lincRNA_Tc13743.2-miR-133-5p-TcGSTm02 regulation pathway mediates cyflumetofen resistance in Tetranychus cinnabarinus

https://doi.org/10.1016/j.ibmb.2020.103413Get rights and content

Highlights

  • A total of 4454 lncRNAs were identified in the Tetranychus cinnabarinus mite.

  • An up-regulated lincRNA_Tc13743.2 increased TcGSTm02 expression by competing for miR-133-5p binding in T. cinnabarinus.

  • Down-regulation of miR-133-5p increased the expression of TcGSTm02 in the cyflumetofen resistant strain.

  • A competing endogenous RNA pathway plays an important role in the acaricide resistance in mite pests.

Abstract

Differential expression of metabolic detoxification enzymes is an important mechanism involved in pesticide/acaricide resistance of mite pests. The competing endogenous RNA hypothesis offers a new opportunity to investigate post-transcriptional regulation of those genes. In this study, 4454 long non-coding RNAs were identified in the carmine spider mite Tetranychus cinnabarinus by transcriptome sequencing. Software-based predictions indicated that a long intergenic non-coding RNA, (lincRNA)_Tc13743.2 and a detoxification enzyme gene, TcGSTm02, both contained a microRNA (miR-133-5p) response element. Over-expression of lincRNA_Tc13743.2 and TcGSTm02 were detected in a cyflumetofen-resistant T. cinnabarinus strain (CyR), whereas down-regulation of miR-133-5p was observed in this strain. Conversely, up-regulation of miR-133-5p could inhibit TcGSTm02 expression levels, and both lincRNA_Tc13743.2 and TcGSTm02 were significantly enriched in miR-133-5p biotin–avidin pull-down assays. RNA-binding protein immunoprecipitation assay showed that lincRNA_Tc13743.2 and TcGSTm02 bound to a silencing complex containing miR-133-5p. Moreover, a luciferase reporter assay based on a human cell line revealed that over-expression of lincRNA_Tc13743.2 could significantly reduce the inhibition exerted by miR-133-5p through the TcGSTm02 3′UTR. In addition, co-localization of lincRNA_Tc13743.2 and miR-133-5p was detected using fluorescence in situ hybridization, suggesting that lincRNA_Tc13743.2 interacts directly with miR-133-5p in spider mites. More importantly, silencing the expression of lincRNA_Tc13743.2 significantly reduced the expression levels of TcGSTm02 and increased the sensitivity of spider mites to cyflumetofen. Our data show that lincRNA_Tc13743.2 up-regulates TcGSTm02 expression by competing for miR-133-5p binding, demonstrating that a lincRNA_Tc13743.2–miR-133-5p–TcGSTm02 pathway mediates cyflumetofen resistance in mites.

Introduction

Tetranychus cinnabarinus is a major agricultural mite pest with over 1000 plant documented hosts (Snoeck et al., 2019). In addition to its strong hosts adaptability, T. cinnabarinus also exhibits extraordinary ability to develop pesticide/acaricide resistance. Currently, species in the genus Tetranychus, including T. cinnabarinus, are reported to be one of the “most resistant species” in terms of their resistance to the total number of pesticides (Van Leeuwen and Dermauw, 2016). Therefore, the study of resistant mechanisms in these mites is crucial to carry out rational use of acaricides and to control the resistance in mites. Morphologically and physiologically, T. cinnabarinus is similar to Tetranychus urticae and some researchers consider that T. cinnabarinus is a red form of T. urticae (Auger et al., 2013). Nevertheless, other studies consider these mites as an allied species based on some unique features (Li et al., 2009; Lozano-Fernandez et al., 2019).

Cyflumetofen is mitochondrial electron transport chain inhibitor (METI), whose de-esterified metabolite AB-1 inhibits complex II much more strongly than the parent compound (Hayashi et al., 2013). The mechanisms of cyflumetofen resistance have been investigated in spider mites. For example, the enhanced glutathione S-transferase (GST) activity and overexpression of the gene, TuGSTd05, are considered key factors mediating cyflumetofen resistance in T. urticae (Pavlidi et al., 2017). In addition, GSTs are primary contributors to the development of cyflumetofen resistance in T. cinnabarinus (Feng et al., 2019b), while cytochrome P450 monooxygenases, as well as carboxylesterases (CCEs), can also contribute to the development of cyflumetofen resistance via various metabolic pathways (Feng et al., 2019a; Wei et al., 2019).

In addition to the identification of resistant genes and their functions, determining and elucidating regulation pathways of these genes are important tasks in pesticide resistance research (Belles, 2017; Hong et al., 2014). As members of the non-coding RNA family, microRNAs (miRNAs) are important post-transcriptional regulatory players (Bartel, 2009) that down-regulate the expressions of metabolic detoxification genes by binding to the three prime untranslated regions (3′ UTRs) of their messenger RNAs. This mode of action has been examined in the context of pyrethroid resistance in Culex pipiens (Tian et al., 2016), chlorantraniliprole resistance in Plutella xylostella (Zhu et al., 2017a), and cyflumetofen resistance in T. cinnabarinus (Zhang et al., 2018b).

Long non-coding RNAs (lncRNAs), a class of transcripts exceeding 200 nucleotides (nt), were originally thought to be transcriptional “noise” (Ponting et al., 2009). However, recent research has demonstrated that these molecules can compete with messenger RNAs (mRNAs) for miRNA binding through miRNA response elements, a mechanism referred as the competing endogenous RNA (ceRNA) hypothesis (Salmena et al., 2011). In this case, lncRNAs acting as “sponges” for miRNAs, and provide an additional level of post-transcriptional regulation of downstream mRNAs by absorbing and isolating miRNAs (Tay et al., 2014). ceRNAs first garnered close attention in mammalian study, where it has allowed researchers to explain numerous pathological phenomena, particularly in fields such as cancer cell migration and expansion (Liu et al., 2014; Shi et al., 2013).

The investigation of insect lncRNAs remains at an early stage. Many studies are primarily focused on the identification of lncRNAs and measurement of their differential expression (Li et al., 2019; Zhou et al., 2018; Zhu et al., 2017b). A recent study showed that lincRNA-IBIN is involved in the immune response of Drosophila melanogaster against bacteria (Valanne et al., 2019). However, systematic investigations examining whether lncRNAs have specific roles in post-transcriptional regulation are lacking.

In this study, through transcriptome sequencing, combined with software-based predictions, we found that both lincRNA_Tc13743.2 and TcGSTm02, an important gene associated with cyflumetofen resistance (Feng et al., 2019b), contained miR-133-5p response elements. Subsequently, biotin-avidin, RNA-binding protein immunoprecipitation (RIP), dual-luciferase reporter assay, fluorescence in situ hybridization and RNA interference (RNAi) results showed that lincRNA_Tc13743.2 could function as a ceRNA to regulate the expression of TcGSTm02 and cyflumetofen resistance through competition for miR-133-5p in T. cinnabarinus. This study also demonstrated that a lncRNA-miRNA-mRNA regulation pathway is involved in pesticide/acaricide resistance of arthropods.

Section snippets

T. cinnabarinus strains

The susceptible strain (SS) was collected from a field in Beibei, Chongqing, China, and maintained on fresh cowpea seedlings in incubators at 26 °C ± 1 °C, 55%–75% relative humidity (RH), with a photoperiod of 14:10 h (L:D). The SS had been maintained for >20 years without any pesticide exposure (He et al., 2009). The cyflumetofen-resistant strain (CyR) was selected from the SS by continuous selection with cyflumetofen until the resistance ratio reached 104.7-fold (Feng et al., 2018).

RNA extraction and lncRNA sequencing

Total RNA

Selection of miRNAs targeting TcGSTm02 and their expression patterns in SS and CyR strain

Based on our previous transcriptome data of miRNA (NCBI, in BioProject, ID PRJNA306970), a total of 23 miRNAs were predicted to be potentially capable of binding to TcGSTm02. Among these, only miR-133-5p was predicted by all three pieces of software (MicroTar, RNAhybrid and PITA) (Fig. 1A), suggesting that miR-133-5p was most likely the miRNA to interact with TcGSTm02. Therefore, miR-133-5p was selected to carry out subsequent experiments. The results of RT-qPCR showed that the expression level

Discussion

An increase in metabolic detoxification capacity and mutation of target sites are believed to be the primary factors that cause acaricide resistance in spider mites. Selection or field collection of cyflumetofen-resistant spider mites have been reported in several previous studies. However, none of these studies detected complex II mutation-mediated cyflumetofen resistance (Khalighi et al., 2014, 2016; Wang et al., 2014b). This suggests that an increase in detoxification capacity may be the

Author contributions

All authors conceived and designed research. FKY and LJ performed experiments. FKY and HL analyzed data. FKY, WP, XQ and HL wrote the manuscript. All authors reviewed the manuscript.

Data linking

The transcriptome data of lncRNA reported in this study have been deposited in National Center for Biotechnology Information (NCBI), in BioProject (ID PRJNA593307). The previous transcriptome data of miRNA had been deposited in NCBI in BioProject (ID PRJNA306970).

Acknowledgements

We are very grateful to Prof. Wang, Dr. Zhang and Dr. Xu (Jinjun Wang, Qiang Zhang and Li Xu, Academy of Agricultural Sciences, Southwest University, Chongqing, China) for their kind help (provide the HEK293T cell line) in this work. The National Natural Sciences Foundation (no. 31972297) and the Chongqing Research Program of Basic Research and Frontier Technology (no. cstc2017jcyiBx0061) funded this research. This work was also supported by Abilene Christian University SEED Award.

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