Journal: Viruses

Article Title: A Linear Surface Epitope in a Proline-Rich Region of ORF3 Product of Genotype 1 Hepatitis E Virus

doi: 10.3390/v8080227

Figure Lengend Snippet: Genotype 1 VP13 protein reacts with the monoclonal antibody (Mab) in western blotting (WB). ( A ) Detection of purified maltose-binding protein (MBP)-VP13, but not MBP-X or MBP-Helicase. MBP detection was done as a control; ( B ) Detection of only genotype 1 VP13 in lysate of HEK293 cells transiently transfected with YFP-VP13 plasmids or empty vector (EV). Purified MBP-VP13 was included as a positive control. Ab: antibody.

Article Snippet: Antibodies used in the WB were rabbit anti-MBP antibody (Rockland Immunochemicals Inc.), mouse anti-YFP (yellow fluorescent protein) monoclonal antibody (Rockland Immunochemicals Inc.), mouse anti-tubulin monoclonal antibody (Sigma-Aldrich Corp., St. Louis, MO, USA), homemade monoclonal anti-VP13 clone 3C3 (this study) and goat anti-mouse IgG or goat anti-rabbit IgG conjugated with horseradish peroxidase (Rockland Immunochemicals Inc.).

Techniques: Western Blot, Purification, Binding Assay, Control, Transfection, Plasmid Preparation, Positive Control

Journal: Journal of Experimental Botany

Article Title: Chilli veinal mottle virus HCPro interacts with catalase to facilitate virus infection in Nicotiana tabacum

doi: 10.1093/jxb/eraa304

Figure Lengend Snippet: ChiVMV HCPro directly interacts with CAT1 and CAT3 in vitro and in vivo . (A) Yeast two-hybrid assay. The ability of cells to grow on synthetic dropout medium lacking Leu, Trp, His, and Ade (-LWHA) suggested the interaction. (B) GST pull-down assay showing the interaction among HCPro, CAT1, and CAT3 in vitro . Purified CAT1–MBP, CAT2–MBP, CAT3–MBP, or MBP was incubated with HCPro–GST. After being immunoprecipitated with GST beads, the proteins were detected by protein gel blot analysis with anti-MBP or anti-GST antibodies. (C) BiFC assay. HCPro interacted with CAT1 and CAT3 in N. benthamiana leaves.Scale bars=30 μm.

Article Snippet: HCPro , CAT1 , CAT2 , and CAT3 were amplified by PCR and then inserted into the pGEX-glutathione S -transferase (GST) tag and pMAL-C2X maltose-binding protein (MBP) tag.

Techniques: In Vitro, In Vivo, Y2H Assay, Pull Down Assay, Purification, Incubation, Immunoprecipitation, Western Blot, Bimolecular Fluorescence Complementation Assay

Journal: Journal of Experimental Botany

Article Title: Chilli veinal mottle virus HCPro interacts with catalase to facilitate virus infection in Nicotiana tabacum

doi: 10.1093/jxb/eraa304

Figure Lengend Snippet: Identification of HCPro domains responsible for the interaction between host factors and HCPro. (A) Schematic description of deletion mutants of ChiVMV HCPro. HCPro can be divided schematically into three regions: the N-terminus (residues 1–100), central region (residues 101–300), and C-terminus (residues 301–457). (B) Identification of the interaction specificity between CAT1 and HCPro from different sources. (C) The C-terminus of HCPro is necessary for the interaction. Different purified deletion mutants of HCPro–MBP or MBP were incubated with CAT1–GST or CAT3–GST. After being immunoprecipitated with GST beads, the proteins were detected by protein gel blot analysis with anti-MBP or anti-GST antibodies.

Article Snippet: HCPro , CAT1 , CAT2 , and CAT3 were amplified by PCR and then inserted into the pGEX-glutathione S -transferase (GST) tag and pMAL-C2X maltose-binding protein (MBP) tag.

Techniques: Purification, Incubation, Immunoprecipitation, Western Blot

Journal: Frontiers in Plant Science

Article Title: MeABL5 , an ABA Insensitive 5-Like Basic Leucine Zipper Transcription Factor, Positively Regulates MeCWINV3 in Cassava ( Manihot esculenta Crantz )

doi: 10.3389/fpls.2019.00772

Figure Lengend Snippet: Activation of the MeCWINV3 promoter in yeast by MeABL5. pGADT7-MeABL5 + pMeCWINV3-AbAi shows that MeABL5 can bind to promoter of MeCWINV3; pGADT7-Rec2-53 + p53HIS2-AbAi is the positive control and pGADT7 + pMeCWINV3-AbAi and pGADT7-MeABL5 + pAbAi are the negative controls. Yeast cells were grown in SD/−Leu selective medium containing 0 or 150 ng/ml Aureobasidin A (AbA) for 3 days at 30°C.

Article Snippet: The MBP-tagged MeABL5 protein was expressed in Escherichia coli BL21 (DE3) by inducing with 1.0 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 4 h, followed by purification using a Ni column (GenScript, Nanjing, China) according to the manufacturers’ instructions.

Techniques: Activation Assay, Positive Control

Journal: Frontiers in Plant Science

Article Title: MeABL5 , an ABA Insensitive 5-Like Basic Leucine Zipper Transcription Factor, Positively Regulates MeCWINV3 in Cassava ( Manihot esculenta Crantz )

doi: 10.3389/fpls.2019.00772

Figure Lengend Snippet: Alignment of MeABL5 and other ABI5 proteins. The amino acid sequence of MeABL5 was aligned with AtABI5 (Arabidopsis thaliana, AAD21438.1), BolABI5 ( Brassica oleracea , JX870620.1), OsABI5 ( Oryza sativa , ABM90395.1), and ZmABI5 ( Zea mays , NP_001150949). The squares and circles mark phosphorylation and ubiquitination sites, respectively. C1, C2, and C3, conserved regions; bZIP, basic leucine zipper domain.

Article Snippet: The MBP-tagged MeABL5 protein was expressed in Escherichia coli BL21 (DE3) by inducing with 1.0 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 4 h, followed by purification using a Ni column (GenScript, Nanjing, China) according to the manufacturers’ instructions.

Techniques: Sequencing

Journal: Frontiers in Plant Science

Article Title: MeABL5 , an ABA Insensitive 5-Like Basic Leucine Zipper Transcription Factor, Positively Regulates MeCWINV3 in Cassava ( Manihot esculenta Crantz )

doi: 10.3389/fpls.2019.00772

Figure Lengend Snippet: Phylogenetic analysis of MeABL5 with bZIP proteins from cassava and Arabidopsis. A total of 77 bZIPs from cassava, 75 bZIPs from Arabidopsis, and MeABL5 were used to create the NJ tree with 1,000 bootstraps using MEGA software from CLUSTAL W alignments. The bZIP proteins are grouped into 10 subfamilies (A, B, C, D, E, F, G, H, I, and S), and MeABL5 was classified into subfamily A.

Article Snippet: The MBP-tagged MeABL5 protein was expressed in Escherichia coli BL21 (DE3) by inducing with 1.0 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 4 h, followed by purification using a Ni column (GenScript, Nanjing, China) according to the manufacturers’ instructions.

Techniques: Software

Journal: Frontiers in Plant Science

Article Title: MeABL5 , an ABA Insensitive 5-Like Basic Leucine Zipper Transcription Factor, Positively Regulates MeCWINV3 in Cassava ( Manihot esculenta Crantz )

doi: 10.3389/fpls.2019.00772

Figure Lengend Snippet: Nuclear localization of the MeABL5 protein. Green fluorescent protein (GFP) fluorescence is shown in green and chlorophyll autofluorescence is shown in red as a chloroplast marker. Top row/bottom row: the corresponding bright field, GFP fluorescence, chlorophyll autofluorescence, and merged fluorescent images of MeABL5-GFP/GFP control. Bar = 30 μm.

Article Snippet: The MBP-tagged MeABL5 protein was expressed in Escherichia coli BL21 (DE3) by inducing with 1.0 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 4 h, followed by purification using a Ni column (GenScript, Nanjing, China) according to the manufacturers’ instructions.

Techniques: Fluorescence, Marker

Journal: Frontiers in Plant Science

Article Title: MeABL5 , an ABA Insensitive 5-Like Basic Leucine Zipper Transcription Factor, Positively Regulates MeCWINV3 in Cassava ( Manihot esculenta Crantz )

doi: 10.3389/fpls.2019.00772

Figure Lengend Snippet: The transactivation activity of the MeABL5 protein in yeast. MeABL5 was inserted into the pGBKT7 vector and transformed into the AH109 yeast strain. The empty vector, pGBKT7, and pGBKT7-p53 + pGADT7-largeT were also transformed into AH109 as a negative and positive control, respectively. The yeast cells were spotted on plates with SD/−Trp selective medium containing 10 mg/ml X-α-gal.

Article Snippet: The MBP-tagged MeABL5 protein was expressed in Escherichia coli BL21 (DE3) by inducing with 1.0 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 4 h, followed by purification using a Ni column (GenScript, Nanjing, China) according to the manufacturers’ instructions.

Techniques: Activity Assay, Plasmid Preparation, Transformation Assay, Positive Control

Journal: Frontiers in Plant Science

Article Title: MeABL5 , an ABA Insensitive 5-Like Basic Leucine Zipper Transcription Factor, Positively Regulates MeCWINV3 in Cassava ( Manihot esculenta Crantz )

doi: 10.3389/fpls.2019.00772

Figure Lengend Snippet: EMSA of the interaction between MeABL5 and the MeCWINV3 promoter. (A) Expression of the MeABL5 protein in E. coli BL21 (DE3). M: Protein marker; Lane 1: the MBP-tagged MeABL5 protein (top arrow). Lane 2: the target MeABL5 protein with the MBP affinity tag removed by TEV protease (bottom arrow). (B) Analysis of the MeABL5 interaction with the MeCWINV3 promoter by EMSA. The promoter of MeCWINV3 with the MeABL5 protein was stained with SYBR Green EMA for visualization of DNA. M: DNA marker. Lane 1: the promoter of MeCWINV3 DNA (500 ng) only; Lanes 2–5: the promoter of MeCWINV3 DNA (500 ng) with increasing amounts of MeABL5 protein (100, 150, 200, and 250 ng); Lane 6: 250 ng MeABL5 protein only; Lane 7: the promoter of MeCWINV3 DNA (500 ng) and 250 ng bovine serum albumin (BSA). The arrows indicate the MeABL5–DNA complex or free DNA.

Article Snippet: The MBP-tagged MeABL5 protein was expressed in Escherichia coli BL21 (DE3) by inducing with 1.0 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 4 h, followed by purification using a Ni column (GenScript, Nanjing, China) according to the manufacturers’ instructions.

Techniques: Expressing, Marker, Staining, SYBR Green Assay

Journal: Frontiers in Plant Science

Article Title: MeABL5 , an ABA Insensitive 5-Like Basic Leucine Zipper Transcription Factor, Positively Regulates MeCWINV3 in Cassava ( Manihot esculenta Crantz )

doi: 10.3389/fpls.2019.00772

Figure Lengend Snippet: MeABL5 binds to the ABRE cis-element in the MeCWINV3 promoter. (A) Yeast-one hybrid (Y1H) assay of the core binding site in MeABL5 binds to the MeCWINV3 promoter. The full-length MeABL5 and the bZIP domain deletion (MeABL5ΔbZIP) sequences were inserted into the pGADT7 vector. Yeast cells carried pGADT7-MeABL5/MeABL5ΔbZIP + pMeCWINV3-AbAi, positive control (pGADT7-Rec2–53 + p53HIS2-AbAi), and negative control (pGADT7 + pMeCWINV3-AbAi), were grown in SD/−Leu selective medium containing 0 or 150 ng/ml AbA, for 3 days at 30°C. (B) Analysis of the MeABL5 interaction with the ABRE cis-element of MeCWINV3 promoter by Y1H assay. The different fragments P1 (−1 to −400), P2 (−401 to −800), P2 without the ABRE cis-element, and P3 (−801 to −1,160) of MeCWINV3 promoter were inserted into the pGBKT7 vector. Yeast cells carried pGADT7-MeABL5 + pMeCWINV3 (P1 to P3, P2ΔABRE)-AbAi, positive control (pGADT7-Rec2–53 + p53HIS2-AbAi), and negative control (pGADT7-MeABL5 + pAbAi) were grown in SD/−Leu selective medium containing 0 or 150 ng/ml AbA, for 3 days at 30°C. (C) Analysis of the MeABL5 interaction with the ABRE cis-element of MeCWINV3 promoter by EMSA. pMeCWINV3-WT: 60 bp DNA sequence contained the ABRE cis-element on the MeCWINV3 promoter; the bold CACGTG represents the ABRE cis-element binding site. pMeCWINV3-MT: 54 bp DNA sequence without the ABRE cis-element on the MeCWINV3 promoter. The + and − indicate the presence and absence of 250 ng MeABL5 protein, respectively. The arrows indicate the MeABL5–DNA complex or free DNA.

Article Snippet: The MBP-tagged MeABL5 protein was expressed in Escherichia coli BL21 (DE3) by inducing with 1.0 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 4 h, followed by purification using a Ni column (GenScript, Nanjing, China) according to the manufacturers’ instructions.

Techniques: Y1H Assay, Binding Assay, Plasmid Preparation, Positive Control, Negative Control, Sequencing

Journal: Frontiers in Plant Science

Article Title: MeABL5 , an ABA Insensitive 5-Like Basic Leucine Zipper Transcription Factor, Positively Regulates MeCWINV3 in Cassava ( Manihot esculenta Crantz )

doi: 10.3389/fpls.2019.00772

Figure Lengend Snippet: Regulation of the transcription activity of the MeCWINV3 promoter by MeABL5 in a transient expression system. (A) Schematic diagrams of the vectors used in the transient expression analysis. (B) Transcriptional activity assay of the MeCWINV3 promoter coinfiltrated with the effector vectors pGreen II 62-SK (control) or pGreen II 62-SK-MeABL5 into tobacco leaves. The MeCWINV3 promoter was fused to the LUC reporter and the promoter activity was determined by a transient dual-LUC assay. The relative LUC activities were normalized to the reference Renilla (REN) luciferase. Error bars represent the SD of nine technical replicates. The significant difference was assessed by ANOVA; a and b represent a significant difference at p < 0.01.

Article Snippet: The MBP-tagged MeABL5 protein was expressed in Escherichia coli BL21 (DE3) by inducing with 1.0 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 4 h, followed by purification using a Ni column (GenScript, Nanjing, China) according to the manufacturers’ instructions.

Techniques: Activity Assay, Expressing, Luciferase

Journal: Frontiers in Plant Science

Article Title: MeABL5 , an ABA Insensitive 5-Like Basic Leucine Zipper Transcription Factor, Positively Regulates MeCWINV3 in Cassava ( Manihot esculenta Crantz )

doi: 10.3389/fpls.2019.00772

Figure Lengend Snippet: Expression pattern analysis of MeABL5 and MeCWINV3. (A) Expression analyses of MeABL5 and MeCWINV3 in cassava organs or tissues using qRT-PCR. FR, fibrous roots; SR, storage roots; SP, storage root phloems; SX, storage root xylems; S, stems; L, leaves (the seventh or eighth leaves from the top of the stem); Fl, flowers; Fr, fruits. The expression of MeCWINV3 in storage roots (SR) was used as a calibrator. (B,C) Expression patterns of MeABL5 (B) and MeCWINV3 (C) respond to ABA, SA, GA3, MeJA, PEG2000 (artificial drought condition), cold (4°C), and heat (42°C). Forty-five-day-old SC8 plantlets under different treatments were used for the analyses. The plantlets without any treatment were used as the control (CK). Each value represents the mean ± SE of three independent biological replicates. Letters a–d on the error bars indicate significant differences, as assessed by ANOVA ( p < 0.05).

Article Snippet: The MBP-tagged MeABL5 protein was expressed in Escherichia coli BL21 (DE3) by inducing with 1.0 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 4 h, followed by purification using a Ni column (GenScript, Nanjing, China) according to the manufacturers’ instructions.

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Plant Science

Article Title: MeABL5 , an ABA Insensitive 5-Like Basic Leucine Zipper Transcription Factor, Positively Regulates MeCWINV3 in Cassava ( Manihot esculenta Crantz )

doi: 10.3389/fpls.2019.00772

Figure Lengend Snippet: Regulation of the MeCWINV3 expression by MeABL5 in the overexpressing transgenic cassava FECs. (A) Expression analyses of MeABL5 and MeCWINV3 in the MeABL5-OX transgenic cassava FECs using qRT-PCR. Three independent transformation experiments were performed. The expression of MeABL5 in the untransformed cassava wild-type SC8 FECs (WT) was used as a calibrator. (B) Expression analyses of MeCWINV3 in the MeABL5-OX transgenic FECs under ABA, SA, GA3, MeJA, PEG2000, cold (4°C), or heat (42°C) treatment. The WT and MeABL5-OX transgenic FECs under different treatments for 12 h were used for the analyses. WT cassava SC8 FECs were used as control. The data are presented as the mean ± SE values ( n = 3) from three independent experiments. Asterisks indicate statistically significant differences compared with WT. * p < 0.05; ** p < 0.005.

Article Snippet: The MBP-tagged MeABL5 protein was expressed in Escherichia coli BL21 (DE3) by inducing with 1.0 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 4 h, followed by purification using a Ni column (GenScript, Nanjing, China) according to the manufacturers’ instructions.

Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR, Transformation Assay