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BIO-CAT Inc the full length irx1 coding sequence (bc166635)

Structure of <t>IRX1</t> and its methylation analysis in lung cancer. ( a ) The IRX1 gene encodes a polypeptide of 480 aa and the protein contains a homeobox at position 145–184, a HARE-HTH (HB1, ASXL, restriction endonuclease helix-turn-helix) domain at position 188–247 and the Iroquois (IRO) box at position 313–326 (NCBI tool for conserved domain search) ( b ) IRX1 is located on chromosome 5p15.33 in an 8.6 kb CpG island (dark green box) flanked by the two indicated CTCF binding sites (5′- and 3′-CTCFBS) . ( c ) Overview of the analyzed 675 bp sequence located in the proximal IRX1 promoter region. The transcriptional start site (TSS; arrow) and individual CpG sites (black lines) are depicted by Python vs. CoBRA . For methylation analysis, two Taq I sites of CoBRA, nine CpGs sites (1, 2, 3, 4, 5, 6, 7, 8, and 9) of bisulfite pyrosequencing, and three CpG sites (cg05534710, cg06689918, and cg09232937) of 450K array are marked. Positions of guide#1 and guide#2 for promoter deletion are marked as blue boxes upstream of the IRX1 TSS. ( d ) A 272 bp fragment of the IRX1 promoter was analyzed by CoBRA in five NSCLC (A549, A427, HCC15, H322, and H358), 11 SCLC (HTB171, HTB175, SCLC21H, SCLC22H, SCLC24H, H82, HTB187, H209, H510, H1092, and H1184), HeLa and in vitro methylated DNA (ivm). PCR products were mock (−) or Taq I (+) digested and analyzed on 2% agarose gels together with a 100 bp ladder. Unmethylated (u), partially methylated (pm), and methylated (m) samples are indicated.

The Full Length Irx1 Coding Sequence (Bc166635), supplied by BIO-CAT Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Epigenetic Inactivation of the Tumor Suppressor IRX1 Occurs Frequently in Lung Adenocarcinoma and Its Silencing Is Associated with Impaired Prognosis"

Article Title: Epigenetic Inactivation of the Tumor Suppressor IRX1 Occurs Frequently in Lung Adenocarcinoma and Its Silencing Is Associated with Impaired Prognosis

Journal: Cancers

doi: 10.3390/cancers12123528

Structure of IRX1 and its methylation analysis in lung cancer. ( a ) The IRX1 gene encodes a polypeptide of 480 aa and the protein contains a homeobox at position 145–184, a HARE-HTH (HB1, ASXL, restriction endonuclease helix-turn-helix) domain at position 188–247 and the Iroquois (IRO) box at position 313–326 (NCBI tool for conserved domain search) ( b ) IRX1 is located on chromosome 5p15.33 in an 8.6 kb CpG island (dark green box) flanked by the two indicated CTCF binding sites (5′- and 3′-CTCFBS) . ( c ) Overview of the analyzed 675 bp sequence located in the proximal IRX1 promoter region. The transcriptional start site (TSS; arrow) and individual CpG sites (black lines) are depicted by Python vs. CoBRA . For methylation analysis, two Taq I sites of CoBRA, nine CpGs sites (1, 2, 3, 4, 5, 6, 7, 8, and 9) of bisulfite pyrosequencing, and three CpG sites (cg05534710, cg06689918, and cg09232937) of 450K array are marked. Positions of guide#1 and guide#2 for promoter deletion are marked as blue boxes upstream of the IRX1 TSS. ( d ) A 272 bp fragment of the IRX1 promoter was analyzed by CoBRA in five NSCLC (A549, A427, HCC15, H322, and H358), 11 SCLC (HTB171, HTB175, SCLC21H, SCLC22H, SCLC24H, H82, HTB187, H209, H510, H1092, and H1184), HeLa and in vitro methylated DNA (ivm). PCR products were mock (−) or Taq I (+) digested and analyzed on 2% agarose gels together with a 100 bp ladder. Unmethylated (u), partially methylated (pm), and methylated (m) samples are indicated.

Figure Legend Snippet: Structure of IRX1 and its methylation analysis in lung cancer. ( a ) The IRX1 gene encodes a polypeptide of 480 aa and the protein contains a homeobox at position 145–184, a HARE-HTH (HB1, ASXL, restriction endonuclease helix-turn-helix) domain at position 188–247 and the Iroquois (IRO) box at position 313–326 (NCBI tool for conserved domain search) ( b ) IRX1 is located on chromosome 5p15.33 in an 8.6 kb CpG island (dark green box) flanked by the two indicated CTCF binding sites (5′- and 3′-CTCFBS) . ( c ) Overview of the analyzed 675 bp sequence located in the proximal IRX1 promoter region. The transcriptional start site (TSS; arrow) and individual CpG sites (black lines) are depicted by Python vs. CoBRA . For methylation analysis, two Taq I sites of CoBRA, nine CpGs sites (1, 2, 3, 4, 5, 6, 7, 8, and 9) of bisulfite pyrosequencing, and three CpG sites (cg05534710, cg06689918, and cg09232937) of 450K array are marked. Positions of guide#1 and guide#2 for promoter deletion are marked as blue boxes upstream of the IRX1 TSS. ( d ) A 272 bp fragment of the IRX1 promoter was analyzed by CoBRA in five NSCLC (A549, A427, HCC15, H322, and H358), 11 SCLC (HTB171, HTB175, SCLC21H, SCLC22H, SCLC24H, H82, HTB187, H209, H510, H1092, and H1184), HeLa and in vitro methylated DNA (ivm). PCR products were mock (−) or Taq I (+) digested and analyzed on 2% agarose gels together with a 100 bp ladder. Unmethylated (u), partially methylated (pm), and methylated (m) samples are indicated.

Techniques Used: Methylation, Binding Assay, Sequencing, Combined Bisulfite Restriction Analysis Assay, In Vitro

Methylation and expression of IRX1 in cancer cell lines. ( a ) Methylation level of the IRX1 promoter at the three CpG sites (cg05534710, cg06689918, and cg09232937) were analyzed by Infinium Human Methylation 450 BeadChip (ilmnhm450K array) through the R2 Genomics Analysis and Visualization Platform . Methylation of 244 normal lung samples (GSE52401) were compared to methylation of 1028 cancer cell lines (GSE68379). IRX1 methylation levels are plotted in beta-value (1 = 100% methylation) and p -value was calculated by one-way ANOVA. ( b ) Correlation analysis of IRX1 promoter methylation (beta-value) and IRX1 expression (2log ps). Methylation level of the three CpG sites of cancer cell lines (GSE68379) was plotted for IRX1 expression (11725275_at) in the corresponding mRNA 1017 of cancer cell lines . Significance of correlation was calculated by the R2 platform. p < 0.05 was considered significant.

Figure Legend Snippet: Methylation and expression of IRX1 in cancer cell lines. ( a ) Methylation level of the IRX1 promoter at the three CpG sites (cg05534710, cg06689918, and cg09232937) were analyzed by Infinium Human Methylation 450 BeadChip (ilmnhm450K array) through the R2 Genomics Analysis and Visualization Platform . Methylation of 244 normal lung samples (GSE52401) were compared to methylation of 1028 cancer cell lines (GSE68379). IRX1 methylation levels are plotted in beta-value (1 = 100% methylation) and p -value was calculated by one-way ANOVA. ( b ) Correlation analysis of IRX1 promoter methylation (beta-value) and IRX1 expression (2log ps). Methylation level of the three CpG sites of cancer cell lines (GSE68379) was plotted for IRX1 expression (11725275_at) in the corresponding mRNA 1017 of cancer cell lines . Significance of correlation was calculated by the R2 platform. p < 0.05 was considered significant.

Techniques Used: Methylation, Expressing

Epigenetic inactivation of IRX1 in primary lung cancers compared to matching lung. ( a ) Methylation level of the IRX1 promoter at the nine CpG sites was analyzed in 100 adenocarcinoma (ADC), 100 squamous cell carcinoma (SQCC), and 41 small cell lung cancer (SCLC) samples by bisulfite pyrosequencing. Average CpG methylation levels are depicted by box plot. ( b ) IRX1 methylation of tumors and corresponding matching lung samples were revealed by pyrosequencing. Average methylation was compared to tumor tissue by box plot and p -value was calculated. ( c ) Relative IRX1 expression in primary tumors and corresponding lung samples. mRNA levels were analyzed by qRTPCR normalized to the housekeeping genes ESD and RPS18 (ΔCt). Note that a higher ΔCt value indicates lower IRX1 expression. Median expression levels of IRX1 of NSCLC, ADC, and SQCC and corresponding lung samples are shown and significances ( p -values) were calculated. p < 0.05 was considered significant.

Figure Legend Snippet: Epigenetic inactivation of IRX1 in primary lung cancers compared to matching lung. ( a ) Methylation level of the IRX1 promoter at the nine CpG sites was analyzed in 100 adenocarcinoma (ADC), 100 squamous cell carcinoma (SQCC), and 41 small cell lung cancer (SCLC) samples by bisulfite pyrosequencing. Average CpG methylation levels are depicted by box plot. ( b ) IRX1 methylation of tumors and corresponding matching lung samples were revealed by pyrosequencing. Average methylation was compared to tumor tissue by box plot and p -value was calculated. ( c ) Relative IRX1 expression in primary tumors and corresponding lung samples. mRNA levels were analyzed by qRTPCR normalized to the housekeeping genes ESD and RPS18 (ΔCt). Note that a higher ΔCt value indicates lower IRX1 expression. Median expression levels of IRX1 of NSCLC, ADC, and SQCC and corresponding lung samples are shown and significances ( p -values) were calculated. p < 0.05 was considered significant.

Techniques Used: Methylation, CpG Methylation Assay, Expressing

Impaired survival probability of ADC patients is associated epigenetic silencing of IRX1 . ( a ) To correlate high and low IRX1 expression with survival probabilities, we performed the Kaplan–Meier estimator for all patients with non-small cell lung cancer (NSCLC) ( n = 416) and adenocarcinoma (ADC) ( n = 226) shown in months. ( b ). Survival probability of NSCLC and ADC patients ( n = 189 and 91, respectively) was correlated with IRX1 methylation level at CpG site 8 (CpG8). ( c ) Correlation analysis of CpG8 methylation and relative IRX1 expression ( p < 0.001). p < 0.05 was considered significant.

Figure Legend Snippet: Impaired survival probability of ADC patients is associated epigenetic silencing of IRX1 . ( a ) To correlate high and low IRX1 expression with survival probabilities, we performed the Kaplan–Meier estimator for all patients with non-small cell lung cancer (NSCLC) ( n = 416) and adenocarcinoma (ADC) ( n = 226) shown in months. ( b ). Survival probability of NSCLC and ADC patients ( n = 189 and 91, respectively) was correlated with IRX1 methylation level at CpG site 8 (CpG8). ( c ) Correlation analysis of CpG8 methylation and relative IRX1 expression ( p < 0.001). p < 0.05 was considered significant.

Techniques Used: Expressing, Methylation

IRX1 is regulated by DNA methyltransferase and p300 HAT activity. ( a ) Deletion (Δ) of 5′- and 3′-CTCF binding site in U251 cell by CRISPR/Cas9 system. We generated two control clones, three Δ5′-CTCFBS, four Δ3′-CTCFBS, and two Δ5′+3′-CTCFBS clones and analyzed IRX1 expression for each clone. RNA levels were determined by qRTPCR in triplicates and normalized to ACTB level. IRX1 expression of control clones was set 1 for comparison. ( b ) Deletion of the IRX1 promoter (ΔIRX1) by CRISPR/Cas9 system. Expression of IRX1 was analyzed in 13 ΔIRX1 clones by qRTPCR and normalized to ACTB . p -value was calculated by unpaired t-test. ( c ) A427 and A549 cell lines were treated with the indicated concentration of 5-Aza-2′-deoxycytidine (Aza) and trichostatin A (TSA) for 4 days and RNA was isolated. IRX1 mRNA levels were analyzed in technical triplicates and normalized to ACTB . Then, 0 µM Aza treatment was set 1. ( d ) p300 HAT induced expression of endogenous IRX1 . HeLa cells were transfected with IRX1 guide RNA constructs and p300-dCas9 or pcDNA-dCas (control). IRX1 expression was analyzed by qRTPCR after 1 µM Aza and 0.3 µM TSA treatment for 72 h and normalized to ACTB levels. ( e , f ) Correlation analysis of IRX1 and ( e ) DNMT3A or ( f ) EZH2 expression in primary NSCLC samples (GSE33532 data set, n = 100). Analysis was performed by R2 . ( g ) Impaired survival probability of adenocarcinoma patients is associated with high EZH2 expression. Survival probability of ADC patients ( n = 719) was correlated with EZH2 expression (low and high) through the Kaplan–Meier plotter . p < 0.05 was considered significant.

Figure Legend Snippet: IRX1 is regulated by DNA methyltransferase and p300 HAT activity. ( a ) Deletion (Δ) of 5′- and 3′-CTCF binding site in U251 cell by CRISPR/Cas9 system. We generated two control clones, three Δ5′-CTCFBS, four Δ3′-CTCFBS, and two Δ5′+3′-CTCFBS clones and analyzed IRX1 expression for each clone. RNA levels were determined by qRTPCR in triplicates and normalized to ACTB level. IRX1 expression of control clones was set 1 for comparison. ( b ) Deletion of the IRX1 promoter (ΔIRX1) by CRISPR/Cas9 system. Expression of IRX1 was analyzed in 13 ΔIRX1 clones by qRTPCR and normalized to ACTB . p -value was calculated by unpaired t-test. ( c ) A427 and A549 cell lines were treated with the indicated concentration of 5-Aza-2′-deoxycytidine (Aza) and trichostatin A (TSA) for 4 days and RNA was isolated. IRX1 mRNA levels were analyzed in technical triplicates and normalized to ACTB . Then, 0 µM Aza treatment was set 1. ( d ) p300 HAT induced expression of endogenous IRX1 . HeLa cells were transfected with IRX1 guide RNA constructs and p300-dCas9 or pcDNA-dCas (control). IRX1 expression was analyzed by qRTPCR after 1 µM Aza and 0.3 µM TSA treatment for 72 h and normalized to ACTB levels. ( e , f ) Correlation analysis of IRX1 and ( e ) DNMT3A or ( f ) EZH2 expression in primary NSCLC samples (GSE33532 data set, n = 100). Analysis was performed by R2 . ( g ) Impaired survival probability of adenocarcinoma patients is associated with high EZH2 expression. Survival probability of ADC patients ( n = 719) was correlated with EZH2 expression (low and high) through the Kaplan–Meier plotter . p < 0.05 was considered significant.

Techniques Used: Activity Assay, Binding Assay, CRISPR, Generated, Clone Assay, Expressing, Concentration Assay, Isolation, Transfection, Construct

Figure Legend Snippet: Significances of hallmarks correlated with IRX1 expression in NSCLC data sets by gene set enrichment analysis (GSEA).

Techniques Used: Expressing

Nuclear localization of IRX1 and irregular shape of IRX1 expressing nuclei. IRX1 was fused to EYFP and transfected in A549 ( a ) HeLa ( b ) and HEK293T ( c ) cells. Localization of IRX1 in the nucleus was analyzed by DAPI co-staining and fluorescent microscopy. IRX1 deletion construct of homeobox (ΔHomeo), HARE-HTH domain (ΔHare), and IRO box (ΔIro) were generated and transfected. The nuclear shape of transfected cells was analyzed after 24 h in A549 (80–160 cells analyzed), HeLa (200–350 cells), and HEK293 (150–300 cells). Normal nuclei exhibited a round shape and fragmented nuclei showed an irregular, lobed shape. Significance was calculated by Fisher’s exact test. White bar represents the length standard of 5 µM.

Figure Legend Snippet: Nuclear localization of IRX1 and irregular shape of IRX1 expressing nuclei. IRX1 was fused to EYFP and transfected in A549 ( a ) HeLa ( b ) and HEK293T ( c ) cells. Localization of IRX1 in the nucleus was analyzed by DAPI co-staining and fluorescent microscopy. IRX1 deletion construct of homeobox (ΔHomeo), HARE-HTH domain (ΔHare), and IRO box (ΔIro) were generated and transfected. The nuclear shape of transfected cells was analyzed after 24 h in A549 (80–160 cells analyzed), HeLa (200–350 cells), and HEK293 (150–300 cells). Normal nuclei exhibited a round shape and fragmented nuclei showed an irregular, lobed shape. Significance was calculated by Fisher’s exact test. White bar represents the length standard of 5 µM.

Techniques Used: Expressing, Transfection, Staining, Microscopy, Construct, Generated

IRX1 expression is associated with expression of the apoptotic regulator BAX. ( a ) IRX1 wt and IRX1 deletion construct of homeobox (ΔHomeo), HARE-HTH domain (ΔHare), and IRO box (ΔIro) were transfected in HeLa cells. RNA was isolated after 48 h and BAX mRNA levels were analyzed in technical triplicates and normalized to ACTB . BAX expression was plotted relative to EYFP-control transfected HeLa cells (set = 1). ( b ) BAX expression values (208478_s_at) were analyzed in pcDNA3.1-GFP (control) and pcDNA3.1-IRX1-GFP transfected HEK293T cells in the data set GSE75376 by GEO2R [ , ]. ( c ) Expression of BAX (NM_004324) was analyzed in 143B cells after IRX1 (shIRX1) and control (shCtrl) knock down in technical triplicates in the data set GSE56255 by GEO2R [ , ].

Figure Legend Snippet: IRX1 expression is associated with expression of the apoptotic regulator BAX. ( a ) IRX1 wt and IRX1 deletion construct of homeobox (ΔHomeo), HARE-HTH domain (ΔHare), and IRO box (ΔIro) were transfected in HeLa cells. RNA was isolated after 48 h and BAX mRNA levels were analyzed in technical triplicates and normalized to ACTB . BAX expression was plotted relative to EYFP-control transfected HeLa cells (set = 1). ( b ) BAX expression values (208478_s_at) were analyzed in pcDNA3.1-GFP (control) and pcDNA3.1-IRX1-GFP transfected HEK293T cells in the data set GSE75376 by GEO2R [ , ]. ( c ) Expression of BAX (NM_004324) was analyzed in 143B cells after IRX1 (shIRX1) and control (shCtrl) knock down in technical triplicates in the data set GSE56255 by GEO2R [ , ].

Techniques Used: Expressing, Construct, Transfection, Isolation

Image Search Results

Silencing of GmMEKK2 by virus‐induced gene silencing (VIGS) increased soybean mosaic virus (SMV) susceptibility. (A) Efficiency of GmMEKK2 silencing in empty vector control (EV) and GmMEKK2 ‐silenced mekk2 i1 and mekk2 i2 plants at 0, 7, 14 and 21 days post‐inoculation (dpi). (B) Phenotypes of soybean after SMV infection: EV and GmMEKK2 ‐silenced lines generated using VIGS. Images were taken at 21 dpi. (C) Disease indices of plants at 21 dpi. Lowercase letters denote statistically significant differences among groups at the same time point ( p < 0.05, one‐way ANOVA with Duncan's test). (D) Relative SMV accumulation in top new leaves of EV and GmMEKK2 ‐silenced plants at 7, 14 and 21 dpi, quantified by reverse transcription‐quantitative PCR using SMV coat protein‐specific primers.

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: Silencing of GmMEKK2 by virus‐induced gene silencing (VIGS) increased soybean mosaic virus (SMV) susceptibility. (A) Efficiency of GmMEKK2 silencing in empty vector control (EV) and GmMEKK2 ‐silenced mekk2 i1 and mekk2 i2 plants at 0, 7, 14 and 21 days post‐inoculation (dpi). (B) Phenotypes of soybean after SMV infection: EV and GmMEKK2 ‐silenced lines generated using VIGS. Images were taken at 21 dpi. (C) Disease indices of plants at 21 dpi. Lowercase letters denote statistically significant differences among groups at the same time point ( p < 0.05, one‐way ANOVA with Duncan's test). (D) Relative SMV accumulation in top new leaves of EV and GmMEKK2 ‐silenced plants at 7, 14 and 21 dpi, quantified by reverse transcription‐quantitative PCR using SMV coat protein‐specific primers.

Article Snippet: The GmMEKK2 full‐length coding DNA sequence (CDS) was inserted into pDONOR221 (Invitrogen) and then transferred to a pB7FWG2 vector via an LR recombination reaction in the Gateway system.

Techniques: Virus, Plasmid Preparation, Control, Infection, Generated, Reverse Transcription, Real-time Polymerase Chain Reaction

Overexpression of GmMEKK2 improved soybean mosaic virus (SMV) resistance in soybean. (A) Infection symptoms on soybean leaves after SMV inoculation. NT, nontransgenic plants; ZMP1, 3, 6 and 7 indicate GmMEKK2 ‐overexpression lines 1, 3, 6 and 7, respectively. (B) Disease indices of NT and each GmMEKK2 ‐overexpression line. The disease index was investigated at 21 days post‐SMV‐inoculation. (C) Quantification of SMV content in soybean leaves. SMV‐susceptible line 1138‐2 was used as a positive control. (D) The GmMEKK2 expression pattern in NT plants after SMV inoculation. (E) Comparison of yield traits between NT and overexpression plants after SMV infection. Mock‐inoculated plants served as the control. Values labelled with different lowercase letters (a–e) are significantly different at p < 0.05 as determined by Duncan's test.

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: Overexpression of GmMEKK2 improved soybean mosaic virus (SMV) resistance in soybean. (A) Infection symptoms on soybean leaves after SMV inoculation. NT, nontransgenic plants; ZMP1, 3, 6 and 7 indicate GmMEKK2 ‐overexpression lines 1, 3, 6 and 7, respectively. (B) Disease indices of NT and each GmMEKK2 ‐overexpression line. The disease index was investigated at 21 days post‐SMV‐inoculation. (C) Quantification of SMV content in soybean leaves. SMV‐susceptible line 1138‐2 was used as a positive control. (D) The GmMEKK2 expression pattern in NT plants after SMV inoculation. (E) Comparison of yield traits between NT and overexpression plants after SMV infection. Mock‐inoculated plants served as the control. Values labelled with different lowercase letters (a–e) are significantly different at p < 0.05 as determined by Duncan's test.

Techniques: Over Expression, Virus, Infection, Positive Control, Expressing, Comparison, Control

Expression profiles of key differentially expressed genes (DEGs) between nontransgenic (NT) and GmMEKK2 ‐overexpression lines (ZMP) involved in the reactive oxygen species (ROS)‐ and salicylic acid (SA)‐related pathways. (A) KEGG enrichment analysis of DEGs between NT and ZMP plants. Left: NT_CK versus ZMP_CK (uninfected controls); Right: NT_7d versus ZMP_7d (7 days post‐SMV‐inoculation [dpi]). Points represent enriched pathways, with size indicating gene count and colour reflecting −log 10 (adjusted p ‐value). Red arrows highlight defence‐related pathways. (B) Expression dynamics of key components among MAPK, plant hormone signalling and plant–pathogen interaction pathways. Schematic depicts signal transduction from apoplast to cytoplasm, including Ca 2+ sensors (CNGCs and CDPKs), ROS producers (Rbohs) and SA‐induced defence protein (PR1). Heatmaps show expression levels across conditions (NT and ZMP at 0, 7 and 14 dpi), with gene IDs labelled.

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: Expression profiles of key differentially expressed genes (DEGs) between nontransgenic (NT) and GmMEKK2 ‐overexpression lines (ZMP) involved in the reactive oxygen species (ROS)‐ and salicylic acid (SA)‐related pathways. (A) KEGG enrichment analysis of DEGs between NT and ZMP plants. Left: NT_CK versus ZMP_CK (uninfected controls); Right: NT_7d versus ZMP_7d (7 days post‐SMV‐inoculation [dpi]). Points represent enriched pathways, with size indicating gene count and colour reflecting −log 10 (adjusted p ‐value). Red arrows highlight defence‐related pathways. (B) Expression dynamics of key components among MAPK, plant hormone signalling and plant–pathogen interaction pathways. Schematic depicts signal transduction from apoplast to cytoplasm, including Ca 2+ sensors (CNGCs and CDPKs), ROS producers (Rbohs) and SA‐induced defence protein (PR1). Heatmaps show expression levels across conditions (NT and ZMP at 0, 7 and 14 dpi), with gene IDs labelled.

Techniques: Expressing, Over Expression, Transduction

Kinase activity of GmMEKK2 is dispensable for its function in mediating defence signalling. (A–E) Relative expression levels of (A) GmMKK1 , (B) GmMPK4A , (C) GmMPK13‐like , (D) GmSUMM2 and (E) GmCRCK3 in nontransgenic control (NT), GmMEKK2 ‐overexpression lines (ZMP1, ZMP3 and ZMP7), empty vector control (EV) and GmMEKK2‐ silenced lines ( mekk2 i1 and mekk2 i2 ). Lowercase letters denote significant differences at p < 0.05 as determined by one‐way ANOVA with Duncan's test. (F) Domain architecture of GmMEKK2 highlighting the kinase domain (6–264 amino acids) and ATP‐binding site (K36). Autophosphorylation of GmMEKK2 was assessed by immunoblotting with α‐pSer/Thr antibody. Recombinant proteins GmMEKK1‐FLAG and GmMEKK1 K321M ‐FLAG were used as positive and negative controls, respectively. Coomassie brilliant blue staining validated the equal loading of recombinant proteins. (G) Yeast two‐hybrid analysis of GmMEKK2 interaction with GmMKK1, GmMPK4A and GmMPK13‐like. Transformants expressing pGADT7 and pGBKT7 constructs were grown on SD/−Leu/−Trp (control) and SD/−Leu/−Trp/−Ade/−His (selection) media. (H–J) Glutathione S‐transferase (GST) pull‐down assays with anti‐His and anti‐GST antibodies demonstrating direct binding between GST‐GmMEKK2 and (H) GmMKK1‐His, (I) GmMPK4A‐His and (J) GmMPK13‐like‐His.

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: Kinase activity of GmMEKK2 is dispensable for its function in mediating defence signalling. (A–E) Relative expression levels of (A) GmMKK1 , (B) GmMPK4A , (C) GmMPK13‐like , (D) GmSUMM2 and (E) GmCRCK3 in nontransgenic control (NT), GmMEKK2 ‐overexpression lines (ZMP1, ZMP3 and ZMP7), empty vector control (EV) and GmMEKK2‐ silenced lines ( mekk2 i1 and mekk2 i2 ). Lowercase letters denote significant differences at p < 0.05 as determined by one‐way ANOVA with Duncan's test. (F) Domain architecture of GmMEKK2 highlighting the kinase domain (6–264 amino acids) and ATP‐binding site (K36). Autophosphorylation of GmMEKK2 was assessed by immunoblotting with α‐pSer/Thr antibody. Recombinant proteins GmMEKK1‐FLAG and GmMEKK1 K321M ‐FLAG were used as positive and negative controls, respectively. Coomassie brilliant blue staining validated the equal loading of recombinant proteins. (G) Yeast two‐hybrid analysis of GmMEKK2 interaction with GmMKK1, GmMPK4A and GmMPK13‐like. Transformants expressing pGADT7 and pGBKT7 constructs were grown on SD/−Leu/−Trp (control) and SD/−Leu/−Trp/−Ade/−His (selection) media. (H–J) Glutathione S‐transferase (GST) pull‐down assays with anti‐His and anti‐GST antibodies demonstrating direct binding between GST‐GmMEKK2 and (H) GmMKK1‐His, (I) GmMPK4A‐His and (J) GmMPK13‐like‐His.

Techniques: Activity Assay, Expressing, Control, Over Expression, Plasmid Preparation, Binding Assay, Western Blot, Recombinant, Staining, Construct, Selection

GmMEKK2 promotes the immune response induced by salicylic acid (SA). (A) Contents of free (SA) and bound salicylic acid (SAG) in nontransgenic (NT) and GmMEKK2 ‐overexpression (ZMP) lines. (B) GmMEKK2 expression in NT plants after exogenous hormone treatments. ETH, ethylene; ABA, abscisic acid (C–H) Expression of pivotal genes in the SA signalling pathway in NT, GmMEKK2 ‐overexpression and GmMEKK2 ‐silenced ( mekk2 i1 and mekk2 i2 ) plants at 7 days post‐inoculation. EV, empty vector. Values labelled with different lowercase letters (a–c) are significantly different at p < 0.05 as determined by Duncan's test.

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: GmMEKK2 promotes the immune response induced by salicylic acid (SA). (A) Contents of free (SA) and bound salicylic acid (SAG) in nontransgenic (NT) and GmMEKK2 ‐overexpression (ZMP) lines. (B) GmMEKK2 expression in NT plants after exogenous hormone treatments. ETH, ethylene; ABA, abscisic acid (C–H) Expression of pivotal genes in the SA signalling pathway in NT, GmMEKK2 ‐overexpression and GmMEKK2 ‐silenced ( mekk2 i1 and mekk2 i2 ) plants at 7 days post‐inoculation. EV, empty vector. Values labelled with different lowercase letters (a–c) are significantly different at p < 0.05 as determined by Duncan's test.

Techniques: Over Expression, Expressing, Plasmid Preparation

GmMEKK2 is involved in the regulation of reactive oxygen species homeostasis in soybean. (A, B) H 2 O 2 and O 2− levels in leaves were detected at 7 days post‐inoculation (dpi) using 3,3′‐diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining, respectively. The mock‐inoculated leaves were sampled as controls. (C–G) Trends in the gene expression of antioxidases were measured after soybean mosaic virus (SMV) infection. CK, noninoculated control (H–J) Antioxidase activities were measured. POD, peroxidase; CAT, catalase; SOD, superoxide dismutase. The statistical analysis was independently performed for GmMEKK2 ‐overexpression lines ZMP1, ZMP3 and ZMP7, and gene‐silenced lines mekk2 i1 , mekk2 i2 and nontransgenic (NT) plants at each stage. Values labelled with different lowercase letters are significantly different at p < 0.05 as determined by Duncan's test.

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: GmMEKK2 is involved in the regulation of reactive oxygen species homeostasis in soybean. (A, B) H 2 O 2 and O 2− levels in leaves were detected at 7 days post‐inoculation (dpi) using 3,3′‐diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining, respectively. The mock‐inoculated leaves were sampled as controls. (C–G) Trends in the gene expression of antioxidases were measured after soybean mosaic virus (SMV) infection. CK, noninoculated control (H–J) Antioxidase activities were measured. POD, peroxidase; CAT, catalase; SOD, superoxide dismutase. The statistical analysis was independently performed for GmMEKK2 ‐overexpression lines ZMP1, ZMP3 and ZMP7, and gene‐silenced lines mekk2 i1 , mekk2 i2 and nontransgenic (NT) plants at each stage. Values labelled with different lowercase letters are significantly different at p < 0.05 as determined by Duncan's test.

Techniques: Staining, Gene Expression, Virus, Infection, Control, Over Expression

Molecular mechanisms underlying the GmMEKK2‐mediated regulation of soybean mosaic virus (SMV) resistance in soybean. (A) Phenotype and regulatory mechanism of GmMEKK2 ‐overexpression plants under SMV inoculation. Left: GmMEKK2 ‐overexpression plants (ZMP) show no visible SMV symptoms with autoimmunity phenotype such as leaf yellowing. Right: In ZMP plants, GmMEKK2 (orange ellipses) interacts with GmMKK1 and GmMPK4A, blocking the phosphorylation (letter P in a blue circle) of the GmMEKK1‐GmMKK1‐GmMPK4A cascade. This inhibition represses (cross in a red circle) WRKY transcription factors and leads to non‐phosphorylated CRCK3 releasing SUMM2. This then triggers defence responses such as salicylic acid (SA)‐induced gene expression and basal reactive oxygen species (ROS) accumulation. The elevated ROS constitutivly results in autoimmunity in ZMP plants. (B) Left: Nontransgenic (NT) plants exhibit severe SMV symptoms such as mosaic leaves and mottled pods. Right: In NT plants, GmMEKK2 expression is low, so the GmMEKK1‐GmMKK1‐GmMPK4A cascade remains active. GmMPK4A phosphorylates CRCK3, which binds with and represses SUMM2. This suppresses defence responses, and leads to a ROS burst.

Journal: Molecular Plant Pathology

Article Title: GmMEKK2 Disrupts the MKK1 /2– MPK4 Cascade to Amplify Immune Signalling and Confer Enhanced Resistance to Soybean Mosaic Virus

doi: 10.1111/mpp.70184

Figure Lengend Snippet: Molecular mechanisms underlying the GmMEKK2‐mediated regulation of soybean mosaic virus (SMV) resistance in soybean. (A) Phenotype and regulatory mechanism of GmMEKK2 ‐overexpression plants under SMV inoculation. Left: GmMEKK2 ‐overexpression plants (ZMP) show no visible SMV symptoms with autoimmunity phenotype such as leaf yellowing. Right: In ZMP plants, GmMEKK2 (orange ellipses) interacts with GmMKK1 and GmMPK4A, blocking the phosphorylation (letter P in a blue circle) of the GmMEKK1‐GmMKK1‐GmMPK4A cascade. This inhibition represses (cross in a red circle) WRKY transcription factors and leads to non‐phosphorylated CRCK3 releasing SUMM2. This then triggers defence responses such as salicylic acid (SA)‐induced gene expression and basal reactive oxygen species (ROS) accumulation. The elevated ROS constitutivly results in autoimmunity in ZMP plants. (B) Left: Nontransgenic (NT) plants exhibit severe SMV symptoms such as mosaic leaves and mottled pods. Right: In NT plants, GmMEKK2 expression is low, so the GmMEKK1‐GmMKK1‐GmMPK4A cascade remains active. GmMPK4A phosphorylates CRCK3, which binds with and represses SUMM2. This suppresses defence responses, and leads to a ROS burst.

Techniques: Virus, Over Expression, Blocking Assay, Phospho-proteomics, Inhibition, Gene Expression, Expressing

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