DOI: 10.5061/dryad.z08kprrts
title: Target genes, interacting proteins, ubiquitination sites, and primers for OsWRI1a functional analysis

description: Nitrogen (N) deficiency-induced increases in the root-to-shoot biomass
 ratio in plants are adaptive in nature but suboptimal for agriculture.
 Understanding the regulatory mechanisms governing this developmental
 plasticity could help improve crop performance while reducing fertilizer
 application. We identified OsWRI1a (WRINKLED1a) as a regulatory hub
 coordinating rice root and shoot growth in response to external N supply,
 thereby stabilizing the root-to-shoot ratio. In roots, OsWRI1a enhances
 N-responsive development by promoting auxin accumulation. Meanwhile, shoot
 OsWRI1a stimulates tiller development and therefore shoot growth. We
 identified an elite OsWRI1a haplotype that minimizes root-to-shoot ratio
 fluctuation under N deficiency, improving N-use efficiency (NUE) and grain
 yield. Our findings reveal a central mechanism coordinating N-responsive
 growth allocation for sustainable agriculture.
description: # Target genes, interacting proteins, ubiquitination sites, and primers
 for OsWRI1a functional analysis Dataset DOI:
 [10.5061/dryad.z08kprrts](https://doi.org/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 &lt; 0.01, fold change &gt; 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 &gt; 2 and *P*-value
 &lt; 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::*O*sWRI1a-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::*O*sWRI1a-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 BC~1~F~2~ and
 724 BC~2~F~2~ 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 ## Access information Other
 publicly accessible locations of the data: * 
 
 Data was derived from the following sources: *