Multi gauge software bas 1000

For examining RSS activity of CPs and CRPs in N. benthamiana and onion plants, GFP, CP and CRP-FLAG genes were cloned into the pBE2113 binary vector, and Agrobacterium was transformed with those constructs. Bacterial cultures were adjusted to OD₆₀₀ of 1.0 for N. benthamiana and 0.2 for onion in the resuspension buffer described above. The bacterial suspensions of GFP and CP/CRP were mixed at 1:2 with the resuspension buffer for N. benthamiana and 1:1 for onion. Plants were agroinfiltrated as described above, and GFP fluorescence was assessed using blue light at 5 dpa. GFP was detected in western blots using anti-GFP antibodies and quantified using Multi Gauge Software (BAS-1000, Fujifilm, Tokyo, Japan). For the assay using onion cells, 0.2~0.3 mL of the bacterial inocula were used to infiltrate onion bulbs with a 1-mL syringe and 0.5 × 25 mm needle. Cells were checked for GFP fluorescence at 3 dpa using an epifluorescence microscope. The fluorescence intensity was quantified using LAS AF software (Leica Microsystems).

Total protein extracts from the infiltration patches on N. benthamiana leaves were separated in 12% polyacrylamide gel by SDS-PAGE. The GFP level in each sample was detected by western blot using anti-GFP antibodies. The relative accumulation level of GFP was calculated using Multi Gauge Software (BAS-1000, Fujifilm, Tokyo, Japan).