Sp8 laser confocal microscope

For protoplast transient expression assay, protoplasts were transfected with pHBT-35S::GFP, pHBT-35S::NUT-GFP or pHBT-35S::FLS2-GFP and incubated for 12 h. For N. benthamiana transient expression assays, A. tumefaciens strain GV3101 containing pMDC32-35S::GFP, pMDC32-35S::SCREW2-GFP, or pMDC32-35S::NUT-GFP was cultured overnight in LB medium (10 g l⁻¹ tryptone, 5 g l⁻¹ yeast extract, 5 g l⁻¹ NaCl) at 28 °C. The bacterial suspension containing 10 mM MES, pH 5.7, 10 mM MgCl₂ and 200 μM acetosyringone at an optical density at 600 nm (OD_{600 nm}) of 1.0 was infiltrated into leaves of four-week-old soil-grown N. benthamiana using a needleless syringe and incubated for 72 h. To detect SCREW1-induced NUT endocytosis, true leaves of seven-day-old Arabidopsis transgenic seedlings carrying pMDC32-35S::NUT-GFP were treated with water or 100 nM SCREW1 for 5 and 45 min and imaged using the Leica SP8 confocal laser microscope. The cotyledons of ten-day-old transgenic seedlings carrying p35S::GFP or p35S::NUT-GFP grown on ½MS plates were used to observe GFP and NUT–GFP subcellular localization. Fluorescence images were taken with the Leica SP8 confocal laser microscope. The excitation laser of 488 nm and 561 nm was used for imaging GFP and chloroplast fluorescence, respectively.