Silencing of NbCMT3s has Pleiotropic Effects on Development by Interfering with Autophagy-Related Genes in Nicotiana benthamiana.

DNA methylation is a chromatin mark that has a crucial role in regulating gene expression. The chromomethylase (CMT) protein family is a plant-specific DNA methyltransferase that mediates growth and development. However, the roles of CMT3 in autophagy remain to be elucidated. Here, we identified the potential targets of CMT3 in Nicotiana benthamiana (NbCMT3) during developmental programs. Virus-induced gene silencing of NbCMT3/3-2 in N. benthamiana had pleiotropic effects on plant morphology, which indicates its indispensible role in development. Genome-wide transcriptome analysis of NbCMT3/3-2-silenced plants revealed interference with genes related to autophagy and ubiquitination. The expression of NbBeclin 1 and NbHRD1B was higher in NbCMT3/3-2-silenced than control plants. The formation of autophagosomes and starch degradation was disrupted in NbCMT3/3-2-silenced plants, which implies a perturbed autophagic processes. We further generated transgenic N. benthamiana plants carrying a chimeric promoter-reporter construct linking the NbBeclin 1 promoter region and β-glucuronidase (GUS) reporter (pNbBeclin::GUS). NbBeclin 1 promoter activity was significantly enhanced in NbCMT3/3-2-silenced plants. Thus, NbCMT3/3-2 silencing had pleiotropic effects on development by interfering with NbBeclin 1 expression and autophagy-related processes.