C. elegans. PAN-1 localizes on cell membrane and binds with MYRF, a membrane-bound transcription factor indispensable for promoting synaptic rewiring. Full-length MYRF was known to undergo self-cleavage on ER membrane and release its transcriptional N-terminal fragment in cultured cells. We surprisingly find that MYRF trafficking to cell membrane before cleavage is pivotal for C. elegans development and the timing of N-MYRF release coincides with the onset of synaptic rewiring. On cell membrane PAN-1 and MYRF interact with each other via their extracellular regions. Loss of PAN-1 abolishes MYRF cell membrane localization, consequently blocking myrf-dependent neuronal rewiring process. Thus, through interactions with a cooperating factor on the cell membrane, MYRF may link cell surface activities to transcriptional cascades required for development.Neural circuits develop through a plastic phase orchestrated by genetic programs and environmental signals. We have identified a leucine-rich-repeat domain transmembrane protein PAN-1 as a factor required for synaptic rewiring in  C. elegans nervous system, in which six GABAergic motor neurons, known as dorsal D (DD) neurons, reverse their axonal and dendritic domains at the end of larval stage 1 (L1), with no overt changes in morphology   collectively indicates an essential role for Myrf in animal development. In mice, germline deletion of Myrf led to early embryonic lethality  . Myrf faethality , and oliormation . Deletiontiation . The nulelopment . Over seand eyes . In thesMyrf family, mouse Myrf, transcriptionally regulates expression of myelinogenesis-related genes in oligodendrocytes (Immunoglobulin (Ig)-fold like DNA-binding domain (DBD) homologous to the DBD of Ndt80, a pleiotropic transcription factor in yeast  without increasing in the nucleus  was pulled down using similar procedures, MYRF-1 and MYRF-2 were found among the most specific interactors of PAN-1. The results of the three IP-MS analyses indicate that MYRF and PAN-1 can form a protein complex in vivo.To identify factors that potentially interact with MYRF and regulate MYRF’s activity, we performed mass-spectrometry analysis of co-immunoprecipitation (IP-MS) of GFP::MYRF-1 as well as GFP::MYRF-2 from extracts of transgenic animals expressing romoters . We idenromoters . Inversein vitro. When both proteins were expressed in HEK293T cells, MYRF-1 could be co-immunoprecipitated with PAN-1 , a null deletion allele (pan-1(gk142) mutants (pan-1 mutants. Axon morphology of DDs in pan-1(gk142) was no different from control animals; if anything, the GFP intensity of dorsal processes in pan-1(gk142) appeared to be weaker (pan-1(gk142), labeled by synpatic vesicle clusters, appeared to be similar in puncta brightness and number, compared to wild-type animals. The presence of juvenile DD synapses in pan-1 mutants suggests that the loss of pan-1 does not generally affect synaptogenesis in DDs. Because pan-1 mutants are the first generation from heterozygous parent, it remains possible that maternal PAN-1 may contribute to embryonic and early L1 development of pan-1 mutants. Nevertheless, the absence of zygotic pan-1 results in synaptic remodeling defects that are similar to those when myrf is lost  protein containing 14 leucine rich repeat (LRR) domains, a transmembrane domain, and an N-terminal ER-targeting signal peptide (SP)  restored the synaptic remodeling defects in pan-1 mutants, while the other two isoforms, pan-1a or pan-1c could not (pan-1 (referring to pan-1b hereafter) driven by proflp-13  also rescued synaptic rewiring defects in pan-1 mutants  (d larvae . To conf neurons . Among tide (SP) . The panould not . Further mutants . In contpan-1(0) . Togethepan-1 mutants exhibit progressive larval arrest and never grow to L4 or fertile adults. Arrested animals feed, move, and live a normal life span that is similar to wild type (pan-1(0) is caused by general developmental arrest, we generated a cell-type-specific deletion of pan-1 by combining the LoxPpan-1 allele and a DD-expressing Cre line , such that some DD neurons may have acquired sufficient amount of PAN-1 for synaptic remodeling. Nevertheless, the data strongly suggest that the neuronal remodeling deficiency in pan-1(0) is neither a secondary result of overall developmental arrest in mutants, nor due to developmental delay. The results also further support a cell autonomous function of pan-1 in DDs.ild type . To addrCre line . The ani altered . Because altered , the lac altered  and adulGFPpan-1 knock-in allele the GFP tag was inserted at the C-terminal of the pan-1 coding sequence. PAN-1GFP signal was detected broadly in embryos and early larvae, but decreased substantially in late larvae and adults   . DeletinC. elegans. In GFPmyrf-1 knock-in allele, the GFP was inserted into the N-terminal region of MYRF, such that GFP labels both the full-length and N-terminal MYRF , a putative null indel allele, although the synaptic rewiring in S483A K488A GFPmyrf-1 appeared to be less complete than myrf-1(ybq6)  –6. When  –6. When blocked –12, consWe next characterized how MYRF and PAN-1 interact with each other using human cell lines . PAN-1 iTo further test which part of PAN-1 is required for MYRF interaction, we generated a series of truncated PAN-1 mutants and analyzed their ability to bind MYRF . The LRR1-700 GFPmyrf-1 that removed the entire extracellular domain from MYRF-1 . We tested if the C2 domain is particularly important for MYRF function (1-790 GFPmyrf-1 that removed the C2 domain (791-926). 1-790 GFPmyrf-1 mutant also displayed larval arrest at L2 . In contrast, 1-700 GFPmyrf-1 allele, in which the whole extracellular region of MYRF is lost, exhibited precocious synaptic rewiring, that is clearly detectable synaptic puncta formed in the dorsal processes of DDs at 12, 14 post-hatch hours, time points by which synaptic rewiring has not occurred yet in wild type animals  background. Notably, the precocious dorsal synapses in 1-700 GFPmyrf-1 mutants were not uniformly distributed along the dorsal cord, that is in some DDs the remodeling occurred while in the rest the remodeling did not, even though they were in the same animal, implying that the observed precocity in 1-700 GFPmyrf-1 mutants was not a strictly regulated event.While the loss of extracellular domains of MYRF resulted in larval arrest, we examined if such aberrations in MYRF led to defective synaptic rewiring in DDs. Remarkably, the two deletion alleles of ckground –12, a ph animals –12. The 1-700 GFPmyrf-1 mutants, we analyzed the M cell division, which undergoes larval stage-specific lineage progression ; even more so, M-cell division was delayed in 1-700 GFP; myrf-2(0)myrf-1 double mutants myrf-1 double mutants because many animals exhibited precocious synaptic rewiring while the M-cell division was delayed ; however, the process was incomplete because prominent GFP signals remained in cytoplasm  mutants was comparable to that in wild type animals  was not due to failed transcription of myrf genes, as myrf-1 and myrf-2 transcript levels were not significantly changed in pan-1(0) mutants  animals, MYRFGFP signals re-emerged in many cells, indicating that MYRF proteins are subject to proteasome degradation in the absence of PAN-1. Importantly, restored MYRFGFP in pan-1 mutants was not localized to the cell membrane or nucleus; rather, the signal appeared in ER-like structures in the cytoplasm, suggesting that interaction between PAN-1 and MYRF initiates when they are in ER. In conclusion, PAN-1 is necessary for normal localization and function of MYRF.One explanation for the disappearance of MYRF in e system  and test mutants . When crpan-1 can restore the cell-membrane localization of MYRF in pan-1 mutants. To this end, we used a seam cell-specific promoter to drive pan-1 expression  animals, indicating that MYRF had been normally localized and processed in those cells. This result also supports a cell-autonomous function for pan-1.We asked if exogenously expressed pression  and obse1-700 GFP, the mutant protein was predicted to be able to evade the degradation surveillance even when PAN-1 was unavailable. The prediction proved to be correct because MYRF-11-700 GFP signals remained strong in pan-1(0) mutants , resembling wild-type MYRF in pan-1(0). Therefore, PAN-1 is essential for stabilizing MYRF and trafficking it to cell membrane.The propensity of MYRF being degraded in the absence of PAN-1 implies that the non-cytoplasmic region of MYRF is propone to degradation. Since the whole extracellular region of MYRF was removed in MYRF-1 mutants . In cont681-931): one fused to TMD of NEP-2, a type II transmembrane metallopeptidase Neprilysin (681-931 and SP:: MYRF-1681-931) in DD neurons blocked synaptic remodeling, supporting that the interaction between PAN-1 and MYRF is important for MYRF activity.To further test if MYRF functionally interacts with PAN-1 at the cell membrane, we aimed to disrupt their interaction by overexpressing the extracellular region of MYRF-1 and analyzed how the perturbation affected MYRF’s activity . To thisprilysin , and theprilysin . OverexpGFPmyrf-1, we observed much brighter MYRFGFP signals in target cells than in neighbor cells, and importantly, the signals were primarily cytoplasmic showing a pattern similar to ER structures. These results support that PAN-1 promotes MYRF’s transport to cell membrane, and that PAN-1 interacting with MYRF in ER is insufficient for MYRF’s cleavage.Since the interaction of PAN-1 and MYRF likely begins in ER, we seek a way to trap MYRF in ER so that MYRF does not traffick to cell membrane. We expressed a modified PAN-1 without transmembrane domain and fused with KDEL motif, the ER retention signal, in DDs . This al1-482 GFPmyrf-1 that removed the entire carboxyl region from the cleavage point to the end of MYRF. This mutant MYRF-11-482 GFP concentrated in the nucleus throughout the body  (The synaptic rewiring in both -1(ybq6) –12 There-1(ybq6) .C. elegans. PAN-1 interacts with MYRF on the cell membrane, and the interaction is required for MYRF’s cell membrane localization and nuclear translocation  concluded that the full-length MYRF resides on ER before undergoing constitutive self-cleavage; but none of the studies has described if MYRF could be detected on the cell membrane . Our obs1-700 GFP indicate that the ectodomain of MYRF is indispensable, further suggesting a regulatory role for PAN-1. The extracellular domain of PAN-1 is highly homologous to the ectodomain of human Toll-like receptor 5 (TLR5), a member of the toll-like receptor (TLR) family  family . TLR5 is) family . AlthougC. elegans and mouse , lacking the transmembrane domain and non-cytoplasmic region, was much less competent at promoting oligodendrocyte maturation than full-length MYRF, even though the truncated MYRF(1-756), when overexpressed, can be processed to release N-MYRF in cultured cells  appeared to be deficient in MYRF function, even though N-MYRF-1 in all cases concentrated in the nucleus. We reasoned that overexpressed transgene N-MYRF-1 could sufficiently form trimers, a prerequisite for its full transcriptional activity, whereas endogenously expressed N-MYRF-1 could not. To test the hypothesis, we generated two single-copy transgene lines, expressing full-length MYRF-1 and N-MYRF-1, respectively. We found that the full-length MYRF-1 could rescue the defective synaptic rewiring in myrf-1; myrf-2 double mutants, while N-MYRF-1 could not  is insufficient for MYRF’s cleavage.We previously determined that overexpressing N-MYRF-1rewiring . Howeverould not . This reIn cultured cells, human MYRF is found to carry post-translational modifications, for example glycosylation . MembranC. elegans were Bristol N2 strain. Strains were cultured on NGM plates using standard procedures (Wild-type ocedures  at 20–23Human embryonic kidney cells 293 (HEK293T) were obtained from cell bank of CAS (Shanghai). The cells were verified by STR profiling and tested to be free of mycoplasma contamination by stand PCR methods.All alleles generated in Y.B.Q. lab are designated as ‘ybq’ alleles, and all strains, as ‘BLW’ strains. ‘Ex’ denotes transgene alleles of exchromosomal array. ‘Is’ denotes integrated transgene. ‘syb’ alleles (in ‘PHX’ strains) are generated by genomic editing using CRISPR-Cas9 technique. ‘syb’ alleles were designed by Y.B.Q. and produced by SunyBiotech .For the following described alleles generated by CRISPR-Cas9 editing, Cas9 and gRNA were expressed from plasmids. The positive clones were identified using PCR screening to test singled F1 resulted from microinjection.S483A K488A GFP(syb1487) allele was generated in the background of strain BLW889, GFP::myrf-1::3xFlag(ybq14). The resulted allele changes Ser 483 to Ala; Lys 488 to Ala. gRNA targets are:myrf-1CCGATATTCGACTCAAGGAAGCA;Sg1:TCCATCCGATATTCGACTCAAGGSg2:TCCGATATTCGACTCAAGGAAGCAATTACTGAACGGGAAACTGCTGAAATTTATATGAGTGGCCGAATAATCAATCCAybq14(background): …GCTGATATCAGACTTGCAGAAGCAATTACTGAACGGGAAACTGCTGAAATTTATATGAGTGGCCGAATAATCAATCCAsyb1487: …1-700 GFP(syb1313) allele was generated in the background of strain BLW889, GFP::myrf-1::3xFlag(ybq14). The resulted allele removes G701-T927 of MYRF-1 coding sequence. gRNA targets are:myrf-1CCACTAAAGGAGAACTCGCCAAT;Sg1:CCTATCATCTTCAGACTACAAGGSg2:CATTTCGAAACGAACACACCATCCACTAAA-(951 bp)- CTATCATCTTCAGACTACAAGGACCACGACybq14(background): …CATTTCGAAACGAACACACCATCCACTAAA-(delete 951 bp)-CTATCATCTTCAGACTACAAGGACCACGACsyb1313: …1-790 GFP(syb1333) allele was generated in the background of strain BLW889, GFP::myrf-1::3xFlag(ybq14). The resulted allele removes I791-C926 of MYRF-1 coding sequence. gRNA targets are:myrf-1CCTGCACTGAATGTGACACTTGA;Sg1:CCACGACGGTGACTACAAGGACCSg2:GAGCACATGGCTTTTGAAACTGGAGTTGAG-(551 bp)-ACCCTATCATCTTCAGACTACAAGGACCACybq14(background): …GAGCACATGGCTTTTGAAACTGGAGTTGAG-(delete 551 bp) CCTATCATCTTCAGACTACAAGGACCACsyb1333: …1-482 GFP(syb1491) allele was generated in the background of strain BLW889, GFP::myrf-1::3xFlag(ybq14). The resulted allele removes Ser483-Ser931 and 3xFlag. gRNA targets are:myrf-1CCGATATTCGACTCAAGGAAGCA;Sg1:CCACGACGGTGACTACAAGGACCSg2:ATTTATATGAGTGGCCGAATAATCAATCCA-(1683 bp)-TAAgctaatgcttcatatatcatttttgtt…ybq14(background): …ATTTATATGAGTGGCCGAATAATCAATCCA-(delete 1683 bp)-TAAgctaatgcttcatatatcatttttgtt…syb1491: …1-656 GFP(syb1468) allele was generated in the background of strain BLW889, GFP::myrf-1::3xFlag(ybq14). The resulted allele removes Gly657-Ser931 and 3xFlag. gRNA targets are:myrf-1CCGACTTAGTCAAGGAACAGTTG;Sg1:CCACGACGGTGACTACAAGGACCSg2:GCACAATCCTGTGGAAGCCGACTTAGTCAA-(1161 bp)-TAAgctaatgcttcatatatcatttttgtt…ybq14(background): …GCACAATCCTGTGGAAGCCGACTTAGTCAA- (delete 1161 bp)- TAAgctaatgcttcatatatcatttttgtt…syb1468: …1-728 GFP(syb1454) allele was generated in the background of strain BLW1111, myrf-2(ybq46)GFP. The resulted allele removes Leu729-Val949 of MYRF-2 coding sequence. gRNA targets are:myrf-2CGTCGTTATTCCATTGGgtaagg;Sg1:CCGCGCGTGCAATAGAACAAACTSg2:GTCGTTATTCCAAAGGAAGGTCCAGGAAAC-(1244 bp)-TAAcatcagagataatcacacaatagatac…ybq46(background): …GTGGTTATTCCAAAGGAAGGTCCAGGAAAC-(delete 1244 bp)-TAAcatcagagataatcacacaatagatac…syb1454: …pan-1GFP(ybq47) was generated in the background of wild-type (N2). GFP is inserted before stop codon (TAA) of the PAN-1 coding sequence. gRNA target:GGAATTTTCAGCACGGTCAAAGG. sgRNA#2, GGGCCGAAGAAGACTGTACGATTCCAGAATTTTAATACT-(insertion point)-TAAagaagaaaaatttcataacctcaaatcaWT: …GGACGAAGAAGACTGTACGATTCCAGAATTTTAATACT-GFP-TAAagaagaaaaatttcataacctcaaatca.ybq47: …LoxP(syb1217) allele was generated in the background of wild-type (N2). Two LoxP sites are inserted into flanking introns of the third exon of pan-1b gene. gRNA target sites: Sg1:CCAGTATGCATCTGCGCCGACAA; Sg2:GTATGCATCTGCGCCGACAACGG; Sg3:CAAGTTAAACTCGAACGATGTGG; Sg4:CCAAATTCCTTCCAATCCCTTGG; Cas9 and gRNA were expressed from plasmids. The repair template is plasmid-based. The positive clones were identified using PCR screening to test singled F1 resulted from microinjection.pan-1WT: …TGCGCCGACAACGGAATTTTCAGCACGGTCAAAGGATTTACTATAGAgtatgttgaattttactatacctgtaacttttcagCCAGTATGCATC-(insertion point)-cgactataaaacttcttgaaactgaaattttcttttcagATGCGAATCCGCTTCAATTGCATCTGTATCTGAAAATTTGGCTTCACTGAATGGCACAGAATTGGGACGTCTCACAATAAGAGATTCTACAGTTAATGTTCTTCCACAGGATCTTTTTGAGAATgtaagttgaattacaataatgccaatcaggacagaaacgtatatc-(insertion point)-AAACTCGAACGATGTGGTCTTTCAACTCTTCAACCAAATTCCTTCCAATCCCTTGGAGGATCAGCTGAAactagttaacttttttctcatcgcggcgagacccacttaaatcaatccaattttcagGTCTTCGCTAAACAAGTTsyb1217: …TGTGCCGACAACGGAATTTTCAGCACGGTCAAAGGATTTACTATAGAgtatgttgaattttactatacctgtaacttttcagCCAGTATGCATC-(ATAACTTCGTATAGCATACATTATACGAAGTTAT)-cgactataaaacttcttgaaactgaaattttcttttcagATGCGAATCCGCTTCAATTGCATCTGTATCTGAAAATTTGGCTTCACTGAATGGCACAGAATTGGGACGTCTCACAATAAGAGATTCTACAGTTAATGTTCTTCCACAGGATCTTTTTGAGAATgtaagttgaattacaataatgccaatcaggacagaaacgtatatc-(ATAACTTCGTATAGCATACATTATACGAAGTTAT)-AAGCTCGAACGATGTGGTCTTTCAACTCTTCAACCAAACTCCTTCCAATCCCTTGGAGGATCAGCTGAA.actagttaacttttttctcatcgcggcgagacccacttaaatcaatccaattttcagGTCTTCGCTAAACAAGTTpro-pan-1b(ybqIs130): The vector pQA1237 was injected into N2 at 20 ng/μl in N2. The resulted Ex array was integrated to generate ybqIs130. unc-25pro-pan-1a(ybqEx624): The vector pQA1239 was injected into BLW1167  at 20 ng/μl. unc-25pro-pan-1c(ybqEx626): The vector pQA1240 was injected into BLW1167 at 20 ng/μl. unc-25pro-pan-1b(ybqEx821): The vector pQA1284 was injected into BLW1056 , at 5 ng/μl.flp-13pro-pan-1b(ybqEx741): The vector pQA1298 was injected into BLW1056 at 10 ng/μl.dpy-7pro-pan-1b(ybqEx737): The vector pQA1297 was BLW1056 at 10 ng/μl.myo-3pro-pan-1(ybqEx739): The vector pQA1583 was injected into BLW1056 at 10 ng/μl. vha-6pro-NLS-tagRFP(ybqIs138): The vector pQA1311 was injected into N2 at 20 ng/μl, and the Ex array transgene was integrated to generate ybqIs138. pan-1pro-pan-1b::mCherry(ybq Ex778).The vector pQA1534 was injected into BLW1465  at 20 ng/μl. seam-cellpro-nCre(tmIs1073) was sent by National Bioresource Project for the Experimental Animal ‘Nematode C. elegans’ ( unc-25elegans’ .pro-pan-1ΔLRR(ybqEx642)unc-25; pQA1323 Punc-25-pan-1::GFP-ΔR2-S18 (SP) for pro-pan-1ΔSP(ybqEx643)unc-25; pQA1324 Punc-25-pan-1::GFP-ΔG513-F538 (TM) for pro-pan-1ΔTM (ybqEx644)unc-25; pQA1337 Punc-25-pan-1::GFP-ΔK539-F594 (Cyto) for pro-pan-1ΔCyto(ybqEx645)unc-25.The following vectors were injected into BLW1056 at 50 ng/μl. pQA1322 Punc-25-pan-1::GFP-ΔE130-S442  for pro-SP-MYRF-1Ex.C.unc-25 (ybqEx752); pQA1540 Punc-25-nep-2 (1–51)::myrf-1 (681–931) for pro-TMD-MYRF-1Ex.C.unc-25 (ybqEx754).The following vectors were injected into CZ8656 [Punc-25-mCherry::Rab-3(juIs236)] at 50 ng/μl. pQA1539 Punc-25-mig-17(1-40)::myrf-1 (681–931) for The vector pQA1654 was injected into BLW1419 [Pflp-13-GFP::rab-3(ybqIs47)] at 20 ng/μl to make Punc-25-pan-1(1-G513)::mCherry::KDEL(ybqEx815); Punc-25-pan-1(1-G513)::mCherry::KDEL(ybqEx817). The vector pQA1654 was injected into CZ8761  to make Punc-25-pan-1(1-G513)::mCherry::KDEL(ybqEx807), and ybqEx807 was crossed into BLW889 to make BLW1890. The vector pQA1652 was injected into BLW889 to make Pscm-Pan-1(1-G513)::mCherry::KDEL(ybqEx820).C. elegans of RNAi library generated by M. Vidal et al.) were cultured in LB with 100 μg/ml of Ampicillin for 16–18 hr at 37°C. About 50 μl of bacteria liquid culture was seeded onto a NGM plate with 100 μg/ml of Ampicillin and 1 mM of IPTG (Takara) for induction of dsRNA expression. Stage-synchronized early L1 animals were cultured on RNAi plates at 20°C for 24 hr. The animals were then examined and imaged under OLYMPUS BX63 microscope.HT115(DE3) strains carrying empty vector L4440, ubq-2, or rpt-2 plasmids (from pro-mCherry::rab-3(juIs236); pan-1(gk142); hlh-8pro-GFP(ayIs6)) of L2 that were hand-picked, together with 50 μl of the stage-matched control animals  were collected and frozen in the RNAiso plus reagents (Takara). Total RNAs were extracted and the cDNAs were synthesized using the reverse transcription system (Takara). Quantitative PCR reactions were performed in triplicates on a Quantstudio 7 PCR machine using the SYBR Green dye (Vazyme). Relative gene expression levels were calculated using the 2−ΔΔCt method and plotted as median with range48M. RT-qPCR experiments were performed at least three times using RNA samples derived from animal tissues collected independently. The data were analyzed in GraphPad Prism. qPCR primers include: myrf-1, gacgatggctcaatggtgtc(F), ttgggtccgaatgatgtgat(R), 214 bp; myrf-2, ccgatgacatcccaaatcgcaaac(F), gaagagttgggtcgtctccaag(R), 204 bp, pan-1, cctcccaagtccgtactccattc(F), ccattccttccaatgcgcgag(R), 174 bp; pmp-3, tggccggatgatggtgtcgc(F), acgaacaatgccaaaggccagc(R), 190 bp.About 600 synchronized pan-1(0) mutants  were taken by a sCMOS camera (PrimΣ Photometrics camera (model 2)) mounted on OLYMPUS BX63, which is driven by CellSens Dimension software (Olympus). Images of live animals were also acquired on Zeiss LSM880 with Airyscan. The thickness of the optical slices was typically 0.8 μm.For quantification of patterns of GFP::MYRF, images of stage-synchronized animals were acquired using wide-field microscopy described as above. The same parameters were used, including the power of excitation light, identical objective, exposure duration, and screen display setting. The images are examined and the patterns of GFP::MYRF were categorized based on how consistently the signals are observed at particular subcellular localization throughout the animal body. ‘Weak or unclear signal’ means that no clear signals are detected, or there are some weak signals but they are inconsistent throughout the animal body. Three independent rounds of culture, imaging, and scoring were carried out, and the data were pooled and presented in the percentage column graphs.GFP (and variants) and RFP-Rab-3 colabeling, each image acquired from Zeiss Airyscan was opened in ImageJ in color mode, a rectangular ROI was selected to contain a complete DD soma, a plug-in program ‘3-D surface plot’ in Analyze menu was run with the following parameters, Filled; Grid Size 128; Smoothing 1.5; Perspective 0; Lighting 0.2 to generate the plot image.For generation of the 3-D plot of MYRF-11-700 GFP and MYRF-1(syb1333)1-790 GFP, each image stack acquired from Zeiss Airyscan was opened in ImageJ and a single slice with focused seam cell was selected for further analysis. A 20 pixel (~1 μm)-thick, 12-μm-long ROI line was drawn to cross a seam cell in head-to-tail direction. The middle point of the ROI line was positioned at the center of the nucleus. A ROI for analyzing single ventral cord neuron was similarly drawn except for that the line was 5μm-long. A program ‘plot profile’ located in Analyze menu was run and plot data was recorded. The final graph was generated in GraphPad Prism 6. Each plot line represented the intensity distribution across a single cell from an independent animal.For generation of the line plot of MYRF-1(syb1313)GFP were carried out similarly to GFP::MYRF. Whether PAN-1GFP are consistently observed on cell membrane throughout the animal body was examined and quantified. For quantification of intensity of PAN-1GFP in myrf-1(ju1121) mutants, images of PAN-1GFP in myrf-1 mutants and controls were acquired by wide-field microcopy similarly to GFP::MYRF, and then analyzed in ImageJ. A ROI of 5 pixel (~0.8 μm)-thick line was drawn along the PAN-1GFP signals at the cell membrane of seam cells. For images acquired from Zeiss Airyscan, each image was opened in ImageJ, and a ROI of 5 pixel (~250 nm)-thick line was drawn along the PAN-1GFP signals at the cell membrane of seam cells. The mean intensity of the ROI was measured and recorded. At least two seam cells were measured in each animal. A single data point represented the mean intensity of PAN-1GFP signals for each animal.Quantification of patterns of PAN-1Bleach-synchronized arrest young L1 were cultured at 20°C to appropriate post-hatch hours. Animals were anesthetized, and imaged on OLYMPUS BX63 microscope using wide field sCMOS camera. The z-stack was maximally projected to produce a single image in ImageJ, or single section was analyzed when Z-maximum-projection did not produce clear image . The complete ventral and dorsal cords were selected using Segmented Line tool to generate a line ROI of 30 pixel-wide (~5 μm). Run Plot Profile under ‘Analyze’ menu. Run Find Peaks under BAR-Data Analyze, set values in ‘Find Local Maxima/Minima’ window so that the program detects all visible synapses while excluding minor peaks, and execute the counting. Signals of each cord were also examined by eyes to confirm there was no major discrepancy between human evaluation and computer counting. The values and statistics are then processed in GraphPad Prism. The mean number of puncta for individual group was presented in the scatter dot plot graphs.For the analysis of the growth of the animals, DIC images of animals were acquired on OLYMPUS BX63 microscope using x10 objective. The lengths of animals in the images were measured using polyline tool in the OLYMPUS imaging software. The data were further analyzed in GraphPad Prism.myrf-1 was mammalian codon optimized and encodes C. elegans myrf-1 protein. The DNA V5::myrf-1::3xFlag (in pQA1228) was synthesized by Genscript. 6xHis::3xFlag::myrf-1 (1–482) (in pQA1250) and myrf-1 (483–931)::3xFlag (in pQA1296) were made by mutagenesis from pQA1228. myrf-1 (680–931)::3xFlag (in pQA1525) and myrf-1 (586–931): :3xFlag (in pQA1581) were made by mutagenesis from pQA1296.All constructs used in transient expression in HEK293T cells were built in pcDNA3.1 with CMV promoter. Full-length pan-1b was cloned by PCR from C. elegans cDNA and inserted into pcDNA3.1 . pan-1 (19–594) (ΔSP) (in pQA1527), pan-1   (in pQA1580), pan-1 (1–512)  (in pQA1528), and pan-1  (ΔLRR) (in pQA1535) were made by mutagenesis from pQA1524 and they all have HA-His tag at C’.Full-length 5 per well were seeded in 500 μl antibiotic-free medium on the day before transfection. The cells were let grown to 90–95% confluence by the time of transfection. Discard the old medium before transfection. Prepare transfection mix following the recipe: For each well, dilute plasmid DNA (2.5 μg) in 150 μl DMEM serum-free medium and mix gently. The following was an example of set up: (1) Plasmid A(1.25 μg)+Control plasmid(1.25 μg); (2) Plasmid B(1.25 μg)+Control plasmid(1.25 μg); (3) Plasmid A(1.25 μg)+Plasmid B(1.25 μg). Gently shake Lipofectamine-2000(Thermo Fisher) well before use. Take an appropriate amount of Lipofectamine-2000 (6 μl) and dilute it in 150 μl DMEM medium. Incubate at room temperature for 5 min. Mix the DNA and Lipofectamine-2000 to make the total volume 300 μl, gently mix and leave at room temperature for 20 min. Add 300 μl of transfection solution to each well of cells and shake gently. The cells were returned to 37°C, and the medium was changed after 4–6 hr of transfection.HEK293T cells were cultured in DMEM supplied with 10% Foundation FBS (Gemini) and 100 units/ml penicillin/streptomycin (HyClone) antibiotics. in six well-plates. For transient transfection, adherent cells of 0.5–2 × 10The transfected cells were incubated at 37°C for 24 hr. The cells were lysed in 1 mL of cell lysis buffer . The lysate was incubated on ice for 30 min and centrifuged at 14,000Xg for 30 min at 4°C. The supernatant was collected and incubated with addition of primary antibodies, including anti-HA  and rabbit IgG  controls at the same molar concentration as primary antibodies, respectively, at 4°C for overnight. The next day, Dynabeads protein A  were added to the reactions and incubated at 4°C for 1 hr to precipitate the IgGs. Beads were washed and treated in SDS sample loading buffer. The samples were denatured at 95°C for 10 min and analyzed by SDS-PAGE and western blot. The antibodies used in western blot included: mouse anti-V5 ; mouse anti-HA ; Rabbit Anti-Actin ; Mouse Anti-Mouse IgG Polyclonal Antibody, HRP Conjugated , Mouse Anti-Rabbit IgG Polyclonal Antibody, HRP Conjugated .Worms expressing the GFP-tagged proteins were cultured to the indicated stage, collected, and washed three times with M9 buffer. The volumes of the worm pellets obtained after centrifugation and sedimentation on ice was recorded before the worm pellets were frozen in liquid nitrogen. The frozen worm pellets were cryo-milled using Mixer Mill MM 400 (Retsch) at 30 Hz for 60 s each time for a total of four times. The resulting powder for each sample was transferred to a new tube, mixed with an equal volume of 2 × pre cool lysis buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 1% Triton-X100, 10% glycerol, 1 mM PMSF, 2 × Roche cOmplete EDTA-free Protease Inhibitor Cocktail, 10 mM NaF, and let rotate at 4°C for 30 min. The lysate was then centrifuged at 13,000 rpm for 30 min at 4°C and the supernatant was incubated with homemade anti-GFP beads for one hour at 4°C before the beads were washed three times with cold lysis buffer. The anti-GFP immunoprecipitates were eluted with chilled elution buffer (100 mM glycine-HCl pH 2.5) and precipitated with 25% TCA. After cold acetone wash and drying in air, the immunoprecipitated proteins were dissolved in 100 mM Tris, pH 8.5, 8 M urea, reduced with 5 mM TCEP, alkylated with 10 mM iodoacetamide, and digested with trypsin overnight at 37°C.LC-MS/MS analysis of the resulting peptides was conducted on a Q Exactive mass spectrometer (ThermoFisher Scientific) interfaced with an Easy-nLC1000 liquid chromatography system (ThermoFisher Scientific). Peptides were loaded on a pre-column  and separated on an analytical column  using an 0–30% (v/v) acetonitrile gradient over 78 min at a flow rate of 200 nl/min, with 0.1% (v/v) formic acid added to the mobile phase throughout. The top 15 most intense precursor ions from each full scan  were isolated for HCD MS2 , with a dynamic exclusion time of 30 s. Precursors with unassigned charge states or charge states of 1+, 7+, or > 7+ were excluded. Each sample was analyzed in technical duplicates.http://pfind.ict.ac.cn/) against the UniProt C. elegans protein database (WS235). The filtering criteria were: 1% FDR at both the peptide level and the protein level; precursor mass tolerance, 20 ppm; fragment mass tolerance, 20 ppm; the peptide length, 6–100 aa. To identify high-confidence candidate interacting proteins, The WD-Scores of proteins identified in each IP-MS sample were calculated against the identification results of 71 unrelated IP-MS samples, according to the formulas from the CompPASS tutorial  and were intrigued by the implication of a novel protein in the control of the very unusual MYRF transcription factor. The reviewers and editors also appreciated the many advances that you made, as well as the very solid nature of some the reagents that you used . However, all reviewers identified a substantial list of shortcomings. All these shortcomings are relatively straight-forward to address, but do amount to a substantial body of work. We will therefore need to reject the manuscript in its present form. However, should you be willing to engage with these specific requests, we may want to consider a substantially revised version of this manuscript sometime in the future.Reviewer #1:C. elegans. A previous study  showed that animals lacking the very unusual, but conserved transcription factor MYRF-1 show defects in DD rewiring at the late L1 stage. The present study finds that an LRR-containing transmembrane protein, PAN-1, interacts with the membrane bound MYRF-1. Both proteins localize to the cell membrane, and their interaction seems to be required for DD-rewiring. pan-1 mutants also show defects in DD-rewiring, and loss of PAN-1 affects MYRF-1 localization. The interaction between these two proteins occurs through their extracellular domains, and the extracellular portion of MYRF-1 is required for its activity.In this manuscript, the authors show molecular events underpinning rewiring of DD neurons in I find this to be a very interesting paper that takes us a step further in understand how this very unusual activation mechanisms for a membrane bound (!) transcription factor works. However, some conclusions should be supported by more data and/or better data presentation. Moreover, some results are poorly described and there is general lack of detail throughout the manuscript. Specific points are listed below:1. The manuscript shows that PAN-1 and MYRF-1 interact, and that mutants of each gene result in DD-rewiring defects . However, it does not adequately show that several perturbations leading to changes in PAN-1 and MYRF-1 localization result in DD-rewiring defects, the relevant paradigm of the study. For example, the authors make several truncations in PAN-1 and MYRF-1 to test their interaction using Co-IP , summarizing that their "results provide the first direct evidence that the non-cytoplasmic part of MYRF protein is indispensable for its function". However, they do not show what happens to DD-specific dorsal synapses in animals with truncations in MYRF-1 .2. The presentation of data for DD-rewiring defects in pan-1 mutants in Figures 1D, 1E and 5B is not convincing. Representative images in Figure 1C (middle) and 1F (bottom-right) clearly show that synapses are present, albeit much dimmer or with gaps, but this is not reflected in the quantification. Perhaps providing several representative images of pan-1 animals (and of pan-1a and pan-1c rescue in pan-1 mutants), like those shown in Figures 1F and S1A, would allow the reader to better interpret the DD-rewiring phenotype. Representative images should also be provided for PAN-1 variant animals quantified in Figure 5B.3. Throughout the manuscript, the authors show changes in subcellular localization using seam cells as an example. These images are great and clearly convey the point, but serve as controls at best. Since the manuscript is framed around DD-rewiring, it is critical to show similar spatiotemporal changes in MYRF-1 localization in WT  and pan-1 mutants  in DD neurons. This region, with existing reporters, may not look as clear-cut, but an overall distribution of signal in cell-membrane / cytoplasm / nucleus may be evident using a confocal microscope and would allow the reader to better assess the results.Reviewer #2:C. elegans. They find that PAN-1, a TM-LRR protein, can interact in vitro and in vivo with MYRF-1/2, a transmembrane-tethered DNA-binding protein previously shown to be required for DD remodeling. They present an interesting model in which PAN-1 localizes MYRF-1/2 to the plasma membrane (a novel finding) and that regulated nuclear entry of the MYRF-1/2 N-terminal fragment is important for its function. However, there are some gaps and over-interpretations that, for me, undermine confidence in the model.The paper by Li et al. makes some intriguing findings about the mechanisms underlying DD synaptic remodeling in 1. For a few reasons, I have concerns about the specificity of the pan-1 phenotype. They don't show us whether overall DD anatomy is normal in pan-1 mutants. The DD/VD-specific rescue is nice, but isn't it possible that pan-1 expression in VDs (which are born during L1) has a role in DD rewiring?  Similarly, it's great to see the use of a pan-1 conditional allele, but its phenotype is really weak compared to the null, raising concern that some of the mutant defects could arise from pleiotropic functions .2. Along these lines, there's some confusion about what's meant by pan-1 and myrf-1 "activity." Because they're both expressed broadly, it's possible that they have different functions in different tissues and/or at different times. But the model doesn't consider this, and all phenotypes  are interpreted in terms of the same molecular model. Many of the key conclusions about what's going on in DD remodeling are drawn from looking in other cell types or at larval arrest . This makes me less confident that we really understand what's going on in DDs themselves.3. According to the simplest model, expression of the myrf-1 N-terminal fragment should cause premature DD remodeling. Unexpectedly, it causes early (L1) larval arrest, a gain-of-function phenotype, since the null arrests during L2. This is consistent with the idea that transient membrane localization of MYRF-1 is necessary, but it could also be that it's simply that temporally regulated production/activity of the N-terminal fragment is important. In either case, it seems important to know whether premature DD remodeling has occurred in the arrested L1s.4. In the final set of experiments, the MYRF-1 extracellular region is overexpressed, which blocks synaptic remodeling. Authors conclude that this demonstrates that the MYRF-1-PAN-1 interaction is essential. However, many other interactions (or other process) could also be disrupted by overexpressing this fragment, so it's not clear that the defect is truly a specific consequence of the disrupted interaction.Reviewer #3:In a previous genetic screen, the authors identified the MYRF transcription factors as key-components required for synaptic rewiring of the DD neurons. By pulling down MYRF interacting partners, the authors here identified the membrane protein PAN-1 as a necessary factor required for MYRF activity. Previous models for MYRF activation suggested that MYRF undergo self-cleavage on ER membrane. The authors convincingly demonstrate that MYRF and PAN-1 physically and functionally interact, which is new and well documented, even if I have concerns with some data.1. Because PAN-1 is found at the plasma membrane, the authors propose, first, that the localization at the PM is necessary for MYRF activation and, second, that "MYRF links cell surface activities to transcriptional cascades required for development". My main concern is that, at this stage, this remains an appealing hypothesis, which is not directly supported by experimental evidence. PAN-1 is also detected in intracellular compartments, at least in DD neurons , and might as well act by promoting MYRF oligomerization and cleavage early during the secretion pathway. Non-cleaved MYRF might then travel with PAN-1 to the membrane, but the functional relevance of this distribution is not demonstrated so far. One way to test this hypothesis would be to force the retention of PAN-1 in the ER and analyze MYRF processing and MYRF-dependent phenotypes.2. In their previous article , the authors demonstrated that they could rescue DD rewiring by expressing the N' 1-482 domain of MYRF-1 in D neurons. In the present manuscript, they show that the same fragment no longer rescues myrf-1  and conclude that "MYRF membrane association is indispensable to its activity". This apparent contradiction needs to be solved.3) At a technical level, imaging of PAN-1 and MYRF-1/2-GFP fusions are analyzed in a mixture of cell populations. Except for seam cells, the images are not very demonstrative and the quantification performed in the figures 3 and 4 appears quite subjective. To correlate DD rewiring with MYRF-1 relocalization, it should be necessary to focus on these neurons, use co-markers of ER, PM and nuclei, and perform quantitative analyses.Altogether, it is not clear if PAN-1 is constitutively permissive for MYRF function in any tissue or can regulate MYRF activity, especially in the context of DD rewiring. Although a definite answer cannot be provided in this article, the first possibility cannot be dismissed and should be discussed. [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]All reviewers very much appreciated the very interesting biological problem  and were intrigued by the implication of a novel protein in the control of the very unusual MYRF transcription factor. The reviewers and editors also appreciated the many advances that you made, as well as the very solid nature of some the reagents that you used . However, all reviewers identified a substantial list of shortcomings. All these shortcomings are relatively straight-forward to address, but do amount to a substantial body of work. We will therefore need to reject the manuscript in its present form. However, should you be willing to engage with these specific requests, we may want to consider a substantially revised version of this manuscript sometime in the future.Common key points:1. While all new myrf-1 truncation alleles exhibit larval arrest, there is a lack of characterization of DD rewiring phenotype in those mutants.1-700 GFP, in which the whole extracellular region of MYRF-1 is deleted. myrf1(syb1313) exhibits precocious yet discordant DD rewiring. We have also observed that MYRF-11-700 GFP(syb1313) can be partially processed in ER to produce N-MYRF while the majority of MYRF-11-700 GFP(syb1313) protein appears to be in ER. MYRF-11-700 GFP(syb1313) signals remained strong in pan-1(0) mutants, while the signals of MYRF-11-790 GFP(syb1333), in which part of extracellular region was removed, were undetectable in pan-1(0), resembling wild type MYRF in pan-1(0). Thus, from an unexpected angle, these new results support that the extracellular region of MYRF is indispensable for MYRF’s processing, and the cleavage of wild type MYRF is under developmental regulation. See Figure 6; 8, 9; Figure S5-16.We have now added the analysis of DD synaptic rewiring for all new myrf-1 truncation alleles, and corresponding myrf-1(truncation); myrf-2(null) double mutants. All new myrf-1 alleles, except for myrf-1(syb1313), behave similarly to previously characterized myrf-1 loss of function mutants, i.e., they exhibit blocked synaptic rewiring in myrf-2 null background. The exception is myrf-1(syb1313)GFP in DD neurons.2. There is a lack of localization analysis for truncated MYRF-1GFP variants with DD-specifically expressed mCherry::Rab-3. Rab-3 signals are localized to presynaptic vesicles, but also often in cytoplasm, presumably labeling a subset of ER, Golgi, or endosomes. With contrastive localization of MYRF-1GFP variants and mCherry::Rab-3, one can appreciate the differential localization of MYRF-1GFP variants on cell membrane, cytoplasm, and nucleus. Because DDs are small in size (3µm in diameter), further colocalization analysis using specific organelle markers are often unproductive due to the resolution limitation. See Figure 3D, 4F, 5G, 8F, 12G.MYRF-1-GFP signals are very weak, e.g., they cannot be detected by conventional confocal. Fortunately, we were able to image them under Zeiss Airyscan. For certain imaging experiments, myrf-1 alleles were crossed into glo-1 mutant background to reduce gut auto fluorescence. We have added new sets of images to display signal patterns of MYRF-1 variants. We have also added images of colabeling MYRF-13. The choice of flp-13 and unc-25 promoterspro driven synaptic marker also label an unidentified head neuron that extend its axon to the anterior half of ventral cord, which only becomes clear until L3-L4. Together, unc-25 and flp-13 have its pro and con as synaptic marker for DDs.unc-25 encodes Glutamic Acid Decarboxylase, and the particular unc-25 promoter that we use labels 4 RMEs (head neurons that form neuromuscular junctions with head muscles to control foraging) and 6 DDs in L1 larvae. The unc-25 promoter also labels 13 VDs, which are born at late L1. flp-13 encodes a FMRF-Like Peptide, and is expressed in 6 DDs not in VDs among ventral cord motor neurons; however, flp-13 is expressed in many other head neurons. We even observed that the flp-13 promoter-driven marker was expressed in intestine, and such expression is particularly strong during early-mid L1, which can obscure the DD signals. flp-13pro is often weak in DD5 and very weak in DD6. flp-13pro-pan-1 worms were thin and pale, and difficult to form lines, while unc25pro-pan-1 worms did not show any signs of sickness. We reasoned that the overexpression of pan-1 caused defects because flp-13pro was expressed in vital tissues.When it comes to experiments of cell-autonomy test, unc-25 is overall a more restricted promoter than flp-13, since flp-13 is expressed much more broadly than unc-25. Take an example of pan-1 rescue experiments, we found that many flp-13Even though the birth of VDs coincides with DD rewiring in timing, VDs are not critical for DD rewiring, because lineage mutants that do not generate VDs exhibits normal DD rewiring .pro marker. We have added pan-1 rescue experiments use flp-13pro driven pan-1. The results were consistent with the conclusion based on unc-25pro. See Figure S1A.We have added analysis for DD rewiring phenotype of pan-1(0) using flp-13pro synpatic marker for analyses of DD rewiring in all myrf-1 truncation alleles. See Figure 6; Figure S5-16.We used flp-131-482 exchromosomal array transgene and myrf-11-482(syb1491) allele4. Phenotypic discrepancy between N-MYRF-11-482 was overexpressed in multi-copy exchromosomal arrays, and it caused early DD rewiring . In contrast, the present myrf-11-482(syb1491) allele exhibit deficient DD rewiring.Previously, MYRF-11-482 can be readily detected in nucleus , while MYRF-11-482(syb1491) was very weak .One notable difference between the two experiments was that the expression level driven by exchromosomal array was much higher than endogenous level, considering that the transgene GFP::MYRF-11-482. We found that full length MYRF-1 rescued the rewiring defects in myrf-1(0); myrf-2(0) double mutants, while MYRF-11-482 did not. This results support the hypothesis that the low expression of N-MYRF is insufficient for DD rewiring.We have performed new experiment in which we generated single copy insertion transgene to express full length MYRF-1 and MYRF-1Studies of mammalian MYRF show that monomer N-MYRF does not function as efficiently as trimer N-MYRF. When full length MYRF is trimerized and cleaved, the N-MYRF is released as form of trimer. The C-tail of N-MYRF is required for N-MYRF maintain trimer form (Kim D. 2017 Nucleic Acids Res).It is conceivable that the membrane localization is necessary for MYRF (of endogenous level) to oligomerize, because the 2-dimension greatly increases the probability of interaction between protomers.1-700(syb1313) (with no extracellular domain) can indeed generate N-MYRF that are sufficient for early rewiring in a subset of DDs, even though the cleavage process is inefficient. Comparing to MYRF-11-700(syb1313), MYRF-11656(syb1468) is only short of TMD. MYRF-11-656(syb1468) produced plenty of NMYRF-1 (which is likely as monomer) but the rewiring is deficient. This support that membrane association greatly facilitates the cleavage of MYRF.MYRF-1We have included the data as Figure S21, and discussed the results. See page 28, line 569.loxp; unc-25pro-Cre.5. DD rewiring defects show incomplete penetrance in pan-1We added an illustration of DD anatomy in Figure 1C. There are processes from six DDs tiled along ventral and dorsal cords. If only one or two DDs are defective in rewiring, one or two segments of synapses would appear absent in the dorsal cord.loxp; unc-25pro-Cre is comparable to pan-1(0) mutants. We observed a sustained rewiring block by L4  in these animals, which indicates the defect is not secondary to developmental arrest.We consider that the rewiring defect in pan-1The incomplete penetrance in DD rewiring was likely because the effects of Cre was not penetrant among individual DDs. Cre is driven by unc-25 promoter, and unc-25 is reported to be expressed during late embryogenesis. Signals of DD rewiring can be detected as early as 16 post-hatch hours. This concludes that the time window between Cre transcription and DD rewiring is about 18 hours. The duration may not be long enough to allow Cre to excise the target with high penetrance. See page 11, line 209.Reviewer #1:C. elegans. A previous study  showed that animals lacking the very unusual, but conserved transcription factor MYRF-1 show defects in DD rewiring at the late L1 stage. The present study finds that an LRR-containing transmembrane protein, PAN-1, interacts with the membrane bound MYRF-1. Both proteins localize to the cell membrane, and their interaction seems to be required for DD-rewiring. pan-1 mutants also show defects in DD-rewiring, and loss of PAN-1 affects MYRF-1 localization. The interaction between these two proteins occurs through their extracellular domains, and the extracellular portion of MYRF-1 is required for its activity.In this manuscript, the authors show molecular events underpinning rewiring of DD neurons in I find this to be a very interesting paper that takes us a step further in understand how this very unusual activation mechanisms for a membrane bound (!) transcription factor works. However, some conclusions should be supported by more data and/or better data presentation. Moreover, some results are poorly described and there is general lack of detail throughout the manuscript. Specific points are listed below:1. The manuscript shows that PAN-1 and MYRF-1 interact, and that mutants of each gene result in DD-rewiring defects . However, it does not adequately show that several perturbations leading to changes in PAN-1 and MYRF-1 localization result in DD-rewiring defects, the relevant paradigm of the study. For example, the authors make several truncations in PAN-1 and MYRF-1 to test their interaction using Co-IP , summarizing that their "results provide the first direct evidence that the non-cytoplasmic part of MYRF protein is indispensable for its function". However, they do not show what happens to DD-specific dorsal synapses in animals with truncations in MYRF-1 .See our response to Common Key Point #1.2. The presentation of data for DD-rewiring defects in pan-1 mutants in Figures 1D, 1E and 5B is not convincing. Representative images in Figure 1C (middle) and 1F (bottom-right) clearly show that synapses are present, albeit much dimmer or with gaps, but this is not reflected in the quantification. Perhaps providing several representative images of pan-1 animals (and of pan-1a and pan-1c rescue in pan-1 mutants), like those shown in Figures 1F and S1A, would allow the reader to better interpret the DD-rewiring phenotype. Representative images should also be provided for PAN-1 variant animals quantified in Figure 5B.We have added new figures, in which multiple ventral, dorsal cords images were vertically tiled. To represent the true synaptic pattern of the animals, the display contrast of ventral and dorsal cord images was adjusted to the same parameters for comparison. The counting of synapse was performed using Find Peaks, a plug-in program in ImageJ. The parameters were set in a way to faithfully detect all clearly visible synapses in wild type.We agree that there were dim, small, puncta present in the dorsal cords of the mutants. It is of our interest to further characterize those structures. We consider that the synaptic rewiring occurs in multiple steps, and plan to analyze how myrf and pan-1 may be involved in specific steps.3. Throughout the manuscript, the authors show changes in subcellular localization using seam cells as an example. These images are great and clearly convey the point, but serve as controls at best. Since the manuscript is framed around DD-rewiring, it is critical to show similar spatiotemporal changes in MYRF-1 localization in WT  and pan-1 mutants  in DD neurons. This region, with existing reporters, may not look as clear-cut, but an overall distribution of signal in cell-membrane / cytoplasm / nucleus may be evident using a confocal microscope and would allow the reader to better assess the results.See our response to Common Key Point #2.Reviewer #2:C. elegans. They find that PAN-1, a TM-LRR protein, can interact in vitro and in vivo with MYRF-1/2, a transmembrane-tethered DNA-binding protein previously shown to be required for DD remodeling. They present an interesting model in which PAN-1 localizes MYRF-1/2 to the plasma membrane (a novel finding) and that regulated nuclear entry of the MYRF-1/2 N-terminal fragment is important for its function. However, there are some gaps and over-interpretations that, for me, undermine confidence in the model.The paper by Li et al. makes some intriguing findings about the mechanisms underlying DD synaptic remodeling in 1. For a few reasons, I have concerns about the specificity of the pan-1 phenotype. They don't show us whether overall DD anatomy is normal in pan-1 mutants. The DD/VD-specific rescue is nice, but isn't it possible that pan-1 expression in VDs (which are born during L1) has a role in DD rewiring?  Similarly, it's great to see the use of a pan-1 conditional allele, but its phenotype is really weak compared to the null, raising concern that some of the mutant defects could arise from pleiotropic functions .loxp; unc-25pro-Cre animals. See Figure 1F.We have added new data that axon morphology of DDs was normal in pan-1 mutants and in pan-1See our response to Common Key Point #3 for choice of promoters and VD’s role in DD rewiring.See our response to Common Key Point #5 about the penetrance of Cre.pro and flp-13pro (two promoters intersect at DDs) in pan-1(0) rescued DD rewiring; in other words, presence of pan-1 in DDs sufficiently promotes DD rewiring in an arrested animal. This implies that pan-1 specifically regulates DD rewiring.Our results indicate that pan-1 is required cell-autonomously in DD rewiring. Expression of pan-1 by unc-25We agree that pan-1 regulates development of other cell types. Certain mechanisms by which pan-1 regulates DD rewiring may apply to development of non-DD cells.In our research, we did not observe direct link between animal arrest and defective DD rewiring. On the contrary, we have seen a great number of L2arrested mutants (many do not have VD), but the DD rewiring was normal. A good example is myrf-1(0) single mutant; it arrests at the end of L1 with largely normal DD rewiring. In contrast, myrf-1(0); myrf-2(0) double mutants can develop further (to the end of L2), but the rewiring is blocked.Based on our screening, the arrest (late L1 or L2) mutants are many, while rewiring-blocked mutants are extremely rare.2. Along these lines, there's some confusion about what's meant by pan-1 and myrf-1 "activity." Because they're both expressed broadly, it's possible that they have different functions in different tissues and/or at different times. But the model doesn't consider this, and all phenotypes  are interpreted in terms of the same molecular model. Many of the key conclusions about what's going on in DD remodeling are drawn from looking in other cell types or at larval arrest . This makes me less confident that we really understand what's going on in DDs themselves.See our response to Common Key Point #1.3. According to the simplest model, expression of the myrf-1 N-terminal fragment should cause premature DD remodeling. Unexpectedly, it causes early (L1) larval arrest, a gain-of-function phenotype, since the null arrests during L2. This is consistent with the idea that transient membrane localization of MYRF-1 is necessary, but it could also be that it's simply that temporally regulated production/activity of the N-terminal fragment is important. In either case, it seems important to know whether premature DD remodeling has occurred in the arrested L1s.See our response to Common Key Point #1 and #4.4. In the final set of experiments, the MYRF-1 extracellular region is overexpressed, which blocks synaptic remodeling. Authors conclude that this demonstrates that the MYRF-1-PAN-1 interaction is essential. However, many other interactions (or other process) could also be disrupted by overexpressing this fragment, so it's not clear that the defect is truly a specific consequence of the disrupted interaction.681-931 overexpression. We took Reviewer #3’s suggestion and performed a new experiment in which an ER-retained PAN-1 was over expressed under unc-25pro. The transgene caused severely deficient DD rewiring. The ER-retained pan-1 also trapped MYRFGFP in ER as MYRFGFP signals were greatly increased in DDs comparing to those in neighboring cells. These results support that PAN-1 interacting MYRF begins in ER, but their interaction merely in ER is insufficient for MYRF processing.We agree that there are more than one possibilities to explain the dominant negative effects of MYRF-1We are further analyzing interaction between PAN-1 and MYRF by protein domain mapping and structural analysis. With additional insights, more precise perturbation of MYRF-PAN-1 interaction could be designed.Reviewer #3:In a previous genetic screen, the authors identified the MYRF transcription factors as key-components required for synaptic rewiring of the DD neurons. By pulling down MYRF interacting partners, the authors here identified the membrane protein PAN-1 as a necessary factor required for MYRF activity. Previous models for MYRF activation suggested that MYRF undergo self-cleavage on ER membrane. The authors convincingly demonstrate that MYRF and PAN-1 physically and functionally interact, which is new and well documented, even if I have concerns with some data.1. Because PAN-1 is found at the plasma membrane, the authors propose, first, that the localization at the PM is necessary for MYRF activation and, second, that "MYRF links cell surface activities to transcriptional cascades required for development". My main concern is that, at this stage, this remains an appealing hypothesis, which is not directly supported by experimental evidence. PAN-1 is also detected in intracellular compartments, at least in DD neurons , and might as well act by promoting MYRF oligomerization and cleavage early during the secretion pathway. Non-cleaved MYRF might then travel with PAN-1 to the membrane, but the functional relevance of this distribution is not demonstrated so far. One way to test this hypothesis would be to force the retention of PAN-1 in the ER and analyze MYRF processing and MYRF-dependent phenotypes.GFP(ybq14). The transgene caused severely deficient DD rewiring. The ERretained pan-1 also trapped MYRFGFP in ER as MYRFGFP signals were greatly increased in DDs comparing to those in neighboring cells. These results support that PAN-1 interacting MYRF begins in ER, but the ER-contained MYRF-PAN-1 interaction is insufficient for MYRF processing. See Figure 11B-D.We overexpressed ER-retained pan-1 under unc-25 promoter in myrf1We agree that further mechanisms remain to be determined.We have changed “MYRF links cell surface activities…” to “MYRF may link cell surface activities..”. See page 1, summary, line 33.2. In their previous article , the authors demonstrated that they could rescue DD rewiring by expressing the N' 1-482 domain of MYRF-1 in D neurons. In the present manuscript, they show that the same fragment no longer rescues myrf-1  and conclude that "MYRF membrane association is indispensable to its activity". This apparent contradiction needs to be solved.See our response to Common Key Point #4.3. At a technical level, imaging of PAN-1 and MYRF-1/2-GFP fusions are analyzed in a mixture of cell populations. Except for seam cells, the images are not very demonstrative and the quantification performed in the figures 3 and 4 appears quite subjective. To correlate DD rewiring with MYRF-1 relocalization, it should be necessary to focus on these neurons, use co-markers of ER, PM and nuclei, and perform quantitative analyses.See our response to Common Key Point #1 and #2.Altogether, it is not clear if PAN-1 is constitutively permissive for MYRF function in any tissue or can regulate MYRF activity, especially in the context of DD rewiring. Although a definite answer cannot be provided in this article, the first possibility cannot be dismissed and should be discussed.1-700 GFP indicate that the extracellular domain of MYRF is indispensable, further suggesting a regulatory role for PAN-1.” See page 26, line 531.We have added the sentences in discussion: “Our results demonstrate that PAN-1 is required for proper function of MYRF. One possibility is PAN-1 is constitutively permissive for MYRF function, e.g., PAN-1 stabilizes the extracellular domain of MYRF; however, aberrant processing and function of MYRF


Proteobacteria (genus Photobacterium), while one was dominated by the phyla Spirochaetes (genus Brevinema) and Tenericutes (unclassified Mycoplasmataceae).We report here 16S rRNA gene amplicon sequence analysis of the gut microbiota in three species of deep-sea fish collected from Suruga Bay, Japan. Of the three species, two were dominated by the phylum  Proteobacteria (genus Photobacterium), while one was dominated by the phyla Spirochaetes (genus Brevinema) and Tenericutes (unclassified Mycoplasmataceae).We report here 16S rRNA gene amplicon sequence analysis of the gut microbiota in three species of deep-sea fish collected from Suruga Bay, Japan. Of the three species, two were dominated by the phylum  The gut microbiota in fish plays a critical role in host physiology , 2 and i3–Chlorophthalmus albatrossis, Glossanodon semifasciatus, and Helicolenus hilgendorfi) were caught with a trawl net at a depth of approximately 300 m in Suruga Bay, Japan . Their gut contents were collected with dissection and stored at −80°C until use. Total DNA was extracted from the gut contents (150 to 200 mg) of three different fish of the same species of deep-sea fish using the DNeasy PowerSoil kit (Qiagen) following homogenization with a personal Minilys homogenizer (Bertin Instruments) at 5,000 rpm for 10 min. The V3-V4 region of the 16S rRNA gene was amplified using primers 341F (5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-NNNNN-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-NNNNN-GACTACHVGGGTATCTAATCC-3′) and MightyAmp DNA polymerase v.3  according to the manufacturer’s instructions except for annealing at 55°C. Library construction and sequencing were performed at the Bioengineering Lab Co., Ltd. The libraries were sequenced using a MiSeq platform (Illumina) to generate 301-bp paired-end reads. The sequence data were analyzed using EzBioCloud 16S rRNA gene-based microbiome taxonomic profiling (MTP)    with theC. albatrossis and H. hilgendorfi, the phylum Proteobacteria dominated, with relative abundances of 66.4 to 98.7% (Photobacterium (Proteobacteria), followed by Vibrio, Enterovibrio, Aliivibrio (Proteobacteria), and Clostridium (Firmicutes)  and Tenericutes (unclassified Mycoplasmataceae)  . Photobaatrossis . These tstridium , suggestataceae)  and B. Tlifornia . In all lifornia , suggestDRR237428 to DRR237436.The 16S rRNA gene amplicon sequence data have been deposited in the DDBJ Sequence Read Archive (DRA) under the accession numbers


Nilaparvata lugens, the most voracious pest of rice, but also leads to elevated production of reactive oxygen species (ROS) through metabolism of imidacloprid and host plant compounds. The inevitable production of ROS incurs a fitness cost to the pest, which drives the increase or fixation of the compensatory modifier allele T65549 within the promoter region of N. lugens peroxiredoxin (NlPrx) in the pest populations. T65549 allele in turn upregulates the expression of NlPrx and thus increases resistant individuals’ ability to clear the cost-incurring ROS of any source. The frequent involvement of P450s in insecticide resistance and their capacity to produce ROS while metabolizing their substrates suggest that peroxiredoxin or other ROS-scavenging genes may be among the common modifier genes for alleviating the fitness cost of insecticide resistance.Chemical insecticides have been heavily employed as the most effective measure for control of agricultural and medical pests, but evolution of resistance by pests threatens the sustainability of this approach. Resistance-conferring mutations sometimes impose fitness costs, which may drive subsequent evolution of compensatory modifier mutations alleviating the costs of resistance. However, how modifier mutations evolve and function to overcome the fitness cost of resistance still remains unknown. Here we show that overexpression of P450s not only confers imidacloprid resistance in the brown planthopper,  This study shows that peroxiredoxin is a modifier gene whose cis-upregulation in the brown planthopper (Nilaparvata lugens) increases the ability of resistant populations to clear the fitness cost-incurring reactive oxygen species that inevitably result from P450-meditated metabolism of imidacloprid insecticide. Bacillus thuringiensis (Bt), have been heavily employed as the most effective measures for control of arthropod pests -T65549 and P[−288/+193]) and tested their constitutive promoter activities in Trichoplusia ni (Hi5) cells. The full-length T65549 allele promoter construct P(−881/+193)-T65549 had the highest promoter activity, followed by the full-length 65549A allele promoter construct P(−881/+193)-65549A , the 65549-site-containing deletion construct P(−373/+193)-T65549, and the 65549-site-free deletion construct P(−288/+193)  (NlPrx.To test if enhancing the expression of  < 0.05) . These rNlPrx expression, ROS production (a possible source of the fitness cost associated with imidacloprid resistance), and survivorship between R-P, a high-resistant population with a T65549 allele frequency of 84.38%, and S-P, a low-resistant population with a T65549 allele frequency of 45.95%  and S-P (ROSS-P) were equal. By contrast, the ROS level was significantly higher during the ascending phase (at 12 h) but significantly lower during the descending phase (at 36 and 48 h) in R-P than in S-P  and the other with homozygous 65549A alleles (R-P_AA line)—from the CYP6ER1-mediated imidacloprid-resistant strain R-P (NlPrx was significantly higher in the R-P_TT line than in the R-P_AA line (1.27-fold)  . In the 27-fold) . In addi27-fold) .Conoidin A (0.1 mmol)  and 1.56-fold at 50 mg/l [P = 0.003]) (NlPrx (45.35% at 10 mg/l and 31.57% at 50 mg/l), compared with those injected with dsGFP (61.43% at 10 mg/l [P = 0.014] and 43.44% at 50 mg/l [P = 0.002])  of the endogenous of dsGFP . We furt 0.003]) . Additio 0.002]) .NlPrx was performed in D. melanogaster. Reverse transcription PCR (RT-PCR)  and presence of imidacloprid (NlPrx progeny line than in the UAS-NlPrx reporter line (0%) and the Act5C-GAL4 driver line (5.0%)   and qRT-(RT-PCR)  confirmeacloprid . By conte (5.0%) .D statistics  and low (GX-P-LR) resistance to imidacloprid from the field-evolved resistant population GX-P yielded 139 candidate selective sweep regions that hosted 69 genes . Furtherhemicals –56,66–68hemicals ,58. This CYP6AY1 –44,69 an CYP6AY1 –64,69–73NlPrx locus between GX-P-HR and GX-P-LR  that have a significantly higher frequency in GX-P-HR than GX-P-LR, the T65549 allele is the adaptive sweep allele selected by field application of imidacloprid, whereas the 20089A and C15914 alleles are the nearby neutral hitchhikers of the T65549 allele. This is evidenced by their locations within NlPrx (promoter versus intron 2 or 3) and the conception that promoters are more likely subject to natural selection than non-first introns [0, and GD-P-2014 F3) with a different level of imidacloprid resistance , a mitogen-activated protein kinase (MAPK) gene. On the one hand, MAPK genes have been recently proved to indirectly confer Cry1Ac resistance by downregulating Cry1Ac receptors in the diamondback moth [CYP6CM1 in the whitefly [Our experiments have validated that ndidates  may alsoack moth ,74 and iwhitefly . One thewhitefly . Additiowhitefly ,76, suggN. lugens results from the inevitable production of ROS by the resistance-conferring P450 CYP6ER1  through metabolism of imidacloprid and/or host plant compounds  and genotyping. The high-resistant R-P (LC50 52.02 mg/l) and low-resistant S-P (LC50 2.49 mg/l) populations provided by Zhejiang Academy of Agricultural Sciences, and a field population (GD-P-2020) collected from Shaoguan, Guangdong Province, China in August 2020, were used for several comparative experiments to link allele frequency with NlPrx expression, ROS level, resistance fold increases, and fitness in the presence and absence of imidacloprid. All these populations/subpopulations were reared on “Fenghuazhan” (a brown-planthopper-susceptible variety) rice plants in a controlled environment (25 ± 2 °C and a 14-h light/10-h dark photoperiod).A total of 5 populations of N = 60). Nymph death was observed and recorded 96 h post-treatment. The lethal concentration–mortality probit regression line (LC-P line) and LC50 of each population were estimated using probit analysis in SAS . The resistance ratio of each population was calculated by dividing the LC50 of each population with the LC50 of the 2 susceptible strains used by Wang et al. [We bioassayed the susceptibilities of GX-P, GD-P, R-P, and S-P to imidacloprid using the rice stem dipping method . Brieflyg et al. ,46 and Wg et al. .50 values of GX-P obtained above, we exposed the third instar nymphs of the second generation of GX-P to 300 mg/l (approximately a LC90 dose) and 5 mg/l imidacloprid (approximately a LC10 dose). The nymphs that survived LC90 treatment were considered highly resistant (GX-P-HR) individuals, whereas those that died from LC10 treatment were considered low-resistant (GX-P-LR) individuals.According to the LC-P line and LCN. lugens (GenBank Accession No. GCF_000757685.1) [FST values in a sliding window manner. Because the scaffold N90 of N. lugens is approximately 45 kb, we calculated the average FST within a nonoverlapping 20-kb window. Contigs less than 10 kb were excluded from our analysis. We then Z-transformed the FST values, and the outliers with a Z(FST) score of >4 were considered as putative sweep regions selected by the application of imidacloprid. The Tajima’s D was calculated for each of GX-P-LR and GX-P-HR using a nonoverlapping 20-kb sliding window size by VCFtools [D between GX-P-HR and GX-P-LR using the standardized difference of D, describing the difference of Tajima’s D between 2 populations, as was described by Bigham et al. [Two replicates of 30 individuals randomly selected from GX-P-HR or GX-P-LR were used to extract genomic DNA samples as previously described . The who57685.1)  using BW57685.1) , and SNP57685.1) . SubsequVCFtools . Becausem et al. .50 dose) imidacloprid in the laboratory. Nymphs randomly picked from the F0 (before selection) and F3 generations were individually genotyped to monitor the dynamics of the SNP alleles at the NlPrx locus by PCR sequencing. The primers for PCR amplification of the 3 SNP sites are listed in NlPrx locus [The third instar nymphs of GD-P were selected for 3 generations with 60 mg/l  according to the manufacturer’s instructions. Template was generated from a mixture of RNA from GX-P, R-P, and S-P. The specific primers used for amplification of NlPrx cDNA are detailed in NlPrx was assembled from all sequencing results and verified by PCR using the primers detailed in 5′ and 3′ RACE (rapid amplification of cDNA ends) was carried out for amplification of the NlPrx promoter region were PCR-amplified from GX-P-HR genomic DNA samples. The fragments ranged from +193 to −288 , −373 (containing T65549), and −881 (containing T65549) (NlPrx promoter constructs P(−881/+193)-T65549, P(−373/+193)-T65549, and P(−288/+193). The obtained P(−881/+193)-T65549 and P(−373/+193)-T65549 constructs were sequenced to verify that they are the sweep allele containing T at the 65549 SNP site. Mutagenesis of the P(−881/+193)-T65549 construct was performed to make the counterpart allele construct, P(−881/+193)-65549A, by IGE Biotechnology .Three stepwise deletion fragments of the  T65549) . The resT. ni(Hi5) cells were transfected with a mixture of 0.1 ng of internal control plasmid , 0.6 μl of Fugent HP (Promega), and 0.2 μg of construct in Grace’s medium without FBS [hout FBS . After 4N. lugens samples using the Total RNA Kit . One microgram of total RNA was reverse transcribed to cDNA in a 20-μl reaction system using PrimeScript Reverse Transcriptase . All primers for the qRT-PCR analysis of NlPrx, CYP6ER1, and CYP6AY1 genes are listed in β-actin was used as endogenous control [CT−Δ.Total RNA was extracted from  control . Three bN. lugens female adults, the time course experiment was done on 3 consecutive days. On day 1, 3 replicates of 50 1-day-old N. lugens female adults each from S-P and R-P were treated with solvent control or 10 mg/l imidacloprid using the rice stem dipping method described above. Ten live insects were randomly taken out from each replicate at 0 (before treatment), 12, and 24 h after treatment and used for measurement of ROS (5 insects) and NlPrx expression (5 insects). The 5 insects for ROS measurement were ground in phosphate buffered saline  and incubated in PBS containing 10 μM DCFH-DA (Invitrogen) for 1 h in a dark room [Due to the limited availability of 1-day-old ark room . Then, tNlPrx expression. For the day 3 insects, we observed and recorded the numbers of live and dead insects at 0, 12, 24, 36, and 48 h, and randomly drew 10 live insects from each replicate at 48 h for measurement of ROS and NlPrx expression.Similarly, the same replicates and numbers of 1-day-old female adults from each population were exposed to the solvent control or imidacloprid on day 2 and day 3. Ten live insects were randomly withdrawn from each replicate of the day 2 insects at 36 h for measurement of ROS and N. lugens female adults from GD-P-2020 to validate the field relevance of the observed phenomenon.Furthermore, the same experiment as described above was conducted on the 1-day-old NlPrx were pooled together to establish the R-P_TT line, whereas the offspring whose parents both had the homozygous 65549A allele of NlPrx were pooled together to yield the R-P_AA line.Two isogenic lines, one with the homozygous sweep allele T65549 (named R-P_TT) and the other with the homozygous counterpart allele 65549A (named R-P_AA), were generated from the highly resistant R-P strain as diagramed in Under normal rearing conditions, we measured the ROS level and life history traits of the 2 isogenic lines. Specifically, ROS level was measured using 1-day-old female adults as described above. For measurement of life history traits, multiple mated female adults were randomly chosen from each of the 2 isogenic lines and kept in 2 separated cages to lay eggs on fresh rice plants. When the eggs started to hatch, we transferred the newly hatching nymphs of each isogenic line to more than 10 vials  with fresh rice plants to record their growth, development, and longevity. When the adults emerged, we randomly paired 10 virgin females and 10 virgin males from the 10 vials and introduced each of the 10 pairs of each isogenic line into a plastic cage with fresh rice plants for oviposition. The total number of offspring of each pair was counted and recorded after 15 days. At least 6 of the 10 pairs successfully mated and produced offspring for both isogenic lines.Conoidin A and 10 mg/l imidacloprid + 0.1 mmol Conoidin A + 5 mg/l PBO. For each treatment and control, 3 replicates of 20 1-day-old female adults were treated. Survival and ROS level of the survivors were checked or measured 24 h post-exposure.We also exposed 1-day-old female adults of the 2 isogenic lines to solvent control, 10 mg/l imidacloprid alone, or combination 10 mg/l imidacloprid + 5 mg/l P450 inhibitor PBO. For the combination treatment, we pretreated the 2 isogenic lines on rice stems dipped with 5 mg/l PBO for 5 h, and then transferred the insects onto rice stems dipped with 10 mg/l imidacloprid + 5 mg/l PBO for 19 h. There were 2 additional combination treatments for the R-P_TT line: 10 mg/l imidacloprid + 0.1 mmol peroxiredoxin II inhibitor NlPrx was cloned into the pMD-18T vector (Takara Bio) and sequenced by IGE Biotechnology before dsRNA synthesis. The sequence-verified plasmid was used to amplify a 355-bp template for production of the dsRNA of NlPrx (dsNlPrx). The fragment of the green fluorescent protein (GFP) gene (ACY56286) was amplified with the primers reported by Chen et al. [NlPrx (dsNlPrx) and GFP (dsGFP) were prepared using the T7 RiboMAX Express RNAi System  following the manufacturer’s instructions.The full coding sequence of n et al.  (S6 TablNlPrx or dsGFP (1 ng/nl) was injected into each of more than 200 2-day-old female adults of R-P according to the method of Zhai et al. [NlPrx transcripts using the method described above. After confirming the RNAi efficiency of dsNlPrx, injected female adults were randomly divided into 3 groups at 24 h post-injection. The 3 groups of female adults were transferred to the cages with rice stems dipped with 0 (solvent control), 10, or 50 mg/l imidacloprid solutions. Each group included 3 replicates of treatment. The ROS level of each replicate of the 3 groups was measured 24 h after insecticide treatment, and the mortality of each replicate was observed 48 h after treatment.One hundred nanoliters of the purified dsi et al. . The injNlPrx cloned from the GX-P-HR sample was inserted into pValium20 vector to make the UAS-NlPrx construct. The UAS-NlPrx construct (1 ng/nl) was microinjected into the embryos  of the y sc v nanos-integrase; attP2 D. melanogaster line using standard techniques. The resulting UAS-NlPrx transgenic line was crossed with the Act5C-GAL4 driver line for production of the GAL4/UAS-NlPrx progeny line that expresses the NlPrx gene.The open reading frame of NlPrx transgenic line, the Act5C-GAL4 driver line, and the GAL4/UAS-NlPrx progeny line, using the methods described above. RT-PCR gel analysis and qRT-PCR were conducted to detect and quantify the expression of NlPrx in the 3 Drosophila lines using the specific primers  from the UAS- primers  for NlPr DmActin .NlPrx transgenic line, or the GAL4/UAS-NlPrx progeny line were placed in vials with 10 ml of corn meal medium containing 300 μg of imidacloprid or solvent control [Every 20 1- to 3-day-old adults  from the Act5C-GAL4 driver line, the UAS- control . The ROSχ2 test. The correlation between the NlPrx T65549 allele frequency and imidacloprid resistance of N. lugens was estimated using odds ratios and Fisher’s exact test as previously described [N. lugens populations in SPSS Statistics version 21 . The correlation between ROS level and the P450 CYP6AY1 and CYP6ER1 expression in GD-P-2020 was calculated according to the Pearson correlation in R. The paired comparisons on normalized mRNA levels, ROS levels, survival rates, and fitness-related traits were analyzed with the Student t test. All multiple comparisons on promoter activities, normalized mRNA levels, ROS levels, and survival rates were tested with 1-way ANOVA, followed by Duncan’s multiple range test (P < 0.05).All paired comparisons on allelic frequencies were analyzed using the escribed , and wasS1 Data(XLSX)Click here for additional data file.S1 Fig10.6084/m9.figshare.14177009).Numerical values are provided in (TIF)Click here for additional data file.S2 Fig10.6084/m9.figshare.14177009).Numerical values are provided in (TIF)Click here for additional data file.S3 FigThe start and stop codons are shaded gray. The catalytic triad sites and peroxidatic sites are boxed.(TIF)Click here for additional data file.S4 FigOriginal images for gels are provided in (TIF)Click here for additional data file.S1 Raw Images(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.