RNA Interference Silences Genes at Post-Transcriptional Level Without Impacting Nascent RNA in Soybean Hairy Roots

RNA interference (RNAi) is an effective mechanism for reducing gene expression, therefore enabling the exploration of gene function. Its application in hairy root transient transformation facilitates rapid preliminary evaluation of gene function, specifically tackling the difficulties linked to steady transformation in soybean. The mechanisms of RNAi have been thoroughly investigated in model plants. Nonetheless, it remains ambiguous whether silencing proceeds at the transcriptional or post-transcriptional level in hairy roots. We analyzed RNA levels and phenotype by RNAi targeting four genes (GmIAA14, GmCPC, GmPHT1, and GmTTG1) in soybean roots. A reduction in mRNA levels is generally associated with an altered lateral root phenotype. However, the analysis of chromatin-bound/nascent RNAs during the identical RNAi events revealed no substantial decrease in their abundance. Moreover, the functional impairment of GmDCL2 or GmDCL3 did not influence RNA interference, indicating either redundancy or the presence of alternate routes for short RNA-mediated RNA interference in soybean hairy roots. Collectively, our findings underscore possible issues associated with the use of the hairy root transformation method for gene transcription studies. The lack of investigation into RNAi effects at the nascent RNA level, even in model plants, requires a re-evaluation of the current knowledge of RNAi mechanisms.