Abstract
Discovering transcription factor (TF) targets is necessary for the study of regulatory pathways, but it is hampered in plants by the lack of highly efficient predictive technology. This study is the first to establish a simple system for predicting TF targets in rice (Oryza sativa) leaf cells based on 10 × Genomics’ single-cell RNA sequencing method. We effectively utilized the transient expression system to create the differential expression of a TF (OsNAC78) in each cell and sequenced all single cell transcriptomes. In total, 35 candidate targets having strong correlations with OsNAC78 expression were captured using expression profiles. Likewise, 78 potential differentially expressed genes were identified between clusters having the lowest and highest expression levels of OsNAC78. A gene overlapping analysis identified 19 genes as final candidate targets, and various assays indicated that Os01g0934800 and Os01g0949900 were OsNAC78 targets. Additionally, the cell profiles showed extremely similar expression trajectories between OsNAC78 and the two targets. The data presented here provide a high-resolution insight into predicting TF targets and offer a new application for single-cell RNA sequencing in plants.
Highlights
DNA-binding proteins mediate gene regulation and are involved in most cellular process
Using the PEG-mediated transfection method, the transcription factor (TF) plasmids were introduced into protoplasts for expression
The interaction between OsNAC78 and the 19 candidate genes cannot be all clearly verified owing to experimental limitation, we found that genes having expression trajectories similar to the TF are more likely to be targets
Summary
DNA-binding proteins mediate gene regulation and are involved in most cellular process. The principle is simple, the technique is challenging, involving many experimental steps and requiring pre-optimization, and it is highly prone to operational errors and artifacts (Perna and Alberi, 2019; Yang et al, 2019) Another technique, microarray-based chromatin immunoprecipitation, requiring a specific antibody, is employed to identify TF targets, but often no significant DNA fragment enrichment is obtained (Mukherjee et al, 2004). For RNA-seq, millions of cells are processed in batches that may harbor potential differences, resulting in the generation of a large number of DEGs (Olsen and Baryawno, 2018) It is, difficult to subsequently verify the DNA-protein interactions. There are still many obstacles in using these technique to discover TF targets
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