ***p<0.001 (CftrF508del/F508del vs CftrWT mice prior gliadin challenge), °°°p<0.001 (CftrF508del/F508del mice vs CftrF508del/F508del mice after gliadin challenge), (ANOVA, Bonferroni post hoc test).(G-I) BALB/c mice (G) fed with a gluten-free diet for at least 3 generations, or (H) NOD or (I) NOD-DQ8 mice orally challenged with vehicle or gliadin for 4 weeks (5 mg/daily for one week and then 5 mg/daily thrice a week for 3 weeks). Representative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-induced increase of chloride current (Isc (μA/cm2)) in small intestines mounted in Ussing chambers; quantification of the peak CFTR inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc) in tissue samples (n=3 independent experiments). Mean±SD of samples assayed; **p<0.01, ***p<0.001 vs challenged with gliadin (Student's t test).
Figure 2(A) Representative traces of CFTR-dependent Cl- secretion measured by forskolin (Fsk)-inducible chloride current (Isc (μA/cm2)) in Caco-2 cells mounted in Ussing chambers after 3 h of incubation with P57-68 or P33-mer or P31-43 peptides (20 µg/ml) and of P31-43 in the presence of up to 30 minutes of pre-treatment of VX-770 (10 µM); quantification of the peak CFTR Inhibitor 172 (CFTRinh172)-sensitive Isc (∆Isc) in Caco-2 cells (n= 3 independent experiments). Mean ± SD of samples assayed. ***p<0.001 vs P31-43 challenge, ##p<0.01 vs Caco-2 challenged with P31-43 in the presence of VX-770 (ANOVA, Bonferroni post hoc test).(B) Incubation of Caco-2 cells with P31-43 or control peptides (p57-68 or PGAV) for 1 h that were preceded or not by 20 min of pre-treatment with VX-770. Immunoprecipitation in non-reducing and non-denaturing conditions of CFTR protein and immunblot with streptavidin-HRP or CFTR antibody. Data information: The blots are representative of one experiment for group of treatment.(E) Surface plasmon resonance (SPR) analysis of increasing concentrations of P31-43 and P57-68 peptides on rhNBD1 covalently bound to the CM5 sensor chip.(F) Surface plasmon resonance (SPR) analysis of rhNBD1 binding to P31-43 and P57-68 biotinylated peptides immobilized on SA sensor chip.Blue native polyacrylamide gel electrophoresis (PAGE) western blotting of P31-43 and P57-68 biotinylated peptides in the presence of rhNBD1 (G) All the recombinant proteins and the indicated peptides were pre-incubated in an appropriate buffer at 4°C for 30 min and then resolved in native conditions to preserve the formation of peptide/protein complexes. Data information: The blots are representative of one experiment for group of treatment.Blue native polyacrylamide gel electrophoresis (PAGE) western blotting of WT and double NBD1 mutants in the presence of biotinylated P31-43 (H). All the recombinant proteins and the indicated peptides were pre-incubated in an appropriate buffer at 4°C for 30 min and then resolved in native conditions to preserve the formation of peptide/protein complexes. Data information: The blots are representative of one experiment for group of treatment.(I) P31-43 induced modifications on NBD1 ATP binding site using the intrinsic W401 fluorescence.(J) P31-43 effect on NBD1 ATPase activity.(K) P31-43 effect on NBD1 ATPase activity, applied at its IC50, compared to that obtained with 5µM of the non-hydrolyzable ATP analogue (P-ATP) as a positive control of inhibition. Mean ± SD of triplicates of independent experiments.
Figure 1(A-B) Expression patterns (blue stain) of GUS reporter genes driven by the SKM1(A) or SKM2(B) promoter in roots of 5-day-old Col-0seedlings.(C) Non-complementation of CLE45 insensitivity of the bam3mutant by BAM3expressed under control of the SKM1promoter (7-day-old seedlings; 2 independent transgenic lines per construct are shown).(D) Full CLE45 sensitivity of skm1or skm2 single and double mutant roots (7-day-old seedlings).(E) Failure of SKM1 or SKM2 expressed under control of the BAM3 promoter to complement CLE45 insensitivity of the bam3 mutant (7-day-old seedlings; 3 independent transgenic lines per construct are shown).
Figure 2(A) Root phenotypes of 7-day-old seedlings of indicated genotypes. (B) Primary root length of 7-day-old seedlings. (C) Root meristem size in 5-day-old seedlings.(D) Root meristem phenotype of 5-day-old seedlings, confocal microscopy, propidium iodide-stained (red).(E) Auxin activity as visualized by the constitutively expressed inverse DII-NLS-VENUS marker (green) in root meristems of 5-day-old seedlings.(F) Gap cell frequency in brxsingle or brxmakr5 double mutants (note: gap cells were absent from Col-0 wild type or makr5single mutants). (G) Illustration of gap cells (arrowheads) in developing protophloem sieve element strands (asterisks) of brxsingle or brxmakr5 double mutants.
Figure 3(A) Primary root length of 7-day-old seedlings of indicated genotypes in response to continuous CLE45 treatments.(B) Root meristem phenotypes of (CLE45-treated) 5-day-old seedlings (asterisks: protophloem sieve element strands). Note absence of protophloem in CLE45-treated Col-0 wild type roots.(C) Transverse sections of (CLE45-treated) 5-day-old roots, just before the onset of protoxylem (asterisks) differentiation. Note failure of protophloem sieve elements (arrowheads) to differentiate in CLE45-treated Col-0 (indicated by dense cytoplasm).(D) Expression pattern of GUS reporter gene driven by the MAKR5 promoter in roots of 5-day-old Col-0seedlings. (E) Corresponding transverse sections from different positions along the root meristem.(F) Expression of a MAKR5-GFP fusion protein under control of the MAKR5 promoter in roots of 5-day-old seedlings.(G) Corresponding optical sections illustrating MAKR5-GFP expression in developing protophloem sieve elements (asterisk) and adjacent cell files.(H) Complementation of makr5CLE45 insensitivity by MAKR5expression under control of a promoter specific for developing sieve elements (CVP2), but not by expression under control of a companion cell-specific promoter (SUC2) (7-day-old seedlings; 3 independent transgenic lines per construct are shown).(I) Subcellular localization of MAKR5-GFP (meristematic zone). Note plasma membrane association (white, green arrowheads) and dissociation from the cell wall (red arrowhead) upon osmosis.
Figure 4(B) Expression of truncated MAKR5 fusion proteins under control of the MAKR5 promoter in 5-day-old roots (asterisks: protophloem sieve element strands).(C) Close up of protophloem sieve element strands expressing truncated MAKR5-CITRINE fusion proteins under control of the UBQ10 promoter.(D) Failure of truncated MAKR5 fusion proteins to complement CLE45 insensitivity of the makr5 mutant (7-day-old seedlings; 1-2 independent transgenic lines per construct are shown).(E) Sensitivity of makr5 mutant roots to continuous brassinolide (BL) treatment (7-day-old seedlings).(F) Expression of MAKR5-BKI1 fusion proteins under control of the MAKR5 promoter in roots of 5-day-old makr5 seedlings.(G) Complementation of CLE45 insensitivity of makr5 roots by BKI1-MAKR5, but not MAKR5-BKI1 fusion protein (7-day-old seedlings; 3 independent transgenic lines per construct are shown).
Figure 5(A) Increased expression of BAM3-CITRINE fusion protein under control of the BAM3 promoter in developing protophloem sieve elements (asterisks) of 5-day-old bam3 roots upon CLE45 treatment.(B) Expression of BAM3-CITRINE fusion protein under control of the BAM3 promoter in 5-day-old Col-0 roots or makr5 roots.(C) Expression of MAKR5-GFP fusion protein under control of the MAKR5 promoter in 5-day-old Col-0 or bam3 roots.(D) GUS reporter gene expression driven by the MAKR5 promoter in roots of 5-day-old Col-0seedlings upon CLE45 treatment.(E) Expression of MAKR5-GFP fusion protein under control of the MAKR5 promoter in 5-day-old roots upon CLE45 treatment (3 h). Note accumulation of MAKR5-GFP in protophloem sieve elements (magnified) and its dependence on functional BAM3 and CLE45. The frequency of representative observations per total observations is indicated.(F) Close up view of MAKR5-GFP accumulation in the protophloem cell file upon CLE45 treatment.(G-H) Accumulation of MAKR5-BKI1 fusion proteins in protophloem cells upon CLE45 treatment. The frequency of representative observations per total observations is indicated. (I-J) Quantification of fusion protein abundance in developing protophloem cells upon expression under control of the MAKR5 promoter, showing mean total fluorescence (I) and plasma membrane to cytosol signal ratio (J).
Figure 1B. Neutrophil gene signature comparing human skin day 3 wounds to human DFUs demonstrating decreased presence of neutrophils in human DFUs.C. qPCR validations of neutrophil genes. n=7 DFUs and n=3 Skin D3 wounds. **P < 0.01 (two-tailed unpaired Student's t-test). Data presented as mean ± SD.
Figure 2A. Representative images of human neutrophils treated with the FOXM1 inhibitor, FDI-6. Vehicle (DMSO) served as a control. Neutrophils undergoing NET formation are visualized in green and live neutrophils are visualized in red. Quantification was performed by normalizing the number of neutrophils undergoing NET formation to the number of live neutrophils. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test). Data presented as mean ± SD. (Scale bar: 100µm).B. Quantification of ROS levels in human neutrophils treated with FDI-6 or in combination with N-acetylcysteine (NAC). Phorbol 12-myristate 13-acetate (PMA) treatment served as a positive control. Data presented as mean ± SD. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test). C. Quantification of NET formation in human neutrophils treated with FDI-6 or in combination with NAC. PMA treatment served as a positive control. Neutrophils from n=3 different blood donors were isolated, pooled and peformed in triplicate. Data presented as mean ± SD. *P < 0.05 (two-way ANOVA followed by Tukey's post-hoc test).
Figure 3A. Upstream regulators found to be activated in human skin wounds that are suppressed or partially regulated in human DFUs involved in neutrophil response.
Figure 4A. Representative images of wounded skin after topical treatment with either vehicle (IgG isotype control) or ⍺-TREM1 activator at 0, 2, 4, 6, and 8 days after wounding. Percent of wound area at each time following vehicle or ⍺-TREM1 activator treatment relative to the original wound area. Quantification of wound areas in n = 10 for vehicle diabetic and 12 wounds for ⍺-TREM1 treated wounds were performed with Fiji software. Data presented as mean ± SD. **P < 0.01, and ****P < 0.0001 (two-way ANOVA followed by Tukey's post-hoc test).B. Representative pictures of vehicle (IgG isotype control) and ⍺-TREM1 treated diabetic wounds at day 4 show basal keratin marker K5, and neutrophil marker Ly6G, FOXM1 and citH3. Treatment of wounds with ⍺-TREM1 resulted in increased FOXM1+ and decreased citH3+ neutrophils compared to vehicle treated diabetic wounds. (Scale bar: 50µm). Quantification of mean fluorescence intensity was performed with Fiji software. n= 7 diabetic vehicle wounds and 6 diabetic ⍺-TREM1 wounds. Data presented as mean ± SD. *P < 0.05, *P < 0.05, and **P < 0.01 (two-tailed unpaired Student's t-test).
Figure 5A. qPCR of TREM1 and genes involved in neutrophil recruitment demonstrate increased expression in healing DFUs compared to nonhealing DFUs (n = 5 healing and n= 6 nonhealing). Data presented as mean ± SD. *P < 0.05. **P < 0.01 (two-tailed unpaired Student's t-test).B. Representative images of healing and nonhealing DFUs show basal keratin marker K5 and TREM1 and corresponding quantification from healing (n=4) and nonhealing (n=4) is shown in the graph. Data presented as mean ± SEM. *P < 0.05 (two-tailed unpaired Student's t-test). (Scale bar: 50µm).C. Representative images of cit-H3 immunohistochemistry show increase staining in nonhealing DFUs when compared to healing DFUs, which was confirmed by corresponding quantification from healing (n=3) and nonhealing (n=3), shown in the graph. Data presented as mean ± SD. *P < 0.05 (two-tailed unpaired Student's t-test). (Scale bar: 50µm).
Figure 1A. 3-OH-C10:0-induced ROS burst is suppressed by A23 and K252a. Two-week-old Col-0 seedlings were treated with 2μM K252a or 40 μM A23. One hour later, 1 μM 3-OH-C10:0 was used to trigger the ROS burst, and the photo count was recorded by a luminometer. Mock is solvent control. RLU, relative light unit. Values are means ± SD (n=3 biological replicates).B. A23 and K252a suppress 3-OH-C10:0-induced PTI marker gene expression. Treatments with 3-OH-C10:0 and inhibitors were same as in (A), and the plant leaves were collected for RT-qPCR 4 hrs later. Values are means ± SD (n=3 biological replicates). (ANOVA, P <0.01).C. MAPK cascade activation was inhibited by A23. Treatments with 3-OH-C10:0 and A23 were same as in (A). The plant leaves were collected at 0, 5, and 10 min for immunoblotting. Activated MAPKs were detected by immunoblotting with anti-phospho-p44/42 MAPK antibody. The corresponding bands are indicated for MPK3 and MPK6. Ponceau staining of Rubisco was used for estimating equal loading in each lane. The experiment was repeated three times with similar results.D. Tyrosine phosphorylation of LORE in vivo. Two-week-old 35S:LORE-FLAG complementation plants were subjected to immunoprecipitation with anti-FLAG agarose beads. A23 (40 μM) was used to inhibit the protein phosphorylation, and the treatments were same as in (A). Protein abundance of precipitated LORE-FLAG was detected by immunoblotting. The tyrosine phosphorylation was probed by anti-pTyrosine (α-pY) antibody. Similar results were observed in three biological replicates.E. LORE is an active tyrosine kinase. The α-pY antibody was used to probe tyrosine phosphorylation in recombinant protein MBP-tagged LORE-CD (cytosolic domain). The LORE activation loop conserved K516 was mutated to E, and D613 was mutated to V. The bottom gel showing protein abundance stained by Coomassie Brilliant Blue (CBB).
Figure 2A. Phosphorylated Y600 of LORE was identified by LC-MS/MS. MBP-tagged LORE-CD was used for in vitro kinase assays. Trypsin digested recombinant proteins were subjected to LC-MS/MS analysis. The doubly-charged peptide with m/z 476.24 matches GLLYLHR of LORE with Y600 phosphorylation. Fragment ions retaining charges in the N and C terminus were denoted by "b" and "y", respectively. pY600 indicated phosphorylated Y600.B. Y600 mutation reduced LORE kinase activity. MBP-tagged LORE-CD and LORE-Y600F-CD (Y600 tyrosine was substituted with phenylalanine, F) were subjected to kinase reactions. The kinase dead mutant LOREK516E served as a negative control. Autophosphorylated LORE was visualized by autoradiography following SDS-PAGE gel electrophoresis. Protein abundance was stained by CBB.C. LORE Y600 is phosphorylated in plants. Two-week-old pLORE:LORE-FLAG complementation plant leaves were subjected to immunoprecipitation (IP) with anti-FLAG agarose beads. Y600 specific phosphorylation antibody was used to detect Y600 phosphorylation in LORE. The experiment was repeated three times with similar results.D. 3-OH-C10:0 induced Y600 phosphorylation. Two-week-old pLORE:LORE-FLAG complementation plants were treated with 1 μM 3-OH-C10:0 at indicated times. Plant leaves were subjected to anti-FLAG IP. Phosphorylation level of Y600 was detected by anti-pY600 antibody. The experiment was repeated three times with similar results.E. Y600 mutation abolished the ROS burst triggered by 3-OH-C10:0. The transgenic plants were treated with 3-OH-C10:0 and ROS burst was measured. WT(6-1) and WT(10-3) are two independent complementation lines with LORE; Y600F(2-6) and Y600F(7-1) are two independent lines complemented with LOREY600F; and kinase-dead LOREK516E was transformed to lore mutant as a negative control. The 2-3 and 5-2 are two independent lines. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).F. Y600 is required for growth inhibition by 3-OH-C10:0. The Arabidopsis seedlings were grown on ½ MS containing 2.5 μM 3-OH-C10:0 for six days, and the root length was measured. The experiment was repeated three times with similar results. WT(6-1) and Y600 (2-6) lines were used for the assay. Scale bar=0.4 cm. Values are means ± SD (n=3 biological replicates). (ANOVA, P <0.01).G. Y600 mutation decreased the disease resistance to Pst DC3000. Plants were spray-inoculated with Pst DC3000 at a concentration of 1×109 cfu/ml, and were subjected to bacterial growth curve analysis at 3 dpi. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).
Figure 3A. LORE interacts with specific RLCKs (PBL34/35/36) in yeast. BD-LORE-CD and AD-PBLs were co-transformed to yeast cells and were screened on synthetic dextrose media lacking leucine and tryptophan (SD/-Leu-Trp). The single yeast colonies were serially diluted onto SD/-Leu-Trp and SD/-Leu-Trp-His (SD media lacking leucine, tryptophan and histidine) to observe yeast cell growth. 3-AT(20 mM) was used to inhibit the autoactivation of LORE. Yeast co-transformed with pGADT7-T+pGBKT7-53(+) served as a positive control, and the yeast co-transformed with pGADT7-T+pGBKT7-lam(-) and AD-PBL39+BD-LORE served as the negative controls. EV, empty vector.B. LORE interacts with PBLs by MBP pull-down assays. The recombinant GST-LORE-CD and the MBP-PBLs were subjected to MBP pull-down analysis. MBP served as a negative control. The interacting proteins were revealed by anti-GST immunoblotting. The experiment was repeated three times with similar results. C. Co-immunoprecipitation (Co-IP) of LORE with PBLs. 35S promoter-driven LORE-T7 and PBLs-FLAG were transiently co-expressed in N. benthamiana by agroinfiltration. Plant leaves were immunoprecipated with anti-FLAG beads and the proteins were immunoblotted by anti-FLAG antibody. Co-IP proteins were immunoblotted by anti-T7 antibody. The experiment was repeated three times with similar results.  D. LORE interacts with PBLs, revealed by split-luciferase assays. N. benthamiana leaves were co-infiltrated with Agrobacteria carrying 35S:LORE-nLUC and 35S:cLUC-PBLs. Images of chemiluminescence were obtained by applying 0.5 mM luciferin 36 h post infiltration. Similar results were observed in three biological replicates.
Figure 4 A. ROS burst in pbl34/35/36 triple mutant, induced by 3-OH-C10:0. Two-week-old seedlings were treated with 1 μM 3-OH-C10:0, and the ROS burst was measured. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).  B. PTI marker gene expression was reduced in pbl34/35/36 triple mutant in response to 3-OH-C10:0. Treatment is same as in A. Samples were collected for RT-qPCR at 4 h post infiltration. Values are means ± SD (n=3 biological replicates). (Student's t-test, **P< 0.01).  C. pbl34/35/36 triple mutant displayed decreased callose depositions in response to 3-OH-C10:0 treatment. Two-week-old seedlings were sprayed with 1 μM 3-OH-C10:0. The leaves were sampled for callose deposition assays 12 hrs later. The stained callose was counted for 1 mm2 of the leaves. Mock is solvent control. Scale bar=100μm.Values are means ± SD (n=6 leaves). (Student's t-test, **P< 0.01).  D. Root length of pbl34/35/36 triple mutant treated with 3-OH-C10:0. The Arabidopsis seedlings were grown on ½ MS containing 2.5 μM 3-OH-C10:0 for six days, and the root length was measured. Scale bar=0.4cm. Values are means ± SD (n=3 biological replicates). (ANOVA, P <0.01).  E. Disease phenotype of pbl34/35 double, pbl34/35/36 triple mutants and PBL34 overexpression plants. 35S:PBL34-FLAG was introduced to the Col-0 to generate the overexpression lines. The homozygous transgenic plants were used for the assays. The plants were sprayed with Pst DC3000 at a concentration of 1×109 cfu/ml. Plants were subjected to bacterial growth curve analysis at 3 dpi. OE-3# and OE-11# are two independent transgenic lines. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).  F. pbl34/35/36 triple mutant phenotype was rescued by PBL34. PBL34-HA was introduced to the triple mutant driven by its native promoter. Four-week-old homozygous seedlings were inoculated with 5×104 cfu/ml Pst DC3000 , and the leaves were subjected to bacterial growth curve analysis at 3 dpi. CM2-5 and CM7-3 are two independent transgenic lines. Values are means ± SD (n=6 biological replicates). (ANOVA, P <0.01).
Figure 6 A. LORE interacts with HopAO1 by MBP pull-down assays. The recombinant GST-HopAO1 and the MBP-LORE-CD were subjected to MBP pull-down analysis. MBP and GST proteins served as the negative controls. The interacting proteins were revealed by anti-GST immunoblotting. The experiment was repeated three times with similar results.  B. LORE interacts with HopAO1 by Co-IP assays. 35S promoter-driven LORE-T7 and HopAO1-FLAG were transiently co-expressed in N. benthamiana by agroinfiltration. Plant leaves were immunoprecipated with anti-FLAG beads and immunoblotted by anti-FLAG. Coimmunoprecipitated proteins were immunoblotted by anti-T7. The experiment was repeated three times with similar results.  C. HopAO1 dephosphorylates LORE Y600 in vitro. The recombinant GST-HopAO1/HopAO1CS and GST-LORE-CD were subjected to in vitro phosphatase assays. Phosphorylation level of Y600 was detected by anti-pY600 immunoblotting. The experiment was repeated three times with similar results.  D. HopAO1 dephosphorylates LORE Y600 in N. benthamiana. LORE-FLAG was transiently co-expressed with GUS-T7, HopAO1-T7 or HopAO1CS-T7 in N. benthamiana leaves mediated by Agrobacteria. The protein was immunoprecipitated and probed by anti-pY600 antibody. The experiment was repeated at least three times with similar results.   E. HopAO1 suppresses 3-OH-C10:0-triggered ROS burst. HopAO1 was ectopically expressed in Col-0 driven by estradiol-inducible promoter. 100μM estradiol was sprayed on Est:HopAO1 plants, and the leaf disc was used for ROS burst assays 12 hrs later. Values are means ± SD (n=6 biological replicates).  F. HopAO1 inhibits 3-OH-C10:0-induced disease resistance. The Est:HopAO1 plants were pretreated with 1 μM 3-OH-C10:0 (C10) and100 μM estradiol, and then the plants were inoculated with 5×104 cfu/ml Pst DC3000 24 hrs later. The bacterial titer was analyzed at 3 dpi. Values are means ± SD (n=6; ANOVA; p < 0.01