AUTHOR=Xu Yingru , Ji Xiaotong , Xu Zhuangzhuang , Yuan Yanping , Chen Xiling , Kong Derong , Zhang Yanlong , Sun Daoyang 

TITLE=Transcriptome Profiling Reveals a Petunia Transcription Factor, PhCOL4, Contributing to Antiviral RNA Silencing

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.876428

DOI=10.3389/fpls.2022.876428

ISSN=1664-462X

ABSTRACT=<p>RNA silencing is a common antiviral mechanism in eukaryotic organisms. However, the transcriptional regulatory mechanism that controls the RNA silencing process remains elusive. Here, we performed high-depth transcriptome analysis on petunia (<italic>Petunia hybrida</italic>) leaves infected with tobacco rattle virus (TRV) strain PPK20. A total of 7,402 differentially expressed genes (DEGs) were identified. Of them, some RNA silencing-related transcripts, such as <italic>RNA-dependent RNA polymerases</italic> (<italic>RDR</italic>s), <italic>Dicer-like RNase III enzymes</italic> (<italic>DCL</italic>s), and <italic>Argonautes</italic> (<italic>AGO</italic>s), were induced by viral attack. Furthermore, we performed TRV-based virus-induced gene silencing (VIGS) assay on 39 DEGs encoding putative transcription factors (TFs), using green fluorescent protein (GFP) and <italic>phytoene desaturase</italic> (<italic>PhPDS</italic>) as reporters. Results showed that the down-regulation of <italic>PhbHLH41</italic>, <italic>PhbHLH93</italic>, <italic>PhZPT4-3</italic>, <italic>PhCOL4</italic>, <italic>PhHSF</italic>-<italic>B3A</italic>, <italic>PhNAC90</italic>, and <italic>PhWRKY75</italic> led to enhanced TRV accumulation and inhibited <italic>PhPDS</italic>-silenced photobleaching phenotype. In contrast, silencing of <italic>PhERF22</italic> repressed virus accumulation and promoted photobleaching development. Thus, these TFs were identified as potential positive and negative regulators of antiviral RNA silencing, respectively. One positive regulator <italic>PhCOL4</italic>, belonging to the B-box zinc finger family, was selected for further functional characterization. Silencing and transient overexpression of <italic>PhCOL4</italic> resulted in decreased and increased expression of several RNA silencing-related genes. DNA affinity purification sequencing analysis revealed that PhCOL4 targeted <italic>PhRDR6</italic> and <italic>PhAGO4</italic>. Dual luciferase and yeast one-hybrid assays determined the binding of PhCOL4 to the <italic>PhRDR6</italic> and <italic>PhAGO4</italic> promoters. Our findings suggest that TRV-GFP-<italic>PhPDS</italic>-based VIGS could be helpful to identify transcriptional regulators of antiviral RNA silencing.</p>