Arabidopsis thaliana requires a series of processing events in consecutive compartments of the secretory pathway. Following cleavage of the signal peptide upon entry into the endoplasmic reticulum (ER), the peptide precursors are processed in the cis-Golgi by the subtilase SBT6.1. SBT6.1-mediated cleavage within the variable domain allows for continued passage of the partially processed precursors through the secretory pathway, and for subsequent post-translational modifications including tyrosine sulfation and proline hydroxylation within, and proteolytic maturation after exit from the Golgi. Activation by subtilases including SBT3.8 in post-Golgi compartments depends on the N-terminal aspartate of the mature peptides. Our work highlights the complexity of post-translational precursor maturation allowing for stringent control of peptide biogenesis.Post-translationally modified peptides are involved in many aspects of plant growth and development. The maturation of these peptides from their larger precursors is still poorly understood. We show here that the biogenesis of CLEL6 and CLEL9 peptides in  Arabidopsis thaliana (hereafter ‘Arabidopsis’), there are more than 1000 genes potentially encoding signaling peptides, apparently involved in all aspects of plant growth and development . HPAT is encoded by three genes in Arabidopsis that are at least partially functionally redundant  for Arabidopsis SBTs constitute a large family of mostly extracellular proteases including e.g. 56 members in Arabidopsis , 86 in tWith its predominant Golgi localization , AtSBT6.sbt mutants suppressing the CLEL6-overexpression phenotype (agravitropic root growth and increased hypocotyl elongation)  . The prongation) . Surprisngation)  suggestiPhytophthora infestans were expressed in transgenic Arabidopsis plants under control of the CLEL6 or CLEL9 promoters (CLEL6 and CLEL9 are expressed is expected to phenocopy the CLEL6/9 loss-of-function phenotype if SBT activity is required for precursor processing and peptide maturation.In order to confirm the involvement of SBTs in the maturation of CLEL6 (GLV1/RGF6), we used the inhibitor-based loss-of-function approach that was previously employed to demonstrate a role for redundant SBTs in the maturation of IDA (Inflorescence Deficient in Abscission) resulting in the shedding of Arabidopsis flower organs after pollination . CLEL9 , and becromoters . InhibitCLEL6 or the CLEL9 promoter were impaired in the gravitropic response of the hypocotyl  cleavage sites, RRLR and RRAL , and theN. benthamiana. The sfGFP tag was linked to the N-terminus of the precursor, just downstream of the signal peptide .ER retention by the KDEL-motif is mediated by Golgi-resident K/HDEL-receptors, which effect retrograde transport of soluble ER proteins from the Golgi back to the ER . Cleavagal Golgi . The samal Golgi  indicatim’ in To assess the relevance of cleavage by SBT6.1 for processing and secretion, we masked both cleavage sites m’ in . The cenm  lacking both SBT6.1 processing sites . Again, m , is thusm , suggestm , may conIn contrast to Δ-Sec, the unprocessed precursors were observed for both Δ-KDEL and Δ-XylT as single larger bands on the immunoblot . RetentiAll CLEL peptide precursors including both CLEL6 and 9 share a conserved aspartate upstream of the sulfated tyrosine . To testN. benthamiana and any peptides produced from these precursors were extracted in apoplastic washes. The activity of these peptides was tested in the tpst-1 mutant, which is devoid of endogenous sulfated peptides , the gravitropic response was restored to wild-type levels indicating the formation of bioactive CLEL6 and CLEL9 peptides (A bioassay was then used to assess whether the aspartate and aspartate-dependent processing are required for the formation of bioactive CLEL peptides in planta. The full-length Sec CLEL6 and CLEL9 constructs were transiently expressed in peptides . When tppeptides . In contpeptides . The datCLEL6 or the CLEL9 promoter (N. benthamiana (The protease(s) required in addition to SBT6.1 for the final maturation step and activation of CLEL6 and CLEL9 was hitherto unknown. To test a potential involvement of SBTs, as suggested by the impaired gravitropic response of the hypocotyl in seedlings expressing EPI1a or EPI10 under the control of either the promoter , the flathamiana . As compthamiana . The dattpst-1 mutant, as a compensatory response to the deficiency in bioactive sulfated peptides. We thus analyzed the expression of the 56 Arabidopsis SBT genes in tpst-1 hypocotyls in comparison to the wild type and found that four  were up-regulated in the tpst-1 background . However, the subcellular site of SBT6.1-mediated processing remained unresolved. We show here that SBT6.1 cleaves CLEL precursors soon after exit from the ER in an early Golgi compartment , indicatSite-directed mutagenesis of SBT6.1 cleavage sites impaired secretion of the CLEL precursors, as some of the signal got stuck in the ER and the Golgi . The varThe requirement of SBT6.1-mediated processing for secretion provides an explanation for the perplexing finding that SBT6.1 cleavage sites and, by inference, cleavage by SBT6.1 are required for CLEL6 activity , despiteen route. Nonetheless, cleavage does not occur before the partially processed precursor leaves the Golgi . All experiments were carried out at least three times with similar results. If indicated, media were supplemented with synthetic CLEL6 (DY(SO3H)PQPHRKPPIHNE) or CLEL9 (DMDY(SO3H)NSANKKRPIHNR) peptides  at the indicated concentrations. The sbt3.8 loss-of-function mutant has been described before For growth experiments in axenic culture, Arabidopsis seeds were surface-sterilized in 70% ethanol for 15 min, washed in 100% ethanol and laid out in rows on square plates containing 0.5 x MS (Murashige-Skoog) medium, 1% sucrose and 0.38% gelrite. Seeds were stratified for two days at 4°C and grown for 5 days in the dark. For quantitative analysis of gravitropic responses, 5 day old vertically grown seedlings were rotated for 90° in the dark and grown for further two days. The bending angle was measured using ImageJ . CLEL6 and CLEL9 were N-terminally coupled to sfGFP using EcoRI and BamHI. For constructs expressing EPI1a and EPI10 under the control of the CLEL6 or CLEL9 promoters, EPI1a and EPI10 constructs described by EcoRI and XhoI restriction sites and ligated into the corresponding restriction sites of pGreen0229, upstream of the nptII terminator sequence. CLEL6 and CLEL9 promoters . After purification, PCR-products were digested with Eco31I-HF (Thermo Scientific) to open module specific overhangs. Fragments were then ligated in Eco31I-opened and purified entry vectors. Presence and sequence of inserts were verified via restriction digest and sequencing.The expression construct for the VMA12-mRFP ER marker has been described previously . The expg system . GreenGaE. coli, the ORF of CLEL6 lacking the predicted signal peptide was amplified by PCR from a previous construct and cloned into the NcoI restriction site of pETDuet1 . Correct orientation and translational fusion with the C–terminal His-tag was verified by sequencing. Expression in E. coli BL21 was induced by 1 mM IPTG for two hours. His-tagged CLEL6 precursor was purified from bacterial extracts by metal chelate affinity chromatography on Ni-NTA Agarose  according to the manufacturer’s recommendations. After ultrafiltration (30 kDa molecular weight cutoff), the recombinant protein in the filtrate was further purified by size exclusion chromatography  in 50 mM NaH2PO4 /Na2HPO4, pH 5.5, 10 mM NaCl. The D71A mutant was created by site-directed mutagenesis, confirmed by sequencing, and purified as above. For co-translational incorporation of sulfo-tyrosin, the Y72 codon of the proCLEL6 ORF in pETDuet1 was replaced with an amber stop (UAG) by site-directed mutagenesis and confirmed by sequencing. The sulfoCLEL6 expression construct was transformed into electro-competent BL21 pEVOL SYRS carrying an amber suppressor t-RNA and a cognant sulfo-tyrosine specific aminoacyl-tRNA synthetase  at 28°C and were washed off the plates in 10 mM MES, pH 5.6 containing 10 mM MgCl2. A blunt syringe was used to infiltrate the bacterial suspension supplemented with 150 µM acetosyringone into the leaves. For total protein extraction, leaves were harvested two to three days after infiltration into liquid nitrogen and ground to a fine powder. The powder was thawed in 50 mM Tris/HCl, pH 7.5, 100 mM NaCl and 10 mM β-mercaptoethanol containing 0.5% Triton X-100 and proteinase inhibitor mix P . The extracts were centrifuged  and the supernatant was kept at 4°C until usage at the same day, or frozen at −20°C.2PO4/Na2HPO4, pH 7, 300 mM NaCl. Apoplastic washes were harvested by centrifugation at 1100 x g. In order to obtain SBT3.8 in sufficient amounts and purity for assays, metal chelate affinity chromatography on Ni-NTA Agarose (Qiagen) was performed according to the manufacturer’s recommendations. The eluates were dialyzed against 50 mM sodium phosphate buffer pH 7.0 or 5.5, 10 mM NaCl and used for enzyme activity measurements. For the empty-vector control, apoplastic extracts from mock-infiltrated plants were subjected to the same purification scheme. To collect exudates from wild type plants and the sbt3.8 mutant, seedlings were grown in submerged culture in 0.5 x MS (Murashige-Skoog) medium with 1% sucrose for ten days as described by Five days after agro-infiltration, the leaves were harvested and vacuum (70 mbar)-infiltrated with 50 mM NaH2PO4/Na2HPO4, pH 5.5, 10 mM NaCl for the time indicated and separated by SDS-PAGE.Extended CLEL6 peptide  was digested with recombinant SBT3.8 or the negative control for 2 hr until the reaction was stopped by addition of 1% TFA. Reactions were performed at pH 5.5 and pH 7.0 in 50 mM potassium phosphate buffer, 10 mM NaCl. 1.5 µl of the samples were mixed with an equal volume of the crystallization matrix  on the MALDI target, and mass spectra were recorded with a AutoflexIII mass spectrometer  in the reflector mode with external calibration . Flex Analysis 3.0 was used for data analysis with a mass tolerance of 50 ppm for ions. Recombinant CLEL6 and the D71A mutant were digested with SBT3.8 in 50 mM NaHC-terminally sfGFP-tagged CLEL6/9 expression constructs were infiltrated into tobacco leaves. Leaves were harvested after five days and apoplastic extracts were obtained as above. GFP concentration was determined spectro-fluorimetrically using a Spark microplate reader . Equivalent amounts of cell wall extract  were directly applied to the growth media of etiolated seedlings, and the gravitropic response was analyzed as described before. For the control, cell wall extracts were prepared from empty-vector infiltrated plants and used at the largest volume of experimental samples.Proteins were separated by SDS-PAGE or Tris Tricine PAGE. For western blots, proteins were transferred to nitrocellulose membranes using standard procedures. Polyclonal anti-GFP antibodies , monoclonal anti-His  or anti-Flag antibodies  directly coupled to horseradish peroxidase were used for immunodetection, followed by enhanced chemiluminescence detection with an Odyssey Fc imager .N. benthamiana were observed with a TCS SP5 II inverted Confocal Laser Scanning Microscope  using a HCX PL APO lambda blue 63.0 × 1.20 water immersion objective (Leica Microsystems). sfGFP was excited with the 488 nm line of the VIS-Argon laser; for mRFP/mCherry the 561 nm line generated by a VIS-DPSS 561 laser was used. Emission was detected at 500–550 nm for sfGFP and 610–670 nm for mRFP/mCherry with HyD hybrid detectors (Leica Microsystems) in standard operation-mode. Autofluorescence was detected between 700–800 nm with identical laser settings as used for sfGFP-mRFP/mCherry image recording. Images were adjusted in brightness and processed using ‘Mean’ Filter with a pixel radius of 0.1 with ImageJ software version 1.51 s . Propidium iodide  was vacuum-infiltrated into leaves five min prior to imaging (extinction 515 nm/emission 595 nm).Agro-infiltrated leaves of E. coli and SYBR-Green . Relative SBT mRNA levels were determined after normalization to three reference genes  using the optimized ΔCT method by RNA was isolated from approximately 50 hypocotyls of 5 day-old etiolated seedlings as previously described with minor modifications . cDNA wa In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:In Arabidopsis and other plants, CLEL peptides serve as ligands for major signaling pathways. Bioactive CLEL peptides must be processed from larger precursor proteins. In this work, the authors identify multiple cleavage steps occurring in consecutive compartments of the secretory pathway, and candidate subtilisin-like proteases, SBT6.1 and SBT3.8, required for these steps. This work highlights the complexity of post-translational precursor maturation allowing for stringent control of peptide biogenesis in plants.Decision letter after peer review:[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your work entitled "The biogenesis of CLEL peptides involves several processing events in consecutive compartments of the secretory pathway" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in There was consensus that the context and rationale for the study was well laid out and that there is a need for better mechanistic understanding of how CLEL peptides are processed. The extent to which this study definitively advanced our knowledge beyond that of previous studies, however, was considered by all reviewers to be fairly modest. Certainly identification of the SBT capable of driving the next processing reaction would have made this work much more compelling.Reviewer #1:This is an engagingly written article about CLEL processing. The CLE-like family of peptides have been known for about 20 years; initially via genetics in Arabidopsis. There are many biological functions ascribed to this family, particularly during development-shoot meristems, root meristems, embryos all employ family members, and these proteins have also been found and studied in Maize, tomato, etc. The challenge of this family is that the active peptide are only 12 AA long and are processed and heavily modified. What I didn't appreciate until this manuscript was that, although many of the modification enzymes have been identified, the initial proteolytic processing is fairly uncharacterized. For a family as critical as this one, missing the first step is a big deal!Subtilisin proteases (SBT) are found in all forms of life. In plants, there is a large family of SBTs, but most are extracellular. This manuscript focuses on SBT6.1, one of the few TM-domain forms, and one that is also thought to have emerged before the plant/animal split. SBT6.1 was recently shown to be required for activity of CLEL6 . So, this manuscript must add something new. What's new is that they show where the first SBT6.1 processing takes place and, they tracked down a second processing step that need to take place in the Golgi and they found the residue in CLEL that is needed for this second step. They then do an assay that suggests another SBT  is needed later on, but don't know what it is.The approach relies on several technical innovations, some of which I find very clever and others  I am less convinced of the specificity of.e.g., I like that they set up a transient cleavage assay (N. benthi) with GFP attached to the CLEL precursors + various tags in which they could monitor process through the secretory system with live cell imaging and also by Immunoblot against GFP to see where processing occurred.I am a little worried about overexpression of a pathogen-derived EPI, in that its specificity isn't completely clear. I do like the genetic experiments of showing that EPIs driven with CLEL6 or CLEL9 promoters leads to a phenotype similar to loss of the CLEL6/9 peptides themselves, and can be suppressed by supplying synthesized, processed CLEL.Strengths: This is well written and I am now convinced that it's important to understand the processing of the CLEL peptides and that the processing is going to be complicated. The assays are clever and the experiments appear to be done rigorously. The data are presented in a clear and straightforward manner in the figures.Weaknesses: Relies on EPIs being highly specific . Biochemical assays are in a non-native system, although I recognize that it is likely not technically feasible to do the assays in Arabidopsis and they also include bioassays in Arabidopsis, so I am less concerned about this leading to completely artifactual results.Although they demonstrated that it IS important for the peptides to be processed at the site they identified, is this a surprise based on previous work?Life paper.The manuscript does a nice job of demonstrating that a late Golgi SBT is likely to be involved, but then can't supply an answer as to which one it is, and I felt like there was no attempt to use other data sources  to narrow down the possibilities. All in all, I found this a bit too preliminary for an eThe Discussion was quite long and speculative relative to the compact data section. It seems as if the authors are addressing some points of contention in the field, and I (as an outsider) don't come to the story with a particular bias that needs to be overcome. I will defer to others who are closer in the field.Reviewer #2:In this manuscript, the authors studied biogenesis mechanisms of Arabidopsis CLEL6 and CLEL9 peptides by expressing their GFP fusion proteins using N. benthamiana transient expression system followed by Western blotting. Cumulative results suggested that SBT6.1-mediated cleavage within the variable domain is a prerequisite for continued passage through the secretory pathway and subsequent proteolytic maturation after exit from the Golgi. Furthermore, the authors proposed that the cleavage for final activation occurs in a post-Golgi compartment by an aspartate dependent subtilases.Because the maturation mechanisms of plant peptide hormones is still poorly understood, the authors' efforts will contribute to expand our knowledge of the peptide hormone biogenesis. Indeed, their findings that cleavage within the variable domain is a prerequisite for continued passage through the secretory pathway and subsequent proteolytic maturation are interesting. However, some of the conclusions are not supported by the results, especially the assumed involvement of aspartate residue in the cleavage for final activation.1) The authors concluded that aspartate-dependent processing is required for peptide activation of CLEL6 and CLEL9 based on a bioassay data using N. benthamiana leaf extracts transiently expressing GFP-CLEL6 and GFP-CLEL9 fusion proteins. This approach is, however, not quantitative and cannot exclude the possibility that structurally immature (larger or unmodified) peptide products are detected if present at high concentrations. Indeed, extracts from leaves expressing D-to-A mutants of CLEL6 and CLEL9  still shows considerable activities . Therefore, in addition to the bioassay experiments, mass spectroscopic analysis which can evaluate both actual structure and abundance of mature peptides is prerequisite to justify the conclusion.2) Related to above points, there is no detailed explanation of methods for extraction of apoplastic peptides and bioassay. Explain in the Materials and methods section.3) The authors used hypocotyl for bioassay in Figure 1, but used roots in Figure 4. This is confusing for readers. Use hypocotyl bioassay throughout the manuscript.4) In Figure 2B, it is better to indicate theoretical molecular sizes for each possible fragments. This information greatly helps understanding of SDS-PAGE bands shown in Figure 2C, E, and F. The same is true for Figure 3B.Reviewer #3:The paper is sufficiently well written. It provides incremental data about subsequent steps in the maturation of the plant secreted signal peptides CLEL6 and 9  and analyzes the subcellular compartments where proteolytic events take place. The work is overall sound, except for problems explained here below . But it has a narrow scope and shows some weakness in the interpretation of the results. The manuscript is worth publishing, but I do not feel that it is a major contribution to its field.Subsection “The cleavage for final activation occurs in a post-Golgi compartment by an aspartate-dependent SBT” fourth paragraph. Provided additional explanations are given (see below), I will trust that the experiments illustrated in Figure 4C to E show that CLEL extracts have an impact on root growth, and that there is a differential response between the wt and D>A extracts. However, the explanation of the root growth referring to the "waving phenotype" is incorrect. On dense agar plates slanted backward, wild-type untreated roots "wave", that is their normal growth pattern which is explained as a recurrent alternation between obstacle avoidance response and gravitropic response. In Whitford et al., 2012, CLEL peptides added to the medium do not induce waving, but they alter the waving pattern. In fact, the peptides induce "coiling" rather than waving: because of a reduction in gravitropism: roots often form complete loops as they grow away from the vertical.In the experiments presented here, the control roots do not wave, but simply grow down . The lack of waving may be due to the angle of the plate , the composition of the medium, the concentration of the agar. What is reported in this manuscript is that treated roots do not follow as strictly the vertical direction compared to the controls, they meander… but they don't wave. If my interpretation is right, the text should be corrected by removing all references to "waving" and simply state that treated roots have a reduced gravitropic response.Related to these experiments, the authors should address the following questions that are not covered in the Results or Materials and methods sections?How were the roots grown  light and light cycles)? How exactly was the variable "changes in root growth direction" calculated? Can they mention other papers describing this method to measure gravitropism? How many independent primary roots were analyzed per treatment, in how many independent experiments and with how many independently prepared cell wall extracts? How where the apoplastic washes prepared? What does the expression "treated with equal amount of these cell wall extracts" exactly mean? There is not sufficient information in the manuscript about these experiments.Is there not a contradiction to state that the variable pro-region of the precursor, containing the known SBT6.1 cleavage sites, is important for secretion and to show that a truncated version of the precursor NOT containing this pro-region is processed just fine ? Furthermore, I understand that the authors assume  that the two parts of a precursor protein stay in the same subcellular compartment(s) after proteolytic cleavage, as the presence of the GFP signal corresponding to the protein segment on the amino terminus is interpreted as indicating where the CLEL processed peptide (on the other side of the cleavage site) also resides . Is this assumption warranted?eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Christian Hardtke as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Yoshikatsu Matsubayashi (Reviewer #2).Thank you for submitting your article "The biogenesis of CLEL peptides involves several processing events in consecutive compartments of the secretory pathway" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Although many processes in plants rely on information transmitted via small secreted proteins, peptides and/or hormones, the maturation and processing mechanisms of plant peptide hormones are poorly understood. In this manuscript, the authors show that several CLE-like peptides are processed several times, with early proteolytic cleavage within the pro-peptides variable domain is a prerequisite for continued passage through the secretory pathway and subsequent proteolytic maturation. The identification of candidate proteases for these two events was also shown.Essential revisions:sbt3.8 mutant. The data also indicate that sbt3.8 should have a gravitropic phenotype. The authors must provide evidence for these two to be able to conclude that SBT3.8 processes CLEL in vivo. The editor expects that these experiments would be performed; if the results are that there is still some cleavage of CLELs in the mutant and/or that there is no gravitropic phenotype, this will not rule out the paper being published, but it will require that the text and summary figures are modified to accurately convey the results.The major issue is that the claim that SBT3.8 processes CLEL in vivo is not clearly supported by the available data. Although it is shown in this revised manuscript that SBT3.8 can cleave CLEL and that SBT3.8 is expressed, secreted and active at the right conditions, the key missing evidence is the absence of cleavage in the  [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]There was consensus that the context and rationale for the study was well laid out and that there is a need for better mechanistic understanding of how CLEL peptides are processed. The extent to which this study definitively advanced our knowledge beyond that of previous studies, however, was considered by all reviewers to be fairly modest. Certainly identification of the SBT capable of driving the next processing reaction would have made this work much more compelling.We agree with the reviewers and the editor’s judgement that our story is incomplete as long as we cannot present the enzyme responsible for the final processing step. Using the subtilase-specific EPI inhibitor, we had shown in the original manuscript that the protease responsible for the final processing step is a subtilase. We had also shown that this processing event depends on an aspartic acid residue at the cleavage site within the precursor, and that it occurs in a post Golgi compartment. However, we had not yet identified the proposed asp-dependent protease. In this revised manuscript we show that the missing protease is subtilase SBT3.8. SBT3.8 cleaves the recombinant precursor in an asp-dependent manner. Cleavage by SBT3.8 was further shown to be pH dependent. Cleavage was only observed at acidic pH, which is consistent with the proposed localization of this cleavage event late in the secretory pathway, in a post-Golgi compartment.Senior editor (post-decision communication)If the authors have identified an SBT that they can show has the needed activity against a CLEL precursor and they can show, in vivo, where this enzyme is located , then I would be happy to reconsider this manuscript.N. benthamiana as well as stable Arabidopsis lines we show that SBT3 is targeted to the apoplast. Extracellular localization of SBT3 is fully consistent with the proposed site of precursor processing in a post-Golgi compartment, and with the acidic pH requirements of SBT3.We show that CLEL precursors are processed at the predicted site by SBT3.8, that cleavage depends on the critical aspartate residue at the cleavage site, and that cleavage by SBT3.8 only occurs at acidic pH as it is found in late compartments of the secretory pathway including the cell wall. Using both, transient expression in Reviewer #1:This is an engagingly written article about CLEL processing. The CLE-like family of peptides have been known for about 20 years; initially via genetics in Arabidopsis. There are many biological functions ascribed to this family, particularly during development-shoot meristems, root meristems, embryos all employ family members, and these proteins have also been found and studied in Maize, tomato, etc. The challenge of this family is that the active peptide are only 12 AA long and are processed and heavily modified. What I didn't appreciate until this manuscript was that, although many of the modification enzymes have been identified, the initial proteolytic processing is fairly uncharacterized. For a family as critical as this one, missing the first step is a big deal!Subtilisin proteases (SBT) are found in all forms of life. In plants, there is a large family of SBTs, but most are extracellular. This manuscript focuses on SBT6.1, one of the few TM-domain forms, and one that is also thought to have emerged before the plant/animal split. SBT6.1 was recently shown to be required for activity of CLEL6 . So, this manuscript must add something new. What's new is that they show where the first SBT6.1 processing takes place and, they tracked down a second processing step that need to take place in the Golgi and they found the residue in CLEL that is needed for this second step. They then do an assay that suggests another SBT  is needed later on, but don't know what it is.The approach relies on several technical innovations, some of which I find very clever and others  I am less convinced of the specificity of.e.g., I like that they set up a transient cleavage assay (N. benthi) with GFP attached to the CLEL precursors + various tags in which they could monitor process through the secretory system with live cell imaging and also by Immunoblot against GFP to see where processing occurred.I am a little worried about overexpression of a pathogen-derived EPI, in that its specificity isn't completely clear.The EPIs were identified and characterized in the lab of Sophien Kamoun . EPI1 is a two domain inhibitor, consisting of one typical (EPI1b) and one atypical (EPI1a) Kazal inhibitor domain. EPI10 comprises three domains, two typical and one atypical Kazal domain. Tian et al. (doi:10.1186/1471-2091-6-15) reported that the so-called ‘atypical Kazal domain’ of EPI1 and EPI10 is highly specific for subtilases, while the ‘typical’ Kazal domains show less specificity and may also inhibit other proteases. This is why we used EPI1a  in addition to EPI10 in our experiments. The same phenotype (reduced gravitropic response of the hypocotyl in EPI expressing transgenics) was observed for both inhibitors. Because of the specificity reported for EPI1a, we conclude that the phenotype is caused by the inhibition of subtilase activity. Nonetheless, we agree with the reviewer. We cannot formally exclude the possibility that EPI1a may exert some additional, unknown effects in plants. On the other hand, with the identification of SBT3.8 as the final processing protease, we confirm the predicted involvement of subtilase(s).I do like the genetic experiments of showing that EPIs driven with CLEL6 or CLEL9 promoters leads to a phenotype similar to loss of the CLEL6/9 peptides themselves, and can be suppressed by supplying synthesized, processed CLEL.Strengths: This is well written and I am now convinced that it's important to understand the processing of the CLEL peptides and that the processing is going to be complicated. The assays are clever and the experiments appear to be done rigorously. The data are presented in a clear and straightforward manner in the figures.Weaknesses: Relies on EPIs being highly specific .50) by EPI1a was observed at 0.8 and 6.4 nM, respectively (doi:10.1126/science.aai8550). Because of the low-nM binding/inhibition of different plant subtilases, we considered the EPIs suitable for targeting subtilase activity in vivo. The phenotype we observe for EPI1a and EPI10 over-expressors  suggests that concentration/affinity of the EPIs was in fact high enough to exert an effect.With respect to specificity, please see our response above. This is certainly true. We cannot expect to see an effect, unless the activity of endogenous subtilases is sufficiently inhibited. Therefore, expression level and binding affinity of the EPIs must be high enough to inhibit potentially redundant subtilases that are present in the respective tissue. In a previous study, we had analyzed the Kd of inhibitor binding to a plant subtilase (SBT4.13) and found it to be in the low nM range . For two other subtilases (SBT4.12 and SBT5.2) half-maximal inhibition  and substrate (the CLEL6 precursor), as well as synthetic peptide substrates. With respect to cleavage by SBT6.1 you are absolutely right; previous work had shown that SBT6.1 is required for CLEL6 function (CLEL9 was not addressed in this study). Therefore, it was not surprising to us to see that cleavage by SBT6.1 at the predicted site is important. What is new here is not the fact that SBT6.1 is important, but why it is important. SBT6.1 does not generate the bioactive peptide; it is important because cleavage by SBT6.1 is necessary for continued passage through the secretory pathway. Therefore, all subsequent maturation events in consecutive compartments of the secretory pathway depend on the initial cleavage by SBT6.1. Other novel aspects include the characterization of the subsequent N-terminal processing event, its localization and cleavage specificity, and the identification of the protease responsible for it.The manuscript does a nice job of demonstrating that a late Golgi SBT is likely to be involved, but then can't supply an answer as to which one it is, and I felt like there was no attempt to use other data sources  to narrow down the possibilities. All in all, I found this a bit too preliminary for an eLife paper.tpst mutant, which cannot produce sulfated peptides including CLEL6 and 9. Four SBTs were found to be upregulated, including SBT3.8. Further analysis showed that SBT3.8 is able to cleave the precursor at the predicted site in an aspartate-dependent manner (please see our response to the editor for further details on this point). With these data included, the story is much more complete and we hope that you will not consider it preliminary anymore.We completely agree with this point – we should have addressed the identity of the SBT predicted to contribute to peptide maturation late in the secretory pathway. We followed your suggestion and used expression data to narrow down on the identity of the SBT responsible for N-terminal processing in the late Golgi. We reasoned that SBTs that are involved in the production of bioactive CLEL peptides might be upregulated in mutants that are deficient in such peptides. Therefore, we analyzed the expression of all 56 Arabidopsis SBTs in hypocotyls of the The Discussion was quite long and speculative relative to the compact data section. It seems as if the authors are addressing some points of contention in the field, and I (as an outsider) don't come to the story with a particular bias that needs to be overcome. I will defer to others who are closer in the field.We tried to be more concise in the Discussion; parts which may have been distracting to readers not directly involved in the field have been removed (about 300 words).Reviewer #2:[…] Because the maturation mechanisms of plant peptide hormones is still poorly understood, the authors' efforts will contribute to expand our knowledge of the peptide hormone biogenesis. Indeed, their findings that cleavage within the variable domain is a prerequisite for continued passage through the secretory pathway and subsequent proteolytic maturation are interesting. However, some of the conclusions are not supported by the results, especially the assumed involvement of aspartate residue in the cleavage for final activation.1) The authors concluded that aspartate-dependent processing is required for peptide activation of CLEL6 and CLEL9 based on a bioassay data using N. benthamiana leaf extracts transiently expressing GFP-CLEL6 and GFP-CLEL9 fusion proteins. This approach is, however, not quantitative and cannot exclude the possibility that structurally immature (larger or unmodified) peptide products are detected if present at high concentrations. Indeed, extracts from leaves expressing D-to-A mutants of CLEL6 and CLEL9  still shows considerable activities .We repeated this experiment using the hypocotyl bioassay . The effect of the D-to-A substitution was stronger in this experiment. No bioactivity was observed for the D-to-A substituted CLEL6 precursor, and that of the CLEL9 precursor was much reduced. Nevertheless, we agree with your point. There is some residual activity (at least for CLEL9) and, therefore, the aspartate is not absolutely required for bioactivity. For processing on the other hand, the aspartate is essential. SBT3.8 was found to cleave recombinant CLEL6 and a synthetic, N-terminally extended CLEL6 peptide that included 7 precursor-derived amino acids . Substitution of Asp by Ala abolished processing of the recombinant precursor.As you suggested, the residual activity observed for the CLEL9 precursor in the bioassay may very well be due to immature (longer or unmodified) peptides, that retain some (probably reduced) receptor binding activity. We thus agree in that the aspartate, while required for processing by SBT3.8, is only important but not absolutely required for bioactivity. In our revisions, we paid attention not to overstate our results.Therefore, in addition to the bioassay experiments, mass spectroscopic analysis which can evaluate both actual structure and abundance of mature peptides is prerequisite to justify the conclusion.We tried to do this but, unfortunately, failed. We were unable to detect the fully processed and post-translationally modified CLEL6 or CLEL9 peptides in cell wall extracts by mass spectrometry. In fact, this is why we developed the bioassay as a tool for the detection of the bioactive peptides.2) Related to above points, there is no detailed explanation of methods for extraction of apoplastic peptides and bioassay. Explain in the Materials and methods section.We apologize for this omission. In the revised manuscript, we added some more detail in the figure legend, and the full procedure in the Materials and methods section.3) The authors used hypocotyl for bioassay in Figure 1, but used roots in Figure 4. This is confusing for readers. Use hypocotyl bioassay throughout the manuscript.Thank you for this suggestion. We agree that the use of two different bioassays may cause some confusion. Therefore, we repeated the experiment shown in Figure 4 using the hypocotyl bioassay. The result is fully consistent with the previous root bioassay; in fact it is even more clear. The D-to-A substitution abolished bioactivity for the CLEL6 precursor; only for the D-to-A mutant of CLEL9 precursor, some residual activity was observed .4) In Figure 2B, it is better to indicate theoretical molecular sizes for each possible fragments. This information greatly helps understanding of SDS-PAGE bands shown in Figure 2C, E, and F. The same is true for Figure 3B.Thank you for this suggestion. This point was also raised by reviewer #3, who requested to indicate more clearly what forms in Figure 2A correspond to the bands in Figure 2C, E and F. We tried different ways of how to help readers to interpret the SDS-PAGE bands and came up with the following solution. Rather than indicating the theoretical masses, we added a scheme to panel A of Figures 2 and 3, showing each of the possible fragments in a different color. Asterisks of the same color are used to mark the SDS-PAGE bands.Reviewer #3:The paper is sufficiently well written. It provides incremental data about subsequent steps in the maturation of the plant secreted signal peptides CLEL6 and 9  and analyzes the subcellular compartments where proteolytic events take place. The work is overall sound, except for problems explained here below . But it has a narrow scope and shows some weakness in the interpretation of the results. The manuscript is worth publishing, but I do not feel that it is a major contribution to its field.By adding the identification and characterization of SBT3.8 as the protease responsible for aspartate- and pH-dependent processing at the N–terminus of CLEL peptides we extended the scope considerably. Considering the limited knowledge we have about the proteases and factors that govern proteolytic maturation of peptide hormones, we feel that our study now makes an important contribution to the field.Subsection “The cleavage for final activation occurs in a post-Golgi compartment by an aspartate-dependent SBT” fourth paragraph. Provided additional explanations are given (see below), I will trust that the experiments illustrated in Figure 4C to E show that CLEL extracts have an impact on root growth, and that there is a differential response between the wt and D>A extracts. However, the explanation of the root growth referring to the "waving phenotype" is incorrect. On dense agar plates slanted backward, wild-type untreated roots "wave", that is their normal growth pattern which is explained as a recurrent alternation between obstacle avoidance response and gravitropic response. In Whitford et al., 2012, CLEL peptides added to the medium do not induce waving, but they alter the waving pattern. In fact, the peptides induce "coiling" rather than waving: because of a reduction in gravitropism: roots often form complete loops as they grow away from the vertical.In the experiments presented here, the control roots do not wave, but simply grow down . The lack of waving may be due to the angle of the plate , the composition of the medium, the concentration of the agar. What is reported in this manuscript is that treated roots do not follow as strictly the vertical direction compared to the controls, they meander… but they don't wave. If my interpretation is right, the text should be corrected by removing all references to "waving" and simply state that treated roots have a reduced gravitropic response.We fully agree with this interpretation. Since ‘waving’ is the normal behavior of wild type roots, we cannot refer to the difference between roots treated with wt and D>A extracts as the ‘waving phenotype’. The suggested wording that ‘treated roots show a reduced gravitropic response’ is much more appropriate. Thank you for this suggestion. However, on request of reviewer #2, we replaced the root growth bioassay by a hypocotyl bioassay in the revised manuscript. Therefore, the changes to the subsection “The cleavage for final activation occurs in a post-Golgi compartment by aspartate dependent subtilase SBT3.8” and Figure 4—figure supplement 2 legend, are now obsolete.Related to these experiments, the authors should address the following questions that are not covered in the Results or Materials and methods sections?How were the roots grown  light and light cycles)? How exactly was the variable "changes in root growth direction" calculated? Can they mention other papers describing this method to measure gravitropism? How many independent primary roots were analyzed per treatment, in how many independent experiments and with how many independently prepared cell wall extracts?Some of the points raised here (those directly related to the root growth bioassay) are obsolete (see point above). The hypocotyl bioassay was performed in three independent experiments using independent cell wall extracts. The data shown in Figure 4 are the representative result of one of these experiments. This information was included in the figure legend.How where the apoplastic washes prepared? What does the expression "treated with equal amount of these cell wall extracts" exactly mean?The CLEL precursors and D-to-A mutants were expressed as GFP fusion proteins. Therefore, we used GFP fluorescence to quantify expression levels. ‘equal amounts of cell wall extracts’ referred to equal amounts of GFP fluorescence as a measure of protein expression. We agree that this statement is unclear; it was rephrased in the revised manuscript .There is not sufficient information in the manuscript about these experiments.We apologize for this omission. In the revised manuscript, we added some more detail in the figure legend, and the full procedure in the Materials and methods section.Is there not a contradiction to state that the variable pro-region of the precursor, containing the known SBT6.1 cleavage sites, is important for secretion and to show that a truncated version of the precursor NOT containing this pro-region is processed just fine ?This is a good observation. The fact that the truncated precursor lacking most of the prodomain is efficiently processed  does indeed suggest that it is a minor fraction that is retained intracellularly . The effect caused by mutation of the SBT6.1 cleavage sites is clearly more pronounced, as both secretion and processing are affected. We therefore toned down the relevance of the prodomain as compared to cleavage by SBT6.1 for secretion.Furthermore, I understand that the authors assume  that the two parts of a precursor protein stay in the same subcellular compartment(s) after proteolytic cleavage, as the presence of the GFP signal corresponding to the protein segment on the amino terminus is interpreted as indicating where the CLEL processed peptide (on the other side of the cleavage site) also resides . Is this assumption warranted?In this example (the Δ-Sec construct), most of the GFP signal is extracellular , and most of the precursor is processed . Therefore, in this particular case, the two parts of the precursor are expected to be in the same compartment . However, we do not assume that this is generally the case. For the KDEL or XylT-tagged precursors, for example, GFP fluorescence indicates that the N-terminal part of the protein is retained in the ER or Golgi, respectively. Cleavage of these precursors would separate the C-terminus from the retention signals and, therefore, any cleavage product would be expected to continue along the secretory pathway, potentially ending up in the cell wall.[Editors’ note: what follows is the authors’ response to the second round of review.]Essential revisions:The major issue is that the claim that SBT3.8 processes CLEL in vivo is not clearly supported by the available data. Although it is shown in this revised manuscript that SBT3.8 can cleave CLEL and that SBT3.8 is expressed, secreted and active at the right conditions, the key missing evidence is the absence of cleavage in the sbt3.8 mutant. The data also indicate that sbt3.8 should have a gravitropic phenotype. The authors must provide evidence for these two to be able to conclude that SBT3.8 processes CLEL in vivo. The editor expects that these experiments would be performed; if the results are that there is still some cleavage of CLELs in the mutant and/or that there is no gravitropic phenotype, this will not rule out the paper being published, but it will require that the text and summary figures are modified to accurately convey the results.sbt3.8 loss-of-function mutant. Since we have no way of analyzing the processing of endogenous peptide precursors in planta, we prepared cell wall exudates of wild type plants and sbt3.8 mutants. Wild-type exudates efficiently cleaved the CLEL6 precursor, sbt3.8 mutant exudates did not. CLEL6 precursor cleavage thus depends on SBT3.8 under these conditions. The data were added as Figure 5—figure supplement 2B. In order not to overstate our results, we write in the text:’ these data support a role for CLEL6 maturation in vivo’ . We do not claim that SBT3.8 is the only protease capable of CLEL6 precursor processing. In fact, evidence shows that there must be redundant activities: the gravitropic response of the hypocotyl turned out to be unaffected in the sbt3.8 mutant . Therefore, to acknowledge the presence of redundant activities, we changed the wording in the Abstract and Discussion. Also, we revised the model in Figure 6, including ‘other D-dependent subtilases’ in addition to SBT3.8, for the final processing event.We compared CLEL6 precursor processing in wild type and the


Leishmania donovani, an intracellular protozoan parasite upon infection, encounters a range of antimicrobial factors within the host cells. Consequently, the parasite has evolved mechanisms to evade this hostile defense system through inhibition of macrophage activation that, in turn, enables parasite replication and survival. There is growing evidence that epigenetic down-regulation of the host genome by intracellular pathogens leads to acute infection. Epigenetic modification is mediated by chromatin remodeling, histone modifications, or DNA methylation. Histone deacetylases (HDACs) removes acetyl groups from lysine residues on histones, thereby leading to chromatin remodeling and gene silencing. Here, using L. donovani infected macrophages differentiated from THP-1 human monocytic cells, we report a link between host chromatin modifications, transcription of defense genes and intracellular infection with L. donovani. Infection with L. donovani led to the silencing of host defense gene expression. Histone deacetylase 1 (HDAC1) transcript levels, protein expression, and enzyme activity showed a significant increase upon infection. HDAC1 occupancy at the promoters of the defense genes significantly increased upon infection, which in turn resulted in decreased histone H3 acetylation in infected cells, resulting in the down-regulation of mRNA expression of host defense genes. Small molecule mediated inhibition and siRNA mediated down-regulation of HDAC1 increased the expression levels of host defense genes. Interestingly, in this study, we demonstrate that the silencing of HDAC1 by both siRNA and pharmacological inhibitors resulted in decreased intracellular parasite survival. The present data not only demonstrate that up-regulation of HDAC1 and epigenetic silencing of host cell defense genes is essential for L. donovani infection but also provides novel therapeutic strategies against leishmaniasis. Leishmania donovani. These intracellular parasites replicate inside phagolysosomes of the macrophages and have evolved mechanisms that allow survival within the hostile environment of their hosts. They have evolved strategies to dramatically modify the transcriptome and proteome content of the host cells they infect, facilitating their survival within the host cell. Here, we have defined a mechanism by which Leishmania donovani subverts host cell defense genes by manipulating the epigenetic control of defense gene expression. The intracellular protozoan parasite, Leishmania, results in gene silencing of innate host defense genes. The underlying molecular mechanisms assisting such modifications were inferred from the results. An increase in the transcript, protein levels and activity of host HDAC1 was observed at 6 h post-infection. This data confirms the role of HDAC1 in the silencing of the host defense genes and enabling the intracellular survival of the parasite. Our results decode a new insight into epigenetic regulation of host cell defense mechanisms. These findings will facilitate the identification of novel strategies and will promise newer ways to control parasite growth.Visceral leishmaniasis is a neglected tropical disease caused by the protozoan parasite  Leishmania donovani. Visceral leishmaniasis continues to be a major public health concern, especially in Africa, Asia, and South America . Error bars used in the figures stipulate standard error of the deviation (SD).GraphPad Prism (version 5.0) software  was used for plotting data. Statistical analysis was measured using ANOVA. A L. donovani infection in THP-1 cells. We focused on the transcript levels of antimicrobial factors such as MPO, hepcidin antimicrobial peptide (HAMP), cathelicidin-type antimicrobial peptide (CAMP), and granulysin (GNLY). This set of 4 genes was designated as cluster I , and cathepsin C (CTSL) as part of cluster III. IL8, a proinflammatory gene known to be up-regulated during Leishmania infection [L. donovani at 1:20 multiplicity of infection (MOI), and the host cells were harvested at 6 and 24 h post-infection. The expression of the 15 defense genes was analysed by qRT-PCR and compared to the expression levels in uninfected controls. Among the 15 defense genes studied, down-regulation was observed in the majority of the defense genes at 6 h post-infection compared to that observed at 24 h post-infection.We also checked the expression levels of phosphatase and tensin homolog nfection , was incMPO [MPO, a key host defense gene [CAMP, which is reported to have high leishmanicidal activity [HAMP, which has been reported to be up-regulated in L. amazonensis infected macrophages [-L. donovani infection. However, at 24 h post-infection, the expression of HAMP showed down-regulation. The expression levels of GNLY, another important antimicrobial peptide [It has been reported previously that leishmanial infection results in the down-regulation of MPO . In the nse gene , was dowactivity , showed rophages , showed  peptide , showed DEFB4. At 24 h post-infection, the down-regulation of DEFB4 was observed. DEFA5 and DEFA6 expression showed up-regulation (~4.8-fold and ~ 6.7-fold respectively) while the other defense genes  were down-regulated at 24 h post-infection,Antiparasitic activities of various defensins have been reported in different leishmanial studies , 30, 31.PTEN, which has been reported to get suppressed upon L. donovani infection [LEP and CTSB remained unchanged at 6 h post-infection, whereas CTSL showed down-regulation, which is in agreement with earlier reports [LEP and CTSL were up-regulated (~ 2.1-fold and ~ 1.4-fold respectively). Parasite infection led to the up-regulation of the expression of the pro-inflammatory gene, IL-8  followed by qRT-PCR using promoter specific primers as reported in the methods section.The change in the pattern of histone-post-translational modifications is likely to have a direct effect on gene expression by affecting chromatin structure, thereby leading to highly compact chromatin conformation and limited access to transcriptional activators . EpigeneL. donovani was observed in 8 out of 10 defense gene promoters at 6 h post-infection  infected with L. donovani (20:1 MOI) were harvested at 6 and 24 h post-infection. The HDAC1 protein was immunoprecipitated from the total cell lysates of these experimental samples, using an HDAC1 specific antibody. The HDAC1 enzyme activity of the immunoprecipitated protein was analysed. We observed a significant increase in HDAC1 activity at 6 h post-infection when compared to the HDAC1 activity in uninfected THP1 cells (P = 0.00001). HDAC1 activity at 24 h post-infection was down to basal levels as in the uninfected THP1 cells  higher compared to the levels in the uninfected cells (UI). A decrease in HDAC1 expression was observed at 12 and 24 h post-infection in comparison to the values obtained at 6 h post-infection  at 24 h before infection with L. donovani. After 6 h post-infection, the infected and uninfected cells were harvested, followed by RNA isolation, cDNA synthesis, and qRT-PCR, as mentioned in the methods section. The transcript level of HDAC1 in Sc-siRNA transfected cells was higher (~ 1.5-fold) in infected cells in comparison to the uninfected cells  in comparison to cells transfected with scrambled-siRNA  in the parasite load in cells transfected with HDAC1-siRNA as compared to the cells transfected with Sc-siRNA. The result suggests an important role of HDAC1 in the intracellular survival of the parasite.We next studied the parasite load within the infected host cells transfected with Sc-siRNA and C1-siRNA . ParasitHDAC1 has a direct role in silencing host defense genes. To this end, the expressions of five defense genes  were analyzed in the infected cells transfected with HDAC1-siRNA. Expression of DEFA1 , DEFA5 , DEFA6 , DEFB4  and MPO  were up-regulated in infected THP-1 cells transfected with HDAC1-siRNA as compared to the levels in cells transfected with scrambled-siRNA  (HDAC1 (P<0.001) and total HDAC activity (P<0.001) was observed with 5 mM of NaB in comparison to untreated infected cells. Infected cells treated with NaB were further analysed for parasite load by visually counting the amastigotes within the THP1 cells after Giemsa staining. A decrease in parasite load within infected macrophages was observed with increasing concentrations of NaB (8 cells) with 10 mM of the inhibitor. NaB treatment had no inhibitory effect on the promastigotes (Sodium butyrate (NaB), a short chain fatty acid that blocks class I and II HDACs (36), was examined for its role in HDAC1 inhibition and 5–20 mM) . Total H5–20 mM) . A decres of NaB . To checstigotes .DEFA1 , DEFA5 , DEFA6 , DEFB4  and MPO  in infected macrophages in the presence of 5 mM of NaB in comparison to untreated THP-1 cells  is a known class I and class II HDAC inhibitor . It is FL. donovani followed by treatment with the increasing concentration of SAHA (0–10 μM) for 6 h. The cells were then harvested and the transcript levels of host HDAC1 were quantitated by qRT-PCR. As in the case of NaB, the presence of SAHA also resulted in a concentration dependent (0–10 μM) decrease in HDAC1 transcript levels (P = 0.0005), with 5 μM SAHA  with 5 μM SAHA (8 cells) were incubated with 5 μM of SAHA to check the effect of SAHA on the parasite. No inhibitory effect of 5 μM SAHA was observed on the promastigotes , DEFA5 , DEFA6 , DEFB4  and MPO  were observed in the infected THP-1 cells in the presence of 5 μM SAHA as compared to the levels in the absence of SAHA  increases histone deacetylase (HDAC) levels leading to decreased acetylation. Decreased acetylation resulted in down-regulation in the expression of antimicrobial peptides leading to the establishment of parasite infection in the host cells Click here for additional data file.S1 FigA. Promastigotes were pre-treated with 0 or 10 mM of NaB prior to incubation with THP-1 cells for parasite infection (MOI– 20:1). B. Promastigotes were pre-treated with 0 or 5 μM of SAHA prior to incubation with THP-1 cells for parasite infection (MOI– 20:1).(DOCX)Click here for additional data file. 14 Nov 2019http://journals.plos.org/plosntds/s/content-license (NOTE: we cannot publish copyrighted images). (4) If applicable, we encourage you to add a list of accession numbers/ID numbers for genes and proteins mentioned in the text (these should be listed as a paragraph at the end of the manuscript). You can supply accession numbers for any database, so long as the database is publicly accessible and stable. Examples include LocusLink and SwissProt.(5) To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-methodsPlease note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.We hope to receive your revised manuscript by Jan 13 2020 11:59PM. If you anticipate any delay in its return, we ask that you let us know the expected resubmission date by replying to this email.To submit a revision, go to https://www.editorialmanager.com/pntd/ and log in as an Author. You will see a menu item call Submission Needing Revision. You will find your submission record there. Sincerely,Armando Jardim, PhDAssociate EditorPLOS Neglected Tropical DiseasesHans-Peter FuehrerDeputy EditorPLOS Neglected Tropical Diseases***********************Reviewer's Responses to QuestionsKey Review Criteria Required for Acceptance?Dear Dr. Muthuswami:Thank you very much for submitting your manuscript "Epigenetic Regulation of Defense Genes by Histone Deacetylase 1 in Human Cell Line-Derived Macrophages Promotes Intracellular Survival of Leishmania donovani." (#PNTD-D-19-01539) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer. When you are ready to resubmit, please be prepared to upload the following:(1) A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.(2) Two versions of the manuscript: one with either highlights or tracked changes denoting where the text has been changed (uploaded as a "Revised Article with Changes Highlighted" file); the other a clean version (uploaded as the article file).(3) If available, a striking still image (a new image if one is available or an existing one from within your manuscript). If your manuscript is accepted for publication, this image may be featured on our website. Images should ideally be high resolution, eye-catching, single panel images; where one is available, please use 'add file' at the time of resubmission and select 'striking image' as the file type. Please provide a short caption, including credits, uploaded as a separate "Other" file. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License at As you describe the new analyses required for acceptance, please consider the following:Methods-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?-Is the study design appropriate to address the stated objectives?-Is the population clearly described and appropriate for the hypothesis being tested?-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?-Were correct statistical analysis used to support conclusions?-Are there concerns about ethical or regulatory requirements being met?ResultsReviewer #1: The authors have addressed the role of the chromatin remodeling enzyme HDAC1 in the downregulation of defensin genes. Overall, the experiments are well designed, although there are some important concerns on the methodology applied to the study and the statistics applied by the authors.Reviewer #2: it is very well designed, only I have a comment; why you didn't use ATCC organism.Is the strain you used typed already or no?Reviewer #3: When taken at face-value the results presented in this manuscript support the hypothesis that Leishmania infection of macrophages triggers histone deacetylase 1 (HDAC1)-mediated down-regulation of defense genes, thereby promoting intracellular parasite survival and growth. In general, the methodology used  are suitable to address the questions asked.---------------------Does the analysis presented match the analysis plan?-Are the results clearly and completely presented?-Are the figures  of sufficient quality for clarity?ConclusionsReviewer #1: The overall quality of the figures is not good. There is an evident lack of resolution in the graphics.The authors should improve the figures. Importantly, the statistical interpretation based on T student test seems not to be correct. ANOVA is highly recommended.Reviewer #2: I recumbent the author to add a picture related to the mechanism of action of siRNAReviewer #3: However, numerous problems with data presentation and the use of imprecise language throughout significantly undermine confidence in the validity of the results obtained. In particular, several figures are mis-labeled and there are numerous questions as to whether appropriate controls are used. An over-reliance in using the ratio of infected to uninfected samples to present most data is of particular concern (see below). In addition, several examples of inconsistent results between experiments are ignored.---------------------Are the conclusions supported by the data presented?-Are the limitations of analysis clearly described?-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?-Is public health relevance addressed?Editorial and Data Presentation Modifications?Reviewer #1: The authors discuss very poorly their results. They should discuss the observed modulation of defensin gene expression resulted from the experimental time course and the possible impact in the infection.Reviewer #2: very well writtenReviewer #3: There is very little discussion of alternative explanations for the observed results. However, the authors do a good job discussing the possible relevance of the findings to public health.--------------------Summary and General CommentsUse this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”. Reviewer #1: The authors based the work on previously published studies, that demonstrated the correlation of HDAC1 induced expression by pathogens and the repression of some innate responsive genes.The figures have poor quality and the statistical analysis seems inappropriate. Limes 63 and 64, the authors make a general assumption regarding the gene silencing induced by pathogens. This assumption is too general and it is not applied in many models.Regarding the methodology, the authors use a general acetyl lysine antibody in the experiments. They should have used a more precise mark for Histone acetylation in the assays. By no means, Figure 3 presents specific HDAC1 activity data. Did the have immunoprecipitated HDAC1 prior to the activity measurement?There is no reason for the use of NAB in the experiments. NaB is a pan HDAC inhibitor and the results are not really useful. Instead, they should have used another HADC1 specific inhibitor. The authors should have shown the level of transfection, the percentage of transfected cells when they proceeded with the si experiments. Figure 4 shows an important effect of scrambled si on the parasite load. Please, explain.Reviewer #2: To elaborate more about the strain that has been used from Dr Dr StephenBeverlyTo add a picture related to the mechanism of action of siRNAReviewer #3: Much of the results section is spent merely re-stating the results from the figures, rather than summarizing what they (might) mean. In addition, the much of the language used lacks precision. I am particularly concerned about the relative modest fold-change value (often less than 2-fold) and the lack of negative controls (i.e. genes that are not expected to change expression). How can were be sure the results shown don't merely represent noise. In this context, it is important to know whether the three replicates used in most experiments represent three independent samples or merely technical replicates from the same sample? The following comments are meant to illustrate some  of the problems with the Results section.Line 280: there appears to be text missing after "upon pathogen"Lines 285-291: what is the logic behind the using the terms Cluster I, Cluster II and Cluster III to describe the genes studied? They certainly don't cluster by their behavior.Figure 1: How do fold-change values of <1 indicate up-regulation and >1 indicate down-regulation (lines 297-298)? The y-axis indicates that the fold-change is presented on a log2-scale, but this is clearly not the case. the results would be much clearly if that were the case. On the x-axis, one gene is labeled LKP, while in the text it is called LEP. Why is there no discussion of the  lack of correlation between the fold-changes at 6 hr and 24 hr. Line 307: DEFB4 is not down-regulated Line 319-321: there is no experimental evidence that down-regulation of the defense gene mRNA levels allows sustained inhibition of antimicrobial defense and facilitates intracellular survival of the parasite. This is merely the hypothesis.Line 324-326: It would be better to provide some context/background for this statement and hypothesis.Figure 2: The same scale (preferably log2) should be used for all plotsFigure 3: While it is obvious that + and - in panel B present uninfected and infected cells, respectively, this is not explicitly stated in the legend. I find panel D (and its description in the text) to be very confusing. It is not clear how you can have a negative value for HDAC enzyme activity. In my option, it would be much better to present the HDAC levels relative to the 0 hr sample in panel D (as it is in panel C). It is also not clear what sample values were compared in the statistical analyses. Figure 4: In general, I find use of the infected/uninfected ratio confusing in presenting these data. In my option, it is more important to directly compare the results obtained in with and without siRNA. This is particularly true for panel B, where the value in the uninfected sample is very low (since they contain no kDNA) and could introduce large errors. It would be better to present these data as ratio to those obtained with a know number of parasites. I am also concerned about the use of the "scrambled-siRNA" as the control since it is clearly different from the no siRNA (negative) control. Figure 5 and Figure 6: Once again, use of the infected/uninfected ratio is confusing, since the inhibitors are almost certainly doing something in the uninfected cells. In my option, it would be much more informative to present all the date relative to untreated uninfected cells. The data presented in panel D of both figures is not entirely consistent with the values presented in Fig 1. Indeed, the results in panel 5A and 6A show considerable variation in the (relative) HDAC1 levels in the absence of inhibitor. Line 510: "hallmark" is one word Line 527: I hesitate to call the down-regulation of defense gene mRNA levels as "major", since it was only 3-fold at most .--------------------Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.Reviewer #1: The authors based the work on previously published studies, that demonstrated the correlation of HDAC1 induced expression by pathogens and the repression of some innate responsive genes.The figures have poor quality and the statistical analysis seems inappropriate. Limes 63 and 64, the authors make a general assumption regarding the gene silencing induced by pathogens. This assumption is too general and it is not applied in many models.Regarding the methodology, the authors use a general acetyl lysine antibody in the experiments. They should have used a more precise mark for Histone acetylation in the assays. By no means, Figure 3 presents specific HDAC1 activity data. Did the have immunoprecipitated HDAC1 prior to the activity measurement?There is no reason for the use of NAB in the experiments. NaB is a pan HDAC inhibitor and the results are not really useful. Instead, they should have used another HADC1 specific inhibitor. The authors should have shown the level of transfection, the percentage of transfected cells when they proceeded with the si experiments. Figure 4 shows an important effect of scrambled si on the parasite load. Please, explain.Reviewer #2: 1-To elaborate more about the strain that has been used from Dr Dr StephenBeverly2-To add a picture related to the mechanism of action of siRNAReviewer #3: In general, the hypothesis is provocative, and the data are (on the surface) quite convinci 8 Feb 2020AttachmentResponse PlosNTD.docxSubmitted filename: Click here for additional data file. 24 Feb 2020Dear Dr. Muthuswami,We are pleased to inform you that your manuscript 'Epigenetic Regulation of Defense Genes by Histone Deacetylase 1 in Human Cell Line-Derived Macrophages Promotes Intracellular Survival of Leishmania donovani.' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. 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Surface-to-volume ratio in two-dimensional (2D) materials highlights among their characteristics as an inherent and intrinsic advantage taking into account their strong sensitivity to surface effects. For this reason, we have proposed in this work micromechanically exfoliated 2D nanosheets of InSe as an optical vapour sensor. As a proof of concept, we used 2-mercaptoethanol as the chemical analyte in vapour phase to monitor the change of the InSe photoluminescence (PL) before and after exposure to the analyte. For short vapour exposure times , we found a PL enhancement of InSe nanosheets attributed to the surface localization of Se defects. For long vapour exposure times (or higher concentrations) a PL reduction is observed, probably due to the diffusion of molecules within the nanosheet. These results confirm the capability of 2D InSe as a photoluminescent sensor of vapours, because of its sensitivity to surface passivation or volume diffusion of molecules. Two-dimensional 2D) materials have been vastly studied in the last decade since graphene was first obtained  by microD materiaAmong 2D materials, indium selenide (InSe), a III-VI semiconductor with high electron mobility compared to other layered materials and potential applications in photovoltaics ,20,21 ac2) are of interest in the detection of explosives and analogues that has become a strategic priority in homeland security, land mine detection, and military issues [Focusing, then, on vapour sensing due to its more direct applicability, there are many interesting analytes for sensing. Among them, sensing of sulphur containing compounds are of particular interest for monitoring organic decomposition ,34 or bay issues .In this work exfoliated InSe nanosheets have been demonstrated as a vapour sensor, which is validated by using 2-mercaptoethanol (MET) as an analyte, because thiols show a very strong affinity to metals ,31,32. A1.05Se0.95 melt. Tin was added to act as a n-dopant in a content 0.01% previously to growth. MET was purchased from commercial suppliers and used as received .Bulk InSe monocrystals used to obtain the nanosheets were acquired through a perpendicular cleave of the (001) direction from an ingot grown by the Bridgman method  from a nInSe nanosheets were obtained through mechanical exfoliation of 3D bulk InSe crystals. Once exfoliated, they were directly transferred from the scotch tape upon a 285 nm layer of silicon dioxide over silicon substrate, previously cleaned with acetone, ethanol, and isopropanol in an ultrasound bath.2. It is interesting to note here that we have maintained the same focal distance for PL spectra acquired in the 2D semiconductor before and after the vapour exposure in order to be able to perform a more direct quantitative comparison between intensities.To characterize the samples, two methods were used. Starting with the optical contrast analysis of the samples ,38,39 toa = p*a · xa, where pa is the partial pressure of the analyte in the solution, p*a is the vapour pressure of the pure analyte, and xa is the mole fraction of the analyte in the mixture), assuming that 0.1 M MET solution behaves as a thermodynamically ideal solution. Once prepared, all the sensing test follows the same protocol: 2D InSe samples (nanosheets deposited on the Si/SiO2 substrate) were attached to the inner side of the cap of a sealed vessel leaving the substrate faced-down and exposed directly for a given time. The exposure times to the analyte in vapour phase were systematically varied from 30 to 300 s. Given that the liquid-vapour equilibrium is broken each time that an exposure is carried out, so for every sample and measurement it was necessary to have an individual vessel with the same analyte in the required concentration, in order to assure the equilibrium vapour pressure, that needs hours to be reached. Furthermore, a constant concentration of the analyte in solution is assumed throughout the experiment because the analyte solution was saturated. The InSe nanosheets were characterized by µ-PL before and after exposure to the analyte by using the same PL setup and experimental conditions. Although InSe is stable in air [2D InSe nanosheets prepared by micromechanical exfoliation were tested as optical sensors, in order to be sensitive to local changes in the environment atmosphere when the target analyte molecules are chemisorbed on their surface. To validate the use of 2D InSe as a potential chemosensor, MET was selected as analyte. Thiol groups (–SH) of MET are very well-known chelating ligands. They exhibit a very strong affinity via their lone pair electrons of S towards metals such as indium atoms ,41. The e in air , in ordet/I0, where I0 and It is the PL maximum intensity before and after exposure at a given time. Real-time sensor response  in Our results clearly demonstrate that the sensing response of the 2D InSe to the MET vapour depends on the nanosheet thickness. In These two different effects can be observed in thicker nanosheets a in contThe defect-inhibition process and subsequent vapour diffusion into the InSe layers described above place the relevant question related to the possibility that vapour diffusion could take place in a layer-by-layer fashion. This possibility is particularly relevant for fine-tuning of quantum confinement effects ,47,48,49Results reported here allow us to propose 2D InSe nanosheets as nanometric vapour sensors for sulphur-containing compounds like MET, as a representative example. Besides, we also found promising sensing response of 2D InSe nanosheets to nitro-containing compound characteristics of explosives like 3-nitrotoluene (3-NT) with very fast response as shown in In this work, we have studied the ability of 2D InSe nanosheets to act as fluorescent sulphur sensors. A change in the photoluminescence of exfoliated InSe nanosheets has been observed by the exposure to 2-mercaptoethanol due to the reactivity of surface exfoliated Se free radicals in InSe. Our results reveal that, at lower concentrations and exposure times, the vapour preferably bonds to surface defects or Se vacancies improving the electronic surface quality and enhancing the luminescent response of the nanosheet. However, higher vapour exposure times or concentrations, tends to favour vapour diffusion into the InSe nanosheets, which hampers its luminescent response. Our preliminary results show that vapour diffusion into 2D InSe tends to occur in a layer-by-layer fashion that introduces significant changes as a function of the nanosheet thickness.