Journal: bioRxiv

Article Title: An ordered pattern of Ana2 phosphorylation by Plk4 is required for centriole assembly

doi: 10.1101/240374

Figure Lengend Snippet: (A) Linear map of Drosophila Ana2 showing the functional and structural domains, including the Sas4-binding domain (red) , cytoplasmic dynein light chain LC8-binding regions (amino acids 159–168 and 237–246; orange) , coiled-coil region (CC, blue), and the STil/ANa2 (STAN) motif (purple). (B) The anti-Ana2 antibodies used in this study are specific. Three different rabbit polyclonal anti-Ana2 antibodies were raised against purified recombinant full-length GST-Ana2 and then affinity-purified with MBP-Ana2. The specificity of these antibodies (R1-3) on Western blots of S2 cell lysates is shown. All antibodies react with the identical ~50 kDa protein which migrates on SDS-PAGE as a tight doublet. (C) Ana2 RNAi efficiently diminishes Ana2 levels in cells. Ana2 dsRNA was generated against sequence of the 5’ and 3’ UTRs. S2 cells were treated with control dsRNA or Ana2 UTR dsRNA for 7 days. On day 5, cells were transfected with GFP-Ana2, allowed to recover for 24 hours, and then induced to express transgenic GFP-Ana2 for an additional 24 hours. Anti-Ana2 immunoblots demonstrate effective endogenous Ana2 depletion (~90%). Anti-GFP immunoblots show that Ana2 UTR RNAi does not diminish expression of exogenous GFP-Ana2. (D) Ana2 but not Ana2 ΔCC co-immunoprecipitates (IPs) with Plk4. S2 cells were RNAi treated for 7 days to deplete endogenous Ana2. On day 5, cells were co-transfected with Plk4-GFP (or control GFP) and either V5-Ana2 wild-type or V5-Ana2 ΔCC; the following day, transgene expression was induced for 24 hrs. Anti-GFP IPs were then prepared from lysates, and blots of the inputs and IPs probed for GFP, V5, and α-tubulin. (E) Ana2 interacts specifically with Plk4 1-381 and PB3 by yeast two-hybrid (Y2H) analysis. Full-length (FL) and fragments of Ana2 and Plk4 were screened by Y2H. In each image, colonies from replica plating are shown and growth indicates the presence of both bait and prey. Left panel, no selection; middle panel, growth selection on QDO; right panel, growth and color selection on DDOXA (blue color indicates an interaction). AA indicates that one or both protein fragments autoactivated the Y2H reporters on their own and could not be tested. Note that in this subfigure only, the Ana2-CT is defined as spanning residues 177-420 and contains the CC. Ana2-CT interacts strongly with Plk4 1-381 (containing the CC-DRE region) and moderately with PB3, but not with PB1-PB2. (F) In vitro GST-pulldown assays demonstrate direct binding between purified GST-Ana2 and FLAG-tagged Plk4 1-317-His 6 . Western blots of input and washed glutathione bead pellet samples were probed with anti-GST and FLAG antibodies. (G) Coiled–coil (CC) regions of Plk4 family members predicted by software from Pole Bioiformatique Lyonnaise (PBIL). The probability of CC structure was determined for the first 300 amino acids, using a 14 amino acid window and a 2.5 weight on positions ‘a’ and ‘d’. (H) Alignment of the 34 amino acid within the predicted CC encoded by Plk4 family members. Identical residues in the alignment are highlighted green, and aligned residues with similar side chains are highlighted yellow.

Article Snippet: Yeast two-hybrid (Y2H) experiments were carried out using the Matchmaker Gold Y2H system (Clontech) with significant modifications. pDEST-GADT7 and pDEST-GBKT7, modified versions of Matchmaker vectors compatible with the Gateway cloning system (Life Technologies), were used ( ). pDEST-GADT7 and pDEST-GBKT7 contain the 2 μ and pUC ori for growth in yeast and bacteria.

Techniques: Functional Assay, Binding Assay, Purification, Recombinant, Affinity Purification, Western Blot, SDS Page, Generated, Sequencing, Transfection, Transgenic Assay, Expressing, Selection, In Vitro, Software

Journal: bioRxiv

Article Title: An ordered pattern of Ana2 phosphorylation by Plk4 is required for centriole assembly

doi: 10.1101/240374

Figure Lengend Snippet: (A) A single non-phosphorylatable alanine substitution of S38 in Ana2 disrupts centriole duplication. S2 cells were control-dsRNA treated (Ctrl) or depleted of endogenous Ana2 (UTR) by RNAi for 12 days. Cells were transfected with Ana2-UTR dsRNA on days 0, 4 and 8. On days 4 and 8, cells were additionally transfected with V5-Ana2 or GFP and induced with 0.1 mM CuSO4. Cells were immunostained for PLP and Asterless to mark centrioles, and the number of centrioles per cell was counted. n = 100 cells in each of three experiments. Asterisks indicate significant differences. Error bars, SEM. (B, C) Phospho-mutants of S38 in Ana2 do not affect binding to Sas4 (B) or Plk4 (C). S2 cells were depleted of endogenous Ana2 by RNAi for 7 days. On day 5, cells were co-transfected with the indicated inducible V5-Ana2 construct and either Sas4-GFP (B) or Plk4-GFP (C), and the next day induced to express for 24 hours. Anti-GFP IPs were then prepared from lysates, and Western blots of the inputs and IPs probed for GFP and V5. (D)Ana2 NT interacts specifically with Ana2 CT by yeast two-hybrid (Y2H) analysis. Fragments of Ana2, NT (1-176) and CT (177-420) were screened by Y2H. In each image, colonies from replica plating are shown and growth indicates the presence of both bait and prey. Left panel, no selection; middle panel, growth selection on QDO; right panel, growth and color selection on DDOXA (blue color indicates an interaction). Note that in this subfigure only, the Ana2-CT is defined as spanning residues 177-420 and contains the CC. Ana2-CT interacts strongly with Ana2-NT in 4 out of 11 interactions, weakly in 1 out of 11, and no interaction was detected in 6 out of 11 interactions. (E) Ana2-5PM does not rescue centriole duplication. S2 cells were control-dsRNA treated (Ctrl) or depleted of endogenous Ana2 (UTR) by RNAi for 12 days. Cells were transfected with Ana2-UTR dsRNA on days 0, 4 and 8. On days 4 and 8, cells were additionally transfected with V5-Ana2 or GFP and induced with 0.1 mM CuSO4. Cells were immunostained for PLP and Asterless to mark centrioles, and the number of centrioles per cell was counted. n = 100 cells in each of three experiments. Asterisks indicate significant differences. Error bars, SEM.

Article Snippet: Yeast two-hybrid (Y2H) experiments were carried out using the Matchmaker Gold Y2H system (Clontech) with significant modifications. pDEST-GADT7 and pDEST-GBKT7, modified versions of Matchmaker vectors compatible with the Gateway cloning system (Life Technologies), were used ( ). pDEST-GADT7 and pDEST-GBKT7 contain the 2 μ and pUC ori for growth in yeast and bacteria.

Techniques: Transfection, Binding Assay, Construct, Western Blot, Selection

Journal: The Journal of biological chemistry

Article Title: Hepatitis C virus core protein binds to a DEAD box RNA helicase.

doi: 10.1074/jbc.274.22.15751

Figure Lengend Snippet: FIG. 1. Primary structures of DBX, PL10, and Ded1 and their interac- tions with HCV core protein in the yeast two-hybrid assay. A, alignment of deduced amino acid sequences of DBX (Gen- Banky accession number AF000982), PL10 (GenBanky accession number J04847), and Ded1p (GenBanky accession number X57278) is shown. Identical amino acids are shown as white on cyan. Conserved substi- tutions are shown as black on magenta. Dots represent gaps to optimize alignments, which were obtained using the Pileup pro- gram. B, two-hybrid assays showing interac- tion of HCV core protein with DBX and PL10 but not with Ded1p. Yeast strain Y190 was co-transformed with a plasmid express- ing the cytoplasmic domain of HCV fused to the GAL4 DNA binding domain and plas- mids expressing either a portion of DBX or the corresponding portions of PL10 or Ded1p fused to the GAL4 transcriptional activation domain. Transformants giving b-galactosid- ase activity (positive interactions) are blue. Control reactions of DBX, PL10, and Dep1p GAL 4 activation domain fusion proteins with GAL4 DNA binding domain alone were negative (data not shown).

Article Snippet: The amplified DNA was cloned into the GAL4 DNA binding domain fusion vector pAS2–1 (CLONTECH) to yield pAS2–1-HCV-core1–123.

Techniques: Y2H Assay, Transformation Assay, Plasmid Preparation, Binding Assay, Expressing, Activation Assay, Activity Assay, Control

Journal: Frontiers in Plant Science

Article Title: The Rice Abscisic Acid-Responsive RING Finger E3 Ligase OsRF1 Targets OsPP2C09 for Degradation and Confers Drought and Salinity Tolerance in Rice

doi: 10.3389/fpls.2021.797940

Figure Lengend Snippet: OsRF1 interacts with clade A OsPP2Cs. (A) Yeast two hybrid (Y2H) assay. AH109 yeast cells were co-transformed with the bait (pGBKT7-OsRF1) and prey (pGADT7-OsPP2Cs) constructs. Transformed cells were plated in color change selection media (SD-LT/X-α-Gal) or growth selection media (SD-LTH) and grown at 30°C for 3 days. (B) BiFC assay using pVYCE-OsRF1 and pVYNE-OsPP2Cs in rice protoplasts. The rice protoplast co-expressing OsRF1-VYCE and OsPP2C09-VYNE or OsPP2C50-VYNE in the presence or absence of MG132 was visualized using a confocal laser scanning microscope. YFP signals are represented in green, and chloroplast auto-fluorescence is represented in red. YFP; yellow fluorescent protein; BF, bright field. Bar = 10 μm.

Article Snippet: For yeast Y2H assay, full-length CDSs of synthetic OsRF1 was cloned into the pGBKT7 vector of Matchmaker Gal4 Two-Hybrid System 3 (Clontech, Palo Alto, CA, United States). pGADT7 vectors fused with nine OsPP2CAs (OsPP2C06, OsPP2C08, OsPP2C09, OsPP2C30, OsPP2C49, OsPP2C50, OsPP2C51, OsPP2C53, and OsPP2C68) were kindly provided by Dr. B-G. Kim (National Institute of Agricultural Sciences, South Korea).

Techniques: Y2H Assay, Transformation Assay, Construct, Selection, Bimolecular Fluorescence Complementation Assay, Expressing, Laser-Scanning Microscopy, Fluorescence

Journal: Molecular Plant Pathology

Article Title: Phytoplasma effector SWP1 induces witches’ broom symptom by destabilizing the TCP transcription factor BRANCHED1

doi: 10.1111/mpp.12733

Figure Lengend Snippet: SWP1 targets the shoot branching integrators BRC1 and BRC2. (a) Yeast two‐hybrid (Y2H) assay was used to examine the interaction of BD‐SWP1 with AD‐BRC1 and AD‐BRC2. Blue yeast colonies on SD/‐Trp‐Leu‐His‐Ade medium containing 40 μg/mL X‐α‐Gal (‐LWHA+X‐α‐Gal) indicate positive interaction between the two proteins. BD‐53/AD‐T, positive control; BD‐Lam/AD‐T, negative control; BD‐SWP1/AD, BD/AD‐BRC1 and BD/AD‐BRC2, vector controls. (b) Bimolecular fluorescence complementation (BiFC) assays confirm the interaction of SWP1 with BRC1 and BRC2 in Nicotiana benthamiana. Fluorescence signals were observed at 60 h after agro‐infiltration using a confocal laser scanning microscope. The plasmid combinations NYFP/CYFP, NYFP‐SWP1/CYFP, NYFP/CYFP‐BRC1 and NYFP/CYFP‐BRC2 were used as negative controls. Bars, 30 μm. (c, d) Y2H assays of SWP1 mutants (c) and BRC1 mutants (d). [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Interactions were determined by dropping 2 μL of yeast suspension (OD 600 = 0.3) onto SD/‐Trp‐Leu‐His‐Ade plates containing 40 μg/mL X‐α‐Gal (Clontech) (‐LWHA+X‐α‐Gal).

Techniques: Y2H Assay, Positive Control, Negative Control, Plasmid Preparation, Fluorescence, Laser-Scanning Microscopy

Journal: BMC Plant Biology

Article Title: Uncovering the involvement of DoDELLA1-interacting proteins in development by characterizing the DoDELLA gene family in Dendrobium officinale

doi: 10.1186/s12870-023-04099-w

Figure Lengend Snippet: Transcriptional activation analysis of fragments of the DoDELLA1 protein. A Schematic showing the DoDELLA1 deletion constructs tested in the transcriptional activation analysis. The DoDELLA1 protein contains the N-terminal DELLA regulatory domain (1–756 aa) and the C-terminal GRAS functional domain (766–1935 aa). B Transcriptional activity analysis of the full length of DoDELLA1, the DoDELLA1-active domain and the DoDELLA1-GRAS domain in yeast. BD: pGBKT7 (a yeast two-hybrid prey expression vector) was used as the negative control; DELLA1-BD: DoDELLA1-pGBKT7; DELLA1-BD-active domain: DoDELLA1-active domain-pGBKT7; DELLA1-BD-GRAS domain: DoDELLA1-GRAS domain-pGBKT7. SD/-Trp: yeast culture medium without tryptophan; SD/-Trp/-His/-Ade: yeast culture medium without tryptophan, histidine and adenine

Article Snippet: The amplified full-length DELLA1 and truncated DELLA1 were separately connected to the vector pGBKT7 (Clontech, Palo Alto, CA, USA) by homologous recombination to obtain the pGBKT7-DoDELLA1 and truncated pGBKT7- DoDELLA1 - X (pGBKT7-DoDELLA1-GRAS and pGBKT7-DoDELLA1-active) vectors.

Techniques: Activation Assay, Construct, Functional Assay, Activity Assay, Expressing, Plasmid Preparation, Negative Control

Journal: BMC Plant Biology

Article Title: Uncovering the involvement of DoDELLA1-interacting proteins in development by characterizing the DoDELLA gene family in Dendrobium officinale

doi: 10.1186/s12870-023-04099-w

Figure Lengend Snippet: DoDELLA1 yeast two-hybrid (Y2H) library screening assay. The N-terminus of the GRAS domain of DoDELLA1 with a binding function was used for Y2H screening. A Y2H screening and positive clone identification. Preliminary screening of 27 transformant colonies on SD medium without Trp and Leu. An additional 18 positive clones were obtained from the interaction between DoDELLA1 and other proteins in an in vitro X-α-gal activity assay, detected on SD medium without Trp, Leu, His and Ade. B Analysis of the interaction between DoDELLA1 and DoMYB39, DoDELLA1 and DoMYB308, and DoDELLA1 and DoWAT1, using a Y2H assay. All 18 positive clones were used for comparing sequences, and three key proteins (DoMYB39, DoMYB308 and DoWAT1) were used for Y2H rotary validation. An empty vector was used as the control. AD: pGADT7, a Y2H bait expression vector; BD: pGBKT7, a Y2H prey expression vector; empty, a negative control. SD/-Trp/-Leu: yeast culture medium without tryptophan and leucine; SD/-Trp/-Leu/-His/-Ade: yeast culture medium without tryptophan, leucine, histidine, and adenine; SD/-Trp/-Leu/-His/-Ade + X-α-Gal: yeast culture medium without tryptophan, leucine, histidine, and adenine, to which X-α-Gal was added

Article Snippet: The amplified full-length DELLA1 and truncated DELLA1 were separately connected to the vector pGBKT7 (Clontech, Palo Alto, CA, USA) by homologous recombination to obtain the pGBKT7-DoDELLA1 and truncated pGBKT7- DoDELLA1 - X (pGBKT7-DoDELLA1-GRAS and pGBKT7-DoDELLA1-active) vectors.

Techniques: Library Screening, Binding Assay, Clone Assay, In Vitro, Activity Assay, Y2H Assay, Plasmid Preparation, Control, Expressing, Negative Control