ebfp2 sequence  (Addgene inc)

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

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.

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

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.

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: Expressing, Over Expression, Transduction

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.

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

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.

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: Over Expression, Expressing, Plasmid Preparation

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.

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

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.

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, Over Expression, Blocking Assay, Phospho-proteomics, Inhibition, Gene Expression, Expressing