**2. Results**

#### *2.1. BSR1 Contributes to Bacterial MAMP-Induced Oxidative Bursts*

To assess the contribution of BSR1 to bacterium-derived MAMP-induced defense responses, we evaluated the effects of BSR1 knockout on defense responses using three independent *BSR1*-knockout lines. These lines were generated in our previous study and contain homozygous frameshift mutations in exon 1 of *BSR1* [39]. Suspension-cultured cells were derived from knockout and non-transgenic (wild-type) lines and treated with the bacterium-derived MAMPs peptidoglycan and LPS. After treatment with peptidoglycan, suspension-cultured cells derived from all three *BSR1*-knockout lines produced lower H2O2 concentrations than wild-type cells (Figure 1a; Supplementary Materials Table S2a). At 180 min after addition, 59%–71% of H2O2 production was lost in knockout cells. The LPS treatment also induced impaired oxidative bursts in *BSR1*-knockout cells (Figure 1b; Supplementary Materials Table S2b). These cells accumulated 38%–45% lower amounts of H2O2 compared with wild-type at 60 min after treatment. Knockout mutations in *BSR1* significantly suppressed the oxidative bursts but they were not completely abolished, indicating functional redundancy for BSR1. Thus, BSR1 plays a role in the induction of oxidative bursts in response to peptidoglycan and LPS.

**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.

To further provide support for the involvement of BSR1 in oxidative bursts against bacterial infections, autoclaved *X. oryzae* pv. *oryzae* cells were used as the elicitor. Knocking out *BSR1* reduced the production of H2O2 by 39%–58% at 60 min after treatment with the autoclaved cells (Figure 1c; Supplementary Materials Table S2c). Thus, BSR1 should contribute to defense responses against not only MAMPs purified from nonpathogenic microbes but also against the cellular components of pathogenic bacteria.
