Misregulation of the signaling axis formed by the receptor tyrosine kinase (RTK) EphA2 and its ligand, ephrinA1, causes aberrant cell-cell contacts that contribute to metastasis. Solid tumors are characterized by an acidic extracellular medium. We intend to take advantage of this tumor feature to design new molecules that specifically target tumors. We created a novel pH-dependent transmembrane peptide, TYPE7, by altering the sequence of the transmembrane domain of EphA2. TYPE7 is highly soluble and interacts with the surface of lipid membranes at neutral pH, while acidity triggers transmembrane insertion. TYPE7 binds to endogenous EphA2 and reduces Akt phosphorylation and cell migration as effectively as ephrinA1. Interestingly, we found large differences in juxtamembrane tyrosine phosphorylation and the extent of EphA2 clustering when comparing TYPE7 with activation by ephrinA1. This work shows that it is possible to design new pH-triggered membrane peptides to activate RTK and gain insights on its activation mechanism. Eph receptors are the largest sub-group of the transmembrane receptor tyrosine kinase (RTK) family  and are erythropoietin-producing hepatoma cell line  connected to the intracellular domain (ICD) by a single transmembrane (TM) helix. Information from ephrinA1 binding to the extracellular ligand-binding domain is transmitted across the membrane by the TM helix in the form of a conformational change in the ICD. As a result, the intracellular kinase domain is activated and auto-phosphorylates multiple tyrosine residues, which triggers a signaling cascade . Full acacidity-triggered rational membrane) peptide. ATRAM is a highly soluble synthetic peptide that is capable of pH-dependent interaction with lipid membranes: at neutral pH, ATRAM binds to the membrane surface, while a decrease in pH triggers insertion into the lipid bilayer as a TM helix (transmembrane tyrosine kinase peptide for Eph). TYPE7 is a highly soluble peptide in aqueous solution that inserts into cellular membranes in a pH-dependent fashion. The TM state of TYPE7 interacts with EphA2 to induce receptor oligomerization and phosphorylation, which causes inhibition of cell migration. The observed mechanistic differences between EphA2 activation by eprhinA1 and TYPE7 provide new insights into the activation mechanism of EphA2.We have recently designed the ATRAM (TM helix . The pH-TM helix . AcidosiTM helix . Tumor aTM helix . We reasp) at pH 8 was 0.8 × 106 (±0.4×106), at pH 5 it increased to 2.9 × 106 (±0.4×106) . This result is in agreement with our expectation of TYPE7 being more hydrophobic at acidic pH, as a result of side chain protonation of glutamic acids. Next, we performed a complete pH titration in the presence of POPC vesicles, and observed that TYPE7-NBD fluorescence changed in a sigmoidal fashion (50) of 6.18. We used CD to determine the conformation that TYPE7 adopts in the presence of lipids at neutral and acidic pH. At close to neutral pH TYPE7 was unfolded (see TYPE7 is comprised of the sequence of the TM region of EphA2 and flanking residues . We intrOriented circular dichroism (OCD) can determine the alignment of an α-helix with respect to the plane of hydrated supported bilayers. We explored if TYPE7 was also able to bind to cellular membranes. To this end, we studied the cellular interaction of a fluorescently labeled version of TYPE7 at different pH values . We obse563-EphA2 . Interestingly, 563-EphA2 by GWALP23, an unrelated peptide that also forms a transmembrane helix   increased significantly upon receptor activation with EA1  . Next, we performed a co-precipitation assay to confirm the interaction between TYPE7 and endogenous EphA2. To this end, we treated H358 cells with TYPE7 labelled with a near-IR fluorophore, DyLight 680 (TYPE7-DL). After EphA2 immuno-precipitation, SDS-PAGE gels showed a fluorescent band corresponding to the molecular weight of TYPE7-DL (5.2 KDa) . EA1 uses a Fc group (heavy chain of human IgG1) to crosslink ephrinA1. Incubation with EA1 recapitulates EphA2 trans-activation by membrane clusters of ephrinA1 . The resr)  (Mandersr) , which s5.2 KDa) . InteresNext, we determined the functional significance of TYPE7 binding to EphA2. EphA2 controls cell-cell contact, and EphA2 activation inhibits cell migration. The effect of TYPE7 on EphA2-mediated cell migration was tested by using a Boyden chamber assay. EA1 was used as a positive control of ligand-induced inhibition of cell migration . Figure Activation of EphA2 by EA1 causes phosphorylation of tyrosine residues in the juxtamembrane segment (JMS) of the ICD (Y588 and Y594) and the kinase domain activation loop (Y772). Phosphorylation of these residues is followed by a signaling cascade that inhibits cell migration and invasion . To undeIntriguingly, TYPE7 and EA1 caused different JMS phosphorylation, as TYPE7 did not promote phosphorylation of Y588 and Y594 . AdditioThe activation of EphA2 by ephrins elicits downstream signaling that inhibits cell migration . Akt is In the absence of ligand, EphA2 is found in a monomer-dimer equilibrium . However2/s  to study EphA2 lateral organization in live cells. FCS is more sensitive than SIM for detecting changes in oligomerization status, particularly for small oligomers. FCS records the time-resolved fluorescence fluctuations within a confocal detection volume caused by diffusion of EphA2. By performing correlation analysis on the recorded fluctuation signals, auto-correlation function (ACF) curves are obtained . From th2/s (2/s . However2/s (2/s . Additio2/s (2/s). This control experiment suggests that TYPE7 does not affect the diffusion of transmembrane proteins in a non-specific manner.To demonstrate that TYPE7 is specifically targeting EphA2 without affecting other single-pass transmembrane receptors, we tested the effect of TYPE7 on Plexin A4. Plexin A4 is a cell surface protein that has a similar domain structure as EphA2: one transmembrane domain, a large ectodomain, and an enzymatic cytoplasmic domain. Previous work showed that Plexin A4 forms an inactive dimer prior to ligand stimulation . COS-7 c2/s , similar2/s . There w563-EphA2 suggested that TYPE7 interacts with the membrane-proximal region of EphA2. However, TMJM563-EphA2 encompasses not only the TM helix of EphA2 but also the first five basic JMS residues  be 563-EphA2, the pH50 of TYPE7 membrane insertion shifted to a less acidic value, and the transition started at neutral pH  were synthesized by Thermo Fisher Scientific  at ≥95% purity. Peptide purity was confirmed by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry and high performance liquid chromatography (HPLC). The matrix α-cyano-4-hydroxycinnamic acid (α-HCCA) and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich . Sodium phosphate and sodium acetate buffers were also purchased from Sigma-Aldrich . HPLC-grade water and methanol were purchased from Fisher Chemical . Succinimidyl 6-amino) hexanoate  was purchased from AnaSpec, Inc . BODIPY FL-X SE, Alexa Fluor 568 SE, and DyLight 680 maleimide were purchased from Thermo-Fisher Scientific . Anti-EphA2 polyclonal antibody (EphA2 D4A2 XP), phospho-EphA2 (Y588-D7 × 2L), phospho-EphA2 (Y594), phospho-EphA2 (Y772), phospho-EphA2 (Y897-D9A1) and EphA2 (8B6) mouse antibody, Akt pan, phospho-Akt T308 and phospho-Akt S473 were purchased from Cell Signaling Technology . The anti-β-actin antibody was purchased from Abcam .Peptides  using the dried droplet method . The BruTo check purity, analytes  were dissolved in methanol and injected into a semi-preparative Agilent Zorbax 300 SB-C18 column on an Agilent 1200 series HPLC system . The gradient from solvent A (water +0.05% TFA) to solvent B (methanol +0.05% TFA) was 50 min from 5% B to 100% B. Peptides typically eluted near 95 – 100% B.TYPE7 was labeled at the N-terminus with NBD-X SE, DyLight 680 maleimide, and BODIPY FL-X SE. Unreacted dye was removed using HPLC or gel filtration through a PD-10 column , and MALDI-TOF was used to determine that a single dye molecule was bound per peptide molecule with α-HCCA matrix.Lipids were purchased from Avanti Polar Lipids, Alabaster, AL. POPC  and 22:1-PC  stocks were prepared in chloroform. Aliquots of lipids were dried under a steady stream of argon gas and then placed in a vacuum overnight. The lipid films were resuspended with 10 mM sodium phosphate buffer (pH 7.9) and were then extruded with a Mini-Extruder  through a 100 nm pore size membrane  to form large unilamellar vesicles (LUVs).i pH 8 with a final concentration of 5 μM or 50 μM. The appropriate buffer backgrounds were subtracted.The sample was prepared by incubation of TYPE7 with POPC LUVs, for a lipid to peptide molar ratio of 200:1. To reach the desired experimental pH, the pH of the samples was adjusted with the addition of either 100 mM sodium phosphate pH 8 or 100 mM sodium acetate pH 4. CD spectra were recorded on a Jasco J-815 spectropolarimeter at room temperature. For the solubility study, peptide samples were prepared in either PBS (pH 7.4) or 10 mM NaP563-EphA2 and GWALP23 stocks were prepared in trifluoroethanol. Dried films of POPC, POPC:TMJM563-EphA2 (molar ratio of 500:1), and POPC:GWALP23 (molar ratio of 500:1) were resuspended in 1 mM NaPi pH 8. The POPC liposomes and proteo-liposomes were prepared via extrusion using a Mini Extruder to form ~100 nm large unilamellar vesicles. Lyophilized TYPE7 conjugated with NBD-X FL was also rehydrated with 1 mM NaPi pH 8 and was incubated with the liposomes and proteo-liposomes with a final concentration of 0.2 μM. The POPC:TYPE7 molar ratio was 2000:1. For the titrations, a series of 100 mM buffers (sodium acetate and sodium phosphate) were used to achieve the desired pH, while keeping the total buffer concentration constant. The final pH of each individual well was measured. Fluorescence spectra were recorded at 25°C with excitation at 470 nm and an emission range of 520 – 600 nm using a Cytation five imaging plate reader . Appropriate lipid blanks were prepared at the lowest and highest pH. The specific blanks were averaged and subtracted accordingly. Data were analyzed by calculating the center of mass (CM) of the fluorescence spectrum using the following equation:Ii is the fluorescence intensity measured at a wavelength λi. (F) values at different pH were fitted to determine the pH50, using AF is the acidic baseline, BF is the basic baseline, m is the slope of the transition, and 50pH is the midpoint of the curve.TMJMngth λi. . The flu563-EphA2 were prepared in chloroform, methanol and TFE, respectively. Appropriate aliquots of lipid and peptide (50:1 lipid to peptide molar ratio) were first dried with argon gas and then placed under vacuum overnight. The lipid-peptide film was resuspended with methanol and spread on two circular quartz slides . To ensure complete methanol evaporation, the slides were placed in a vacuum for 24 hr. After allowing the solvent to evaporate, the samples were hydrated with 150 μL of 100 mM sodium acetate buffer pH 4 – 5 overnight in 96% relative humidity, to obtain supported bilayers. The hydrated slides were assembled into the OCD cell, which had its inner cavity filled with saturated K2SO4 to keep the samples humidified. The OCD spectra were averaged for eight different rotations at 45° angles of the cell and recorded on a Jasco J-815 spectropolarimeter at room temperature. Appropriate lipid backgrounds were subtracted.Stocks of POPC, TYPE7 and TMJMF) were fitted with OriginLab using:0F is the initial fluorescence intensity, ΔF is the change in fluorescence intensity, x is the lipid concentration, and 55.3 is the molar concentration of water. Kp, defined as the ratio of concentrations of a compound in a mixture of two phases.Lyophilized samples of TYPE7-NBD were rehydrated in 10 mM NaPi (pH 8) at a final concentration of 0.8 μM and incubated with increasing concentrations of POPC LUVs. Emission spectra were recorded on a BioTek Cytation5 Cell Imaging Multi-Mode Reader. Three titration curves were averaged, and the resulting fluorescence intensity at 540 nm was plotted against the concentration of POPC in molar units. Fluorescence data . Free calcein was removed by gel filtration through a PD-10 column. TYPE7 was added to the calcein/LUVs suspensions at different concentrations to achieve final peptide:lipid molar ratios of 0.0025 – 0.5% and incubated for 30 min at room temperature. The calcein leakage was tracked by measuring fluorescence using a Synergy two microplate reader  at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Complete calcein release was reached by adding 20% Triton X-100, and melittin was used as a control for a leakage-inducing peptide.2 in RPMI (H358), DMEM (A375) and alpha-MEM (COS-7) media  supplemented with 10% fetal bovine serum, 50 U/mL penicillin and 50 μg/ml streptomycin. Cells were incubated overnight in serum free medium in presence or absence of TYPE7 and treated the next day with recombinant IgG1 Fc  as a control or 0.5 μg/mL of recombinant mouse EphrinA1-Fc chimera (EA1)  for 5 or 10 min. Cell line authentication and mycoplasma-free certification was performed by ATCC for all cell lines.H358, A375, DU-145 and COS-7 cells from ATCC  were cultured in a humidified incubator under 5% CO3 cells per well for proliferation and 5 × 104 for toxicity) 2 days prior the experiments in a 96 well plate, and exposed to vehicle or TYPE7 at different concentrations  and 3 μg/mL of Fc or EA1 and incubated 48 hr (toxicity) or 24 hr (proliferation). The MTS assay was performed in 100 μL of DMEM phenol red free medium  in each well and 20 μL of the CellTiter solution was added to the samples, then the plate was placed in the 37°C incubator with 5% CO2 until it reached the desired color. The absorbance at 490 nm was measured in a plate reader . The results are representative of three independent experiments, performed in triplicate. Cell proliferation was expressed as the percentage of vehicle control.H358 cell viability was measured using the CellTiter 96 Aqueous One Solution  according to the manufacturer’s protocol, which uses the reagent MTS −5-(3-carboxymethoxyphenyl)−2-(4-sulfophenyl)−2H-tetrazolium, inner salt). Briefly, cells were seeded  coated with 50 µg/mL rat tail collagen I . Cells were serum starved ON. In order to block the slide surface, samples were pre-treated with DMEM containing 2 µM unlabeled TYPE7 for 1 hr at 37°C. Samples were then treated with 0.5 µg/mL EphrinA1-Fc  and/or with 0.2 µM of TYPE7-Alexa 568 in PBS containing 1 mM MgCl2 and 100 microM CaCl2 (PBS++) for 5 min at room temperature followed by a 2 min wash with PBS++ and immediately fixed in 4% PFA. After blocking and permeabilizing, samples were incubated with rabbit anti-EphA2 primary antibody followed by secondary antibody labelling with goat-anti rabbit IgG Alexa488 .A375 cells were plated at a seeding density of 1 × 10r, was determined using the ImageJ Co-localization Threshold plugin. The r value can range from −1 for perfect exclusion to +1 for perfect co-localization, and 0 corresponds to random localization. We calculated r for whole images to reduce biases associated to selecting ROIs. However, we expect r to be higher at the plasma membrane, since a population of EphA2 was internalized, while TYPE7 remained at the plasma membrane, precluding co-localization. A second factor that reduced the measured correlation was the heterogeneous expression of EphA2, since some cells have negligible receptor levels .Cells were imaged on a confocal laser scanning microscope (Zeiss LSM 710) with 63x and 100x objectives using Zen2 blue edition software. The Pearson correlation coefficient, H358 cells were incubated with lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 5 mM EDTA and 1% NP-40 with protease inhibitors and phosphatase inhibitors for 30 min at 4°C. The insoluble fraction was eliminated through centrifugation at 10,000 × g for 30 min at 4°C. After the centrifugation, the lysates were incubated with anti-EphA2 antibody and protein A conjugated to Sepharose  for 8 hr at 4°C. To quantify the total amount of protein loaded, 20 μL of the lysates was saved. Beads were washed four times with lysis buffer. Proteins were eluted in SDS-PAGE sample buffer, separated by SDS-PAGE electrophoresis, and analyzed by Western blotting of 16.5% tricine gel to detect TYPE7-DL that was precipitated with endogenous EphA2. Equal amounts of immuno-precipitate were resolved on a 10% SDS-polyacrylamide gel, and then electrophoretically transferred to 0.45 μm nitrocellulose membranes . Total cell lysates were also subjected to immunoblot. Membranes were blocked with a milk solution , 0.1% Tween (v/v), pH 7.5) and successively probed with primary (diluted 1:1000) and IR-dye-conjugated secondary antibodies . Immunoreactive bands and TYPE7-DL were detected using an Odyssey Infrared Scanner .The human Proteome Profiler Phospho-RTK Array Kit, which covers 49 different RTKs in duplicate , and the 43-protein Proteome Profiler Human Phospho-Kinase Array Kit , were purchased from R and D systems. H358 cells were starved O.N and treated with Fc, 2 μM of TYPE7 or 0.5 μg/mL of EA1 for 10 min. The assay was performed accordingly to the manufacturer protocol. Briefly, H358 cells were lysed in the provided lysis buffer with protease and phosphatase inhibitors, then incubated overnight with the nitrocellulose membranes containing the immobilized RTK tested. The membranes were then incubated with the anti-Phospho-Tyrosine-HRP detection antibody and visualized with the kit Chemi Reagent Mix.++ containing 1 mg/mL bovine serum albumin and 0.1% Triton X-100. Nonspecific binding was blocked using goat serum dilution buffer GSDB . Anti-Epha2 rabbit primary and Alexa Fluor-conjugated secondary  antibodies were diluted in GSDB and incubated for 1 hr at room temperature. Cells were visualized on a laser scanning microscope . Contrast and brightness settings were chosen so that all pixels were in the linear range. Images are the product of eightfold line averaging.After the specific treatment, cells were fixed with 4% paraformaldehyde and subsequently permeabilized with PBS5 A375 or H358 cells were starved for 24 hr before the experiment, then treated in serum-free medium. Cells were seeded on the top chamber of polycarbonate 8 μm pore size membrane costar trans-well chambers . EA1, Fc (1 μg/mL) or TYPE7 were added to the lower chamber together with 5% FBS. Cells were allowed to migrate for 24 hr after which the cells on top of the chamber were removed with a cotton swab, and the bottom chamber was fixed with 4% PFA. After staining with eosin and hematoxylin, the cells that passed through the filter and stayed on the undersides of inserts were counted under a bight field microscope with 20x objective. Images are representative of three independent experiments, with an average of 4 images per sample condition.1 × 10EphA2FL (residues 1 – 971) and EphA2ΔJ (residues 1 – 558) were amplified via PCR from human EphA2 cDNA, and cloned into pEGFP-C1 plasmid. The resulting EGFP fusion genes were inserted into a LZRS-Pac retrovirus vector and then transfected into Phoenix retroviral packaging cells for retrovirus production. DU145 cells were infected with retroviral-mediated gene transfer in the presence of 6 μg/mL polybrene and selected with 1 μg/mL of puromycin. The DU145 cells with stable EphA2 expression were cultured in a collagen-coated 10 cm dish with DMEM (10% FBS). EphA2 ICD (residues 559 – 971) was amplified via PCR from human EphA2 cDNA and cloned into pEGFP-N1 vector. The c-Src membrane localization sequence was inserted before the EphA2 gene. The resulting Myr-EphA2 ICD-GFP was transfected into COS-7  cells using Lipofectamine2000 . COS-7 cells were cultured in a 10 cm dish with DMEM (10% FBS). All constructs lack the PDZ domain, as described elsewhere . ExperimFCS measurements were performed with a customized inverted confocal fluorescence microscopy  equipped with a 100x TIRF objective . The 488 nm excitation laser beam was separated from a continuum white light laser (9.7 MHz)  using a narrow-band excitation filter (488: LL01-488-12.5) . The beam was focused onto the live cell samples sitting in an on-stage incubator by the objective. The emission light from the sample was collected through the same objective and directed passing a 520/44 nm emission filter (FF01-520/44-25) . The photons from the emission beam were collected by a single photon avalanche diode (SPAD) detector  and recorded with a time-correlated single photon counting (TCSPC) module . Data was processed and analyzed with a Matlab script.N is average number of fluorescent particles, F is the fraction of molecules in the triplet state, D) was calculated based on N with the detection area that was calibrated with standard dye molecule with known diffusion coefficient.Excitation laser beam at 300 nW was focused on the live cells samples at 37°C. Laser was always parked at the edge of a flat membrane area where there was only homogenous fluorescence . Five 15t-tests or ANOVA were performed. We used IBM SPSS (version 25) and Origin 9.1 to perform t-tests. For each t-test homogeneity was checked and the correct test assuming or not assuming equal variance was applied. The same software package was used for to perform the Mann-Whitney U test to the co-precipitation data. Statistical significance was considered as p<0.05. Where multiple comparisons were performed, significance was determined by t-tests followed by the Benjamini-Hochberg procedure using a false discovery rate of 0.05. Effect sizes in standard deviations were determined by Hedge’s g values as calculated in Excel 2016.Unless indicated otherwise, data are reported as mean ± standard deviation (S.D), and resulted from three or more independent experiments. To evaluate differences between sample means, Student’s  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article is being evaluated by three peer reviewers, and the evaluation is being overseen by a Reviewing Editor and Jonathan Cooper as the Senior Editor.Thank you for sending your article entitled "A novel pH-dependent membrane peptide that binds to EphA2 and inhibits cell migration" for peer review at This manuscript describes a thorough investigation of a novel peptide that inhibits migration of malignant cells by its binding to the transmembrane segment (TMS) of the receptor tyrosine kinase EphA2. Thorough biophysical studies of peptide structure, membrane integration and binding are juxtaposed with a comprehensive cell biological analysis of the effects the pH-dependent TYPE2 binding to EphA2. The discovery of a peptide that can be used to activate EphA2 through an unconventional mechanism is interesting for potential therapeutic applications.The reviewers were positively impressed by the combination of a large body of biophysical, cell biological and modeling work that you have been able to build on a difficult system. They consider that your study will represent a significant advance to the field, but a significant number of additional evidences to support your conclusions and changes to consolidate the paper are nevertheless required.These major points are summarized below:1) To support their hypothesis, the authors must verify that TYPE7 does increase EphA2 kinase activity, for example, by monitoring phosphorylation of a substrate  and/or by using the phospho-kinase antibody array in Figure 4—figure supplement 3, which contains a number of EphA2 downstream targets.2) The authors should add more experimental evidence that TYPE7 interacts with an EphA2 TM interface but not with another interface.3) They should show that TYPE7 does not affect the clustering of EphA2-deltaJ-GFP if the TM domain is replaced by an unrelated TM domain and that TYPE7 does not affect the clustering of Myr-EphA2-ICD-GFP if the positively charged amino acids are eliminated.4) Some control experiments are missing: in order to conclude that TYPE7 increases pY772 and pHLIP does not, the effects of TYPE 7 and pHLIP should be compared in the same cells and preferably in the same experiment.5) Reviewers have concerns with the mathematical model of the kinetics and wonder if the model should be kept in the manuscript. First, it is not clear whether the model is not simply custom-built to produce the observed results and has actually provided new insight. This point should be clarified. Moreover, some predictions of the model are not consistent with published data: for instance, literature does not support the presence of a substantial population of inactive EphA2 dimers in the absence of ephrin-As . According to the proposed model, ephrinA1 stimulation disrupts the EphA2 inactive dimers and promotes formation of active dimers that use a different TM interface. What are the predictions of the model in regard to the behavior the two EphA2 mutants with a disrupted TM interface and are they consistent with the data in Sharonov et al., 2014, where mutation of each TM interface affects EphA2 activity in both unstimulated and ephrinA1-stimulated cells?Reviewer #1:This is an impressive and thorough study of an interesting engineered peptide that builds off of the pHLIP concept. The authors are to be commended for the breadth and scope of the study, which includes a wide-range of both biophysical and cellular experiments, complemented with mathematical modeling. Relevant controls are run, including WALP and pHLIP. The paper offers several suggestions as to the mechanism through which their peptide activates EphA2, although the actual mechanism is left unresolved. I will say that I was surprised that the peptide acts to activate, rather than inhibit, the receptor. If true, the finding is very exciting and opens the door to understanding a new mechanism of activation that couples conformational changes in the TM domain to activation through oligomerization. This is an exciting mechanism that deserves publication in a high impact journal.eLife.I have two major comments, which if adequately addressed will convince me that the paper should be published in 1) The authors state in reference to Figure 3—figure supplement 4D-E that there is no significant increase in EphA2 levels upon treatment with TYPE7. However, the figure does appear to indicate that there is at least some increase in expression (approximately 30%). Since we don't know if there is a relationship between expression levels of EphA2 and activation, it would be necessary to rule out the possibility that the increased  expression induced by TYPE7 isn't causing activation. Overexpression of other membrane proteins, such as TNF receptors, can cause ligand-free clustering and activation in cell membranes. The authors should test the effect in their cell lines of overexpression at various levels of DNA transfection to reassure the reader that this isn't affecting their core findings.2) The use of computational modeling is commendable, but it is not clear to me that the model has actually provided new insight. More specifically, I am not convinced that the model results aren't a trivial consequence of the assumptions used to build the model. The authors should do a better job of explaining the model and making transparent how the model results differ from those that are trivial consequences of the assumptions they have started with. I will do my best to explain, and if I'm simply confused please correct the text to clarify these points:"The model proposes that binding of saturating levels of EA1 causes a conformational switch that disfavors the inactive dimers, completely blocking the inactive interface." Then, not surprisingly, the results show an increase in the number of monomers upon ligand addition, and rapidly an increase in clusters from rapid formation of active dimers . Then, adding Type 7  slows everything down. Is this not simply the consequence of the fact that, unlike the EA1 ligand, the TYPE7 effect is slowed by your choice to slow its reactivity by way of its diffusion? That is, given this difference between EA1 (instantaneous/saturating) and TYPE7 (Diffusion-slowed/concentration dependent), could you not have predicted a priori (i.e. without solving the equations) that the process would be slowed with TYPE7? I'm not saying that the mechanism for slowing isn't correct (ligand binding instantly activates pre-assembled dimers vs TYPE7 has to diffuse), I'm simply asking whether you needed a complex kinetic model to know that would be the case.Reviewer #2:This manuscript describes a novel peptide (TYPE7) that was designed based on the transmembrane segment of the EphA2 receptor tyrosine kinase. TYPE7 contains strategically located glutamic acid residues that make it soluble in aqueous solutions but able to insert into lipid membranes at acidic pH. Treatment of cells with TYPE7 increases phosphorylation of Y772 in the activation loop of the EphA2 kinase domain. This is attributed to the interaction of TYPE7 with the transmembrane (TM) helix and/or the juxtamembrane segment (JMS) of EphA2, which promotes EphA2 clustering and activation. The authors also developed a mathematical model of EphA2 activation based on results in the manuscript and information from the literature.The discovery of a peptide that can be used to activate EphA2 through an unconventional mechanism is potentially interesting and could have therapeutic applications. However, additional experimental evidence is needed to support the conclusions presented and the mathematical model of EphA2 clustering.The authors found that TYPE7 increases EphA2 phosphorylation on Y772 but not Y588 and Y594. They hypothesize that this is due to a new mechanism of EphA2 activation that does not involve Y588 and Y594 phosphorylation. To support this hypothesis, the authors should verify that TYPE7 does in fact increase EphA2 kinase activity  and/or by using the phospho-kinase antibody array in Figure 4—figure supplement 3, which contains a number of EphA2 downstream targets). Of note, pY588 and pY594 are major EphA2 autophosphorylation sites, and thus they would be expected to be phosphorylated in the active receptor even if their phosphorylation does not play a role in receptor activation. It should also be noted that Locard‐Paulet et al., 2016, implicated pY772 in cancer cell migration/invasiveness, but this effect may not be linked to EphA2 kinase activity.Additional controls are needed to support the proposed mechanism of EphA2 activation by TYPE7. For example, additional controls for Figure 2A, B should include clustering of an unrelated transmembrane receptor, to show that TYPE7 specifically co-clusters with EphA2. Additional controls for Figure 3A and Figure 3—figure supplement 3 should demonstrate no effects of TYPE7 on the migration of cells that do not express EphA2, to exclude non-specific effects of TYPE7 on cell migration. Furthermore, the experiments with the pHLIP peptide in Figure 3—figure supplements 2 and 3 may not be sufficient as controls if they were performed at neutral pH, since under these conditions TYPE7 but not pHLIP would insert into the membrane of cells expressing EphA2.The authors propose that TYPE7 interacts with an EphA2 TM interface observed by NMR (hypothesized to mediate dimerization of inactive EphA2 in cells) but not another interface (hypothesized to mediate dimerization of active EphA2 in cells). However, additional experimental evidence should be included to support this model. For example Sharonov et al., 2014, reports mutations that selectively destabilize each TM interface. If the mechanism presented by the authors is correct, TYPE7 should not associate with an EphA2 mutant where the interface hypothesized to mediate formation of inactive dimers and to interact with TYPE7 is mutated.The authors propose that TYPE7 affects EphA2 clustering by interacting with both the TM domain and the positively charged portion at the N-terminus of the JMS segment. To support this idea, the authors should show that (i) TYPE7 does not affect the clustering of EphA2-deltaJ-GFP if the TM domain is replaced by an unrelated TM domain and (ii) TYPE7 does not affect the clustering of Myr-EphA2-ICD-GFP if the positively charged amino acids are eliminated. Could an alternative explanation of the effects observed in Figure 4 be that insertion of TYPE7 changes the properties of membranes, slowing down lateral diffusion of proteins?Different types of experiments and their controls should be carried out in the same cell line, with possible verification of some results in other cell lines. For example, the controls in Figure 3—figure supplement 2 with the pHLIP peptide should be performed in the same cell line used to examine the effects of TYPE7 (i.e. A375). There is also some concern about the use of H358 cells, in which EphA2 activation by ephrinA1 does not seem to have robust signaling effects .The authors present a detailed mathematical model of EphA2 clustering. However, some of the predictions of the model do not seem to be consistent with published data. For example, evidence in the literature does not does not support the presence of a substantial population of inactive EphA2 dimers in the absence of ephrin-As . According to the proposed model, ephrinA1 stimulation disrupts the EphA2 inactive dimers and promotes formation of active dimers that use a different TM interface. What are the predictions of the model in regard to the behavior the two EphA2 mutants with a disrupted TM interface? Are these predictions consistent with the previous data in Sharonov et al., 2014, where mutation of each TM interface affected EphA2 activity in both unstimulated and ephrinA1-stimulated cells?Reviewer #3:This manuscript by Alves et al. describes a thorough investigation of a novel peptide that inhibits cell migration of malignant cells by its binding to the transmembrane segment (TMS) of the receptor tyrosine kinase EphA2. The peptide can be delivered in a soluble form, then integrates into the membrane upon lowering the pH, and eventually binds to EphA2 at its TMS and the adjacent juxtamembrane segment (JMS). The authors also show that one of three phosphorylation sites in the adjacent intracellular regulatory domain of EphA2 is phosphorylated upon binding of the designed peptide TYPE7, which induces dimerization of the receptor, but no further clustering that happens only upon Ephrin A1 ligand binding and phosphorylation of all three sites. Despite its stimulation of phosphorylation of only the Y772 single site, TYPE2 is as active as Ephrin A1 in inhibiting cell migration and therefore may have future therapeutic utility.The study is overall very comprehensive and makes a convincing case for the proposed mechanism. Thorough biophysical studies of peptide structure, membrane integration and binding are juxtaposed with a comprehensive cell biological analysis of the effects the pH-dependent TYPE2 binding to EphA2.The comments below are suggestions on how the authors could and should further improve an already very good and carefully written manuscript.Figure 1: A) please align the sequences for easier comparison. D) There are too many binding curves superimposed on each other so that they cannot be individually distinguished. I suggest to group them in different panels or display some of them with a shifted pH scale. The bundled overlays could still be shown in a supplementary figure to make the point of the good superposition. E) In the cartoon to the right, I am not sure what the evidence is that TYPE2 binds to a dimer of EphA2. Wouldn't it be more realistic to show TYPE2 binding to a monomer displacing the other monomer of EphA2?OCD experiments described in the last paragraph of the subsection “Sharonov et al., 2014”: Is the dashed red line really closer to the theoretical curve for a TM helix? The data look a little ambiguous to me. Are these data really representing the pH 5 situation? Reading the relevant Materials and methods section, it appears that peptide and lipids were first dried and then rehydrated to 96% relative humidity. The pH may actually be undefined in this situation and membranes at 96% RH are not the same as in bulk water or buffer and this may drive the peptide deeper into the membrane and thereby change its orientation relative to fully hydrated bilayers. This should be acknowledged. If the authors think this is not a concern, they should present comparative data at pH 5 and pH 7.Subsection “TYPE7 shows no toxicity and binds to cells in a pH-dependent manner”: define MTSSubsection “TYPE7 interacts with EphA2”, first paragraph: What is the evidence in Figure 1D that a fraction of TYPE7 is already in the TM state at pH 7? – It may also be helpful to state somewhere in this paragraph that TM-EphA2 was in 4-fold molar excess over TYPT7 in these experiments .The sentence “Incubation with EA1 recapitulates EphA2 trans-activation by membrane clusters of ephrinA1”, needs a literature reference.There seems to be some confusion about the definition of the JMS and other parts (kinase domain) of the ICD. In the first paragraph of the subsection “TYPE7 interacts with EphA2”, JMS appears to be defined as the few residues adjacent to the TMS that directly abut the membrane surface, which is a common definition in many other single-span membrane proteins. However, in the second paragraph of the subsection “TYPE7 inhibits cell migration by specific EphA2 phosphorylation at Y772”, the authors seem to include many more residues including all three phosphorylation sites that are as far as 30 residues away from the TMS in their definition of JMS. Please make definitions consistent.Figure 4: A) Is too small to see clusters vs. no clusters that authors want to highlight. B) Does the blue FCS curve possibly represent more than one diffusing component? Its shape is clearly different from the other two. E) It is true that TYPE7 binding decreased diffusion of the myristylated receptor like it did with the native receptor , but all diffusion coefficients are much higher in the lipid-anchored compared to the protein-anchored cases. I.e. the scale is different in E vs. C and D. Nothing wrong with this, but it should be mentioned.Mathematical model: Does a stochastic model, which this is, have a real time scale like the minutes shown in all modeling figures, or does it not have arbitrary time steps, and therefore, should the time scale in these figures not be better called arbitrary? Of course, this does not take away from the modeling, but it does not tie the figures directly to an actual experimental time scale that may be quite different.The model as depicted in Supplementary Figure 11A also needs more explanation. There seem to be 3 different types of k(I),(on and off) and 2 different types k(A),(on and off) as shown in the different panels. Or are they all constrained to the same respective values in the model? So, how many free fit parameters are there in the model? Clarification of this point is important to better understand the main text. The Supplementary Figure 11A legend will also benefit from a more detailed panel-by-panel explanation. The reviewers were positively impressed by the combination of a large body of biophysical, cell biological and modeling work that you have been able to build on a difficult system. They consider that your study will represent a significant advance to the field, but a significant number of additional evidences to support your conclusions and changes to consolidate the paper are nevertheless required.These major points are summarized below:1) To support their hypothesis, the authors must verify that TYPE7 does increase EphA2 kinase activity, for example, by monitoring phosphorylation of a substrate  and/or by using the phospho-kinase antibody array in Figure 4—figure supplement 3, which contains a number of EphA2 downstream targets.To address the request of monitoring phosphorylation of an EphA2 substrate, we have studied the effect of TYPE7 on the phosphorylation of Akt. Akt is an important downstream target of EphA2, and Akt phosphorylation promotes cell migration. We provide new figure panels  that show that TYPE7 causes an Akt de-phosphorylation that is comparable to the effect of ephrinA1-Fc (EA1), a ligand of EphA2. Akt de-phosphorylation is a well-stablished mechanism to reduce cell migration. Thus, this new finding adds on the TYPE7 mechanistic information we had already collected. We were prompted to study Akt after re-evaluating the kinase array, where we observed that TYPE7 promoted de-phosphorylation of Akt , now mentioned in the last paragraph of the subsection “TYPE7 promotes limited self-assembly of EphA2”. To verify the results of the kinase array, we decided to perform Western blots for two activation sites of Akt (S473 and T308), confirming the effect of TYPE7 on this kinase crucial for cell migration. The identification of the effect of TYPE7 on a crucial EphA2 downstream target is a significant addition to the manuscript.We originally proposed to study Rac1, a separate downstream target of EphA2. We performed those experiments, and we did not detect an effect of TYPE7 on Rac1 phosphorylation. This result is not surprising, since Rac1 activation occurs when EphA2 is phosphorylated at Y735, a residue which we have not evaluated in our manuscript for lack of commercially available phospho-specific antibody.2) They should show that TYPE7 does not affect the clustering of EphA2-deltaJ-GFP if the TM domain is replaced by an unrelated TM domain and that TYPE7 does not affect the clustering of Myr-EphA2-ICD-GFP if the positively charged amino acids are eliminated.New FCS experiments have been performed in live cells, where the diffusion coefficient of Plexin A4 receptor has been determined in control conditions and in the presence of TYPE7. The new figure  shows that TYPE7 does not affect the lateral diffusion of Plexin A4-GFP. A new paragraph discussing these results has been added . This result suggests that TYPE7 does not affect the membrane diffusion of single-pass membrane receptors in a non-specific manner, and that the effect of TYPE7 on EphA2 diffusion results from direct binding.3) Some control experiments are missing: in order to conclude that TYPE7 increases pY772 and pHLIP does not, the effects of TYPE 7 and pHLIP should be compared in the same cells and preferably in the same experiment.Tthe manuscript already contained on the original submission data performed with pHLIP and TYPE7, carried at the same time and with the same cell line, H358 . We have performed additional experiments where we study the effect on Y772 phosphorylation of TYPE7 and pHLIP. These experiments are again performed at the same time, but now in A375 cells. Additionally, experiments with pHLIP performed at acidic pH (pH 4.2), where pHLIP is expected to be fully in the transmembrane configuration, showed no effect . In the new data, TYPE7 also increases the phosphorylation of EphA2 at Y772, although to a lower extent than in H358 cells. We also include an additional figure , where cell migration was studied in H358 cells . TYPE7 reduces cell migration in both cell lines. However, the effect seems even larger in H358 cells, which seems to agree with the larger increase in Y772 phosphorylation in this cell line compared to A375.4) Reviewers have concerns with the mathematical model of the kinetics and wonder if the model should be kept in the manuscript. First, it is not clear whether the model is not simply custom-built to produce the observed results and has actually provided new insight. This point should be clarified. Moreover, some predictions of the model are not consistent with published data: for instance, literature does not support the presence of a substantial population of inactive EphA2 dimers in the absence of ephrin-As . According to the proposed model, ephrinA1 stimulation disrupts the EphA2 inactive dimers and promotes formation of active dimers that use a different TM interface. What are the predictions of the model in regard to the behavior the two EphA2 mutants with a disrupted TM interface and are they consistent with the data in Sharonov et al., 2014, where mutation of each TM interface affects EphA2 activity in both unstimulated and ephrinA1-stimulated cells?As a result of your suggestion, the latest version of the manuscript no longer contains the mathematical modeling sections. The concerns raised in this point, and also in the next one, are solely pertinent to the model. After removal of this non-central part of the manuscript, these concerns should have been addressed.5) The authors should add more experimental evidence that TYPE7 interacts with an EphA2 TM interface but not with another interface.Please see response to point 4.Reviewer #1:[…] I have two major comments, which if adequately addressed will convince me that the paper should be published in eLife.1) The authors state in reference to Figure 3—figure supplement 4D-E that there is no significant increase in EphA2 levels upon treatment with TYPE7. However, the figure does appear to indicate that there is at least some increase in expression (approximately 30%). Since we don't know if there is a relationship between expression levels of EphA2 and activation, it would be necessary to rule out the possibility that the increased  expression induced by TYPE7 isn't causing activation. Overexpression of other membrane proteins, such as TNF receptors, can cause ligand-free clustering and activation in cell membranes. The authors should test the effect in their cell lines of overexpression at various levels of DNA transfection to reassure the reader that this isn't affecting their core findings.We have included additional FCS data that confirms the previous Western blot EphA2 quantification. These results show that TYPE7 does not change the plasma membrane levels of EphA2FL-GFP or EphA2DJ-GFP . We now mention this finding in the second paragraph of the subsection “TYPE7 promotes limited self-assembly of EphA2”.2) The use of computational modeling is commendable, but it is not clear to me that the model has actually provided new insight. More specifically, I am not convinced that the model results aren't a trivial consequence of the assumptions used to build the model. The authors should do a better job of explaining the model and making transparent how the model results differ from those that are trivial consequences of the assumptions they have started with. I will do my best to explain, and if I'm simply confused please correct the text to clarify these points:"The model proposes that binding of saturating levels of EA1 causes a conformational switch that disfavors the inactive dimers, completely blocking the inactive interface." Then, not surprisingly, the results show an increase in the number of monomers upon ligand addition, and rapidly an increase in clusters from rapid formation of active dimers . Then, adding Type 7  slows everything down. Is this not simply the consequence of the fact that, unlike the EA1 ligand, the TYPE7 effect is slowed by your choice to slow its reactivity by way of its diffusion? That is, given this difference between EA1 (instantaneous/saturating) and TYPE7 (Diffusion-slowed/concentration dependent), could you not have predicted a priori (i.e. without solving the equations) that the process would be slowed with TYPE7? I'm not saying that the mechanism for slowing isn't correct (ligand binding instantly activates pre-assembled dimers vs TYPE7 has to diffuse), I'm simply asking whether you needed a complex kinetic model to know that would be the case.To address this concern, the mathematical model is no longer in the manuscript.Reviewer #2:[…] The discovery of a peptide that can be used to activate EphA2 through an unconventional mechanism is potentially interesting and could have therapeutic applications. However, additional experimental evidence is needed to support the conclusions presented and the mathematical model of EphA2 clustering.The authors found that TYPE7 increases EphA2 phosphorylation on Y772 but not Y588 and Y594. They hypothesize that this is due to a new mechanism of EphA2 activation that does not involve Y588 and Y594 phosphorylation. To support this hypothesis, the authors should verify that TYPE7 does in fact increase EphA2 kinase activity  and/or by using the phospho-kinase antibody array in Figure 4—figure supplement 3, which contains a number of EphA2 downstream targets). Of note, pY588 and pY594 are major EphA2 autophosphorylation sites, and thus they would be expected to be phosphorylated in the active receptor even if their phosphorylation does not play a role in receptor activation. It should also be noted that Locard‐Paulet et al., 2016, implicated pY772 in cancer cell migration/invasiveness, but this effect may not be linked to EphA2 kinase activity.We thank the reviewer for raising this concern. This prompted us to study the downstream effects of TYPE7. As a result we discovered that TYPE7 causes de-phosphorylation of Akt, a protein that promotes cell migration. The effect of TYPE7 was similar to ephrinA1-Fc. These new data  shows that TYPE7 affects the phosphorylation of a downstream target of EphA2 that is key for cell migration.Additional controls are needed to support the proposed mechanism of EphA2 activation by TYPE7. For example, additional controls for Figure 2A, B should include clustering of an unrelated transmembrane receptor, to show that TYPE7 specifically co-clusters with EphA2.We studied if TYPE7 affected the diffusive properties of Plexin A4, an unrelated single-pass receptor. The data in Figure 4—figure supplement 11 show that TYPE7 does not affect the diffusion of this receptor, which further suggests that the effect of TYPE7 on EphA2 is specific.Additional controls for Figure 3A and Figure 3—figure supplement 3 should demonstrate no effects of TYPE7 on the migration of cells that do not express EphA2, to exclude non-specific effects of TYPE7 on cell migration.The manuscript now contains cell migration data in two different cells lines: A375, Figure 3A; and H358, Figure 3—figure supplement 5, which reinforces our previous cell migration data. However, we think that performing experiments with migratory cells that do not express EphA2 falls outside the scope of the manuscript, which already contains extensive evidence of the specificity of the effect of TYPE7.Furthermore, the experiments with the pHLIP peptide in Figure 3—figure supplements 2 and 3 may not be sufficient as controls if they were performed at neutral pH, since under these conditions TYPE7 but not pHLIP would insert into the membrane of cells expressing EphA2.Figure 3—figure supplement 6 now contains experiments performed with pHLIP at acidic pH. These results show that pHLIP does not induce changes in the phosphorylation of EphA2 Y772, at neutral pH  or an acidic pH . These results support that data already present in Figure 3—figure supplement 7, showing that pHLIP does not alter cell migration.The authors propose that TYPE7 interacts with an EphA2 TM interface observed by NMR (hypothesized to mediate dimerization of inactive EphA2 in cells) but not another interface (hypothesized to mediate dimerization of active EphA2 in cells). However, additional experimental evidence should be included to support this model. For example Sharonov et al., 2014, reports mutations that selectively destabilize each TM interface. If the mechanism presented by the authors is correct, TYPE7 should not associate with an EphA2 mutant where the interface hypothesized to mediate formation of inactive dimers and to interact with TYPE7 is mutated.To address this concern, the mathematical model is no longer in the manuscript.The authors propose that TYPE7 affects EphA2 clustering by interacting with both the TM domain and the positively charged portion at the N-terminus of the JMS segment. To support this idea, the authors should show that (i) TYPE7 does not affect the clustering of EphA2-deltaJ-GFP if the TM domain is replaced by an unrelated TM domain and (ii) TYPE7 does not affect the clustering of Myr-EphA2-ICD-GFP if the positively charged amino acids are eliminated. Could an alternative explanation of the effects observed in Figure 4 be that insertion of TYPE7 changes the properties of membranes, slowing down lateral diffusion of proteins?We thank the reviewer for this comment, raising the intriguing possibility that TYPE7 might alter the physical properties of the membrane in such a way that slowed protein diffusion. To rule out this possibility we have performed FCS experiments with a different receptor, Plexin A4. The new data contained in Figure 4—figure supplement 11 shows that TYPE7 does not affect the diffusion coefficient of Plexin A4, which suggests that TYPE7 does not increase the microviscosity of the plasma membrane.Different types of experiments and their controls should be carried out in the same cell line, with possible verification of some results in other cell lines. For example, the controls in Figure 3—figure supplement 2 with the pHLIP peptide should be performed in the same cell line used to examine the effects of TYPE7 (i.e. A375). There is also some concern about the use of H358 cells, in which EphA2 activation by ephrinA1 does not seem to have robust signaling effects .Experiments performed at the same time with TYPE7 and pHLIP in A375 cells  and H358 cells  show that while TYPE7 does increase P-Y772, pHLIP does not. Additional data in H358 cells support the notion that these cells are valid to study EphA2, since EA1 robustly reduces cell migration , and promotes phosphorylation of EphA2 Y588 , and de-phosphorylates Akt .The authors present a detailed mathematical model of EphA2 clustering. However, some of the predictions of the model do not seem to be consistent with published data. For example, evidence in the literature does not does not support the presence of a substantial population of inactive EphA2 dimers in the absence of ephrin-As . According to the proposed model, ephrinA1 stimulation disrupts the EphA2 inactive dimers and promotes formation of active dimers that use a different TM interface. What are the predictions of the model in regard to the behavior the two EphA2 mutants with a disrupted TM interface? Are these predictions consistent with the previous data in Sharonov et al., 2014, where mutation of each TM interface affected EphA2 activity in both unstimulated and ephrinA1-stimulated cells?To address this concern, the mathematical model is no longer in the manuscript.Reviewer #3:[…] The comments below are suggestions on how the authors could and should further improve an already very good and carefully written manuscript.Figure 1: A) please align the sequences for easier comparison. D) There are too many binding curves superimposed on each other so that they cannot be individually distinguished. I suggest to group them in different panels or display some of them with a shifted pH scale. The bundled overlays could still be shown in a supplementary figure to make the point of the good superposition. E) In the cartoon to the right, I am not sure what the evidence is that TYPE2 binds to a dimer of EphA2. Wouldn't it be more realistic to show TYPE2 binding to a monomer displacing the other monomer of EphA2?A) The sequences have been aligned as suggested by the reviewer.D) The reviewer is right in pointing out that in the original version of the figures it was hard to individually distinguish some of the overlaid curves. We have changed the shading representing the confidence intervals, and we think it is now easier to discriminate the different experiments.E) The figure was meant to show a model of the binding between TYPE7 and TM-EphA2, without making claims about specific oligomerization state or binding stoichiometry.OCD experiments described in the last paragraph of the subsection “Sharonov et al., 2014”: Is the dashed red line really closer to the theoretical curve for a TM helix? The data look a little ambiguous to me. Are these data really representing the pH 5 situation? Reading the relevant Materials and methods section, it appears that peptide and lipids were first dried and then rehydrated to 96% relative humidity. The pH may actually be undefined in this situation and membranes at 96% RH are not the same as in bulk water or buffer and this may drive the peptide deeper into the membrane and thereby change its orientation relative to fully hydrated bilayers. This should be acknowledged. If the authors think this is not a concern, they should present comparative data at pH 5 and pH 7.The reviewer was rightfully confused, since there was a swap between the colors (grey and black) of the two theoretical lines in the figure legend (it was correct in the main text). We thank the reviewer for spotting this error. The text is fixed in the Figure 1 legend. We have updated the Materials and methods section to clarify the point of the 96% relative humidity (subsection “Oriented circular dichroism (OCD)”). During the overnight incubation the dry sample on the slide was first fully hydrated with abundant acidic buffer. We expect that after overnight incubation the humidity of the lipid bilayers would be close to 100%. To avoid the previously added buffer to dry, a large amount of saturated salt was filling the camber where this step was carried out. That camber would have an atmosphere that was kept humid with the saturated salt (96% relative humidity). However, the sample would still be 100% hydrated. In this situation we expect the pH to be adequately controlled. We thank the reviewer for prompting us to clarify this important point. We are confident that the OCD protocol that we employ yields valid results. Specifically, we have carried out OCD experiments on three different peptides, and our results have yielded membrane orientations independently validated by other techniques using 100% hydration. Those are the GWALP23 peptide , the pHLIP peptide , and the ATRAM peptide .Regarding the suggestion of carrying out OCD experiments at pH 7, we think this could not be a straightforward experiment. On the one hand, at this pH the peptide has little helical structure , and a very small OCD signal is expected. Furthermore, performing this experiment has the additional complication that at neutral pH the membrane affinity of TYPE7 decreases , resulting on a non-negligible amount of peptide not being bound to the membrane, but staying on solution. Any CD signal from this sample will not result from oriented peptide, but from TYPE7 on solution, causing an artifact. In the past this situation has make interpreting OCD data of pHLIP at neutral pH unreliable in our hands.Subsection “TYPE7 shows no toxicity and binds to cells in a pH-dependent manner”: define MTS.The chemical name of the MTS compound has been added to the Materials and methods subsection “Cell proliferation assay (MTS)”.Subsection “TYPE7 interacts with EphA2”, first paragraph: What is the evidence in Figure 1D that a fraction of TYPE7 is already in the TM state at pH 7? It may also be helpful to state somewhere in this paragraph that TM-EphA2 was in 4-fold molar excess over TYPT7 in these experiments .The text said”a fraction of TYPE7 is already in the TM state at neutral pH“, which is based on Figure 1D in the presence of TM-EphA2 (orange symbols), showing that at pH 7 we are beyond the high pH plateau , and already within the sigmoidal transition where α helix is being formed. This has been discussed the first paragraph of the subsection “Cell proliferation assay (MTS)”. To clarify that here we are referring to the presence of α helix, not that this is the dominant species, we have changed the text, replacing “a fraction” with “a non-negligible fraction”. Additionally, we have introduced in the main text the presence of a molar excess of TM-EphA2 (TMJM546-EphA2) over TYPE7, as the reviewer suggested.The sentence “Incubation with EA1 recapitulates EphA2 trans-activation by membrane clusters of ephrinA1”, needs a literature reference.An appropriate reference has been added (subsection “Cell proliferation assay (MTS)”, last paragraph).There seems to be some confusion about the definition of the JMS and other parts (kinase domain) of the ICD. In the first paragraph of the subsection “TYPE7 interacts with EphA2”, JMS appears to be defined as the few residues adjacent to the TMS that directly abut the membrane surface, which is a common definition in many other single-span membrane proteins. However, in the second paragraph of the subsection “TYPE7 inhibits cell migration by specific EphA2 phosphorylation at Y772”, the authors seem to include many more residues including all three phosphorylation sites that are as far as 30 residues away from the TMS in their definition of JMS. Please make definitions consistent.563-EphA2, indicating that includes the JM sequence until residue 563. The name has been updated in the whole manuscript.We have clarified that TM-EphA2 contains a subset of the N-terminal region of the longer JMS (subsection “Cell proliferation assay (MTS)”, first paragraph. In fact, to further clarify this point we have decided to rename TM-EphA2 to recognize it includes JM residues. The new name for TM-EphA2 is TMJMFigure 4: A) Is too small to see clusters vs. no clusters that authors want to highlight. B) Does the blue FCS curve possibly represent more than one diffusing component? Its shape is clearly different from the other two. E) It is true that TYPE7 binding decreased diffusion of the myristylated receptor like it did with the native receptor , but all diffusion coefficients are much higher in the lipid-anchored compared to the protein-anchored cases. I.e. the scale is different in E vs. C and D. Nothing wrong with this, but it should be mentioned.A) New insets have been included highlighting the clusters .B) The reviewer is right in pointing out that our FCS experiments do not allow ruling out the presence of more than one diffusing component. However, the data fitted well to a single relaxation term (Equation 5), suggesting that the approximation we use, which is to compare the average values for the single component fits, is reasonable. We have introduced this important qualification in the second paragraph of the subsection “TYPE7 promotes limited self-assembly of EphA2”.E) We now discuss the increased D value for the myristoylated construct in the last paragraph of the subsection “TYPE7 promotes limited self-assembly of EphA2”.Mathematical model: Does a stochastic model, which this is, have a real time scale like the minutes shown in all modeling figures, or does it not have arbitrary time steps, and therefore, should the time scale in these figures not be better called arbitrary? Of course, this does not take away from the modeling, but it does not tie the figures directly to an actual experimental time scale that may be quite different.The model as depicted in Supplementary Figure 11A also needs more explanation. There seem to be 3 different types of k(I),(on and off) and 2 different types k(A),(on and off) as shown in the different panels. Or are they all constrained to the same respective values in the model? So, how many free fit parameters are there in the model? Clarification of this point is important to better understand the main text. The Supplementary Figure 11A legend will also benefit from a more detailed panel-by-panel explanation.The comment should have been addressed after removal of the mathematical model from the manuscript.


TaSSIV plays an important role in starch synthesis and its main function is granule formation. To mine and stack more favorable alleles, single nucleotide polymorphisms (SNPs) of TaSSIV-A, B, and D were investigated across 362 wheat accessions by Ecotype-Targeting Induced Local Lesions IN Genome (EcoTILLING). As a result, a total of 38 SNPs in the amplified regions of three TaSSIV genes were identified, of which 10, 15, and 13 were in TaSSIV-A, B, and D, respectively. These 38 SNPs were evaluated by using KASP and six SNPs showed an allele frequency >5% whereas the rest were <5%, i.e., considered to be minor alleles. In the Chinese mini core collection, three haplotypes were detected for TaSSIV–A and three for TaSSIV–B. The results of an association study in the Chinese mini core collection with thousand grain weight (TGW) and spike length (SPL) showed that Hap-2-1A was significantly associated with TGW and Hap-3-1B with SPL. Allelic frequency and geographic distribution indicated that the favored haplotype (Hap-2-1A) has been positively selected in Chinese wheat breeding. These results suggested that the Kompetitive Allele Specific PCR (KASP) markers can be applied in starch improvement to ultimately improve wheat yield by marker assisted selection in wheat breeding.Wheat is a staple food commodity grown worldwide, and wheat starch is a valuable source of energy and carbon that constitutes 80% of the grain weight. Manipulation of genes involved in starch synthesis significantly affects wheat grain weight and yield.  Breeding for yield potential of cereal crops due to a surging population is an important priority. Wheat is a staple food commodity for the ever-growing worldwide population. Wheat is central to food security and has a large share of required calories globally. Starch is a valuable source of carbon and energy. In leaves, starch is synthesized during daytime from photosynthetically-fixed carbon and is mobilized in the night time. It is also synthesized in other transient organs such as root cap cells and meristems, but major storage organs are fruits, tubers, seeds, and roots . At the Arabidopsis [Arabidopsis suggests that starch granules are initiated at a specific area of the chloroplast. Exon and intron regions of SSIII and SSIV are highly conserved in Arabidopsis, rice, and wheat but are different from SSI, SSII, and GBSS [Arabidopsis starch mutant plant (esv1) cannot synthesize or degrade starch at night and was dwarfed [Arabidopsis and potato, which also show higher rates of growth. Overexpression increased starch content in both photosynthetic and sink organs [OsSSIVa and OsSSIVb are mainly expressed in endosperm and leaves, respectively. Starch granule synthesis initiation in rice endosperm does not solely depend on OsSSIIIa and OsSSIVb, because suppression of these genes did not stop the granule initiation process. These results are different from Arabidopsis double mutant in which suppression of these two genes totally stopped granule production [In plants, starch is degraded at night during the process of respiration and helps the formation of sucrose. Starch consists of amylose and amylopectin. Amylopectin is the major part of starch, contributing about 75% of starch granules . SS is cbidopsis ,10. The and GBSS ,12. The  dwarfed . Overexpk organs ,15. OsSSoduction .TaSSIV is closely related to TaSSIII according to phylogenetic analysis and these genes share a similar arrangement of exons and introns [TaSSIV is on the long arm of homoeologous group 1 chromosome and is syntonic with OsSSIVb located on chromosome 5 [TaSSIII and TaSSIV on the same chromosome but opposite arms and phylogenetic analysis indicates that the evolutionary histories of these genes are related [TaSSIV-D mutation of wheat through EMS (ethyl methane sulphonate) showed that the chloroplasts containing 0–2 granules increased significantly while the chloroplasts containing 3–4 granules decreased [In wheat,  introns . The chrmosome 5 . The pre related . TaSSIV-ecreased .Pin a and Pin b [EcoTILLING , is an inexpensive, robust, and reliable allele mining approach. This approach is capable of allele mining and identifying genotypes of novel functional and natural variation of known genes ,23,24,25nd Pin b . In the TaSSIV have not been systematically surveyed in wheat. Natural polymorphism can be used for further breeding programmes and to understand the function of TaSSIV. There is also a need to know the allelic variation of TaSSIV in A, B, and D genomes.Single nucleotide polymorphisms (SNPs) have been widely used in marker assisted selection (MAS). SNPs and deletions/insertions (InDels) are nucleotide variations in natural populations that can be significantly associated with agronomic parameters . ComprehTaSSIV-A, B, and D genomes in 362 wheat accessions collected from diverse geographical areas of China and Pakistan to identify nucleotide diversity. The developed Kompetitive allele-specific PCR (KASP) markers were applied to screen 262 Chinese mini core collection (MCC) and 100 Pakistani wheat accessions for particular nucleotide variation. SNPs were identified with distinguishable haplotypes in the germplasm collection. In addition, association analysis between the haplotypes and phenotypic traits of the MCC was also performed to reveal marker trait associations (MTAs).In this study, EcoTILLING was used to detect allelic variation in the targeted region of A panel of 362 wheat accessions was used for EcoTILLING. This panel mainly consisted of 262 mini core collection (MCC) accessions from China and 100 accessions originating from Pakistan (spring wheat) . MCC repGenomic DNA (gDNA) was isolated from young seedling leaves using the standard CTAB procedure . Extracthttp://blocks.fhcrc.org/proweb/). Genomic sequences of the three TaSSIV genes were obtained from the GenBank database (http://www.wheatgenome.org/). Multiple alignment of nucleotide sequences was conducted by Bioedit 7.0. Primers were designed using Primer Premier 5.0 . Genome-specific primers for TaSSIV-A and TaSSIV-B were designed on the basis of SNPs and/or InDels; primers were already available for TaSSIV-D [The target gene regions with maximum probability for missense variants were predicted using the CODDLE bioinformatics pipeline (TaSSIV-D . Specifi−1 DNA using 0.4 U/reaction of Taq polymerase (TaKaRa Ex TaqTM). The mixture contained a 4:1 ratio of IRD800-labeled to unlabeled reverse primers and 3:2 ratio of IRD700-labeled to unlabeled forward primers. The EcoTILLING method was followed as described in [For EcoTILLING, each accession and Chinese Spring (WT) were pooled in a 1:1 ratio after diluting the samples. PCRs were performed in 10 µL final volumes on 100 ng µLribed in . After thttp://blocks.fhcrc.org/proweb/) and SIFT (Sorting Intolerant from Tolerant) (http://sift.jcvi.org/) programs with default parameters [Resultant PCR products were directly sequenced in both directions by Sangon Biotech Co., Ltd., Beijing, China, to confirm the sequence variations. Identified sequence variants were analyzed by the PARSESNP . The allele-specific primers were developed using the standard FAM (5′ GAAGGTGACCAAGTTCATGCT 3′) and HEX (5′ GAAGGT CGGAGTCAACGGATT 3′) tails with a targeted SNP at the 3′ end. KASP markers were then applied across the entire population. The KASP markers were developed by following standard KASP guidelines [X-(FAM) and Y-(HEX) signals. Detailed information for primers is given in For high-throughput genotyping, KASP primers were developed on the variants of idelines . A scattt-tests at p < 0.05 (even 0.01).Descriptive statistics and estimates of variance were conducted using Microsoft Excel 2013. The effect of haplotypes on agronomic traits were analyzed using Student’s TaSSIV-A consisted of 13 exons and 12 introns while TaSSIV-B, and TaSSIV-D each consisted of 16 exons and 15 introns. Each gene was exploited in the wheat population for sequence polymorphism by EcoTILLING. A total of 38 putative natural variation sites were identified across the population , which was located in the GT-5 domain and may affect the protein function. All other missense polymorphisms of TaSSIV-A, B, and D were present in coiled regions of protein structure and not in functional domains.The SIFT computer program predicted that only one sequence polymorphism in KASP-A1673T, KASP-C5952T, KASP-A2403C, and KASP-C2436T) in TaSSIV-A and two markers (KASP-C1560T and KASP-C6107T) in TaSSIV-B had allele frequencies over 5%. These results were consistent with EcoTILLING. All SNPs of TaSSIV-D had allelic frequencies less than 5% and were therefore excluded from further analysis. For KASP-A11673T, KASP-C5952T, KASP-A2403C, and KASP-C2436T , Hap-2-1A (ACTT), and Hap-3-1A (ACTC) were more than 5%. The other eleven haplotypes were less than 5%, marking them as rare haplotypes. Three haplotypes of TaSSIV-B i.e., Hap-1-1B (CC), Hap-2-1B (TC), and Hap-3-1B (CT) in the wheat population had frequencies more than 5%  in regions I–IV  and the Punjab rainfed region (52%). The frequency of this haplotype in Khyber Pakhtonkhwa was 35%.  were significantly (p < 0.05) associated with yield-related traits in wheat.The starch synthase family in plants encodes many starch synthesis genes that play essential roles in starch synthesis and ultimately increase yield . There iHap-2-1A had higher TGW while accessions having Hap-3-1B had larger SPL. Yield-related traits of cereal crops are governed by multiple genes and are influenced by environmental factors [It has been demonstrated that grain yield in France , Italy , the UK, factors . Among t factors . In thisHap-2-1A favored wheat breeding. Regions I, II, and III account for about 64.8% of total national wheat area and greater turnover of wheat cultivars was also reported from these regions [TaSSIV-1A-2 and TGW in MCC  was selected in major wheat growing regions of Pakistan especially in Punjab regions (rainfed region and irrigated region) (Hap-2-1A) was reasonably high, indicating the positive selection during modern wheat breeding. Although both populations have different population structures, unconscious selection of the favored haplotype is likely due to the high linkage disequilibrium of major yield-related genes selected during selection breeding. However, the frequency analysis in wheat cultivars from other regions like Europe, USA, and Australia would be required to draw the conclusion that selection of favored haplotypes of TaSSIV is likely due to the selection of major yield-related genes [Choice of populations plays a critical role to detect MTAs via association analysis. Accessions from Pakistan were also used to evaluate the  region) . In bothed genes .TaSSIV among 362 wheat accessions collected. Association analysis showed that two haplotypes in TaSSIV were significantly associated with TGW and SPL and these haplotypes can be used in future studies to assess their usefulness as selection criteria for improving these yield-related traits.In conclusion, we have demonstrated that EcoTILLING is an efficient approach for allele mining of wheat candidate genes and KASP is a robust approach to validate SNPs for further analysis. This study identified 38 unique alleles in


Bemisia tabaci), an important invasive pest that causes severe damage to crops worldwide, has developed resistance to a variety of insecticides. Carboxylesterases (COEs) are important multifunctional enzymes involved in the growth, development, and xenobiotic metabolism of insects. However, systematic studies on the COEs of B. tabaci are scarce. Here, 42 putative COEs in different functional categories were identified in the Mediterranean species of B. tabaci (B. tabaci MED) based on a genome database and neighbor-joining phylogeny. The expression patterns of the COEs were affected by the development of B. tabaci. The expression levels of six COEs were positively correlated with the concentration of imidacloprid to which B. tabaci adults were exposed. The mortality of B. tabaci MED adults fed dsBTbe5 (67.5%) and dsBTjhe2 (58.4%) was significantly higher than the adults fed dsEGFP (41.1%) when treated with imidacloprid. Our results provide a basis for functional research on COEs in B. tabaci and provide new insight into the imidacloprid resistance of B. tabaci.The whitefly ( Carboxylesterases (COEs) are multi-gene superfamily enzymes with an α/β-hydrolase fold that can hydrolyze carboxyl esters into corresponding alcohols and acids , and areBemisia tabaci Middle East-Asia Minor 1 (MEAM1) [Aphis gossypii [Plodia interpunctella [Anisopteromalus calandrae [Lucilia cuprina [Musca domestica [B. tabaci MEAM1 [Nilaparvata lugens [A. gossypii [Helicoverpa armigera [Habrobracon hebetor [Culex quinquefasciatus [A. gossypii Glover [Leptinotarsa decemlineata [Aedes aegypti [Tetranychus cinnabarinus [In insects, studies on COEs have mainly focused on their roles in insecticide resistance. The mutation of COE coding sequences is related to organophosphorus resistance in Hemiptera ( (MEAM1)  and Aphigossypii ), Lepidounctella ), Hymenoalandrae ), and Di cuprina  and Muscomestica ). The ovci MEAM1 , Nilapara lugens , and A. gossypii ), Lepidoarmigera ), Hymeno hebetor ), and Diasciatus ). The ini Glover ), Coleopmlineata ), Dipter aegypti ), and Arabarinus ). COEs fabarinus .B. tabaci (Gennadius), is one of the 100 most catastrophic invasive species in the world [B. tabaci [B. tabaci is a species complex that includes at least 30 cryptic species [B. tabaci MEAM1 has been replaced by B. tabaci MED due to the overuse of insecticides [The whitefly, he world . This ty. tabaci . B. taba species . Among t species . In mostcticides ,27,28.B. tabaci was the first insect to develop resistance to imidacloprid [B. tabaci resistance to neonicotinoid insecticides (including imidacloprid) has been reported globally [B. tabaci to imidacloprid [B. tabaci MED tolerance of imidacloprid has not been investigated.Neonicotinoid insecticides are an important class of chemical insecticides that are used worldwide because of their high toxicity to a range of important pests ,30,31. Wacloprid , and B. globally ,35,36,37acloprid ,40,41,42B. tabaci MED [B. tabaci MED genome by constructing a phylogenetic tree with homologous genes from Apis mellifera and Drosophila melanogaster. We then characterized the expression patterns of COE genes affected by developmental stage and investigated the expression profiles of the COE genes in response to imidacloprid challenge. Finally, RNA interference (RNAi) was used to determine which COEs are involved in B. tabaci MED defense against imidacloprid.Recently, our group sequenced the transcriptome and genome of baci MED ,44, provB. tabaci MED  of COE genes are shown in COE family genes are involved in multiple processes of insect growth and development; thus, their expression levels may vary among developmental stages. To determine the expression profiles of COEs across different developmental stages of BTbe6 was highly expressed at all stages of development, while BTun1 and BTace1 were largely undetectable in transcriptional analysis. Examination of the secreted catalytic class showed that BTbe7 expression was significantly lower in adults than at other stages. BTbe4 was downregulated in male and female adults but highly expressed in 1st-2nd-instar nymphs and 4th-instar nymphs. BTbe9 expression was significantly higher in female adults and lower in eggs, males, and 1st-2nd-instar nymphs. The BTbe15 had significantly lower expression in eggs than at other stages. BTjhe genes were most highly expressed in 4th-instar nymphs, followed by female adults. BTun2 exhibited the highest expression in 3rd-instar nymphs and lowest expression in eggs. BTglt was upregulated in male adults. In the intracellular catalytic class, BTae1 was downregulated while BTae2 and BTae3 were upregulated in male and female adults. In the neurodevelopmental class, most of the BTnrl genes had the highest expression level in eggs, except for BTnrl3, BTnrl6, and BTnrl7, and all BTnrl genes were expressed at low levels in male and female adults. BTace genes except for BTace1 were more highly expressed in 4th-instar nymphs than at the other stages.B. tabaci MED development. The expression levels of 7 of the 12 COEs obtained by qRT-PCR were significantly consistent with the FPKM expression pattern (p < 0.05)  .B. tabaci to imidacloprid challenge, we analyzed the expression profiles of all COEs in response to imidacloprid using the leaf-dip bioassay method with imidacloprid concentrations of 0 (control), 25, 50, and 100 mg/L [BTbe8, BTbe10, BTbe12, BTbe14, and BTbe15), all four acetylcholinesterases, and BTjhe3, BTae5, BTglt, and BTnrl8) was induced by imidacloprid, and the results are shown in BTbe5, BTbe3, BTjhe1, BTjhe2, BTae2, and BTun2 induced by 100 mg/L imidacloprid were higher than those induced by 50 mg/L imidacloprid, followed by 25 mg/L imidacloprid induction. The expression levels of these genes were lowest in the control group with no imidacloprid  dsRNA  (37.3%) or dsEGFP (41.1%) when the adults were treated with imidacloprid at 50 mg/L  and BTjhe2 (58.4%) was significantly higher than that of adults fed dsRNA of EGFP (41.1%) when treated with imidacloprid  were involved in B. tabaci MED imidacloprid resistance.COE genes have multiple functions in insects, including neurogenesis, developmental regulation, xenobiotic metabolism, and insecticide detoxification . B. tabaB. tabaci MED and A. pisum had more beta esterase genes but fewer alpha esterase genes than other insects. The alpha esterase genes belong to the intracellular catalytic class of COEs and participate in insect xenobiotic detoxication [B. tabaci MED. We also found that B. tabaci had more nrl genes than other insects. nrl genes are essential for establishing and remodeling central nervous system synapses [nrl genes in B. tabaci MED is related to its developmental characteristics or the evolution of new biological functions for some of these genes needs to be further investigated.In the current study, we found that the hemipteran insects xication ,46,47. Pxication ,48. Somesynapses ,50. WhetB. tabaci MED showed that adult stages clustered together, while eggs and nymphal stages were clustered together. The COE genes of insects participate in many processes of growth and development. Eggs and nymphs of whiteflies are in the process of rapid growth and development, while adults have completed their development. Therefore, the expression of COEs in B. tabaci MED was clustered as described above. The ecdysone and JH in insect hemolymph regulate insect growth and development in a complex manner [Plutella xylostella is highly expressed in 4th-instar larvae and participates in the induction of larval-to-pupal metamorphosis [BTgli and BTnrls genes, which belong to the neurodevelopment clade, were expressed at low levels in the adult stage, and most of these genes were highly expressed in the egg stage. This difference may be related to the developmental characteristics of B. tabaci. During development from the egg to the nymph, whiteflies develop a complex nervous system; therefore, high expression levels of genes involved in neural development are required. In contrast, at the adult stage, the nervous system is fully developed, and as a result, the expression levels of genes related to nerve development will be reduced. RNA-seq and qRT-PCR are the two main methods for gene expression analysis. Previous studies showed that the correlation of ABC transporter expression between RNA-seq and qRT-PCR was approximately 0.684 [The expression profile of the COEs at different developmental stages of x manner . In numex manner . We deteorphosis . JHE genly 0.684 , and 50%ly 0.684  of aminoB. tabaci MED showed that most COEs can be induced by imidacloprid, and the expression levels of six COE genes were positively correlated with the concentration of imidacloprid applied. COEs are among the most important detoxification enzyme systems that participate in the metabolic detoxification of xenobiotics in insects. COEs, which increased in expression with increasing imidacloprid concentration, are more likely to be involved in the resistance of B. tabaci. Alpha esterases and beta esterases are widely recognized as major COE clades involved in the detoxification of xenobiotics and lipids [BTbe3, BTbe5, and BTae2 were located in these clades, suggesting that these genes play a potentially important role in metabolizing insecticides in B. tabaci. JHE genes are mainly involved in the regulation of hormone metabolism, growth, and development [B. tabaci JHE genes participate in insecticide resistance requires verification by subsequent RNAi experiments.The induction of gene expression represents adaptive plasticity between energy conservation and survival in rapidly changing environments ,55,56. Od lipids . BTbe3, elopment . WhetherB. tabaci has developed high levels of resistance to neonicotinoids in the field [B. tabaci against neonicotinoid insecticides is mainly related to detoxification [B. tabaci [CYP6CM1 and CYP4C64 has been found to be involved in imidacloprid resistance in field B. tabaci [GSTd7 gene increased the mortality of whiteflies exposed to imidacloprid [GST14 significantly increased the mortality of thiamethoxam-treated B. tabaci MED [B. tabaci to imidacloprid, and silencing the ABCG3 gene by RNAi increased the lethality of imidacloprid to adults of B. tabaci MED [B. tabaci MED corresponded to the amount of imidacloprid to which individuals were exposed. We silenced COE gene expression using oral feeding of dsRNA and found that knockdown of the BTbe5 and BTjhe2 genes significantly increased the lethal effect of imidacloprid, indicating that these two genes are involved in the resistance of B. tabaci MED to imidacloprid. In insects, the beta esterase clade genes are recognized as important detoxification enzymes involved in resistance to organophosphorus insecticides [BTbe5 was involved in the imidacloprid resistance of B. tabaci MED. The main function of JHE genes is to regulate the growth and development of insects. B. tabaci adults have completed their growth and development. The expression of the JHE genes in the adult stage indicates that these genes may have other biological functions. In this study, we found that a JHE gene participated in the resistance of adult B. tabaci to imidacloprid.Neonicotinoid insecticides are important chemical pesticides that are widely used in the control of agricultural pests worldwide ,30,31. Ihe field ,35,36,37fication ,40,57,58. tabaci ,38,39. Tacloprid , and knocticides ,59,60. IB. tabaci MED, and the expression profiles of COEs at different developmental stages of B. tabaci were analyzed. Moreover, two COE genes, BTbe5 and BTjhe2, were found to facilitate B. tabaci MED resistance to imidacloprid. The present results will contribute to functional research on COEs, enrich our understanding of the resistance mechanism of B. tabaci MED to imidacloprid, and aid in the development of a management strategy for B. tabaci.In summary, all COE family genes were identified in B. tabaci MED was collected on poinsettia (Euphorbia pulcherrima Wild. ex Kl.) in 2009 in Beijing. It was transferred and continuously maintained on cotton (Gossypium herbaceum L. cv. E-Mian 24) without exposure to chemical pesticides in a glasshouse at 27 ± 1 °C, with a relative humidity (RH) of 70% ± 10% and L16:D8 photoperiod. The purity of the colony was monitored by sequencing a fragment of the mitochondrial cytochrome oxidase I (mtCOI) gene every three to five generations [The colony of erations .B. tabaci MED, the predicted proteins containing the conserved functional domain of COE defined by the Pfam hidden Markov model (HMM) profile COesterase (PF00135) were identified in the B. tabaci MED genome-predicted protein data set [A. mellifera and D. melanogaster downloaded from the NCBI (https://www.ncbi.nlm.nih.gov/genome/?term=Apis+mellifera (accessed on 12 March 2018)) and FlyBase (http://flybase.org/ (accessed on 12 March 2018)) were used as queries to search against the B. tabaci MED genome to ensure that all the COEs had been identified. A total of 42 putative B. tabaci COE genes were confirmed by blasting against the NR database with the BLASTX program on the NCBI website (http://blast.ncbi.nlm.nih.gov/ (accessed on 16 March 2018)). Then, the putative COEs were manually corrected by comparison with assembled expressed sequence tags (ESTs) [To identify the putative COEs in data set  using HMdata set . The COEs (ESTs) .B. tabaci MED, a phylogenetic tree was constructed based on the putative COE genes of B. tabaci MED and COE amino acid sequences from D. melanogaster and A. mellifera. All the selected COE amino acid sequences were aligned using MUSCLE, a module of MEGA 6 (http://www.megasoftware.net/ (accessed on 6 May 2018)) [To comprehensively annotate and systematically classify the COE genes of y 2018)) . A phyloB. tabaci MED and imidacloprid-treated adult B. tabaci MED were obtained from our previous report [B. tabaci MED genome with TopHat software [RNA-seq libraries for different developmental stages of s report . Trimmoms report , and thesoftware ,64. The software .B. tabaci MED at different developmental stages  adults). Total RNA was extracted from each sample using TRIzol reagent  following the manufacturer’s instructions. RNA quality was evaluated by agarose gel electrophoresis, and the total RNA was quantified using a spectrophotometer . The RNA was reverse-transcribed to cDNA with a PrimeScript RT kit   for qRT-PCR analysis and a PrimeScript™ II 1st strand cDNA synthesis kit  for COE double-strand RNA (dsRNA) synthesis. The cDNA was stored at −80 °C for later use.Samples were collected from B. tabaci MED were designed using Primer Premier 5.0 and used to verify the expression levels of COEs by qRT-PCR  . The qRT-PCR programme was as follows: 95 °C for 10 min , followed by 40 cycles of 95 °C for 5 s (denaturation), 60 °C for 15 s , and 72 °C for 35 s (elongation). Only the qRT-PCR primers with 90–110% amplification efficiencies were used for the subsequent data analysis.The specific primers for COEs of  qRT-PCR . qRT-PCRCt−ΔΔ method [RPL29) (GenBank accession no. EE596314) and elongation factor 1 alpha (EF1-α) (GenBank accession no. EE600682) was used to normalize the expression of target genes [Relative expression levels were quantified using the 2t method . The geoet genes ,68. ThreBTbe5, BTbe3, BTTjhe1, BTjhe2, BTae2, and BTun2 were selected for RNAi because their expression was positively correlated with the imidacloprid-induced concentration. The dsRNA primers of BTbe5, BTbe3, BTjhe1, BTjhe2, BTae2, BTun2, and EGFP (GenBank: KC896843) with the T7 promoter sequence were designed using Primer Premier 5.0 to clone partial cDNA of those genes . The quality of dsRNA was evaluated by gel electrophoresis, and the dsRNA was quantified using a NanoDrop spectrophotometer.se genes . The dsRBTbe5, BTbe3, BTjhe1, BTjhe2, BTae2, and BTun2 genes was performed by orally feeding dsRNA to B. tabaci MED adults in feeding chambers. The feeding chambers contained 0.2 mL of diet solution, which contained 30% sucrose, 5% yeast extract (weight/volume), with 0.5 μg/μL dsBTbe5, dsBTbe3, dsBTjhe1, dsBTjhe2, dsBTae2, and dsBTun2 [B. tabaci MED adults (mixed sexes) were released into the feeding chambers, and were kept at 25 °C, 80% RH, and an L14:D10 photoperiod. The effectiveness of RNAi was determined by qRT-PCR using cDNA synthesized from isolated total adult RNA after 2 days of feeding. Each RNAi treatment was repeated three times.Knockdown of  dsBTun2 . ApproxiBioassays were performed using feeding chambers for 24 h. The living whitefly adults fed dsRNA for 48 h were transferred to a new feeding chamber containing 50 mg/L imidacloprid in a diet solution. Bioassays were performed with approximately 40 living whitefly adults per treatment, and each bioassay was replicated three times. Significant differences in the bioassays were determined using SPSS 23 with one-way ANOVA and Holm–Sidak test .