Rice Protoplast Cell Death Assay

The rice protoplast cell death assay was performed as a per previously described method [56 (link)]. The open reading frame of full-length candidate effector genes was amplified from the M. oryzae P131 cDNA library with the primers (Table S2) and sub-cloned into the entry vector pENTR^TM1A (A10462, Invitrogen, Waltham, MA, USA). Then, the gateway-compatible vector pIPKb002 was used for the expression of these candidate effector genes [57 (link)]. The stem and sheath of 14-day-old etiolated rice seedlings were cut into 1.5 mm strips and soaked in a protoplast isolation buffer for 3 h in the dark with gentle shaking (50 rpm/min). Then the recombinant vectors were transfected into rice protoplast, which was suspended in transfection buffer 1 to a concentration of 3.5 × 10⁵ cells/mL. A firefly luciferase (LUC) reaction with the lysate was performed using the Luciferase Assay System (E1500, Promega, Madison, GA, USA), and the LUC activity was detected using a luminometer (F200, Tecan, Männedorf, ZRH, CH).