Journal: Science Advances

Article Title: Tissue-specific regulation of a sugar transporter mediates both resistance and tolerance responses to attack from a leafminer in potato

doi: 10.1126/sciadv.adx5951

Figure Lengend Snippet: ( A ) StubSNF1 interacted with MCPI1a and MCPI1b in Y2H assays. -SP, N-terminal signal peptide removed. ( B ) Co-IP assay was performed to investigate interaction between StubSNF1 and MCPI1a/1b in planta. Co-IP was performed in N. benthamiana protoplasts with StubSNF1-GFP and MCPI1a/1b-Myc coexpression. Proteins were detected by immunoblotting. ( C ) Confocal images of BiFC assays revealed that StubSNF1 interacts with MCPI1a and MCPI1b in planta. Scale bars, 10 μm. YFP: signals from BiFC. 4′,6-Diamidino-2-phenylindole (DAPI): signals from DNA. ( D ) GST-pulldown (PD:GST) assays were performed to evaluate the interaction between StubSNF1 and MCPI1a/1b in vitro. The 6×His-StubSNF1 protein was mixed with either GST (negative control) or GST-MCPI1a/1b and incubated with GST agarose beads. Input proteins were visualized by Coomassie blue (CBB) staining after mixing. Proteins were detected by immunoblotting after pulldown. ( E and F ) MCPI1a and MCPI1b negatively regulated herbivore resistance. MCPI1a/1b overexpression significantly increases larval biomass (E; boxplot, n = 41 to 51; top: representative larvae) and accelerated larval development (F; mean ± SEM, n = 56 to 60). In boxplot, triangle and gray point respectively refer to mean value and data point. Asterisks indicate significant difference between genotypes (Student’s t test or Mann-Whitney U test, * P < 0.05, ** P < 0.01, and *** P < 0.001). ( G and H ) StubSNF1 stabilizes MCPI1a and MCPI1b in planta. MCPI1a-Myc (G) and MCPI1b-Myc (H) proteins were analyzed by immunoblotting after coexpression with StubSNF1-GFP for 72 hours in N. benthamiana leaf, with or without treatment of 26 S proteasome inhibitor MG132. Coexpression with GFP served as the control group, and actin served as an internal standard. Left: Representative image. Right: Quantification (mean ± SEM, n = 3). Asterisks indicate significant difference between StubSNF1 and GFP groups (Student’s t test, * P < 0.05, ** P < 0.01, and *** P < 0.001). SNF1, StubSNF1. Experiments (A) to (E) were repeated independently twice with similar results.

Article Snippet: The bacterial cells were then ultrasonically disrupted, and the protein was purified using GST Sepharose 4FF (Sangon Biotech).

Techniques: Co-Immunoprecipitation Assay, Western Blot, In Vitro, Negative Control, Incubation, Staining, Over Expression, MANN-WHITNEY, Control