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Herbicide resistance can be either target-site or non-target-site based. The molecular mechanisms underlying non-target-site resistance (NTSR) are poorly understood, especially at the level of gene expression regulation. MicroRNAs (miRNAs) represent key post-transcriptional regulators of eukaryotic gene expression and play important roles in stress responses. In this study, the  miR397  gene from  Beckmannia syzigachne  (referred to as  bsy-miR397 ) was functionally characterized to determine its role in regulating fenoxaprop- P -ethyl resistance. We showed that (1)  bsy-miR397  transcript level is constitutively higher in resistant than in sensitive  B. syzigachne  plants, whereas  bsy-Laccase  expression and activity show the opposite trend, and (2)  bsy-miR397  suppresses the expression of  bsy-Laccase  in tobacco, indicating that it negatively regulates  bsy-Laccase  at the transcriptional level. We found evidences that miR397/laccase regulation might be involved in fenoxaprop- P -ethyl NTSR. First, the rice transgenic line overexpressing OXmiR397 showed improved fenoxaprop- P -ethyl tolerance. Second, following activation of  bsy-Laccase  gene expression by CuSO 4  treatment, fenoxaprop resistance in  B. syzigachne  tended to decrease. Therefore, we suggest that  bsy-miR397  might play a role in fenoxaprop- P -ethyl NTSR in  B. syzigachne  by down-regulating laccase expression, potentially leading to the enhanced expression of three oxidases/peroxidases genes to introduce an active moiety into herbicide molecules in Phase-2 metabolism.  Bsy-miR397 ,  bsy-Laccase , and other regulatory components might form a regulatory network to detoxify fenoxaprop- P -ethyl in  B. syzigachne , supported by the differential expression of transcription factors and oxidases/peroxidases in the rice transgenic line overexpressing OXmiR397. This implies how down-regulation of a gene (laccase) can enhance NTSR. Our findings shed light on the daunting task of understanding and managing complex NTSR in weedy plant species.

miR397/Laccase Gene Mediated Network Improves Tolerance to Fenoxaprop-P-ethyl in Beckmannia syzigachne and Oryza sativa