25 gal4 activation domain  (Addgene inc)

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: PLOS Pathogens

Article Title: Use of the Puccinia sorghi haustorial transcriptome to identify and characterize AvrRp1-D recognized by the maize Rp1-D resistance protein

doi: 10.1371/journal.ppat.1012662

Figure Lengend Snippet: (A)-(B) All constructs tested were described previously (5). The amino acid position of the recombination site of each construct was indicated above the construct. The red line indicates a mutated amino acid position. The domain swap constructs were fused with the C-terminal tag 3xHA, and AvrRp1-D.1 was fused with the C-terminal tag 4xcMYC. The strength of HR resulting from co-expression in N . benthamiana is shown. 9 individual plants were infiltrated and showed similar results. The table to the right indicates the relative strength of the HR induced by the co-expression of AvrRp1-D.1 with each Rp1 chimera or mutant (+++ is the strongest HR). Rp1-D21 was used as a positive control. (C) Yeast two-hybrid assay using strains co-expressing Rp1-D, -dp2, -dp6, and -dp7 fused to the GAL4 activation domain (AD) with AvrRp1-D.1 fused to the GAL4 DNA binding domain (BD) on control media lacking leucine and tryptophan (-LW) or selective media additionally lacking histidine (-LWH). Growth on selective media indicates protein-protein interactions. Interaction between T antigen with GAL4 activation domain and Lam or p53 with GAL4 DNA binding domain were used as negative/positive control, respectively. AD- or BD-binding proteins were detected using anti-GAL4 and anti-GAL4 (DBD). (D) Schematic diagram of the parts into which Rp1-D was divided for the yeast-two-hybrid assay shown in (E). (E) Yeast two-hybrid assay using strains co-expressing each part of Rp1-D or Rp1-dp7 shown in the schematic above fused to AD with AvrRp1-D.1 or Lam fused to BD on control media lacking leucine and tryptophan (-LW) or selective media additionally lacking histidine (-LWH). Pictures were taken 5 days after plating.

Article Snippet: To generate a yeast expression vector with Rp1 alleles, their deletion constructs, and effector, Rp1 alleles and their deletion constructs were cloned into a yeast expression vector containing GAL4 activation domain (addgene #20161), and the effector gene was cloned the vector containing GAL4 binding domain (addgene #20162).

Techniques: Construct, Expressing, Mutagenesis, Positive Control, Y2H Assay, Activation Assay, Binding Assay, Control, Protein-Protein interactions

Journal: PLOS Pathogens

Article Title: Zinc-finger (ZiF) fold secreted effectors form a functionally diverse family across lineages of the blast fungus Magnaporthe oryzae

doi: 10.1371/journal.ppat.1012277

Figure Lengend Snippet: (A) Yeast two-hybrid assay of AVR-Pii and AVR-Pii CCH with OsExo70F2 or OsExo70F3 host targets. For each combination, 5μl of yeast were spotted and incubated in double dropout plate for yeast growth control (left) and quadruple dropout media supplemented with X-α-gal and 3AT (right). Growth, and development of blue coloration, in the selection plate are both indicative of protein:protein interaction. OsExo70 proteins were fused to the GAL4 DNA binding domain and AVR-Pii to the GAL4 activator domain. Empty vectors were used as negative control in each combination. (B) Co-immunoprecipitation of AVR-Pii and AVR-Pii CCH with OsExo70F2. N-terminally GFP-tagged AVR-Pii effectors were transiently co-expressed with N-terminally 3xFLAG-tagged OsExo70F2 or 3xFLAG-mCherry in N . benthamiana . Immunoprecipitates (IPs) were obtained with anti-FLAG magnetic beads and total protein extracts were probed with appropriate antisera.

Article Snippet: The membranes were probed with anti-GAL4 DNA Binding domain (Sigma) antibody for the OsExo70F3 protein in pGBKT7 and with the anti-GAL4 activation domain (Sigma) antibody for AVR-Pii and ZiF effectors in pGADT7.

Techniques: Y2H Assay, Incubation, Control, Selection, Binding Assay, Negative Control, Immunoprecipitation, Magnetic Beads

Journal: PLOS Pathogens

Article Title: Zinc-finger (ZiF) fold secreted effectors form a functionally diverse family across lineages of the blast fungus Magnaporthe oryzae

doi: 10.1371/journal.ppat.1012277

Figure Lengend Snippet: (A) Y2H assay of wild-type AVR-Pii and ZiF_VIIIc and their corresponding mutants at the host target binding interface (Phe65Glu and Phe66Glu, respectively) to host target OsExo70F3. Left, control plate for yeast growth. Right, quadruple-dropout media supplemented with X-α-gal and aureobasidine A (Au A). Growth and development of blue coloration in the right panel indicates protein-protein interactions. OsExo70F3 was fused to the GAL4 DNA binding domain while effectors were fused to the GAL4 activator domain. Each experiment was repeated a minimum of three times, with similar results. (B) Rice leaf blade spot inoculation of transgenic M . oryzae Sasa2 isolates expressing AVR-Pii or ZiF_VIIIc from wheat blast isolate BTJP 4–1 in rice cultivars Moukoto (Pii-) and Hitomebore (Pii+). The cultivars Moukoto and Hitomebore are denoted by M and H, respectively. For each experiment, a representative image from replicates with independent M . oryzae transformants are shown. Wild-type rice blast isolate Sasa2 and wheat blast Br32 and BTJP 4–1 are included as control. Full images for the three experimental replicates are presented in . (C) Spray inoculation of transgenic M . oryzae isolates in 3-weeks-old rice cultivar Moukoto and Hitomebore. Each experiment was performed four times (two of the leaves are shown for the Sasa2, Br32 and BTJP 4–1 isolates). All leaf images are shown in .

Article Snippet: The membranes were probed with anti-GAL4 DNA Binding domain (Sigma) antibody for the OsExo70F3 protein in pGBKT7 and with the anti-GAL4 activation domain (Sigma) antibody for AVR-Pii and ZiF effectors in pGADT7.

Techniques: Y2H Assay, Binding Assay, Control, Transgenic Assay, Expressing

Journal: PLOS Pathogens

Article Title: Zinc-finger (ZiF) fold secreted effectors form a functionally diverse family across lineages of the blast fungus Magnaporthe oryzae

doi: 10.1371/journal.ppat.1012277

Figure Lengend Snippet: (A) Y2H binding assay of ZiF effectors from rice blast isolates to host target OsExo70F3. For each tribe, the most prevalent allele in rice blast lineages was fused to the GAL4 activator domain and co-expressed in yeast cells with OsExo70F3 fused to GAL4 DNA binding domain. AVR-Pii Phe65Glu was used as negative control as previously reported . (B) Y2H binding assay of ZiF effectors of wheat blast isolates to host target OsExo70F3. The most prevalent effector allele in each ZiF tribe of M . oryzae isolates infecting wheat was fused to the GAL4 activator domain and co-expressed in yeast cells with OsExo70F3 fused to GAL4 DNA binding domain. Rice blast AVR-Pii and AVR-Pii Phe65Glu were used as positive and negative controls, respectively. For both assays, a control plate for yeast growth is presented on the left and a plate with quadruple-dropout media supplemented with X-α-gal and aureobasidine A (Au A) is presented on the right. Growth and development of blue coloration in the right panel indicates protein-protein interactions. Each experiment was repeated a minimum of three times, with similar results.

Article Snippet: The membranes were probed with anti-GAL4 DNA Binding domain (Sigma) antibody for the OsExo70F3 protein in pGBKT7 and with the anti-GAL4 activation domain (Sigma) antibody for AVR-Pii and ZiF effectors in pGADT7.

Techniques: Binding Assay, Negative Control, 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