Barley Protoplast Transfection and Luciferase Assay

The isolation and transfection of barley protoplasts was performed as described in Saur et al., 2019 [33 (link)]. In short, cDNAs of the AVR_as were co-expressed with cDNAs of Mla10, Mla22, Mla10Lrr22, and Mla22Lrr10 using the pIPKb002 vector with a strong ubiquitin promoter or with intron-containing DNA of chimeras M16666, M11166, M61111, or M66111 in a pUBI-NOS-vector (described in Shen et al., 2003 [38 (link)]) in barley cv. Golden Promise protoplasts. Protoplast solution (300 μl of 3.5 x 10⁵ cells/ml) was transfected with 4.5 μg of LUC reporter construct, 10 μg of Mla plasmid, and 6.5 μg of the respective AVR_a effector or an empty vector (EV). The protoplasts were incubated for 16 h at 21°C in a plant growth chamber and then harvested by centrifugation at 1,000 x g. Subsequently, the supernatant was removed, and protoplasts were lysed by addition of 180 μl of cell culture lysis reagent (Promega, E1531). The LUC activity of samples was measured in a luminometer (Centro, LB960) using a 96-well plate in which 50 μl of protoplast lysate were mixed with 50 μl of the LUC substrate (Promega, E1501). The relative LUC units (RLU) were calculated by setting the absolute value of the EV sample to 1.