Extended Data Fig. 2: Forward genetic screen isolates DMBQ non-responsive mutants.
From: Quinone perception in plants via leucine-rich-repeat receptor-like kinases
a, M2 seedlings were grown in vitro in a white 96-well plate, and used to screen against DMBQ. An image of the seedlings is shown on the left and the results obtained after the seedlings were challenged with DMBQ (5 μM) are shown on the right. Red circles indicate wells that did not have any seedlings, or in which seedlings are very small and give no signals. Yellow circles indicate wells that contained viable seedlings and showed a reduction in DMBQ response. Blue circles indicate seedlings that were viable but were not responsive to DMBQ. These plants were rescued and transferred onto the soil. b, Strategy to further narrow down mutant candidates. c, Results of the homozygosity tests and replications for each candidate. d, eATP response is normal in these candidates. For c and d, experiments were repeated three times with similar results. e, BC1F1 population from each candidate showed semi-dominant phenotypes. f, Allelism tests suggest that these candidates most probably have the mutation in the same gene. For e and f, experiments were performed twice with similar results. g, Table indicating which lines were crossed to perform allelism tests. Line 8 and line 10 were not tested as they were late-flowering compared to the other lines. h, Diagram of CARD1 gene structure, showing exons in blue boxes and introns as lines in between them. Point mutation positions in different card1 alleles, and T-DNA insert positions, are indicated. i, CARD1 is predominantly localized to the plasma-membrane-enriched fraction. Plasma membrane (PM), organelle membrane (OM) and soluble fraction (SF) were separated, and each fraction was probed with anti-CARD1, anti-AHA1 (plasma membrane marker), anti SMT1 (endoplasmic reticulum membrane marker), and anti-RbcL (soluble fraction marker). The localization of CARD1 in card1 loss-of functions alleles, which retained some protein expression, was also assessed. Experiments were performed twice with similar results.
