Study On Calcium Signalling Pathway Genes And Target Function Of Scoparone Against Mites

Studying the mechanism of action of plant secondary metabolites against mites is an important strategy to develop new botanical acaricides.Scoparone（isolated from the Chinese herbal medicine Artemisia capillaris）,a bioresource derived from renewable plants,has efficient acaricidal activity against mites,but its action target is still unclear.The purpose of this paper was to systematically explore the potential molecular targets of scoparone against Tetranychus cinnabarinus,and to provide insights to guide the future use of scoparone as acaricide against mites in agriculture globally.This paper employed scientifically advanced experimental techniques,including RT-PCR,q PCR,RNA-Seq,RNAi technology,and[Ca²⁺]_i,GST pull-down,and electrophysiological recording assays,to comprehensively explore the potential targets of scoparone.Taking the novel target-Ca²⁺signaling pathway as the entry point,from three levels of molecular target screening,functional gene analysis and active target verification,this paper systematically revealed the molecular mechanism of the acaricidal activity of scoparone.Moreover,this paper,for the first time,clarified the the molecular target of the scoparone against T.cinnabarinus.The main results were as follows:1 Screening and identification of Ca²⁺signaling genes related to the acaricidal activity of scoparone against T.cinnabaridesThe[Ca²⁺]_i assay showed that scoparone significantly increased the[Ca²⁺]_i levels in insect Sf9（Spodoptera frugiperda）cells with a dose-dependent manner.The RNA-Seq data were analyzed,and the results showed that 251 genes were identified as significantly differentially expressed genes after treatment with scoparone for 48 h,192 of which were up-regulated and 59 of which were down-regulated.q PCR analysis indicated that randomly selected 15 tested genes,including 8up-regulated genes and 7 down-regulated genes,exhibited expression trends consistent with the RNA-Seq data.The top 10 GO（Gene Ontology）terms included the“voltage-gated Ca²⁺channel（VGCC）complex”,“calcium channel complex”,“monooxygenase activity”,and“VGCC activity”terms,among others.The top 10 KEGG（Kyoto Encyclopedia of Genes and Genomes）terms included the“calcium signalling pathway”,“renin secretion”,“phototransduction”,and“aldosterone synthesis and secretion”terms.Among the top 10 terms from the GO and KEGG analyses,the most highly enriched terms were“VGCC complex”and“calcium signalling pathway”,respectively.The PPI（Protein-Protein Interaction）network showed that Ca M1 is the primary relevant signalling molecule for L-,T-,and N-VGCCs.Next,q PCR and RT-PCR analyses indicated that the expression of VGCC and Ca M genes was upregulated in the scoparone(LC₇₀,LC₅₀ and LC₃₀dosages)exposure groups compared with the control group at 48 h post-treatment.In addition,scoparone upregulated the expression levels of the L-VGCC and Ca M1 genes more significantly than other Ca²⁺signalling pathway-related genes.Finally,1 L-VGCC（Tc L-VGCC）,1 T-type VGCC（Tc T-VGCC）,1 N-type VGCC（Tc N-VGCC）,and 5 Ca M（Tc Ca M1–5）genes associated with the“calcium signalling pathway”term were selected as candidate target genes for further investigation.2 The biological function of Ca²⁺channel（Tc L-VGCC,Tc N-VGCC,Tc T-VGCC）and calmodulin（Tc Ca M1-5）genes in T.cinnabarinus2.1 Cloning and sequence analysis of 3 Ca²⁺channel genes（Tc L-,Tc T-,Tc N-VGCC）and 5calmodulin genes（Tc Ca M1-5）of T.cinnabarinusThe sequence alignment results showed that the Tc VGCCs have four functional regions（domains I-IV）,each of which consists of 6 transmembraneα-helix segments（S1-S6）.Additionally,Tc Ca M1 and Tc Ca M5 include 4 and 2 EF-hand domains,respectively,and Tc Ca M3 contains 1transmembrane helix.Next,phylogenetic analysis showed that Tc VGCCs and Tc Ca Ms share the highest sequence similarity with VGCCs and Ca Ms of T.urticae,respectively,indicating evolutionary relatedness and potential homologous physiological mechanisms between Tc VGCCs and Tu VGCCs and between Tc Ca Ms and Tu Ca Ms.q PCR and RT-PCR analyses showed that Tc VGCCs and Tc Ca Ms were expressed throughout all developmental stages（egg,larval,nymph and adult）,demonstrating that Tc VGCCs and Tc Ca Ms are involved in all bioprocesses of development and growth.2.2 RNAi assay of Ca²⁺channels（Tc L-,N-,T-VGCC）and calmodulin（Tc Ca M1-5）genes of T.cinnabarinusRNAi analysis indicated that the transcript levels of L-,T-,N-VGCC,and Ca M1–5 were significantly lower（50–72%lower）in mites fed the separate corresponding candidate ds RNA for 48h than in control mites.Moreover,RNAi of L-VGCC and Ca M1 markedly knocked down the transcription of these candidate genes simultaneously（by 68%and 60%,respectively）.Mortality was significantly lower for the mites in which the Tc L-VGCC and Tc Ca M1 genes were individually silenced than for the control mites.Interestingly,Tc L-VGCC-and Tc Ca M1-targeting ds RNA provided the largest decreases in mortality（7.25-and 5.50-fold,respectively）,and silencing the L-VGCC and Ca M1 genes simultaneously had a more powerful effect on mortality（measured as a fold change;11.43-fold greater）in mites than knocking down the single genes.Together,these results indicated that the Ca M1-and L-VGCC-mediated Ca²⁺signalling pathways were essential for the acaricidal mechanism of scoparone.3 The molecular mechanism of Ca M1-and L-VGCC-mediated Ca²⁺signalling pathways were activated by scoparoneThe Ca M assay showed that scoparone could indirectly activate the Ca M1 protein in vitro,and its EC₅₀ value was 4.92μM.In addition,a[Ca²⁺]_i assay revealed that[Ca²⁺]_i in Ca M1-expressing cells treated with scoparone dramatically exceeded that in GFP-expressing cells,confirming that Ca M1-mediated Ca²⁺signalling pathways in mites were activated by scoparone.The relationship between Ca M1 and scoparone in the modulation of L-VGCC was studied,and several novel findings were acquired.First,unlike Ca M1,scoparone alone did not block Ca²⁺channel activity at high concentrations（>10μM）.Second,the activating effect of scoparone on L-VGCCs was enhanced by Ca M1.Third,the facilitatory and inhibitory effects of Ca M1 were significantly enhanced and blocked by scoparone,respectively.These results demonstrated that activation of Ca M1 and L-VGCC by scoparone likely induced excessive activation of Ca²⁺signalling pathways by promoting CDF or blocking CDI of L-VGCC.4 The regulatory mechanism of scoparone on Ca²⁺signaling mediated by Ca M1and L-VGCC in T.cinnabarinus4.1 The regulation mechanism of calmodulin1（Ca M1）on L-type Ca²⁺channel（L-VGCC）in T.cinnabarinusGST pull-down assays showed that Ca M1 was markedly bound to the NT,CT1,CT1B,Pre IQ,and IQ peptides in a concentration-dependent manner.Additionally,more than one molecule of Ca M1 bound to the CT motif.Meanwhile,the higher[Ca²⁺]_i is required for Ca M binding to the NT of the Ca²⁺channel.Under 1 m M Ca²⁺,the ranking of the K_d values was CT1B<CT1≈IQ<Pre IQ<NT.Under 100 n M Ca²⁺,the ranking of the K_d values was similarly evaluated,and the order was as follows:Pre IQ<NT<CT1B<CT1<IQ.4.2 Targeted regulatory sites of scoparone on Ca M1 and L-VGCC-mediated Ca²⁺signaling pathway in T.cinnabarinusGST pull-down assays demonstrated that scoparone facilitated Ca M1 binding to the IQ domain（a consensus Ca M-binding domain of L-VGCC）of L-VGCC.Moreover,compared with the peptide with the WT-IQ motif（GST-WT-IQ）,the peptide with mutation of the IQ domain（GST-MT-AA）exhibited significantly weaker Ca M1 binding（66.78%）.Electrophysiological experiments showed that the wild-type Ca²⁺channel（L-VGCC）（WT-IQ）displayed significant Ca²⁺-dependent inactivation（CDI）.Also,compared with WT-IQ channel,the CDI value of mutant L-VGCC（MT-AA）was 2.72-fold higher（2.35 vs.0.86）,while the CDF value was 1.51-fold lower（-0.70 vs.-0.46）,indicating that a mutation of the IQ motif that significantly weakened Ca M binding promoted CDI and blocked CDF of the Ca²⁺channel.Next,in oocytes that expressed MT-AA channels and were pretreated with scoparone,CDI was quite robust,demonstrating that scoparone blocked CDI or promoted CDF of L-VGCC mediated by Ca M by strengthening the Ca M1-IQ interaction.5 Location of new targets for the acaricidal effect of scoparone against T.cinnabarinusAccording to the current findings,it can be hypothesized that the mode of action of scoparone involves Ca M-mediated Ca²⁺channels.At low[Ca²⁺]_i,Ca M exists mainly in a Ca²⁺-free form（apo Ca M）and interacts with relatively high affinity with Pre IQ(a CT motif of Ca²⁺channels),producing basal channel activity.Due to the relatively low affinity of apo Ca M for the IQ/NT region,the interaction between them may be minimal.However,when[Ca²⁺]_i increases,Ca²⁺binds with Ca M,forming Ca²⁺/Ca M,which binds with relatively high affinity to the IQ motif,triggering CDF.Scoparone induces Ca²⁺/Ca M binding at this site as an agonist,after which Ca²⁺channel activity is induced in a concentration-dependent manner.This study suggests that the mode of action of scoparone involves targeting of the interface between Ca M1 and L-VGCC and modulation of the downstream Ca²⁺signalling pathways.Interestingly,Ca M enhances the channel-activating effects of scoparone.Scoparone activates the Ca M-binding site,which is located in the IQ motif at the Ca²⁺channel C-terminus.Thus,the IQ motif may be a promising novel“green acaricide”-binding target site of scoparone.Its identification may accelerate the development of novel acaricides to control destructive phytophagous pest mites worldwide.Furthermore,the findings of this study may contribute to the development of target-specific green acaricidal compounds based on L-VGCC.