gal4 activation domain vector  (TaKaRa)

Journal: Frontiers in Plant Science

Article Title: Functional analysis and interaction networks of Rboh in poplar under abiotic stress

doi: 10.3389/fpls.2025.1553057

Figure Lengend Snippet: Interaction proteins of PyRbohs . (A) Predicted network of protein-protein interactions of PyRbohs using STRING. Spheres represented interacting proteins, with the orange proteins in the center representing PyRboh proteins. The colored lines denoted different types of interaction relationships between proteins: interaction relationships verified by curated databases (blue) or experiments (purple); interaction relationships predicted by gene neighborhood location (green), gene fusion (red), or gene co-occurrence (blue violet); possible interactions of proteins by text mining (olive), co-expression (black), or protein homology (gray-purple). (B) Protein-protein interactions detected using Y2H assays. In Y2H assay, the coding sequences of Poyun17687, Poyun27068 and Poyun21683 was ligated to activation domain vectors (AD, as pGADT7-Poyun17687), and coding sequences of Poyun00939 and Poyun19109 was fused to GAL4 DNA-binding domain vectors, (BD, as pGBKT7-Poyun00939). The Y2H yeast strain (AH109) was used in this assay. The pGADT7 without any coding sequences and pGBKT7-Poyun00939, pGBKT7-19109 were used as the negative control of Y2H. SD/-L-T, SD/-H-L-T, and SD/-A-H-L-T represented SD-Leu-Trp, SD-His-Leu-Trp and SD-Ade-His-Leu-Trp medium. Different concentrations of AbA (0, 400, 800 µg/L) were added for screening.

Article Snippet: The coding sequences of the bait proteins (Poyun00939, Poyun19109) were fused to the GAL4 DNA-binding domain vector (pGBKT7, Clontech, USA), while the coding sequences of the predicted proteins (Poyun270678, Poyun17687, Poyun21683) were fused to the GAL4 activation domain vector (pGADT7, Clontech, USA) ( ).

Techniques: Expressing, Y2H Assay, Activation Assay, Binding Assay, Negative Control

Journal: Molecular Plant Pathology

Article Title: SreC ‐dependent adaption to host iron environments regulates the transition of trophic stages and developmental processes of Curvularia lunata

doi: 10.1111/mpp.13444

Figure Lengend Snippet: SreC transcriptionally regulates reductive iron assimilation (RIA) to siderophore‐mediated iron assimilation (SIA) pathway in an iron‐dependent manner. (a) Iron binding of SreC was dependent on the cysteine‐rich central (CRR) domain. Escherichia coli cells with pET28‐ SreC were cultured on media containing 1 mM IPTG, with 1 mM FeCl 3 (+Fe) and 50 μM bathophenanthroline disulfonate (BPS) (−Fe), at 37°C for 4 h. Cells were centrifuged and cell pellets were photographed. (b) Iron was required for SreC binding to promoters of ClFet3p and ClSit1p as visualized by electrophoretic mobility shift assay (EMSA). The DNA probe was amplified using the ClFet3p and ClSit1p promoter region containing the ATGWGATAW element. His‐SreC and His‐SreC‐∆CRR were produced heterologously in E. coli and purified. The DNA probe was incubated with purified His‐SreC, His‐SreC‐∆CRR, and His with or without proteinase K for 20 min at 25°C. (c) SreC interacted with ClGrx4 and ClFra2 in yeast two‐hybrid assay. Serial dilutions of the yeast cells were plated on synthetic dropout (SD) medium lacking leucine (L), tryptophan (T), histidine (H), and adenine (A) (SD−LTHA). The yeast strain containing pGBKT7‐53 and pGADT7 was used as a positive control, whereas that containing pGBKT7‐Lam and pGADT7 was used as a negative control. (d) The interaction of SreC and ClGrx4 or ClFra2 in the nucleus as visualized by bimolecular fluorescence complementation. A pair of constructs SreC‐CYFP+NYFP, and another pair of constructs ClGrx4/ClFra2‐NYFP+CYFP were used as negative controls. Yellow fluorescent protein (YFP) signals were observed using confocal microscopy. The nuclear localization was confirmed by simultaneous nuclear labelling with H2B‐mCherry. DIC, differential interference contrast microscopy. (e) The expression of iron assimilation pathways genes in wild‐type (WT), Δ ClGrx4 , and Δ ClFra2 at 24 h post‐inoculation (hpi). The maize leaves were inoculated with Curvularia lunata conidia at a concentration of 10 6 conidia/mL. The leaves were sampled at the indicated time for reverse transcription‐quantitative PCR assays. C. lunata ClActin was used as the reference gene. Values are means ± SD ( n = 3 biological replicates). An asterisk indicates significant differences based on unpaired two‐tailed Student's t test with the p values marked (** p < 0.01, ns, not significant).

Article Snippet: Each cDNA fragment was cloned into the yeast GAL4‐binding domain vector pGBKT7 and GAL4‐activation domain vector pGADT7 (Clontech).

Techniques: Binding Assay, Cell Culture, Electrophoretic Mobility Shift Assay, Amplification, Produced, Purification, Incubation, Y2H Assay, Positive Control, Negative Control, Fluorescence, Construct, Confocal Microscopy, Microscopy, Expressing, Concentration Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Two Tailed Test