Nitrosourea Compounds

Adult zebrafish were housed according to Institutional Animal Care and Use Committee protocols at Oregon State University on a recirculating system with water temperature of 28±1°C and a 14 h light/10 h dark schedule. Zebrafish embryos carrying a point mutation in ahr2 (ahr2^hu3335 strain) were requested and generously provided by the Hubrecht Institute. The ahr2^hu3335 line was identified from a library of N-ethyl-N-nitrosourea (ENU)-mutagenized zebrafish using the TILLING method as previously described [51] (link). Offspring of heterozygous ahr2^hu3335 carriers were raised to adulthood at the Sinnhuber Aquatic Research Laboratory, and genotyped for the ahr2^hu3335 point mutation with DNA isolated from fin clips [51] (link). PCR amplification was performed with genomic DNA and ahr2 gene-specific primers (Table 4), the product was purified using a QIAquick PCR purification kit (Qiagen) and sequenced with an ABI 3730 capillary sequencer at the Center for Genome Research and Biocomputing at Oregon State University. Homozygous carriers of the T to A point mutation in residue 534 (Figure 1) were identified to create an ahr2^hu3335 population. Because the TILLING method relies on random mutagenesis, mutant lines of interest carry other mutations throughout the genome. F1 fish are predicted to carry 3–6000 mutations and multiple outcrosses are necessary to reduce off-target mutations [52] (link). ahr2^hu3335 carriers were outcrossed to the wild type 5D (ahr2^+/+) line, and homozygous mutants were identified from an incross of their progeny. The ahr2^hu3335 mutant line has been maintained with subsequent outcrosses on the wild type 5D background, which was also used for all ahr2⁺ control experiments in our laboratory.
All developmental toxicity experiments were conducted with fertilized embryos obtained from group spawns of adult zebrafish as described previously [53] (link). Embryos used in experiments are defined as homozygous (ahr2^hu3335), heterozygous (ahr2^hu3335/+) or wild-type (ahr2⁺) for the point mutation in AHR2.

NB is a traditional lowland indica cultivar that originated in India and is resistant to BSR caused by Burkholderia glumae. KO is a modern lowland rice cultivar released in Japan and is susceptible to BSR^{27 (link)}. To analyse RBG1res, by crossing SL535 with KO and using marker-assisted selection to remove nontarget DNA regions, we successfully developed a NIL homozygous for RBG1res. The resulting RBG1res-NIL contains approximately 380 kb from NB on the short arm of chromosome 10 (between simple sequence repeat (SSR) markers RM474 and RM7361-1; Supplementary Table S3). By screening 3072 M₂ lines of KO mutagenized with N-methyl-N-nitrosourea according to a method described previously^{31 (link)}, we identified a null mutant (Mut-W56*) whose sequence encoding tryptophan at position 56 was changed such that a stop codon was introduced that produces a truncated protein (5.5 kD). Genomic DNA of the M₂ plants was screened with the NB51 primer set listed in Supplementary Table S3 by the targeting induced local lesions in genomes (TILLING) method as described earlier^{52 (link)}. All of the experimental research and field studies on plants (either cultivated or wild), including transgenic plant materials, complied with relevant institutional, national, and international guidelines and legislation.

Male and female B6 wild-type (WT) mice, B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (ROSA-td-Tomato) mice, and B6.CXB1-Pde6βrd10/J (rd10) mice were used. ROSA-td-Tomato and rd10 mice were purchased from the Jackson Laboratory. The mice were bred and reared under cyclic light (12 hours light: 12 hours dark). To perform the experiments, mice were anesthetized via intraperitoneal injection of 15 mg/kg ketamine and 7 mg/kg xylazine. The chemical-induced retinal degeneration model was created via intraperitoneal administration of a single dose of N-methyl-N-nitrosourea (MNU) to mice. After anesthesia, the mice were weighed, and injected with MNU (30−75 mg/kg). All mice were treated in accordance with the standards of the Association for Research in Vision and Ophthalmology for the use of animals in ophthalmic and vision research. All animal experiments were reviewed and approved by the Kyushu University Ethics Committee for Animal Experimentation and were conducted in accordance with the relevant guidelines and regulations (A20-360, A21-225).