Transient expression in Nicotiana benthamiana
Agrobacterium tumefaciens , strain GV3103, carrying plasmids with expression constructs (i) pB7WGF2:35S:GFP:SlACRE75; (ii) pB7WGF2:35S:GFP:SlACRE180; (iii) pB7WGF2:35S:GFP:NbACRE180; (iv) pB7WGF2:35S:GFP:NbACRE75 and; (v) pB7WGF2:35S:GFP (empty vector), were grown in YEP medium (containing 50 µg/ ml rifampicin, 100 µg/ ml spectinomycin, and 25 µg/ ml gentamicin) for 24 h with continuous shaking at 28°C. Overnight cultures were collected by centrifugation, resuspended in Agromix/infiltration buffer (10 mM MgCl2: 10 mM MES) and 200 µM acetosyringone (pH 5.7) and diluted to a final volume of 20 ml at OD600 of 0.1. Cultures were infiltrated into leaves of 4-week-old N. benthamiana plants using 1 ml needleless syringes. One day after agroinfiltration, 1-2 leaves per plant were excised for B. cinerea infection assays (as described above). These experiments were repeated once.