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Regulation of GSTu1-mediated insecticide resistance in Plutella xylostella by miRNA and lncRNA

['Bin Zhu', 'Department Of Entomology', 'China Agricultural University', 'Beijing', 'Linhong Li', 'Rui Wei', 'Pei Liang', 'Xiwu Gao']

Date: 2022-01 

The evolution of resistance to insecticides is well known to be closely associated with the overexpression of detoxifying enzymes. Although the role of glutathione S-transferase (GST) genes in insecticide resistance has been widely reported, the underlying regulatory mechanisms are poorly understood. Here, one GST gene (GSTu1) and its antisense transcript (lnc-GSTu1-AS) were identified and cloned, and both of them were upregulated in several chlorantraniliprole-resistant Plutella xylostella populations. GSTu1 was confirmed to be involved in chlorantraniliprole resistance by direct degradation of this insecticide. Furthermore, we demonstrated that lnc-GSTu1-AS interacted with GSTu1 by forming an RNA duplex, which masked the binding site of miR-8525-5p at the GSTu1-3′UTR. In summary, we revealed that lnc-GSTu1-AS maintained the mRNA stability of GSTu1 by preventing its degradation that could have been induced by miR-8525-5p and thus increased the resistance of P. xylostella to chlorantraniliprole. Our findings reveal a new noncoding RNA-mediated pathway that regulates the expression of detoxifying enzymes in insecticide-resistant insects and offer opportunities for the further understanding of the mechanisms of insecticide and drug resistance.

The development of insecticide resistance in insect pests is a worldwide concern and a major problem in agriculture. Understanding the genetics of insecticide resistance is critical for effective crop protection. Plutella. xylostella (L.), a major pest of cruciferous crops, has developed resistance to almost all kinds of insecticide, and has become one of the most resistant pests in the world. Overexpression of detoxification enzymes is closely associated with insecticide resistance, but researches on their regulatory mechanism are still very limited. Here, GSTu1 was identified to be upregulated in several chlorantraniliprole-resistant P. xylostella populations and was confirmed to be involved in chlorantraniliprole resistance by direct degradation of this insecticide. Further, lnc-GSTu1-AS transcribed from the opposite DNA strand to GSTu1 was identified to be able to enhance the mRNA stability of GSTu1 by blocking miRNA activity, and thus increased the resistance of P. xylostella to chlorantraniliprole. The results provide further insights into the mechanisms underlying metabolic resistance.

Funding: The work was funded by the National Natural Science Foundation of China (No.31572023) to PL. The work was supported by the National Natural Science Foundation of China (No.31901915) and China Postdoctoral Science Foundation (2018M641546) to BZ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Zhu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The present study was conducted aiming to investigate the role of GSTu1 in chlorantraniliprole resistance, and the regulatory mechanisms of GSTu1 by its antisense lncRNA (lnc-GSTu1-AS). Our results reveal a new ncRNA-mediated regulatory pathway that is involved in the control of detoxification enzymes in insecticide resistance and offer opportunities for further studies of the underlying mechanisms of insecticide resistance and even drug resistance.

Noncoding RNAs (ncRNAs) are functional RNA molecules that are not translated into proteins and are usually categorized into two main groups: small and long ncRNAs [ 28 , 29 ]. The focus of this study was mainly on microRNAs (miRNAs) and lncRNAs. MiRNAs are short (∼22 nt), endogenous, noncoding RNAs, which regulate gene expression mainly through binding to the 3’-untranslated region (3’-UTR) of target RNA transcripts and eventually cause cleavage, translational repression or mRNA decay [ 30 – 32 ]. Numerous miRNAs have been confirmed to be involved in insecticide resistance by targeting detoxification enzymes or insecticide targets [ 33 , 34 ]. LncRNAs are non-coding RNAs longer than 200 nucleotides that are structurally similar to messenger RNAs (mRNAs), but lack significant open reading frames [ 35 , 36 ]. Generally, lncRNAs are classified into several categories, such as sense, antisense, intronic and intergenic, based on their position and direction of transcription in relation to protein-coding genes. Accumulated studies have shown that lncRNAs could regulate gene expression through a variety of mechanisms, including transcriptional regulation, post-transcriptional control and epigenetic modification [ 37 ]. In transcriptional level, lncRNA could span the promoter region of downstream protein encoding gene, prevent the binding of the transcriptional regulatory factors by acting as “decoy” or repress their activity by direct active-site occlusion or allosteric effects, thus inhibiting the expression of target genes; In contrast, lncRNAs could also promote transcriptional activity as enhancer RNAs or by binding to a protein with enhancer activity [ 38 ]. In post-transcriptional regulation, lncRNAs are implicated in the stability and translation of mRNAs, pre-mRNA splicing, protein activities, and act as precursors of miRNAs and siRNAs [ 39 , 40 ]. In epigenetic level, lncRNAs regulate gene expression through DNA methylation or demethylation, RNA interference (RNAi), histone modification and chromosome remodeling [ 38 , 39 , 41 ]. In the last decades, plenty of studies on lncRNAs have been conducted in mammals. However, researches on the functions of lncRNAs in insecticide resistance, or even in agricultural insects, is still very limited. A lncRNA in intron 20 of the cadherin alleles was recently reported to regulate the transcription of cadherin 1 in the pink bollworm Pectinophora gossypiella, which also underlies its susceptibility to Cry1Ac [ 42 ]. Furthermore, an intergenic lncRNA, lincRNA_Tc13743.2, was reported to be involved in the upregulation of GSTm02 by competing for miR-133-5p binding and thus mediated cyflumetofen resistance in Tetranychus cinnabarinus [ 43 ].

Metabolic detoxification is very important in insecticide resistance [ 23 , 24 ]. In addition to P450, FMO and UGT, glutathione S-transferases (GSTs) may also be involved in chlorantraniliprole resistance. For example, the activity of GSTs was significantly increased 24 h post chlorantraniliprole exposure in Bombyx mori [ 25 ]. Additionally, RNA-seq analysis showed that three GST genes were differentially expressed with increases in resistance to chlorantraniliprole in P. xylostella [ 26 ]. In our previous study, RNA-seq analysis showed that one GST gene (named GSTu1 in this study) was upregulated in a chlorantraniliprole-resistant P. xylostella population. Interestingly, another transcript (predicted as an antisense long noncoding RNA, lncRNA) that had generic exonic overlap with GSTu1 on the opposite strand was also upregulated [ 27 ]. Thus, we inferred that these two transcripts might play a role in chlorantraniliprole resistance in P. xylostella.

Amino acid mutations (G4946E and I4790M/K) in the ryanodine receptor (the target of diamide insecticides) have been considered one of the main causes of chlorantraniliprole resistance [ 13 – 18 ]. The upregulation of detoxification enzymes or xenobiotic transporters is also reported to play important roles in diamide insecticide resistance. To date, several detoxification enzyme genes, including two cytochrome P450 genes (CYP6BG1 and CYP321E1) [ 19 , 20 ], one uridine diphosphate-glycosyltransferase (UGT) gene (UGT33AA4) [ 21 ] and one flavin-dependent monooxgenase (FMO) gene [ 22 ], have been found to play a role in chlorantraniliprole resistance in P. xylostella.

Insect pests cause serious damage to the crop yield directly and indirectly, which costs billions of dollars annually [ 1 ]. Although awareness of the importance of integrated pest management (IPM) is growing, the use of chemical insecticides is still the main strategy for controlling insect pests [ 2 , 3 ]. However, the control efficacy is threatened by the evolution of insecticide resistance, which poses a continuing influence on agricultural production, environmental safety and even human health [ 4 , 5 ]. The diamondback moth, Plutella. xylostella (L.), a major pest of cruciferous crops, is one of the most resistant insects globally [ 6 , 7 ]. Chlorantraniliprole is an anthranilic diamide insecticide that shows remarkable efficacy in the control of several orders of insect pests, especially lepidopteran pests, but has low toxicity towards untargeted organisms [ 8 , 9 ]. However, in recent years, P. xylostella has developed high-level resistance to chlorantraniliprole in many countries [ 10 – 12 ].

Results

Cloning and expression pattern of GSTu1 and lnc-GSTu1-AS between susceptible and chlorantraniliprole-resistant P. xylostella populations Our previous transcriptome data showed that GSTu1 and its antisense transcript lnc-GSTu1-AS were both upregulated in chlorantraniliprole-resistant P. xylostella populations [27]. The nucleotide sequences of GSTu1 and lnc-GSTu1-AS were initially obtained from previous transcriptome sequencing [27] and then confirmed by reverse transcription PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). Four GSTu1 transcripts (GenBank accession number: MZ889082-MZ889085) with different 3’UTR lengths were obtained (Fig 1A). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Structure and location of GSTu1 and lnc-GSTu1-AS and their relative expression in susceptible and three chlorantraniliprole-resistant populations. A: The structure and location of full-length GSTu1 and lnc-GSTu1-AS. Relative expression of GSTu1 in a susceptible population (SS) and three chlorantraniliprole-resistant populations (NIL, BL and HK) by qRT-PCR (B) and Western blotting (D, E). C: Relative expression of lnc-GSTu1-AS in a susceptible population (SS) and three chlorantraniliprole-resistant populations (NIL, BL and HK) by qRT-PCR. Different lowercase letters represent significant differences (one-way ANOVA followed by Tukey’s multiple comparison tests, P < 0.05). https://doi.org/10.1371/journal.pgen.1009888.g001 Two field chlorantraniliprole-resistant P. xylostella populations were initially collected in vegetable fields in Boluo City, Guangdong Province (BL) and Haikou City, Hainan Province (HK) in 2016, respectively. A near-isogenic resistant population NIL was obtained from BL and a laboratory susceptible population SS. NIL, BL and HK showed 465-, 5057- and 6722-fold resistance to chlorantraniliprole compared with SS, respectively (S1 Table). Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that the transcriptional levels of GSTu1 in NIL, BL and HK larvae were 3.7-, 5.5- and 8.0-fold higher than those in SS larvae, respectively (Fig 1B). Moreover, the transcriptional level of the shortest transcript of GSTu1 was much higher than that of the other three longer transcripts (Fig 1B). Based on their lengths, the shortest transcript was named “short”, and the three longer transcripts together were named “long” in this study (Fig 1A). The Western blot assay results showed that the expression of GSTu1 in NIL, BL and HK larvae was also higher than that in SS larvae (Fig 1D and 1E). The nucleotide sequence of lnc-GSTu1-AS was identified by RT-PCR and RACE (GenBank accession number: MZ889086). Lnc-GSTu1-AS was transcribed from the opposite strand of GSTu1 and overlapped almost the entire 3’UTR of GSTu1 (Fig 1A) The qRT-PCR results showed that the transcriptional levels of lnc-GSTu1-AS in NIL, BL and HK larvae were 6.3-, 14.4- and 35.5-fold higher than those in SS larvae, respectively (Fig 1C). The expressional level of both GSTu1 and lnc-GSTu1-AS were gradually up-regulated with the increase of chlorantraniliprole resistance in NIL, BL and HK populations. It can be inferred that the role of GSTu1 in chlorantraniliprole resistance is conserved and a similar regulatory relationship may exist between GSTu1 and lnc-GSTu1-AS in these three resistant populations. Considering the NIL has very similar genetic background with SS, all subsequent in vivo verification experiments were performed using NIL.

Sequence analysis of GSTu1 GSTs usually have two catalytically active sites: the prime GSH substrate binding site (G-site) and a secondary hydrophobic substrate binding site (H-site). The G-site within each GST class is formed by a group of highly conserved amino acid residues in the N-terminal domain of the protein. The H-site is formed by generally fewer conserved residues in the C-terminal domain [44]. Both G-sites and H-sites in the amino acid sequence of GSTu1 were analyzed with the NCBI CD-search program, and the results are shown in S1A Fig. Moreover, a phylogenetic tree was constructed based on the amino acid sequences of GSTu1 of 22 insect species from Lepidoptera, Diptera, Hymenoptera, Orthoptera and Coleoptera using the neighbor-joining method. The results showed that GSTu1 in Lepidoptera, including P. xylostella, was more conserved and clustered into a specific subclass (S1B Fig).

Functional analysis of GSTu1 in chlorantraniliprole resistance The function of GSTu1 in chlorantraniliprole resistance was investigated by RNAi. The qRT-PCR results showed that the relative expression of GSTu1 decreased by 70% and 62% in the NIL and BL populations, respectively, at 48 h postinjection of dsGSTu1 compared with that of the control (injected with dsEGFP) (Fig 3A and 3B). Moreover, dsRNA-injected (dsEGFP and dsGSTu1) larvae from both NIL and BL were exposed to the LC 50 of chlorantraniliprole. The mortality of the dsGSTu1-injected group was significantly increased by 17% and 22% in NIL and by 21% and 36% in BL at 72 h and 96 h postinjection, respectively (Fig 3A and 3B). These results indicated that RNAi-mediated knockdown of GSTu1 increased the susceptibility of P. xylostella larvae to chlorantraniliprole. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Functional analysis of GSTu1 in chlorantraniliprole resistance. RNAi-mediated knockdown of GSTu1 reduced the resistance of P. xylostella to chlorantraniliprole in NIL (A) and BL(B). C: Expression and purification of GSTu1 in the E. coli system (M: protein marker; lane 1: negative control; lane 2: total soluble proteins; lane 3: purified proteins). D: Enzyme kinetics of GSTu1 with a series of ascending chlorantraniliprole (0–1000 μM) and fixed GSH. E: Depletion rate of chlorantraniliprole by recombinant GSTu1. F: The specific activity of purified GSTu1 toward CDNB. Asterisks indicate significant differences between the treatment and the corresponding control (Student’s t-test, * 0.01 < P < 0.05, **P < 0.01). https://doi.org/10.1371/journal.pgen.1009888.g003 Furthermore, recombinant GSTu1 was expressed as a soluble protein in Escherichia coli. The molecular mass of recombinant GSTu1 protein was approximately 30 KD, and more than 1 mg of recombinant protein was obtained after purification (Fig 3C). The specific activity of GSTu1 with CDNB as a substrate was 0.464 μmol/min/mg at pH 6.5 and 37°C (Fig 3F). The competitive assay revealed that chlorantraniliprole (1–1000 μM) was able to reduce the catalytic capability of recombinant GSTu1 by 26% to 82% (Fig 3D). This result indicated that the inhibition efficiency was dependent on the insecticide concentrations. The metabolism of chlorantraniliprole by recombinant GSTu1 was evaluated by High Performance Liquid Chromatography (HPLC). The results showed that approximately 47.4% of chlorantraniliprole was metabolized by recombinant GSTu1 compared with the control group (heat-inactivated GSTu1) (Fig 3E).

Developmental and tissue expression patterns of lnc-GSTu1-AS in P. xylostella The developmental and tissue-specific expression patterns of lnc-GSTu1-AS were also determined. qRT-PCR showed that lnc-GSTu1-AS was highly expressed in the first- to third-instar larval stage and abundant in the midgut and Malpighian tubules (Fig 4A and 4B). These results showed that GSTu1 and lnc-GSTu1-AS had very similar expression patterns in different developmental stages and tissues of P. xylostella. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Lnc-GSTu1-AS is involved in the regulation of chlorantraniliprole resistance. Relative expression of lnc-GSTu1-AS at different developmental stages (A), in different tissues (B), in nuclear RNA and cytoplasmic RNA (C), and in control and lnc-GSTu1-AS knockdown groups (D) of P. xylostella. Relative expression of GSTu1 in the control and lnc-GSTu1-AS knockdown groups by qRT-PCR (E) and Western blot (F, I). G: Mortality of P. xylostella treated with the LC 50 of chlorantraniliprole. H: Analysis of the binding relationship between lnc-GSTu1-AS and GSTu1-3′ UTR by ribonuclease protection assays (RPA). J: Co-localization of lnc-GSTu1-AS and GSTu1 in the midgut of P. xylostella by fluorescence in situ hybridization (FISH) assay. Asterisks indicate significant differences between the treatment and the corresponding control (Student’s t-test, * 0.01 < P < 0.05, **P < 0.01). Different lowercase letters represent significant differences (one-way ANOVA followed by Tukey’s multiple comparison tests, P < 0.05). https://doi.org/10.1371/journal.pgen.1009888.g004

Functional analysis of lnc-GSTu1-AS in resistance to chlorantraniliprole in P. xylostella RNA fractionation of the nucleus and cytoplasm showed that lnc-GSTu1-AS was mainly abundant in the cytoplasm (Fig 4C), indicating its potential role in posttranscriptional regulation. The function of lnc-GSTu1-AS in chlorantraniliprole resistance was then investigated by RNAi in NIL. The qRT-PCR results showed that the relative expression of lnc-GSTu1-AS was suppressed by 31%, 64%, 44% and 40% at 24 h, 48 h, 72 h and 96 h after si-lnc-GSTu1-AS injection compared with that in the si-NC-injected control, respectively (Fig 4D). In response, the transcriptional level of GSTu1 (short+long) decreased by 29% and 20%, and the transcriptional level of GSTu1-long decreased by 53% and 60%, at 72 h and 96 h post si-lnc-GSTu1-AS injection, respectively (Fig 4E). The Western blot assay results showed that the amount of GSTu1 also decreased significantly after silencing lnc-GSTu1-AS, especially 96 h post si-lnc-GSTu1-AS injection (Fig 4F and 4I). siRNA-injected larvae were exposed to the LC 50 of chlorantraniliprole. The mortality in the si-lnc-GSTu1-AS injection group was increased by 28% and 38% at 72 h and 96 h (Fig 4G). These results indicated that RNAi-mediated knockdown of lnc-GSTu1-AS increased the susceptibility of P. xylostella larvae to chlorantraniliprole by reducing the expression of GSTu1. The reverse complementary sequence between lnc-GSTu1-AS and GSTu1 indicated that they might have a binding relationship. Therefore, the potential binding of lnc-GSTu1-AS directly to the 3’UTR of GSTu1 was investigated. To detect the RNA duplex formed by lnc-GSTu1-AS and GSTu1, an RNase protection assay (RPA) was performed. The results showed that multiple sequence fragments (P3/P4, P5/P6) in the overlapping regions were protected from degradation, whereas the nonoverlapping region of lnc-GSTu1-AS and GSTu1 (P1/P2) was almost completely degraded by RNase A (Fig 4H). Moreover, the colocalization signals of lnc-GSTu1-AS and GSTu1 were also detected in the midguts of fourth instar NIL P. xylostella by the fluorescence in situ hybridization (FISH) assay, suggesting that these two transcripts formed a transient duplex (Fig 4J). Previous studies have shown that antisense lncRNAs promote the stability of sense transcripts by transient duplex formation and inhibition of miRNA- or RNA-binding protein (RBP)-induced sense mRNA decay [45]. Therefore, we assumed that lnc-GSTu1-AS might have a similar function in GSTu1 regulation.

Identification of miRNAs targeting GSTu1 in P. xylostella RNA pull-down assays showed that no RBP interacted with GSTu1 (S2 Fig). The results from two miRNA-target prediction software programs, miRanda and RNAhybrid, showed that 13 miRNAs could potentially act on the 3’UTR of GSTu1, and all the binding sites of these miRNAs were located in the overlap region of the sense-antisense transcripts (S3 Fig). To validate the binding of the 13 predicted miRNAs to GSTu1 mRNA, the full-length 3’UTR sequence of the longest GSTu1 transcript containing all the predicted miRNA-binding sites was initially cloned and inserted into a pmirGLO vector (pmirGLO & GSTu1-long-3’UTR). As a result, significant effects on reporter activity were only observed in miR-8525-5p- or miR-8530-5p agomir-transfected cells (Fig 5A). Next, the binding sites complementary to the ‘seed’ sequences of miR-8525-5p or miR-8530-5p were mutated (pmirGLO & GSTu1-long-3’UTR-mut). When the miR-8525-5p agomir was cotransfected with the pmirGLO & GSTu1-short-3’UTR or pmirGLO & GSTu1-long-3’UTR into HEK293T cells, the luciferase activity declined by 46% or 51.3%, respectively, compared with the mutated control (Fig 5B). When the miR-8530-5p agomir was cotransfected with the pmirGLO & GSTu1-long-3’UTR into HEK293T cells, the luciferase activity declined by 45.3% compared with the mutated control (Fig 5C). Furthermore, an RNA binding protein immunoprecipitation (RIP) assay was performed to determine whether GSTu1 bound to miR-8525-5p- or miR-8530-5p-related RNA-induced silencing complexes (RISCs). The results showed that the levels of GSTu1 significantly increased by 6.75-fold and 2.35-fold, respectively, when miR-8525-5p or miR-8530-5p was applied compared with the negative control (Fig 5D). These data indicated that GSTu1 might interact directly with miR-8525-5p and miR-8530-5p. The binding site of miR-8525-5p was located at the overlapping 3’UTR sequence of all four GSTu1 transcripts, while the binding site of miR-8530-5p was located at the overlapping 3’UTR sequence of the three longer GSTu1 transcripts. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Identification of miRNAs targeting GSTu1. A: Validation of the interactions of 13 predicted miRNAs with GSTu1 by dual-luciferase reporter assays. Dual-luciferase reporter assays through co-transfection of miR-8525-5p agomir (B) or miR-8530-5p agomir (C) with recombinant pmirGLO vectors containing either wild-type or mutated (mut) binding sites. D: Interactions between miR-8525-5p or miR-8530-5p and GSTu1 determined by RNA-binding protein immunoprecipitation (RIP) in vivo. E: The relative expression of miR-8525-5p (E) and miR-8530-5p (F) in SS, NIL, BL and HK by RT-qPCR. Asterisks indicate significant differences between the treatment and the corresponding control (Student’s t-test, * 0.01 < P < 0.05, **P < 0.01). Different lowercase letters represent significant differences (one-way ANOVA followed by Tukey’s multiple comparison tests, P < 0.05). https://doi.org/10.1371/journal.pgen.1009888.g005

The expression levels of miR-8525-5p and miR-8530-5p among SS, NIL, BL and HK The expression levels of miR-8525-5p and miR-8530-5p were measured in SS, NIL, BL and HK by qRT-PCR. The results showed that the levels of miR-8525-5p in NIL, BL and HK larvae were 4.2-, 4.6- and 2.4-fold higher than those in SS larvae, respectively (Fig 5E), and the levels of miR-8530-5p in NIL, BL and HK larvae were 2.7-, 4.8- and 5.8-fold higher than those in SS larvae, respectively (Fig 5F).

Lnc-GSTu1-AS enhanced the stability of GSTu1 by preventing its degradation induced by miR-8525-5p in chlorantraniliprole-resistant P. xylostella To further investigate whether the effect of lnc-GSTu1-AS on the stability of GSTu1 was associated with miRNAs, we first inhibited miR-8525-5p or miR-8530-5p by injecting antagomirs (antagomir-NC was used as a control) and then (12 h later) knocked down the expression of lnc-GSTu1-AS by injecting si-lnc-GSTu1-AS (si-NC was used as a control) into 3rd instar NIL larvae. The relative expression of miR-8525-5p was significantly decreased by 58.8%, 60.6% and 42% at 48 h, 72 h and 96 h postinjection of miR-8525-5p-antagomir/si-lnc-GSTu1-AS (Fig 7A). The relative expression of lnc-GSTu1-AS was significantly downregulated in both the miR-8525-5p-antagomir/si-lnc-GSTu1-AS (by 50.5%, 62.1% and 46.1% at 48 h, 72 h and 96 h postinjection) and antagomir-NC/si-lnc-GSTu1-AS (by 47.8.5%, 53.3% and 50.1% at 48 h, 72 h and 96 h postinjection) injection groups (Fig 7B). PPT PowerPoint slide

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TIFF original image Download: Fig 7. Lnc-GSTu1-AS maintains the stability of GSTu1 through prevention of its degradation by masking the binding site of miR-8525-5p in chlorantraniliprole-resistant P. xylostella. Relative expression of miR-8525-5p (A) and lnc-GSTu1-AS (B) after inhibition of miR-8525-5p and knockdown of lnc-GSTu1-AS by qRT-PCR. Relative expression of GSTu1 after inhibition of miR-8525-5p combined with knockdown of lnc-GSTu1-AS by qRT-PCR (C) and Western blot assay (D, F). E: Mortality of P. xylostella treated with the LC 50 of chlorantraniliprole. Asterisks indicate significant differences between the treatment and the corresponding control (Student’s t-test, ns indicates no significant differences, * 0.01 < P < 0.05, **P < 0.01). https://doi.org/10.1371/journal.pgen.1009888.g007 As an influence, the transcriptional level of GSTu1 (short+long) in the group injected with antagomir-NC/si-lnc-GSTu1-AS decreased by 54.0% and 49.8%, and the transcriptional level of GSTu1-long decreased by 54.0% and 49.8%, at 72 h and 96 h, respectively, after siRNA injection compared with that in the control (antagomir-NC/si-NC injection group) (Fig 7C). Similar results were also obtained at the translational level of GSTu1 by Western blot (Fig 7D and 7F). The mortality of larvae exposed to the LC 50 of chlorantraniliprole was 78.7%, which was significantly higher than that in the antagomir-NC/si-NC group (50.9%) (Fig 7E). The transcriptional level of GSTu1 (short+long) in the group injected with miR-8525-5p-antagomir/si-lnc-GSTu1-AS decreased by 35.1%, and the transcriptional level of GSTu1-long decreased by 30.3% (Fig 7C), only at 72 h after siRNA injection, while no significant difference was found at 96 h after siRNA injection compared with the control (antagomir-NC/si-NC-injected group). Similar results were also obtained at the translational level of GSTu1 by Western blot (Fig 7D and 7F). Furthermore, the mortality of the larvae exposed to the LC 50 of chlorantraniliprole was 54.6%, which was significantly lower than that in the antagomir-NC/si-lnc-GSTu1-AS-injected group (78.7%) (Fig 7E). In summary, these results suggested that knockdown of lnc-GSTu1-AS alone increased the susceptibility of larvae to chlorantraniliprole, while suppression of both lnc-GSTu1-AS and miR-8525-5p did not. It means that lnc-GSTu1-AS maintained the stability of GSTu1 by preventing its degradation induced by miR-8525-5p, and thus mediated chlorantraniliprole resistance in P. xylostella.

[END]

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