Journal: bioRxiv

Article Title: Molding the Rice Methylome for Disease Resistance

doi: 10.64898/2026.05.03.722557

Figure Lengend Snippet: (A) Diagram of transformation and selection for obtaining homozygote T 2 transgenic lines containing the XVE:ROS1-YFP vector (XR1) and wild-type Nipponbare null segregant (NB*) from parental transformant line (T 0 ) (B) Validation of β-estradiol-dependent AtROS1 transcriptional expression in leaves of transgenic XR1 seedlings through RT-PCR, while germinated in liquid media containing 150μM β-estradiol (+) or mock (-) for 6 days (6d), and subsequent transcriptional deactivation after additional 11 days of growth in absence of β-estradiol (2.5w). (C) Validation of β-estradiol-dependent AtROS1 protein expression in transgenic XR1+ seedlings through tagged YFP Venus fluorescence imaging. Roots were imaged after 6d of germination in liquid media containing β-estradiol (+) or mock (-), and protein expression disappeared after additional 11 days of growth in absence of β-estradiol (2.5w; scale bar: 100µm) (D) Left panel: seed yields per plant for NB* and XR1 plants treated with β-estradiol (+) or mock (-) at 6d and grown until seed set. Differences in average seed number produced per plant between XR1 lines and NB* control were assessed statistically by two-tailed Student’s t-test ( n = six plants). Right panel: individual seed weight (in mg) across β-estradiol (+) or mock (-) treated NB* and XR1 rice plants. Center line indicates median, with lower and upper quartiles below and above; small crosses indicate mean; red line indicates grand mean seed weight across the six plants. Differences in seed weights between NB*-, XR1- and XR1+ plants were assessed with a linear mixed model, with replicates as a random grouping factor using SPSS ( p = 0.713). Post-hoc comparison were performed using Bonferroni-corrected estimated marginal means between NB*-, XR1- and XR1+ plants (small letters, n. s ). (E) Diagram of experimental setup for induction and analysis of within-generation and transgenerational methylome alterations and pathogen resistance phenotyping. Green dots indicate separate batches (i.e. seedlings/plants) used in each experiment.

Article Snippet: Powdered β-estradiol (TCI chemicals) was dissolved to 100mM stock concentration using DMSO (Fujifilm Wako chemicals).

Techniques: Transformation Assay, Selection, Transgenic Assay, Plasmid Preparation, Biomarker Discovery, Expressing, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Imaging, Produced, Control, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: Molding the Rice Methylome for Disease Resistance

doi: 10.64898/2026.05.03.722557

Figure Lengend Snippet: (A) Metaplots displaying average DNA methylation (all C contexts) in six-days old (6d) wild-type segregant NB* or transgenic XR1 rice seedlings germinated in liquid media containing 150μM β-estradiol (+) or mock (-) across all loci of Nipponbare telomere-to-telomere assembly (NIP-T2T), protein coding genes (PC) or transposable elements (TEs)/repeat elements (AGIS1.0 annotation). (B) Distribution of differentially methylated cytosines (DMCs) density at representative chromosomes 7 and 8 identified in β-estradiol-treated XR1 seedlings (+) vs mock-treated ones (-), separated by hypermethylated (red) or hypomethylated (blue). Above, yellow and grey density plots show distribution frequencies of PC or TEs loci, respectively. (C) Number of differentially methylated regions (DMRs) in 6d XR1+ vs XR1-seedlings, separated by hypermethylated (red) or hypomethylated (blue) (D) Venn diagrams: overlap (∩) between unique hypo- or hypermethylated DMRs in six-days old XR1+ vs XR1-seedlings with PC or TE loci (AGIS1.0 annotation); Table: number of unique genetic loci covered by one or more DMRs (percentage in parenthesis represents the proportion over the total number of respective loci in the annotation). (E) Browser view examples (IGV) of DMRs overlapping intergenic regions (left panels), PC gene (RAP ID Os09g0118666, middle panel) or both PC and TE (Locus ID LOC_Os02g17600, right panel). Blue bars: methylation % at cytosines in CG context, red bars: methylation % at cytosines in CHG contexts, green bars: methylation % at cytosines in CHH contexts. Positive and negative values represent methylation % of cytosines on the positive or negative strand, respectively.

Article Snippet: Powdered β-estradiol (TCI chemicals) was dissolved to 100mM stock concentration using DMSO (Fujifilm Wako chemicals).

Techniques: DNA Methylation Assay, Transgenic Assay, Methylation

Journal: bioRxiv

Article Title: Molding the Rice Methylome for Disease Resistance

doi: 10.64898/2026.05.03.722557

Figure Lengend Snippet: Metaplots displaying average DNA methylation separated by CG, CHG or CHH contexts in six-days old (6d) wild-type segregant NB* or transgenic XR1 rice seedlings germinated in liquid media containing 150μM β-estradiol (+) or mock (-) across all loci of Nipponbare telomere-to-telomere assembly (NIP-T2T), protein coding genes (PC) or transposable elements (TEs)/repeat elements (AGIS1.0 annotation).

Article Snippet: Powdered β-estradiol (TCI chemicals) was dissolved to 100mM stock concentration using DMSO (Fujifilm Wako chemicals).

Techniques: DNA Methylation Assay, Transgenic Assay

Journal: bioRxiv

Article Title: Molding the Rice Methylome for Disease Resistance

doi: 10.64898/2026.05.03.722557

Figure Lengend Snippet: Distribution of differentially methylated cytosines (DMCs) density at all Nipponbare chromosomes identified in β-estradiol-treated XR1 seedlings (+) vs mock-treated ones (-), separated by hypermethylated (red) or hypomethylated (blue). Above, yellow and grey density plots show distribution frequencies of PC or TEs loci, respectively.

Article Snippet: Powdered β-estradiol (TCI chemicals) was dissolved to 100mM stock concentration using DMSO (Fujifilm Wako chemicals).

Techniques: Methylation

Journal: bioRxiv

Article Title: Molding the Rice Methylome for Disease Resistance

doi: 10.64898/2026.05.03.722557

Figure Lengend Snippet: (A) Distribution of methylated cytosines (DMCs) density at representative chromosomes 7 and 8 identified between 2.5 weeks-old (2.5w) XR1 plants treated with 150μM β-estradiol (+) at six days-old (6d) vs mock-treated ones (-), separated by hypermethylated (red) or hypomethylated (blue). Lighter red and blue colors represent DMCs distribution in 6d seedlings (from ) for comparison. Above, yellow and grey density plots show distribution frequencies of PC or TE loci, respectively. (B) Number of differentially methylated regions (DMRs) identified in 2.5w XR1+ plants vs XR1-ones, separated by hypermethylated (red) or hypomethylated (blue). (C) Basal resistance phenotype of 2.5w NB* and XR1 plants against X. oryzae infection. Displayed are lesion lengths (in mm) in rice leaves caused by the pathogen eight days post-inoculation (dpi) though the leaf clipping method. Center line indicates median, with lower and upper quartiles below and above; small crosses indicate mean. Statistically significant differences in lesion length between XR1 lines and NB* control were assessed by two-tailed Student’s t-test ( n =30+ leaves from 15+ plants, *** p- value <0.001). (D) Overlap between DMRs identified in 6d and 2.5w XR1+ plants with 163 genes causally linked with X. oryzae basal resistance genes reported in ( Tonnessen et al ., 2019 ).

Article Snippet: Powdered β-estradiol (TCI chemicals) was dissolved to 100mM stock concentration using DMSO (Fujifilm Wako chemicals).

Techniques: Methylation, Comparison, Infection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Molding the Rice Methylome for Disease Resistance

doi: 10.64898/2026.05.03.722557

Figure Lengend Snippet: Distribution of methylated cytosines (DMCs) density at all Nipponbare chromosomes identified between 2.5 weeks-old (2.5w) XR1 plants treated with 150μM β-estradiol (+) at six days-old (6d) vs mock-treated ones (-), separated by hypermethylated (red) or hypomethylated (blue). Lighter red and blue colors represent DMCs distribution in 6d seedlings (from ) for comparison. Above, yellow and grey density plots show distribution frequencies of PC or TE loci, respectively.

Article Snippet: Powdered β-estradiol (TCI chemicals) was dissolved to 100mM stock concentration using DMSO (Fujifilm Wako chemicals).

Techniques: Methylation, Comparison

Journal: bioRxiv

Article Title: Molding the Rice Methylome for Disease Resistance

doi: 10.64898/2026.05.03.722557

Figure Lengend Snippet: (A) Number of differentially methylated regions (DMRs) identified in 2.5 weeks-old (2.5w) progeny XR1 plants (F 1 ) derived from parental lines treated with β-estradiol at their seedling stage (P 0 +, XR1-1 to XR1-6) vs parental lines treated with mock (P 0 -), separated by hypermethylated (red) or hypomethylated (blue). Gray boxes indicate the number of hypo- or hypermethylated DMRs overlapping between P 0 and F 1 (transgenerational DMRs). For all lines, the number of transgenerational DMRs have been found to be significantly higher than expected than occurring by chance after random shuffling of the genomic regions (10000 permutations, *** p- value <0.001). (B) Basal resistance phenotype of F 1 2.5w NB* and XR1 plants against X. oryzae infection. Displayed are lesion lengths (in mm) in rice leaves caused by the pathogen eight days post-inoculation (dpi) though the leaf clipping method. Center line indicates median, with lower and upper quartiles below and above; small crosses indicate mean. Statistically significant differences in lesion length between XR1 lines and NB* control were assessed in pairwise comparisons by two-tailed Student’s t-test ( n =40+ leaves from 20+ plants, ** p- value <0.01, *** p- value <0.001). (C) Ontology terms enrichment (biological processes) for genes overlapped by transgenerational hypo- and hypermethylated DMRs unique to X. oryzae -resistant lines XR1-1, XR1-4, XR1-4 and XR1-6. (D) Browser view example (IGV) of transgenerational DMRs maintained as DNA hypomethylated epiallele in line XR1-3 and XR1-6 across one generation, located in the promoter of USP37 (RAP ID Os10g0463300, yellow bar below represents the first exon). Blue bars: methylation % at cytosines in CG context, red bars: methylation % at cytosines in CHG contexts, green bars: methylation % at cytosines in CHH contexts. Positive and negative values represent methylation % of cytosines on the positive or negative strand, respectively.

Article Snippet: Powdered β-estradiol (TCI chemicals) was dissolved to 100mM stock concentration using DMSO (Fujifilm Wako chemicals).

Techniques: Methylation, Derivative Assay, Infection, Control, Two Tailed Test