A microRNA‐1 gene, tci‐miR‐1‐3p, is involved in cyflumetofen resistance by targeting a glutathione S‐transferase gene, TCGSTM4, in Tetranychus cinnabarinus

Abstract

microRNA-1 (miR-1) is a well-studied conservative microRNA (miRNA) involved in immune responses in mammals and insects. However, little is known about its role in pesticide resistance in arthropods. In this study, we found that a microRNA belong to miR-1 family (tci-miR-1-3p) was significantly down-regulated in a cyflumetofen-resistant strain (CYR) of Tetranychus cinnabarinus compared with its homologous susceptible strain (SS), indicating an involvement of miR-1 in cyflumetofen resistance in mites. One glutathione S-transferase (GST) gene (TCGSTM4, a mu class GST gene), a candidate target gene of tci-miR-1-3p, was found to be significantly down-regulated when tci-miR-1-3p was over-expressed. The specific interaction between tci-miR-1-3p and the target sequence in the 3' untranslated region of TCGSTM4 was confirmed. A decrease or increase in tci-miR-1-3p abundance through feeding miRNA inhibitors or mimics significantly increased or decreased TCGSTM4 expressions at the mRNA and protein levels, respectively. In addition, an over-expression of tci-miR-1-3p resulted in a decrease in the tolerance of T. cinnabarinus to cyflumetofen in both SS and CYR strains, and vice versa. After decreasing TCGSTM4 transcription via RNA interference, T. cinnabarinus became more sensitive to cyflumetofen in both resistant and susceptible mites, and the change in mortality was greater in CYR than that in SS. Moreover, the recombinant TCGSTM4 could significantly decompose cyflumetofen, indicating that TCGSTM4 is a functional gene responsible for cyflumetofen resistance in mites.

Stability of cyflumetofen resistance in Tetranychus cinnabarinus and its correlation with glutathione‐S‐transferase gene expression

“…All three genes showed the similar tendency for their expressions: increasing with the resistance development and decreasing with the resistance recession, in which the TcGSTm02 emerged the most sensitivity against the resistance change (its expression changed with significance from one resistant stage to the next) (Fig. ) and the part function of TcGSTm04 was investigated in the previous study of cyflumetofen‐resistant regulation by a miRNA . Therefore, TcGSTm02 was selected as the representative candidate to carry out the further functional study.…”

“…Together, these experiments strongly indicated that TcGSTm02 protein could catalyze GS − to bind with cyflumetofen. Another GST gene, TcGSTm04 (46.61% amino acid similarity with TcGSTm02 ), had been studied in T. cinnabarinus, of which the recombinant protein was shown to metabolize cyflumetofen. The decomposition rate against cyflumetofen was higher with TcGSTm02 compared to TcGSTm04, suggesting that TcGSTm02 has a higher relative contributiones to the observed cyflumetofen‐resistance levels in T. cinnabarinus .…”

“…3) and the part function of TcGSTm04 was investigated in the previous study of cyflumetofen-resistant regulation by a miRNA. 21 Therefore, TcGSTm02 was selected as the representative candidate to carry out the further functional study.…”

“…In addition, the cross‐resistance between cyflumetofen and pyridaben, and genetic trait of cyflumetofen resistance had been demonstrated in T. cinnabarinus . In terms of metabolic resistance mechanism, the important role of GSTs conferring cyflumetofen‐resistance and a micRNA regulating cyflumetofen‐resistance by targeting a glutathione s‐transferase gene ( TCGSTM4 ) in T. cinnabarinus had been documented in our previous study . However, systematic and in‐depth study on the relationship between the GSTs' activity (expression of GST genes) and cyflumetofen‐resistance is still urgently needed.…”

“…19 In terms of metabolic resistance mechanism, the important role of GSTs conferring cyflumetofen-resistance and a micRNA regulating cyflumetofen-resistance by targeting a glutathione s-transferase gene (TCGSTM4) in T. cinnabarinus had been documented in our previous study. 20,21 However, systematic and in-depth study on the relationship between the GSTs' activity (expression of GST genes) and cyflumetofen-resistance is still urgently needed. In the present study, the mRNA levels of 13 GST genes were determined in cyflumetofen-resistance strain (CyR) and susceptible strain (SS).…”