Small RNAs, big impact: miRNAs mediated regulation of herbicide resistance in Echinochloa crus-galli and Echinochloa oryzicola

reductions in crop yields and substantial economic losses. Although the physiological and molecular mechanisms underlying herbicide resistance have been widely studied, the role of epigenetics, especially mechanisms regulating the expression of genes involved in herbicide resistance occurrence, has only recently begun to be investigated. This study aims to explore the miRNA-mediated regulation of enzymes involved in herbicide detoxification in two of the most problematic weed species in Northern Italian rice cropping territory: Echinochloa crus-galli (L.) P. Beauv. (barnyardgrass) and Echinochloa oryzicola (Vasinger) Vasinger (late watergrass). This research focuses on miRNAs predicted to target genes encoding cytochrome P450 monooxygenases (CYPs), glutathione S-transferases (GSTs) and eIF4B, key components of herbicide detoxification pathways. Seeds from plants survived herbicide administration were germinated and grown under controlled conditions. Plant susceptibility/resistance was assessed via visual inspection and biomass measurement. The expression profiles of predicted miRNAs and their gene targets were analyzed by RT-qPCR before and after herbicide application in both susceptible (SUS) and resistant (RES) biotypes.In E. crus-galli, after bispyribac-Na treatment, five miRNAs (gra- miR7487c, gma-miR396f, gra-miR8759, osa-miR395f, and ath-miR847) were upregulated in both biotypes, with higher expression in RES plants. This correlated with reduced expression of target genes (CYP72A254, GSTF1, eIF4B, CYP81A68, CYP71AK2), suggesting post- transcriptional silencing. Three other miRNAs (ata-miR166c-5p, ath-miR396b-5p, osa- miR5538) were unaDected by herbicide application and showed no biotype-specific diDerences. In E. oryzicola, profoxydim treatment led to distinct patterns. In RES biotypes, some miRNAs (osa-miR2099-5p, ath-miR396b, osa-miR395f, osa-miR396a-5p, osa-miR166a-5p, osa- miR166d-5p, gra-miR8759, and gma-miR396f) were not triggered, allowing expression of cytochromes P450 of the 81A subfamily, GSTF1 and eIF4B. The other miRNAs (ata-miR166c- 5p, ath-miR847, osa-miR5538, gra-miR7487c) were upregulated, reducing the expression of CYP71AK2, CYP72A254, and EcGST. In SUS biotypes, the herbicide stimulated ata-miR166c- 5p, ath-miR847, osa-miR5538, gra-miR7487c, osa-miR166a-5p, and gra-miR8759, down- regulating their respective target genes (CYP72A122, CYP71AK2, EcGST, CYP72A254, CYP81A12, and eIF4B). These results indicate that miRNA-mediated regulation plays a significant role in modulating herbicide resistance occurrence through gene silencing in Echinochloa species. Elucidating the miRNA-mediated regulation of herbicide resistance can enhance targeted, sustainable weed control and optimize precision weed management (PWM). miRNAs also show promise as biopesticides due to their cross-organism mobility and gene regulatory roles.