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. 2021 Sep 25;38(3):297–304. doi: 10.5511/plantbiotechnology.21.0610a

Figure 2. Agrobacterium culture conditions and concentrations for achieving efficient agroinfiltration and protein expression levels in N. benthamiana. (A) Agrobacterium carrying pBYR2HS-EGFP was agroinfiltrated (with 1-ml syringes) at different OD600 into N. benthamiana. GFP emission was observed 3 days post agroinfiltration (dpa) with an ultraviolet-absorbing filter, Fujifilm SC-52. (B) Agrobacterium carrying pBYR2HS-EGFP was resuspended either in agroinfiltration MES buffer (10 mM MES, pH 5.6, 10 mM MgCl2, 100 μM acetosyringone; MES) or Phosphate buffer (10 mM potassium phosphate, pH 5.6, 10 mM MgCl2, 100 μM acetosyringone; P buf.) at OD600=0.5, and used to agroinfiltrate N. benthamiana. GFP emission was observed with an ultraviolet-absorbing filter, Fujifilm SC-52, 3 dpa. (C) Agrobacterium resuspensions containing either pBYR2HS-EGFP, pTKB2-EGFP, or pTKB3-EGFP were adjusted to OD600=0.5 or 0.1 and agroinfiltrated into N. benthamiana. GFP emission was detected at 3 dpa. (D) Total soluble proteins, which were extracted from 0.2 mg FW N. benthamiana leaves agroinfiltrated with Agrobacterium carrying pBYR2HS-EGFP, pTKB2-EGFP, or pTKB3-EGFP, were separated by SDS-PAGE, and stained with Coomassie Brilliant Blue (CBB) dye. (E) The relative amount of GFP was calculated based on protein band intensities in CBB-stained gels using ImageJ software. Data represent means±SE (n=4–6).

Figure 2. Agrobacterium culture conditions and concentrations for achieving efficient agroinfiltration and protein expression levels in N. benthamiana. (A) Agrobacterium carrying pBYR2HS-EGFP was agroinfiltrated (with 1-ml syringes) at different OD600 into N. benthamiana. GFP emission was observed 3 days post agroinfiltration (dpa) with an ultraviolet-absorbing filter, Fujifilm SC-52. (B) Agrobacterium carrying pBYR2HS-EGFP was resuspended either in agroinfiltration MES buffer (10 mM MES, pH 5.6, 10 mM MgCl2, 100 μM acetosyringone; MES) or Phosphate buffer (10 mM potassium phosphate, pH 5.6, 10 mM MgCl2, 100 μM acetosyringone; P buf.) at OD600=0.5, and used to agroinfiltrate N. benthamiana. GFP emission was observed with an ultraviolet-absorbing filter, Fujifilm SC-52, 3 dpa. (C) Agrobacterium resuspensions containing either pBYR2HS-EGFP, pTKB2-EGFP, or pTKB3-EGFP were adjusted to OD600=0.5 or 0.1 and agroinfiltrated into N. benthamiana. GFP emission was detected at 3 dpa. (D) Total soluble proteins, which were extracted from 0.2 mg FW N. benthamiana leaves agroinfiltrated with Agrobacterium carrying pBYR2HS-EGFP, pTKB2-EGFP, or pTKB3-EGFP, were separated by SDS-PAGE, and stained with Coomassie Brilliant Blue (CBB) dye. (E) The relative amount of GFP was calculated based on protein band intensities in CBB-stained gels using ImageJ software. Data represent means±SE (n=4–6).