RNA polymerase IV is essential for tomato embryo development through mediating non-CG methylation [RNA-Seq]
Background: Plant-unique RNA polymerase IV (Pol IV) governs the establishment of small RNA-directed DNA methylation (RdDM) and contributes to the maintenance of transposable element (TE) silencing. Pol IV-dependent RdDM is highly active during embryonic development, and its absence occasionally reduces seed sets in species with high TE content. However, the mechanism underlying embryonic development failure due to the loss of Pol IV-RdDM remains largely unclear. Results: Here, we report that the loss-of-function of SlNRPD1, encoding the largest subunit of the Pol IV complex leads to aberrant embryogenesis in tomato (Solanum lycopersicum). The slnrpd1 mutants show non-CG hypomethylation and a burst of 21/22-nt sRNA from the distal chromatin region. We found that non-CG hypomethylation at the gene body region is generally associated with the activation of transcription, and knocking out DEMETER-like DNA demethylases cannot reverse slnrpd1 embryo lethality. mCHH hypomethylation in slnrpd1 disrupts gene expression related to auxin and WUSCHEL-related homeobox (WOX) signaling. TE reactivation in slnrpd1 mainly affects the expression of a limited number of nearby genes rather than contributing to excessive sRNA. Conclusion: We conclude that Pol IV-mediated RdDM is essential for tomato embryogenesis via non-CG methylation. mCHH hypomethylation in the slnrpd1 embryo leads to abnormal auxin/WOX signaling and TE reactivation which probably disrupts the tissue and organ initiation. This study provides new insights into the epigenetic regulation of gene and TE expression during embryogenesis. Keywords: DNA methylation, RdDM, NRPD1, Embryogenesis, Tomato Overall design: To investigate the function of NRPD1 in tomato embryogenesis,  we collected 21 days after flowering (DAF) embryos of wild type and slnrpd1 to perform RNA-sequencing.