bait vector pbridge  (TaKaRa)

Journal: Plant Communications

Article Title: A glutamyl–tRNA reductase and its binding protein promote transitory starch biosynthesis and enhance grain quality and yield in rice

doi: 10.1016/j.xplc.2025.101527

Figure Lengend Snippet: GluTRBP interacts with LSD3 and regulates starch biosynthesis in rice leaves. (A) Interaction between GluTRBP and LSD3 detected by yeast two-hybrid (Y2H) assay. The interactions between pGADT7-T and pGBKT7-53 and between pGADT7 and pGBKT7 were used as positive and negative controls, respectively. Yeast transformants were spotted onto control medium (SD/−Leu/−Trp, Double Dropout Medium, DDO) and selective medium (SD/−Leu/−Trp/−His/−Ade, Quadruple Dropout Medium, QDO). (B) Interaction between GluTRBP and LSD3 detected by GST pull-down assays. GST–LSD3 and His–GluTRBP were detected with anti-GST and anti-His antibodies, respectively. IB, immunoblotting. (C) Interaction between GluTRBP and LSD3 detected by bimolecular fluorescence complementation (BiFC) assay in rice protoplasts. Scale bars, 5 μm. (D) Co-localization of GluTRBP–GFP with chloroplast autofluorescence and GBSSII–CFP in rice protoplasts. CFP, cyan fluorescent protein. Scale bars, 5 μm. (E) Iodine staining of flag leaves from wild type and GluTRBP gene-edited mutants ( glutrbp-1 , glutrbp-2 ) at the end of the day. Scale bar, 1 cm. (F) Ultrastructure of chloroplasts in flag leaves at the end of the day. Starch granules (SGs) are indicated by red arrows. Scale bars, 20 μm. (G) Number of SGs per cell in flag leaves of wild type, glutrbp-1 , and glutrbp-2 . (H) Total starch content in flag leaves at the end of the day. DW, dry weight. (I) Percentage of amylose in total starch in flag leaves at the end of the day. (J) Sucrose content in flag leaves at the end of the day. Data in (G)–(J) are presented as means ± SD from three biological replicates. Different letters indicate significant differences at p < 0.05 according to ANOVA and Duncan’s test.

Article Snippet: For Y2H assays, the coding sequences of LSD3 , GluTRBP , and GBSSII were cloned into pGADT7 (prey) or pGBKT7 (bait) vectors, and the fusion constructs were transferred into the GAL4 Y2H system (Clontech, Dalian, China).

Techniques: Starch, Y2H Assay, Control, Western Blot, Bimolecular Fluorescence Complementation Assay, Staining

Journal: Plant Communications

Article Title: A glutamyl–tRNA reductase and its binding protein promote transitory starch biosynthesis and enhance grain quality and yield in rice

doi: 10.1016/j.xplc.2025.101527

Figure Lengend Snippet: The LSD3–GluTRBP module regulates transitory starch biosynthesis by maintaining GBSSII protein stability and enzymatic activity. (A) Interaction between GluTRBP and GBSSII detected by Y2H assay. Interactions between pGADT7-T and pGBKT7-53, and between pGADT7 and pGBKT7, served as positive and negative controls, respectively. Control medium, SD/−Leu/−Trp (DDO); selective medium, SD/−Leu/−Trp/−His/−Ade (QDO). (B) Interaction between GBSSII and GluTRBP detected by BiFC assay in rice protoplasts. Scale bars, 5 μm. (C) Interaction between GBSSII and GluTRBP detected by GST pull-down assays. GST–GBSSII and His–GluTRBP were detected with anti-GST and anti-His antibodies, respectively. IB, immunoblotting. (D) Cell-free degradation assay of LSD3 in the absence or presence of GluTRBP. (E) Cell-free degradation assay of GluTRBP in the absence or presence of LSD3. (F) Cell-free degradation assay of GBSSII in the absence or presence of GluTRBP and/or LSD3. (G) Expression levels of GBSSII in leaves of wild type, lsd3 , and glutrbp . (H) Immunoblot analysis of GBSSII protein abundance in leaves of wild type and mutants. (I) Enzyme activity of GBSS in leaves of wild type and mutants. (J) Iodine staining of flag leaves from wild type and CRISPR–Cas9 gene-edited mutants of GBSSII ( gbssII-1 , gbssII-2 ) at the end of the day. Scale bar, 1 cm. (K) Ultrastructure of chloroplasts in flag leaves at the end of the day. Starch granules (SGs) are indicated by red arrows. Scale bars, 10 μm. (L) Total starch content in flag leaves of wild type and mutants at the end of the day. DW, dry weight. (M) Percentage of amylose in total starch in flag leaves at the end of the day. (N) Sucrose content in flag leaves at the end of the day. (O) Grain appearance of wild type and mutants. Scale bars, 1 cm. (P–R) Chalky-grain rate (P) , total starch content (Q) , and amylose content (R) in the endosperm of wild type and mutants. Data in (G) , (I) , (L)–(N), and (P)–(R) are presented as means ± SD from three biological replicates. n.d. in (M) , not detected. Different letters indicate significant differences at p < 0.05 according to ANOVA and Duncan’s test.

Article Snippet: For Y2H assays, the coding sequences of LSD3 , GluTRBP , and GBSSII were cloned into pGADT7 (prey) or pGBKT7 (bait) vectors, and the fusion constructs were transferred into the GAL4 Y2H system (Clontech, Dalian, China).

Techniques: Starch, Activity Assay, Y2H Assay, Control, Bimolecular Fluorescence Complementation Assay, Western Blot, Degradation Assay, Expressing, Quantitative Proteomics, Staining, CRISPR