Fig. 1. OsRALF26, an Oryza-specific peptide acting as a damage-associated molecular pattern (DAMP), induces immune responses across monocots and dicots. A, OsRALF26 is taxonomically restricted to Oryza genus. Phylogenetic tree showing plant species with OsRALF26 homolog candidates (left). The BLAST E-value and the percentage of sequence identity were calculated using OsRALF26. Conserved motifs in OsRALF26 were identified by MEME software and are schematically displayed (right). In the sequence LOGOs, the X-axis shows motif width, and the Y-axis indicates bit score; letter height reflects position-specific probability. B‒D, Effects of OsRALF26 and OsRALF27 on disease resistance in rice (B), Arabidopsis (C), and tomato (D). In rice (B), recombinant proteins (1 μmol/L of GFP, OsRALF27mat, or OsRALF26mat) were sprayed onto rice leaves 1 d before Xanthomonas oryzae pv. oryzae (Xoo) inoculation. Representative leaves (left) and lesion length/bacterial population number (right) were obtained 14 d after inoculation. Data are mean ± SD (n = 9 for lesion length; 5 for bacterial population number). Rice + Mock served as the Xoo-susceptible control, and XA21-Rice + Mock was used as the Xoo-resistant control. Arrowheads indicate the bottom boundary of the lesion on each leaf. In Arabidopsis (C) and tomato (D), recombinant proteins (1 μmol/L of GFP, OsRALF27mat, or OsRALF26mat) were infiltrated into leaves 1 d before Pst DC3000 inoculation. Representative leaves (up) and bacterial population number (down) were obtained 3 d after inoculation. Data are mean ± SD (n = 5). +Mock (non-pretreated Pst DC3000) served as the susceptible control, and +flg22 (flg22-pretreated Pst DC3000) served as the resistant control. Scale bars, 2 cm. Different lowercase letters above bares indicate significant differences (P < 0.05, one-way analysis of variance, Tukey’s test). E‒I, Effects of OsRALF26 and OsRALF27 on root growth in rice (E), wheat (F), Arabidopsis (G), tomato (H), and Nicotiana benthamiana (I). Seedlings were grown on Murashige and Skoog (MS) medium containing 1 μmol/L of each recombinant protein (OsRALF26mat, OsRALF27mat, or GFP). Representative seedlings (left) and root length (right) were obtained after 5‒12 d of growth. Red arrowheads indicate root tips. Scale bars, 2 cm. Data are mean ± SD (n = 5). Different lowercase letters above bars indicate significant differences (P < 0.05, one-way analysis of variance, Tukey’s test). J‒N, Effects of OsRALF26 and OsRALF27 on reactive oxygen species (ROS) production in rice (J), wheat (K), Arabidopsis (L), tomato (M), and N. benthamiana (N). Time course of ROS production over 120 min in leaf discs treated with 1 μmol/L of each recombinant protein (GFP, OsRALF27mat, or OsRALF26mat). An inset graph displays ROS burst induced by flg22 (positive control) and Mock (negative control), which were included in the same experiment but shown separately to avoid visual complexity. Data are mean ± SD (n = 4). All experiments were repeated three times with consistent results. GFP, Green fluorescent protein; RLUs, Relative light units; cfu, Colony forming units.
Fig. 1. OsRALF26, an Oryza-specific peptide acting as a damage-associated molecular pattern (DAMP), induces immune responses across monocots and dicots. A, OsRALF26 is taxonomically restricted to Oryza genus. Phylogenetic tree showing plant species with OsRALF26 homolog candidates (left). The BLAST E-value and the percentage of sequence identity were calculated using OsRALF26. Conserved motifs in OsRALF26 were identified by MEME software and are schematically displayed (right). In the sequence LOGOs, the X-axis shows motif width, and the Y-axis indicates bit score; letter height reflects position-specific probability. B‒D, Effects of OsRALF26 and OsRALF27 on disease resistance in rice (B), Arabidopsis (C), and tomato (D). In rice (B), recombinant proteins (1 μmol/L of GFP, OsRALF27mat, or OsRALF26mat) were sprayed onto rice leaves 1 d before Xanthomonas oryzae pv. oryzae (Xoo) inoculation. Representative leaves (left) and lesion length/bacterial population number (right) were obtained 14 d after inoculation. Data are mean ± SD (n = 9 for lesion length; 5 for bacterial population number). Rice + Mock served as the Xoo-susceptible control, and XA21-Rice + Mock was used as the Xoo-resistant control. Arrowheads indicate the bottom boundary of the lesion on each leaf. In Arabidopsis (C) and tomato (D), recombinant proteins (1 μmol/L of GFP, OsRALF27mat, or OsRALF26mat) were infiltrated into leaves 1 d before Pst DC3000 inoculation. Representative leaves (up) and bacterial population number (down) were obtained 3 d after inoculation. Data are mean ± SD (n = 5). +Mock (non-pretreated Pst DC3000) served as the susceptible control, and +flg22 (flg22-pretreated Pst DC3000) served as the resistant control. Scale bars, 2 cm. Different lowercase letters above bares indicate significant differences (P < 0.05, one-way analysis of variance, Tukey’s test). E‒I, Effects of OsRALF26 and OsRALF27 on root growth in rice (E), wheat (F), Arabidopsis (G), tomato (H), and Nicotiana benthamiana (I). Seedlings were grown on Murashige and Skoog (MS) medium containing 1 μmol/L of each recombinant protein (OsRALF26mat, OsRALF27mat, or GFP). Representative seedlings (left) and root length (right) were obtained after 5‒12 d of growth. Red arrowheads indicate root tips. Scale bars, 2 cm. Data are mean ± SD (n = 5). Different lowercase letters above bars indicate significant differences (P < 0.05, one-way analysis of variance, Tukey’s test). J‒N, Effects of OsRALF26 and OsRALF27 on reactive oxygen species (ROS) production in rice (J), wheat (K), Arabidopsis (L), tomato (M), and N. benthamiana (N). Time course of ROS production over 120 min in leaf discs treated with 1 μmol/L of each recombinant protein (GFP, OsRALF27mat, or OsRALF26mat). An inset graph displays ROS burst induced by flg22 (positive control) and Mock (negative control), which were included in the same experiment but shown separately to avoid visual complexity. Data are mean ± SD (n = 4). All experiments were repeated three times with consistent results. GFP, Green fluorescent protein; RLUs, Relative light units; cfu, Colony forming units.
