Dataset DOI: 10.5061/dryad.z08kprrts

Description of the data and file structure

Data S1. Common OsWRI1a target genes identified from the CUT&Tag and RNA-seq analyses. 

Target genes were definedfrom CUT&Tag as those associated with significant binding peaks (P-value < 0.01, fold change > 1.5) found in the genomic region encompassing the entire gene and extending 4 kb upstream and downstream. The criteria for defining differentially expressed genes (DEGs) down-regulated in oswri1a are fold change > 2 and P-value < 0.05 in RNA-seq analysis.

Data S2. SNPs and Indels in the promoters of HJX74 OsWRI1a and OsWRI1b.

Data S3. OsWRI1a-interacting proteins. 

OsWRI1a-FLAG protein and its interacting proteins were captured by incubating extracted proteins from pAct::OsWRI1a-Flag transgenic lines using magnetic beads conjugated with the anti-Flag antibody (Sigma, F1804), and then eluted with 3×Flag peptide (Sigma-Aldrich, F4709). Proteins were electrophoretically separated by SDS-PAGE and gels were stained with Coomassie Brilliant Blue. The proteins were digested into peptides through steps such as reduction, alkylation, and enzymatic lysis (usually trypsin). Finally, the interaction proteins were identified through mass spectrometry analysis.

Data S4. Ubiquitination sites on OsWRI1a. 

OsWRI1a-FLAG protein and its interacting proteins were captured by incubating extracted proteins from pAct::OsWRI1a-Flag transgenic lines using magnetic beads conjugated with the anti-Flag antibody (Sigma, F1804), and then eluted with 3×Flag peptide (Sigma-Aldrich, F4709). Proteins were electrophoretically separated by SDS-PAGE and gels were stained with Coomassie Brilliant Blue. The proteins were digested into peptides through steps such as reduction, alkylation, and enzymatic lysis (usually trypsin). Finally, the ubiquitination sites were identified through mass spectrometry analysis.

Data S5. Information about the ubcs mutants.

We performed sequencing analysis on the corresponding UBC sequences for ubc1, ubc8, ubc9, ubc13, ubc21, ubc37, ubc45, and ubc48 mutants, respectively. The corresponding mutation types are detailed in the fourth column.

Data S6. Primer sequences used for map-based cloning and genotyping assays. 

We used these primers to identify OsWRI1a from 98 BC₁F₂ and 724 BC₂F₂ plants produced from the cross between R104 and Nipponbare.

Data S7. Primer sequences used for transgene and vector construction.

These primers were used to confirm the function and elucidate the mechanism of OsWRI1a.

Data S8. Primer sequences used for qPCR assays.

These primers were employed to evaluate the transcriptional abundances of the genes involved in this study.

Data S9. Primer sequences used for ChIP-qPCR assays.

These primers were utilized to assess the enrichment of downstream genes of OsWRI1a in ChIP-qPCR assays.

Data S10. Quantification data of cell-free assays.

The table provides quantitative data for all cell-free experiments illustrated in Figures 1-5 and Figures S1-22.

Data S11. OsWRI1a haplotypes of the 3 KG rice accessions.

For haplotype analyses of OsWRI1a, structural variations (SVs) and a genome-wide SNP dataset (~18 million SNPs) from 3,024 rice accessions were retrieved from the Rice SNP-Seek Database (https://snp-seek.irri.org). Variants were filtered based on the following criteria: (i) the minor allele frequency of at least 0.01, (ii) the proportion of missing calls per variant at most 0.2 SNPs and SVs located within gene bodies and their 2 kb upstream regulatory regions were extracted for phylogenetic reconstruction. A maximum-likelihood phylogeny was inferred using RAxML with 1000 bootstrap replicates. The resulting tree was visualized and annotated using the Interactive Tree of Life (iTOL) platform (https://itol.embl.de).

Data S12. Intersection of OsWRI1a, RNR10 and DNR1 haplotypes.

Haplotype distribution of OsWRI1a, RNR10 and DNR1.

Files and variables

File: Dataset_S1-S12.zip

Description: This file encompasses an analysis of target genes, interacting proteins, and ubiquitination sites of OsWRI1a, the primers for map-based cloning, genotyping, vector construction, qPCR and ChIP-qPCR assays, the quantification data from cell-free assays and the results of haplotype analysis.

Code/software

Excel

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Data was derived from the following sources: