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, 20 (22)

Broad-Spectrum Disease Resistance Conferred by the Overexpression of Rice RLCK BSR1 Results From an Enhanced Immune Response to Multiple MAMPs

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Broad-Spectrum Disease Resistance Conferred by the Overexpression of Rice RLCK BSR1 Results From an Enhanced Immune Response to Multiple MAMPs

Yasukazu Kanda et al. Int J Mol Sci.

Abstract

Plants activate their immune system through intracellular signaling pathways after perceiving microbe-associated molecular patterns (MAMPs). Receptor-like cytoplasmic kinases mediate the intracellular signaling downstream of pattern-recognition receptors. BROAD-SPECTRUM RESISTANCE 1 (BSR1), a rice (Oryza sativa) receptor-like cytoplasmic kinase subfamily-VII protein, contributes to chitin-triggered immune responses. It is valuable for agriculture because its overexpression confers strong disease resistance to fungal and bacterial pathogens. However, it remains unclear how overexpressed BSR1 reinforces plant immunity. Here we analyzed immune responses using rice suspension-cultured cells and sliced leaf blades overexpressing BSR1. BSR1 overexpression enhances MAMP-triggered production of hydrogen peroxide (H2O2) and transcriptional activation of the defense-related gene in cultured cells and leaf strips. Furthermore, the co-cultivation of leaves with conidia of the blast fungus revealed that BSR1 overexpression allowed host plants to produce detectable oxidative bursts against compatible pathogens. BSR1 was also involved in the immune responses triggered by peptidoglycan and lipopolysaccharide. Thus, we concluded that the hyperactivation of MAMP-triggered immune responses confers BSR1-mediated robust resistance to broad-spectrum pathogens.

Keywords: Oryza sativa (rice); Pyricularia oryzae (formerly Magnaporthe oryzae); disease resistance; microbe-associated molecular pattern (MAMP); reactive oxygen species (ROS); receptor-like cytoplasmic kinase (RLCK).

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Knockouts of BSR1 impaired H2O2 production in rice cell cultures treated with MAMPs. Suspension-cultured cells were treated with peptidoglycan (a), LPS (b), or an autoclaved suspension of Xanthomonas oryzae pv. oryzae (Xoo; c). H2O2 concentrations were measured before treatment and at 20, 60, and 180 min after treatment. Values are presented as the means ± standard deviations of three biological replicates. Experiments were conducted twice with similar results. PGN, peptidoglycan; LPS, lipopolysaccharide; KO, knockout line; KO#1, bsr1-1#13-1; KO#2, bsr1-2#16-2; KO#8, bsr1-8#5-1; WT, wild-type; MAMPs, microbe-associated molecular patterns; BSR1, BROAD-SPECTRUM RESISTANCE 1. The statistical analysis was performed as shown in Supplementary Materials Table S2.
Figure 2
Figure 2
MAMP-induced transcript levels of defense-related genes were suppressed in BSR1-knockout suspension-cultured rice cells. The PBZ1, PAL1, KSL4, and DPF transcript levels at 3-h post treatment with peptidoglycan (a) and LPS (b) were normalized against the RUBQ1 internal control levels. Values are presented as the means ± standard deviations of three biological replicates. Experiments were conducted two times with similar results. Different letters indicate significant differences (Tukey’s test; p < 0.05). PGN, peptidoglycan; KO, knockout line; KO#1, bsr1-1#13-1; KO#2, bsr1-2#16-2; KO#8, bsr1-8#5-1; WT, wild-type; LPS, lipopolysaccharide.
Figure 3
Figure 3
The overexpression of BSR1-HPB enhanced peptidoglycan-induced oxidative bursts in suspension-cultured rice cells. Cells treated with peptidoglycan were analyzed for H2O2 production accompanying oxidative bursts (a) and the transcript levels of defense-related genes (b). Values are presented as the means ± standard deviations of three biological replicates. In (a), H2O2 concentrations were measured before treatment and at 20, 60, and 180 min after treatment. The statistical analysis was performed as shown in Figure S2a. Experiments were conducted three times with similar results. In (b), the PBZ1, PAL1, and KSL4 transcript levels were normalized against the RUBQ1 internal control levels. Experiments were conducted two times with similar results. Different letters indicate significant differences (Tukey’s test; p < 0.05). PGN, peptidoglycan; OX, overexpressing line; HPB, HA–PreScission–Biotin; OX#17, BSR1-HPB:OX#17; OX#39, BSR1-HPB:OX#39; GUS, GUS-HPB:OX; WT, wild-type.
Figure 4
Figure 4
The overexpression of BSR1-HPB enhanced chitin-induced defense responses in suspension-cultured rice cells. Values are presented as the means ± standard deviations of three biological replicates. (a) H2O2 concentrations were measured before treatment and at 20, 60, and 180 min after treatment. The statistical analysis was performed as shown in Figure S2b. Experiments were conducted three times with similar results. (b) The PBZ1, PAL1, and KSL4 transcript levels were normalized against the RUBQ1 internal control levels. Experiments were conducted twice with similar results. Different letters indicate significant differences (Tukey’s test; p < 0.05). CE, chitin elicitor; OX, overexpressing line; HPB, HA–PreScission–Biotin; OX#17, BSR1-HPB:OX#17; OX#39, BSR1-HPB:OX#39; GUS, GUS-HPB:OX; WT, wild-type.
Figure 5
Figure 5
Rice leaf strips derived from BSR1-HPB:OX plants caused an enhanced burst of H2O2 when exposed to conidia of the compatible blast fungus. Leaf strips were cultivated with 8 × 104 mL−1 autoclaved conidia (a) or 8 × 103 mL−1 living conidia (b) in wells of a 12-well plate. H2O2 concentrations in wells were measured before treatment and at 60, 180, and 300 min after treatment. Values are presented as the means ± standard deviations of three biological replicates. Asterisks indicate significant differences between the untreated condition (0 min) values and the values at the indicated times in the same line (Student’s t-test; * p < 0.05, ** p < 0.01, and *** p < 0.001). Experiments were conducted twice with similar results. OX, overexpressing line; HPB, HA–PreScission–Biotin; OX#17, BSR1-HPB:OX#17; GUS, GUS-HPB:OX.
Figure 6
Figure 6
Proposed model in which BSR1 regulates defense responses, such as oxidative bursts, after the perception of MAMPs in wild-type (WT; left) and BSR1-overexpressing rice lines (BSR1-OX; right). PGN, peptidoglycan; LPS, lipopolysaccharide; RLPs, receptor-like proteins; ROS, reactive oxygen species; MTI, MAMP-triggered immunity.

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