Additional file 1 of Light Harvesting-like Protein 3 Interacts with Phytoene Synthase and Is Necessary for Carotenoid and Chlorophyll Biosynthesis in Rice
Creators: name: Yang, Feng
name: Debatosh, Das
name: Song, Tao
name: Zhang, Jian-hua
Publisher: name: figshare
Year: 2021
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: Plant Biology

description: Additional file 1 Figure S1. OsLIL3 sequence alignment between oslil3 and WT. (a), Sequence alignment between WT and oslil3 16-#1 mutant at DNA level. The red triangle indicates stop codon in the mutated OsLIL3 sequence that leads to premature translational termination while the blue triangle indicates end of truncated sequence due to translational termination. (b), amino acid sequence alignment between mutated oslil3 and WT. Figure S2. (a), Principal component analysis (PCA) was used to describe the overall transcriptome structure in the RNA-seq data. Normalized counts for all genes in the sequenced samples for both mutant and WT samples were projected onto the first two principal components; (b), Hierarchical clustering was used to group 6 main samples (according to the normalized counts of genes in the 3 mutant and 3 WT samples) onto a heatmap shown with its associated dendrogram. Darker colors denote greater similarity between samples. Figure S3. Gene Ontology (GO) enrichment analysis for oslil3 vs WT DEGs. Three types of GO enrichments are included: (a), Biological process; (b), Cellular components; (c), Molecular function. Figure S4. Top 20 enriched KEGG pathways for up and down-regulated DEGs of oslil3 vs WT. Figure S5. Protein-protein interaction between OsLIL3 and OsPSY1 &amp; OsPSY3. (a), Y2H assays. OsLIL3 without chloroplast transit peptide was fused with BD vector. OsPSY1 and OsPSY3 were fused with pGADKT7 vector. The combinations OsLIL3/empty vector (pGADT7), empty vector (pGABDKT7)/OsPSY1 and empty vector (pGBKT7)/OsPSY3 were included as negative controls. Yeast cells containing various vector combinations were grown on DDO plates for co-transformation selection. Yeast cells showing growth and blue color on high selection TDO/X/A plates confirmed positive interactions between the two indicated proteins. (b), OsLIL3-FLAG coimmunoprecipitated with OsPSY1-HA and OsPSY3-HA. Proteins were purified from tobacco leaves expressing OsLIL3-FLAG with OsPSY1-HA and OsPSY3-HA immunoprecipitated with an anti-Flag or anti-HA antibody. The presence of OsLIL3-FLAG, and OsPSY1-HA and OsPSY3-HA in the complex was determined by immunoblot analysis with Flag anti-bodies. (c), BiFC-visualized interactions between OsLIL3 and OsPSY1 and OsPSY3 in N. benthamiana. Abbreviations: YN, YFPN. YC, YFPC. Chl, chlorophyll autofluorescence; YFP, yellow fluorescent protein; BF, bright field. All bars = 5 μm. Figure S6. Y2H assay validation for interaction between OsLIL3 and other proteins involved in carotenoid and chlorophyll biosynthesis whose gene products were identified as DEGs in oslil3 vs WT RNA-seq. Protein sequence without chloroplast transit peptide was fused with Gal DNA-activation domain. The combinations OsLIL3 fused into BD vector and OsPSY2 fused into AD vector were included as positive controls. Yeast cells containing various vector combinations were grown on DDO plates (SD –Leu –Trp) for co-transformation selection. Yeast cells with blue color colony on high selection TDO/X/A plates confirmed positive interactions between the two indicated proteins while no growth signals in TDO/X/A suggest lack of interaction. Figure S7. Alignment and domain comparison of OsLIL3 and AtLIL3.1 &amp; AtLIL3.2 from rice and Arabidopsis. Protein sequences of these proteins were aligned using Jalview 2.0 (Waterhouse et al. 2009). The first transmembrane helix (THM) shows a conserved LHC motif, that is indicated by a black line below the respective sequence while the double black lines show the conserved functional globular domain. Figure S8. Comparison of the fold changes of 15 selected transcripts using two reference genes. Each blue point represents a chosen gene normalized to two reference genes, and the gene name is showed at the end of each linked line.