pBYR2T-EGFP

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	Briefly, the vectors described above and GFP_pICH18711 kindly provided by Dr. Victor Klimyuk (Icon Genetics GmbH) were transformed into A. tumefaciens GV3101. A. tumefaciens GV3101 harboring the binary vector was grown in L-broth media containing 10 mM MES (pH 5.6), 20 μM acetosyringone, 100 mg/L of kanamycin, 30 mg/L gentamycin, 30 mg/L of rifampin to the stationary phase at 28 °C. After centrifugation, A. tumefaciens was resuspended in the infiltration buffer (10 mM MgCl2, 10 mM MES (pH 5.6), 100 μM acetosyringone) to adjust OD600 = approximately 1.

Protocol tips

Briefly, the vectors described above and GFP_pICH18711 kindly provided by Dr. Victor Klimyuk (Icon Genetics GmbH) were transformed into A. tumefaciens GV3101. A. tumefaciens GV3101 harboring the binary vector was grown in L-broth media containing 10 mM MES (pH 5.6), 20 μM acetosyringone, 100 mg/L of kanamycin, 30 mg/L gentamycin, 30 mg/L of rifampin to the stationary phase at 28 °C. After centrifugation, A. tumefaciens was resuspended in the infiltration buffer (10 mM MgCl2, 10 mM MES (pH 5.6), 100 μM acetosyringone) to adjust OD600 = approximately 1.

Publication protocol

The preparation of Agrobacterium and transient expression in N. benthamiana was performed as previously described35 with modifications. Briefly, the vectors described above and GFP_pICH18711 kindly provided by Dr. Victor Klimyuk (Icon Genetics GmbH) were transformed into A. tumefaciens GV3101. A. tumefaciens GV3101 harboring the binary vector was grown in L-broth media containing 10 mM MES (pH 5.6), 20 μM acetosyringone, 100 mg/L of kanamycin, 30 mg/L gentamycin, 30 mg/L of rifampin to the stationary phase at 28 °C. After centrifugation, A. tumefaciens was resuspended in the infiltration buffer (10 mM MgCl2, 10 mM MES (pH 5.6), 100 μM acetosyringone) to adjust OD600 = approximately 1. The suspension was infiltrated with a 1-mL syringe without a needle into the abaxial air spaces of 4-week-old leaves of N. benthamiana. The suspension was infiltrated into 4-week-old leaves of tomatoes, Solanum lycopersicum cv. ‘Micro-Tom’, 4-week-old leaves of eggplants, Solanum melongena cv. ‘Dewakonasu’, 4-week-old leaves of hot peppers, Capsicum frutescens L., 3-week-old leaves of melons, Cucumis melo cv. ‘Earl’s Favorite Harukei No. 3′, and petals of commercially produced orchids, Phalaenopsis aphrodite and the rose, Rosa sp. ‘Bonheur’. For infiltration into tomato fruits of S. lycopersicum cv. ‘M82’, a 1-mL syringe with a needle was used. Three days after infiltration, blue LED lamp was shed onto plants. GFP emission was detected with an ultraviolet absorbing filter (SC-52, Fujifilm).

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