Nicotiana attenuata plants. By identifying and silencing a malonyltransferase, NaMaT1, involved in DTG malonylation, we found that DTG malonylation percentages are normally remarkably uniform, but when disrupted, result in DTG-dependent reduced floral style lengths, which in turn result from reduced stylar cell sizes, IAA contents, and YUC activity; phenotypes that could be restored by IAA supplementation or by silencing the DTG pathway. Moreover, the Nicotiana genus-specific JA-deficient short-style phenotype also results from alterations in DTG malonylation patterns. Decorations of plant specialized metabolites can be tuned to remarkably uniform levels, and this regulation plays a central but poorly understood role in controlling the development of specific plant parts, such as floral styles.Plants produce many different specialized (secondary) metabolites that function in solving ecological challenges; few are known to function in growth or other primary processes. 17-Hydroxygeranylinalool diterpene glycosides (DTGs) are abundant herbivory-induced, structurally diverse and commonly malonylated defense metabolites in  Plants produce tens of thousands of molecules called secondary metabolites that are thought to help them cope with threats from their environment, such as attack by insects or ultraviolet radiation from the sun. Wild coyote tobacco plants produce large amounts of a particular class of secondary metabolite known as DTGs. Insects feeding on tobacco plants containing DTGs cause less damage and produce fewer offspringPlants modify many secondary metabolites by attaching tags known as malonyl groups to them. Enzymes called malonyl transferases take a malonyl group from another substrate and attach it to the secondary metabolite. This process can be repeated so that an individual secondary metabolite molecule may have many malonyl groups attached to it. Previous studies have shown that insects feeding on tobacco plants trigger more malonyl groups to be attached to DTGs, but it is not clear what effect this has on the plants.To simulate attack by an insect, Li et al. punctured holes in the leaves of tobacco plants and applied saliva from tobacco hornworms. The experiments show that more DTGs modified with malonyl groups accumulated in these plants compared to untreated plants. However, there was no change in the average number of malonyl groups added to individual DTGs during the modification process. Further experiments show that a malonyl transferase enzyme called NaMaT1 adds malonyl groups to DTGs in coyote tobacco plants. The flowers of plants that produce less of this protein have shorter styles (a tube structure that guides pollen to the egg cells at the base of the flower) and are less fertile than flowers in normal plants.These experiments demonstrate that, along with helping plants to defend themselves from herbivores, DTGs regulate how flowers grow and develop. It was generally thought that secondary metabolites do not play important roles in how plants grow when they are not under stress. Indeed, plant breeders frequently select crops that produce lower levels of secondary metabolites in order to increase their nutritional value. Therefore, the findings of Li et al. may help improve the outcomes of crop breeding programs. Arabidopsis thaliana  to the C’6 of the glycosyl moiety of glucoconjugates . The malthaliana . The malthaliana . Althougthaliana , their iNicotiana spp.), pepper (Capsicum annuum) and wolfberry (Lycium chinense)   are abundant (mg/g FW) secondary metabolites in green tissues of many solanaceous plants, including tobacco (hinense) . DTGs cohinense) . So far,DTG pool . In N. aa sexta) . The malignaling . The mald midgut . This obSpecialized metabolites are primarily thought to function in mediating an organism’s ecological interactions, to help optimize Darwinian fitness. Many are produced in response to particular ecological interactions, such as those that are induced in response to specific attackers. However, many secondary metabolites have effects on growth and development which are more specific than those that might result from resource trade-offs between growth and putative defense metabolites . For exaN. attenuata  and CORONATINE INSENSITIVE 1 (irCOI1), and ectopic expression of Arabidopsis JASMONIC ACID METHYL TRANSFERASE (JMT) and simultaneous silencing of METHYL JASMONATE ESTERASE (MJE) (JMT/mje) which dramatically decreased JA signaling, are reported to have short styles, resulting in reduced fertility  genes that have high similarity with NtMaT1  controlling the malonylation process is the most straightforward way to disentangle the function of DTG malonylation and its remarkably uniformity. To do this, h NtMaT1 , were ush NtMaT1 . There ah NtMaT1 . Proteinh NtMaT1 . Notablyh NtMaT1 , indicatN. attenuata MaTs with known DTG biosynthesis genes, using published multi-tissue and -treatment RNA-seq data , and then slightly decreased by 9 hr  was used to silence all three candidate genes. The tobacco rattle virus VIGS vector migrates to growing meristems and thus efficiently silences target genes in new tissues in all parts of plants (N. attenuata phytoene desaturase (NaPDS), which causes photobleaching where the gene is silenced, demonstrated efficient silencing in floral tissues (NaMaT1 in VIGS-NaMaT1 plants (VIGS-MaT1) decreased more than 80% compared with VIGS controls , without affecting the abundance of NaMaT2 or NaMaT3 transcripts (NaGGPPS) (NaGLS)   and gera (NaGLS)  in VIGS 1 leaves .GGPPSs in the N. attenuata genome: the enzyme that controls the flux of substrates into the DTG pathway, and irGGPPS produces only ca. 10 – 15% of the DTGs levels of WT plants , in comparison to VIGS-EV controls  leaves. Styles and stigmas contained the highest levels of IAA among all the measured tissues, about 6.5-fold more than that of the S1 leaf  in styles of both EV and irGGPPS plants inoculated with VIGS-MaT1. While IAA decreased in VIGS-MaT1 of EV plants, Trp levels increased dramatically . These dckground , which aT1 style . We thenC-like 2 , to be hC-like 2 . The traV plants . ExogenoV plants , withoutV plants , resultsN. tabacum, NtMaT1  and in Solanum lycopersicum (tomato), respectively . To test whether brassinosteroids may contribute to the VIGS-MaT1 style phenotype in other ways, we exogenously applied brassinolide to VIGS-MaT1 flower buds. This treatment did not recover the short style phenotype of VIGS-MaT1  contributes significantly to this process. Silencing this gene disturbed 17-hydroxygeranyllinalool diterpene glycoside (DTG) malonylation patterns, and stunted elongation of flower styles during development and fertility by affecting auxin biosynthesis. Abnormal jasmonate signaling could also disturb DTG malonylation patterns, causing similar stylar developmental defects.NaMaT1 dramatically decreased dimalonylated and trimalonylated DTGs in inflorescences and styles : silencing a rhamnosyltransferase gene inhibits the transcript accumulation of all upstream genes, and silencing NaGGPPS also suppresses transcript accumulation of downstream biosynthetic genes. Here, we show that VIGS of NaMaT1 tended to increase both NaMaT2 and NaMaT3 expression, and significantly increased NaGLS expression , although the possibility that a ratio rather than a single compound is required could still result in many combinations that require testing.Our experiments shed light on this phenomenon by demonstrating that the DTG biosynthesis specific NaGGPPS is required for the development of the truncated style phenotype , and thiArabidopsis, Medicago, tomato and apple  (Agrobacterium tumefaciens containing pBINTRA and either pTV-MaT1 or pTV00 (EV control). VIGS experiments were repeated at least three times.The 31 st inbred generation of 7-230-5) , irAOC (7-230-5) , and an 7-230-5)  were use7-230-5) . The VIG7-230-5) . BrieflyManduca sexta W + OS elicitations, rosette-stage leaves were wounded with a pattern wheel, and 20 µL of diluted M. sexta regurgitant (1:5 in distilled water) was gently rubbed into the freshly created puncture wounds using a clean gloved finger as previously described (For methyl jasmonate (MeJA) treatments, leaf petioles of rosette-stage plants were treated with 20 µL lanolin paste containing 150 µg MeJA (Sigma-Aldrich), or with 20 µL of pure lanolin as a control. MeJA treatment of flower buds followed a similar protocol except the lanolin paste volume was 10 µL and applied to the pedicel of each bud. For escribed . Three described . For floSamples were ground in liquid nitrogen and aliquoted to 10 – 100 mg depending on the tissues and their known DTG and flavonoid concentrations (precise mass was recorded). Approximately 100 mg of aliquoted samples were extracted using 1 mL 80% methanol aqueous buffer and analyzed on a micrOTOF-Q II system  as previously described . QuantAnNtMaT1 were identified from the N. attenuata data hub (http://nadh.ice.mpg.de/). Fragments with coding regions of candidate malonyltransferases were amplified from cDNA with gene specific primers as described in TM 15, through entry vector pENTRTM according to the manufacturer’s instructions. The recombinant proteins were expressed in E. coli BL21 (DE3), extracted and purified using Glutathione-Sepharose 6B  in accordance with the manufacturer’s instructions. The in vitro enzyme activity assays were conducted as previously described  and stained with aniline blue as previously described . StainedTM (ThermoFisher Scientific). One µg RNA was used for reverse transcription by First strand cDNA synthesis kit (ThermoFisher Scientific). RT-qPCR was done via Mastermix (Eurogentec) SYBR Green reaction in a Stratagene 500 MX3005P Real-time qPCR machine. The primers used for mRNA detection of target genes by RT-PCR are listed in N. attenuata IF5a-2 mRNA was used as internal control. RNA-seq data for gene expression in all N. attenuata tissues was previously published in NCBI with accession number PRJNA317743 , and the RNA quantity was confirmed by the 260/280 nm absorbance ratio using NanoDropNA317743 . To readNA223344 .6-JA, 20 ng D6-JA-Ile, 3 ng D5-IAA), purified by successive HR-X and HR-XC SPE column chromatography (MACHEREY-NAGEL), and finally analyzed on a EVO-Q EliteTM Triple quadrupole-MS . Prior to SPE purification, 2 µL of the initial extract was diluted into 98 µL aqueous solution containing 255 fmol µL-1 13C9, 15N1-phenylalanine as an internal standard for the quantification of tryptophan.Jasmonates, IAA and tryptophan were measured as previously described . Briefly2EDTA). The reaction for TAA1 enzyme activity assays was incubated at 55°C for 20 min, and other procedures were as previously described  in negative ESI mode.Approximately 20 mg frozen powdered samples were extracted with 200 µL cold extraction buffer . The Student’s t-tests were performed in Microsoft Office Excel 2010. Homogeneity of variance was evaluated in SPSS using Levene’s test, and outliers were assessed by the function of Explor in SPSS with default parameters. The protein sequences were aligned by CLUSTAL W and phylogenetic trees were constructed using the maximum-likelihood method in MEGA6 (http://www.megasoftware.net/).All ANOVAs were performed in SPSS statistic 17.0 , a group of abundant, herbivore induced compounds in tobacco. Silencing this malonyl transferase NaMaT1 leads to reduced cell elongation in the style, and this effect appears to be mediated by a reduction in IAA biosynthesis. Importantly, it can be suppressed by silencing a key enzyme for DTG biosynthesis, arguing that the NaMaT1-VIGS effect is due to altered DTG malonylation levels. The authors also find that the short-style phenotype of JA-deficient plants that is observed in Nicotiana, but has not been described in other plants, is likely due to the influence of JA signaling on DTG malonylation levels.This manuscript describes the role of a group of specialized (secondary) plant metabolites for normal style development and plant fertility. The authors identify the major enzyme from Understanding how the vast diversity of plant specialized metabolites influences the plant's interactions with its environment and other organisms, but also the endogenous growth and developmental programs remains a key challenge in plant biology. In this respect, this paper provides exciting novel insight into the role of one class of specifically decorated metabolites. In particular, both the apparently strict control over the DTG malonylation levels throughout plant development and the highly specific effects of interfering with these malonylation levels highlight the functional importance of these compounds as putative novel regulators of phytohormone levels and plant growth. The manuscript thus makes an important contribution to our understanding of plant secondary metabolites.The following three issues should be addressed to strengthen the manuscript further.Mat1 irGGPPS plants. If this is still reduced to the same extent as in VIGS-Mat1 plants, it would argue that high levels of 'undermalonylated' DTGs interfere with auxin biosynthesis, rather than the malonylation level itself being critical.1) A key finding in this manuscript is that silencing NaMaT1 only results in short styles in a wild-type, but not in a NaGGPPS-silenced background. This experiment provides the main argument that the NaMaT1 effect is indeed mediated by the altered malonylation levels of DTGs, rather than some other undetected compound(s). However, it is not clear whether the style phenotype results from an excess accumulation of 'undermalonylated' DTGs, or from the altered malonylation percentage per se. It should be possible to address this by determining the malonylation percentage on the remaining DTGs in the VIGS-Mat1 plants should be determined in more detail. Is it just the physical separation of stigma and anthers – in which case hand-pollination should rescue fertility – or is there also a functional difference in the styles that interferes with efficient pollen-tube growth or guidance, which might have interesting implications for plant incompatibility systems?2) The reason for the sterility of the VIGS-3) YUCCA activity appears to be reduced in VIGS-NaMaT1 styles, and the authors seem to favor an effect of altered DTG malonylation levels on YUCCA enzymatic activity, rather than on YUCCA gene expression. Are YUCCA mRNA levels altered in VIGS-NaMaT1 styles?Reviewer #2:Nicotiana attenuata. They found that the malonylation percentage of DTGs was uniform in different organs of N. attenuata and after herbivore treatment. A correlation analysis lead them to the identification of three BAHD-acyltransferases (ATs), NaMaT1-3 able to catalyze a mono- and di- malonylation of the DTG, Lyciumoside IV. Virus induced gene silencing (VIGs) of NaMaT1 lead to a change in DTG malonylation patterns. A decrease in style length was the only phenotypic change for those NaMaT1-VIGs plants observed. Using plant hormone application as well as the background of knock-down plants (irGLS and irAOC) this phenotype was identified to be due to a decrease in auxin levels due to the inhibition of YUCCA. Furthermore the dependence of the effect on DTG and JA signaling could be shown.Li and coworkers report on the importance of malonylated hydroxygeranylinalool diterpene glycosides (DTGs) in N. attenuata is dependent on JA signaling, DTGs and auxin and that the identified ATs are somewhat involved in DTGs malonylation. What the authors did not convince me of, yet, is that the phenotype is due to the malonylation percentage rather than key metabolites of the pathway described.Overall the paper tells a nice, relevant and interesting story of the complex regulation of plant metabolism and the linkage of primary and secondary metabolism. I do believe that the observed style phenotype in My main request is to see an analysis including individual DTGs instead of just pooling them into groups. Furthermore some sections require additional data or rephrasing.- The authors look at DTGs based on their malonylation amounts. It would be of interest to look at single compounds. Could the observed effect of style length be due to single compounds rather than a shift in malonylation percentage? If this is not the case data should be provided to support that only the percentage changes. In fact one could test whether the percentage matters by shifting it through the application of DTG.- The authors state that the phenotype is not observed with irGGPPS VIGS-MaT1 plants. No data however characterizing the chemo type of these plants is supplied. How do the DTGs levels compare to VIGS-MaT1 plants? The same goes for silencing efficiencies of MaT1. This holds for irAOC plants too. Please ensure that the reader has access to the full characterization: qRT for silencing,% of malonylation, mono-di-tri malonylation, style length, IAA and JA content, YUCCA activity for all the plants which are of importance to draw a conclusion. Please show whether YUCCA activity is inhibited in irGGPPS (+/- VIGS-MaT1) plants to support involvement of malonylated DTGs in this phenomenon. The authors claim that the mechanism is the same for irAOC plants. How do auxin levels look in those plants and is auxin biosynthesis (YUCCA) inhibited too?Agrobacterium treatment. Those are however not the organs where a phenotype was observed. Explanations about the observation of a phenotype in styles, not the place of VIGS treatment should be included. The fifth paragraph of the Discussion states the style synthesises large amounts of DTGs. Why was no qRT done on styles? AT gene expression in irGGPPS and irAOC with EV and VIGS-MaT1 should be investigated in order to draw a full picture. Please answer the question of how knock down of MaT1 affects the plants in the background of irGGPPS and irAOC more carefully. For example, don't just show malonylation percentage but show at least mono-, di- and tri-malonylation. Especially as irAOC background changes malonylation percentage to be higher.- Expression level of MaT1 in VIGS plants was solely investigated in leaves the place of VIGS/- The NaMaT1-3 enzymes mediate the mono- and di-malonylation of Lyciumoside IV. The mono-mannosylation however is unlikely to be the enzymes in vivo function as MaT1 VIGS plants produce greater amounts of the Nicotianoside I compared to EV plants. The diglycosylation, which the authors showed happens in vitro too, seems to be affected. Statements about enzyme function should be revisited in the paper as they sometimes seem rather blurry. Statements like "controls the last two malonylation steps" cannot be made as observed patterns can also be due to substrate limitations. In addition you cannot throw all the malonylated compounds together as one when talking about enzyme functions . Those compounds do differ greatly biochemically speaking. I would find it necessary to look at individual compounds rather than the pool of compounds categorized by mono-, di-, tri-malonylation, in order to figure out enzyme function. The specific enzyme function might help to prove the hypothesis about malonylation percentage or even show that the effect observed might be due to rather specific compounds. "found that one MaT mediates this process" is not true. Please phrase carefully. Regarding the three acyltransferases, the authors should provide details about similarity of genes/proteins mentioned in the first paragraph of the subsection “NaMaT is responsible for DTG malonylation”. Substrate specificity of NaMaT1 should be accessed especially due to observed decrease in rutin. You might not find malonylated flavonoids, but how about NaMaT1s ability to accept other acyl donors? Statements like those in the second paragraph of the Discussion do not mean anything unless tested with the protein in question. Please state how similar NaMaT1 and NbMaT1 are. Was NbMaT1 tested with DTGs?- I do not find it surprising that the malonylation percentage of DTGs are uniform. From the biosynthesis perspective if you activate the pathway all compounds accumulate (in case of co-regulated genes) and this is what we see. In contrast, if the malonylation percentage of DTGs has the ability to regulate auxin would you not expect the percentage to vary in different organs or developmental stages rather than to be uniform? From my point of view it is necessary to look at individual compounds. Statements in the third paragraph of the Discussion about the uniformity of DTG malonylation status in contrast to strong regulation of specialist metabolites is rather strange. DTGs appear to be regulated after all as they are induced after herbivory. We were able to conduct many new measurements using plants growing in our glasshouse, and existing samples. This allowed us to address most reviewer concerns. However, we do not yet have measurements for the DTG profile of irGGPPS VIGS-MaT1 styles. We agree with the reviewers that the short style phenotype we observe could be due to one or a few individual DTGs, and we agree that analyzing the chemical profile of irGGPPS VIGS-MaT1 styles is a possible way to narrow down the number of candidates. However, based on our data describing the DTG profile in the inflorescence of irGGPPS VIGSMaT1, and stylar DTG profiles of VIGS-MaT1 in the backgrounds of EV and irAOC, we doubt very much that analysis of irGGPPS VIGS-MaT1 styles would allow us to pinpoint these putative “key” DTGs. Even if it did, we would not be able to complement the short style phenotype to definitively test the role of these putative “key” candidates, for several reasons, which we explain below. These explanations are followed by a point-by-point response to editor and reviewer comments.We hope that we can address the editors’ and reviewers’ concerns without conducting the additional experiments required to profile DTGs in irGGPPS VIGS-EV vs. VIGS-MaT1 styles.Why we think that adding data on the DTG profile in VIGS-EV vs. VIGS-MaT1 irGGPPS styles will not substantially increase the explanatory value of our study:25 combinations to test in complementation tests if we tested combinations of 1-2 compounds  – keeping in mind that even a single candidate compound may be perceived as a ratio against one or more other DTGs. Finally, we do not have a candidate mechanism, and so if we were able to test our best guesses based on comparison of the three background genotypes  with VIGS-EV or VIGS-MaT1, and these tests did not produce conclusive results, there would be no clear way to proceed.Except for 3 of the at least 46 different DTGs , the individual compounds are not present in sufficient abundance for purification and complementation assays within a reasonable time frame; structural similarity of many DTGs further increases the difficulty . Additionally, the malonylated compounds are not stable and previous attempts at the serial fractionation by UPLC of bulk extracts have failed regarding the malonylated derivatives . Furthermore, depending on how much we could narrow down the candidate compounds, and starting from the 16 compounds for which we could present relative quantification in this manuscript , we could have up to (16! * 15!) + ((16!/2!) * 14!)) = 2.8 x 10Nicotiana, and this work would be much more rigorous if done using an approach guided by alternative mechanistic hypotheses, rather than screening as many combinations as possible of individual metabolites for their effects on stylar elongation. Currently, the number of possibilities we could rigorously test is one , which is not likely to explain the phenotype .In summary, substantial additional work is required to determine the mechanism behind the role of DTGs in the short style phenotype in The reviewers and the reviewing editor found that your manuscript makes an important and new contribution to plant biology. Both reviewers have a number of comments that may help improve the paper. The reviewers' comments are following below. The two reviewers have discussed their individual reviews as part of our consultation session. The major request of both reviewers is for a more detailed characterisation of the effects of NaMaT1 silencing in the backgrounds silenced for other relevant biosynthesis genes.We agree that the characterization of the effects of VIGS-MaT1 in the backgrounds of irGGPPS and irAOC contained gaps, and we hope the revised text and new data fill these gaps and lend sufficient rigor to our study at the currently feasible level of explanatory power. The specific changes are detailed in the responses to the respective reviewer comments below.Reviewer #1:[…] The following three issues should be addressed to strengthen the manuscript further.1) A key finding in this manuscript is that silencing NaMaT1 only results in short styles in a wild-type, but not in a NaGGPPS-silenced background. This experiment provides the main argument that the NaMaT1 effect is indeed mediated by the altered malonylation levels of DTGs, rather than some other undetected compound(s). However, it is not clear whether the style phenotype results from an excess accumulation of 'undermalonylated' DTGs, or from the altered malonylation percentage per se. It should be possible to address this by determining the malonylation percentage on the remaining DTGs in the VIGS-Mat1 irGGPPS plants. If this is still reduced to the same extent as in VIGS-Mat1 plants, it would argue that high levels of 'undermalonylated' DTGs interfere with auxin biosynthesis, rather than the malonylation level itself being critical.Mat1 irGGPPS plants is intermediate between EV and VIGS-Mat1. This new data has been added as Figure 4—figure supplement 1.We thank the reviewer for this suggestion. We analyzed the DTG profile in irGGPPS plants in existing samples, and found that the malonylation percentage for the remaining DTGs in the VIGS-We feel it is very difficult to experimentally separate the role of “undermalonylated” DTGs from the malonylation percentage. To do so rigorously would require a tool to increase total DTG abundance without affecting malonylation percentage. The best comparison we have is the comparison of irGGPPS to EV plants (VIGS-EV); although EV has a much greater total abundance of “undermalonylated” DTGs, the two genotypes show similar malonylation percentages  have styles of similar length.2) The reason for the sterility of the VIGS-Mat1 plants should be determined in more detail. Is it just the physical separation of stigma and anthers – in which case hand-pollination should rescue fertility – or is there also a functional difference in the styles that interferes with efficient pollen-tube growth or guidance, which might have interesting implications for plant incompatibility systems?We thank the reviewer for this suggestion, and agree that it is important and relevant for our study to provide more information on this phenotype. We conducted this experiment and show that hand-pollination cannot rescue fertility in VIGS-MaT1 plants. We have added the data to the revised manuscript .3) YUCCA activity appears to be reduced in VIGS-NaMaT1 styles, and the authors seem to favor an effect of altered DTG malonylation levels on YUCCA enzymatic activity, rather than on YUCCA gene expression. Are YUCCA mRNA levels altered in VIGS-NaMaT1 styles?YUCCA by RT-qPCR, and the data showed that silencing NaMaT1 did not affect the abundance of YUCCA transcripts in any background .This is an important point. We measured the transcript abundance of the style-expressed Reviewer #2:[…] - The authors look at DTGs based on their malonylation amounts. It would be of interest to look at single compounds. Could the observed effect of style length be due to single compounds rather than a shift in malonylation percentage? If this is not the case data should be provided to support that only the percentage changes. In fact one could test whether the percentage matters by shifting it through the application of DTG.We agree with reviewer’s point, and we have added the data for the individual DTGs .However, combing the phenotypic and DTG profile data for VIGS in the backgrounds of irGGPPS (inflorescences), irAOC (styles) and EV (inflorescences and styles), we could not pinpoint an individual compound which clearly indicates the truncated style phenotype. For example, the dimalonylated compound Nicotianoside VII is the only individual DTG which seems to follow the same pattern as malonylation percentage in the EV and irAOC backgrounds: it is elevated in VIGS-EV irAOC vs. VIGS-EV EV styles, but reduced to similar levels in VIGS-MaT1 styles of irAOC and EV plants. However, Nicotianoside VII is reduced in irGGPPS vs. EV inflorescences, regardless of VIGS treatment (it is reduced by VIGS-MaT1 in both backgrounds). It is difficult to explain how either elevated (irAOC VIGS-EV) or reduced (EV VIGS-MaT1) levels of a single compound can cause the same phenotype. We propose that this indicates that it is a ratio, or relative abundance of multiple compounds, which is decisive. An alternative hypothesis would be that homeostasis of Nicotianoside VII  determines the short style phenotype. Unfortunately, Nicotianoside VII is present in low abundance relative to other DTGs and has high structural similarity to three other low-abundance DTGs: Attenoside, Nicotianoside VI, and Nicotianoside VIII .N. attenuata contains at least 46 different DTGs , myriad DTGs changes could lead to this phenotype. Although total DTGs are as abundant as starch, Lyciumoside IV and its malonylated compounds, Nicotianoside I and Nicotianoside II, comprise more than 80% of total DTGs , meaning that other components are present in extremely low concentration. Together with the structural similarity and instability of the malonylated DTGs over time in solution, purification of some DTGs is challenging . Heiling and colleagues were able to purify sufficient amounts for structural MS analysis, but bioassays require substantially more material and long-term stability under bioassay conditions.Nicotianoside VII is just one candidate. As In summary, all of these considerations make the use of most individual DTGs and mixes of DTGs in gain-of-function tests unfeasible. We found that the malonylation percentage is a uniform parameter, and all the phenotype patterns match very well with disturbances of the uniformity of malonylation percentage. Thus we think “malonylation percentage” is a good proxy for whatever the plant is specifically sensing. We agree that being able to understand exactly how plants sense “malonylation percentage” would be a high value target, it is clearly a question beyond the scope of this study, as it would likely require understanding the putative protein machinery involved in the sensing mechanism.We thank you for these excellent suggestions and as we think that readers will also be thinking along similar lines, we have added a summary of these considerations in the revised Discussion section (fifth paragraph).- The authors state that the phenotype is not observed with irGGPPS VIGS-Mat1 plants. No data however characterizing the chemo type of these plants is supplied. How do the DTGs levels compare to VIGS-Mat1 plants? The same goes for silencing efficiencies of Mat1. This holds for irAOC plants too. Please ensure that the reader has access to the full characterization: qRT for silencing,% of malonylation, mono-di-tri malonylation, style length, IAA and JA content, YUCCA activity for all the plants which are of importance to draw a conclusion. Please show whether YUCCA activity is inhibited in irGGPPS (+/- VIGS-Mat1) plants to support involvement of malonylated DTGs in this phenomenon. The authors claim that the mechanism is the same for irAOC plants. How do auxin levels look in those plants and is auxin biosynthesis (YUCCA) inhibited too?We agree with the reviewer’s point about the importance of the DTG profile in irGGPPS VIGS-MaT1 plants. We reanalyzed our existing data and provided the profile of inflorescences in this version .We also were able to measure the silencing efficiency in the styles of VIGS plants in background of irGGPPS, irAOC and EV, and YUCCA transcript abundance, from existing cDNA.As mentioned above, we currently don’t have irGGPPS (+/- VIGS-MaT1) plants and style samples, and it will take 4 additional months to get flowering plants. However, we showed that irGGPPS (+/-VIGS-MaT1) didn’t affect auxin concentration , we think the YUCCA activity is not crucial to address our conclusion.The auxin levels and YUCCA activity were provided in the previous version .- Expression level of Mat1 in VIGS plants was solely investigated in leaves the place of VIGS/Agrobacterium treatment. Those are however not the organs where a phenotype was observed. Explanations about the observation of a phenotype in styles, not the place of VIGS treatment should be included. The fifth paragraph of the Discussion states the style synthesises large amounts of DTGs. Why was no qRT done on styles? AT gene expression in irGGPPS and irAOC with EV and VIGS-Mat1 should be investigated in order to draw a full picture. Please answer the question of how knock down of Mat1 affects the plants in the background of irGGPPS and irAOC more carefully. For example, don't just show malonylation percentage but show at least mono-, di- and tri-malonylation. Especially as irAOC background changes malonylation percentage to be higher.We agree with reviewer’s points about spatial or tissue specificity, and we conducted further analyses of existing samples and added new figures  to address these points.- The NaMaT1-3 enzymes mediate the mono- and di-malonylation of Lyciumoside IV. The mono-mannosylation however is unlikely to be the enzymes in vivo function as Mat1 VIGS plants produce greater amounts of the Nicotianoside I compared to EV plants. The diglycosylation, which the authors showed happens in vitro too, seems to be affected. Statements about enzyme function should be revisited in the paper as they sometimes seem rather blurry. Statements like "controls the last two malonylation steps" cannot be made as observed patterns can also be due to substrate limitations. In addition you cannot throw all the malonylated compounds together as one when talking about enzyme functions . Those compounds do differ greatly biochemically speaking. I would find it necessary to look at individual compounds rather than the pool of compounds categorized by mono-, di-, tri-malonylation, in order to figure out enzyme function. The specific enzyme function might help to prove the hypothesis about malonylation percentage or even show that the effect observed might be due to rather specific compounds. "found that one MaT mediates this process" is not true. Please phrase carefully. Regarding the three acyltransferases, the authors should provide details about similarity of genes/proteins mentioned in the first paragraph of the subsection “NaMaT is responsible for DTG malonylation”. Substrate specificity of NaMaT1 should be accessed especially due to observed decrease in rutin. You might not find malonylated flavonoids, but how about NaMaT1s ability to accept other acyl donors? Statements like those in the second paragraph of the Discussion do not mean anything unless tested with the protein in question. Please state how similar NaMaT1 and NbMaT1 are. Was NbMaT1 tested with DTGs?We agree with reviewer’s points. We re-wrote the statement about the catalytic ability of NaMaT1 in the Discussion part (second paragraph).For the specific compounds, we performed additional data analysis and added the figure in this version .The statement of “found that one MaT mediates this process” was changed to “found that one MaT contributes significantly to this process”.We reanalyzed the malonyltransferase protein sequence and add these data in this version .DTGs decorated by other acyl moieties have not yet been identified in the Solanaceae , and our experiment clearly showed that the truncated style phenotype is related with DTGs. Thus, whether NaMaT1 could accept other acyl donors or not is outside the scope of this work.NbMaT1 was not tested for catalytic ability to DTGs, because we found the NaMaT2 and NaMaT3, rather than NaMaT1, are homologues of NbMaT1. Thus we think whether NbMaT1 could catalyze malonylation of DTGs is also outside of the scope of this work.- I do not find it surprising that the malonylation percentage of DTGs are uniform. From the biosynthesis perspective if you activate the pathway all compounds accumulate (in case of co-regulated genes) and this is what we see. In contrast, if the malonylation percentage of DTGs has the ability to regulate auxin would you not expect the percentage to vary in different organs or developmental stages rather than to be uniform? From my point of view it is necessary to look at individual compounds. Statements in the third paragraph of the Discussion about the uniformity of DTG malonylation status in contrast to strong regulation of specialist metabolites is rather strange. DTGs appear to be regulated after all as they are induced after herbivory.We agree with the reviewer that comparison of malonylation status with regulation of metabolites does not make sense, and we revised the related statement in this version . We still think it is an interesting observation that the decoration of specialized metabolites seems to be maintained within uniform boundaries, and to our knowledge this phenomenon has not previously been studied.


LPA was quantified before and after PRP fractioning and activation. The fibrin network of F2-clots from patients with a lower platelet concentration had an organized structure with large and distinct fibers while F2-clots from patients in group B revealed a similar structure to those in group A but with a slight increase in density. ELISA results showed a significantly higher plasma level of LPA in patients with a higher platelet concentration (group B) in comparison to those in group A (p < 0.05). This different concentration was evidenced in PRP but not in the clots. Depending on the number of platelets in patient’s blood, a PRP-clot with higher or lower mechanical properties can be obtained. The higher level of LPA in PRP from patients richer in platelets should be considered as responsible for the higher hOB activity in bone regeneration.The objectives of this study were to compare platelet-rich plasma (PRP) from patients with different concentrations of platelets and to assess the influence of these PRP preparations on human osteoblast (hOB) activity. In the literature, growth factors released by activated platelets have been considered responsible for the active role of PRP on bone regeneration but no specific role has been attributed to lysophosphatidic acid (LPA) as a possible effector of biological responses. In this study, patients were grouped into either group A (poor in platelets) or group B (rich in platelets). Clots from PRP fraction 2 (F2-clots), obtained with CaCl In the field of regenerative medicine and tissue engineering, there is great interest in the search for new compounds to be used as fillers and as promoters of tissue regeneration. The use of platelet concentrates in combination with natural and synthetic biomaterials has been widely assessed and examined, with the aim of obtaining a faster and better bone healing . PlateleHistorically, the active role of PRP in biological responses promoting tissue regeneration has been attributed to growth factors released by activated platelets ,8 whilstTherefore, although the active role of PRP has been attributed to the growth factors released by activated platelets, the role of LPA as a possible biologically active compound for bone regeneration should also be considered.The aim of our study was to quantify the concentration of platelets in PRP samples and their role in the determination of PRP morphological quality. A macroscopic, microscopic, and mechanical analysis of clots formed by platelet activation was performed to highlight the morphology, consistency, and structural diversity of the clot matrices; the adhesion capacity of human primary osteoblast-like cells was determined together with their migration activity. Finally, we focused on the remodeling of extracellular matrix and on the induction of cell proliferation. No specific role has been attributed to LPA in PRPs as a possible effector of biological responses in tissue regeneration so we have quantified LPA in the prepared plasma fractions and plasma clots.After reviewing the literature on PRP preparation, our work has been focused on the production of PRP fractions by centrifugation using the Endoret™ technique to obtain coagulated fibrin gels . Human bFew differences were found in the consistency and macroscopic structure of clots obtained by PRP-F2 coagulation with calcium chloride using PRP from patients of group A and patients of group B A,B. In bElectron microscopy of the fibrin scaffold C,D showep > 0.05) although clots from PRP of the patients in group B (higher in platelets) had higher compressive strength (26 ± 5 KPa) than those of group A (16 ± 2 KPa).The mechanical properties of the F2-clots evaluated by compression are shown in Assays of cell adhesion to materials used as fillers are indicative of whether cells find an environment conducive to their local development and, therefore, are useful tests to understand how cells may promote the integration of the material into the location of the damage. Fluorescence microscopy showed that cells on the outside surface of F2-clots were high in density and well distributed, with no differences between the two groups of patients A,B.Cell migration assays in materials used as fillers are an index of an environment conducive to tissue regeneration with matrix remodeling at the site of the damage. Platelet-rich plasma is an autologous biomaterial and is expected to show good cell adhesion; moreover, being reasonably porous and pharmacologically active, thanks to the presence of growth factors, it is also expected to favor internal colonization when hosted in vivo and to develop a host-like tissue. Sagittal cuts of the F2-clots were made to see the 3D scaffold’s internal structure, and images were captured and evaluated to study cell migration. Representative images of DAPI-stained cells are shown A,B.p > 0.05); they also promoted hOB cell migration. As shown in the histogram in Comparing images, the two groups of patients showed similar results with good cells penetration into the scaffolds. Although the clots had different geometric shapes and different thickness, it seems that the fibrin mesh underneath provided a structure that determines the direction of cell accession. After evaluating almost 10 sections from each clot, we have seen that there were more cells populating the inner part of the scaffold in group A (poorer in platelets). The F2-clots produced an increase in human osteoblast proliferation compared to the control which was not statistically significant  was determined, demonstrating the tendency of platelets to be approximately twice as concentrated in the F2 fraction .Platelet number was considered important in the study as it was hypothesized that the number of platelets could affect the quality of the clot, the basal LPA plasma level, and the amount of LPA released during activation and degranulation.p < 0.05). Centrifugation to obtain PRP did not alter the LPA concentration, and no differences were seen between F1 and F2 within a group, as shown in The ELISA results showed a higher plasma LPA level in the group of patients with higher platelet concentrations (group B) with statistical significance when compared to group A  plasma LPA levels in comparison with the initial plasma values was noticed. However, such decrease seemed to be proportional to the initial values for each group and was dependent on the number of platelets in the group. Therefore, LPA levels were not increased by CaCl2 activation.The LPA value in the lysate of PRP was expected to increase after coagulation and therefore platelet activation, with a higher increase in clots of group B being higher in platelets, but this was not found. Levels of LPA in lysates of F2-clots were lower than the levels in F2 before CaClAn initial review of the literature highlighted the great variety of platelet-derived products and the heterogeneity in their nomenclature and processing methods, with confusion over their therapeutic properties and suggested applications ,19,20,21Despite the variety of commercial products offered to obtain PRPs, two major groups can be identified: (1) PRPs (platelets-rich plasma), F1, and F2 in our study as suggested by Anitua et al.  and (2) Most in vitro studies conducted on PRP have focused on the effects of activated PRP rather than on platelet gel and its individual variability. Our results have shown for the first time that PRP-clots can be produced with higher or lower mechanical properties depending on the number of platelets in a patient blood. Patients with higher platelet number produce a clot with a higher density of fibrin fibers that give higher resistance to fracture in compression tests. Semiquantitative results of hOB adhesion to the PRP-clots and hOB migration inside them show that clots obtained from group A (poorer in platelets) are favored to host cells probably due to the lower fibrin density that allows cells to migrate and proliferate easier within the clot. It is known that there is a link between structure and cell movement beyond the effects of pore size of the scaffold so we think it will be interesting to study better cell behavior with different PRP clots. Geometrical and mechanical properties of scaffolds are able to influence the cell behavior and their response to differentiating stimulations ,24. The P. aeruginosa and that exposure of bacteria to this lipid made them increasingly susceptible to certain antibiotics, suggesting LPA/LPA analogues as potential adjuncts in a bone regenerative setting to reduce potential infection risk of implantable devices. Only a few studies have been done on the activity of LPA on bacteria, and relatively scattered data suggest an important contribution of the platelet lipid signaling to sepsis [Aggregation and activation of platelets results in a rapid release of LPA into the extracellular environment ,17 so weo sepsis  to consio sepsis . LPA anto sepsis  whereas,o sepsis . In gliao sepsis  and exero sepsis . Few reso sepsis . Macropho sepsis . DespiteConsidering that no higher LPA concentrations were found in the clots of the two groups but only in the non-clotted fractions (in the residues), they probably are due to different biological effects. This result is not seen in In addition, LPA release time from gels is an important factor that should be considered indeed; we doubt that bioactive phospholipid can be released by α-granules like other growth factors, and therefore, platelet degranulation could not be enough to release it. It may be necessary to have a biological production time longer than the sixty minutes for coagulation. This data is supported by the small difference occurring in LPA concentration among the plasma, the supernatants, and the non-coagulated portion.The clinical implications of the present study’s results are potentially fundamental for the everyday bone regenerative medicine and may be useful for the clinical operator in order to create a scaffold with adjustable properties to each situation. In fact, the possibility that patients with a higher number of platelets could “produce” PRP-clots with a higher density of fibrin fibers and, consequently, with a higher resistance to fracture in compression tests is a crucial point. A better outcome in these patients could also be due to higher levels of growth factor in PRP liquid fraction that probably will be the real responsibility of the biologic activity. This could explain the various outcomes of bone regeneration in different patients. It could eventually determine a relative contraindication of the PRP technique in patients with low level of platelets, or it could become selection criteria for the use of PRP in regenerative medicine.Venous blood from healthy male and female volunteers aged between 20 and 60 years was obtained and used for the preparation of the platelet-rich plasma (PRPs) samples. Inclusion criteria were ages between 20 and 60 years; nonsmokers; and absence of chronic hematologic, neoplastic, and/or infectious diseases . All procedures involving human participants were performed in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All subjects gave their informed consent for inclusion before they participated in the study. The protocol was approved by the Ethics Committee of Novara “Prot. CE-Novara62/2018”, approved on 27 July 2018.® kit using 10% CaCl2 at 37 °C for 1 h to create the platelet concentrate (F2-clot). The obtained clots were analyzed using macroscopic, mechanical, and microscopic methods. Moreover, the in vitro activity of the PRP clots on proliferation and migration of osteoblast-like cells was assessed together with LPA quantification that was done on PRP-F1 (F1), PRP-F2 (F2), and Endoret®-activated PRP-F2 (F2-clots).Samples of collected blood were analyzed using a hematology autoanalyzer Sysmex XN-2000™  to obtain a platelet count. Samples were then centrifuged at 2500 rpm for 8 min at 25 °C without acceleration and without break in order to obtain PRP in the upper half of the tube, separated from the remaining portion of red blood cells by a thin layer of buffy coat. The PRP portion was removed as two fractions of equal volume: the more superficial one, called fraction 1 (PRP-F1), and the part nearest to the leukocyte fraction, called fraction 2 (PRP-F2). Platelet counting of PRP-F1 (F1) and PRP-F2 (F2) was performed using the Sysmex XN-2000™ before placing F2 into sterile vials and activating it according to EndoretPlasma concentrates (F2-clots) prepared from PRP from patients with low platelet number (group A) and PRP from patients with high platelet number (group B) were compared.3 were positioned inside the instrument’s chamber between two pistons. Tests were performed with a constant crosshead speed of 0.05 mm/s. The instrument showed the real-time displacement of the piston and the force acting on the sample, producing a force/displacement curve. Compressive strength was then calculated, using the surface area of the sample, the initial length of the sample, compressed length, and force at breaking point. All samples were tested in triplicate, and data are presented as mean ± standard deviation (SD), comparing F2-clots from patients of group A and patients of group BMacroscopic images of the obtained F2-clots were obtained. The compression properties of clotted PRPs were tested using an Electro Force BioDynamic Test Instrument . Samples of 2 ± 0.5 mmSEM analysis of the coagulated platelet plasma concentrates was performed. After cacodylate buffer washing, F2-clots were fixed in a 4% paraformaldehyde +2.5% glutaraldehyde solution in 0.1 M cacodylate buffer at pH 7.4 for 30 min at 4 °C. Then, samples were dehydrated using a series of solutions of increasing ethanol concentration (50% to 100%) and, afterwards, in hexamethyldisilizane (20). Finally, they were glued onto aluminum stubs using a colloidal silver fluid, gold sputtered , and analyzed by electron microscopy .l-glutamine, and antibiotics. Cells from up to five passages were used for all the experiments. Prior to culturing cells in the presence of a PRP test sample, cells were starved for 24 h and cultured in serum free media supplemented with 250 mg/mL essentially fatty acid-free human serum albumin.Bone trabecular fragments were taken during surgical interventions from patients in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All subjects gave their informed consent for inclusion before they participated in the study. The protocol was approved by the Ethics Committee of Novara “Prot. CE-Novara 62/2018 on 27 July 2018. Fragments were digested using collagenase/elastase as described previously  to obtaiProliferation was measured using a luminescent-based ATP quantification Kit . Cells were lysed using the cell lysis reagent and treated with ATP monitoring reagent at time 0 and following 1, 3, 6, and 12 days of culture of cells with PRP. The light produced was measured in a luminometer and expressed as relative luminescence units (RLUs).5 cells were plated in 24-well plates in 500 μL of medium and incubated at 37 °C in 5% CO2 until confluence; then, serum was starved for 20–24 h. Monolayers were wounded by the introduction of linear scratches with a sterile pipette tip, rinsed with PBS , and cultured in medium with and without the tested PRP groups. For each experiment, cells in six selected fields were tracked for their distance travelled. The selected fields (30–40 µm) were maintained at a fixed distance from the border of the culture plate, and for each experiment, images were acquired using a Leica digital camera connected to an inverted microscope (Leica). Images were obtained at time 0, 6 h later, and 1–3 days after the addition of the PRP. The distances travelled by the cells were measured using the Qwin Image Analysis system (Leica).To determine the migration activity of cells in response to PRPs, 1.5 × 105 hOB. The multi-well was then put in a shaking incubator (SanyoCO2) at 37 °C, 5% of CO2, and 80–85% humidity. Following 48 h of incubation with hOB, PRP clots were treated with DAPI , which marked the hOB DNA, so that cell penetration could be estimated using fluorescence microscopy.To observe the adhesion and migration of cells in PRPs, clots were placed in the wells of a low adhesion multi-well plate with 1 mL of IMDM containing 10R2 value of above 95%); the average of the duplicates was used as the results.A commercial enzyme-linked immunosorbent assay (ELISA) kit was used for LPA measurement  according to the manufacturer’s instructions. Briefly, standards and samples (50 μL) were aliquoted to the pre-coated plates in duplicates. After 1 h of incubation and washing, 50 μL of conjugate was added. After 1 h of incubation and washing, 100 μL of enzyme substrate was added to each well and incubated for 15 min before adding 100 μL of solution. The optical density of each microwell was read using a microplate reader at 450 nm, and the level of LPA in each well was calculated using a logarithmic standard curve  to compare the groups. Statistical significance was set at p < 0.05. All test samples were assayed in duplicate, and experiments were repeated between two and four times.An unpaired Student’s More studies are needed to better address the application of the clinical work and to deepen how the morphologic characteristics of clots can affect the patient’s outcome. According to the literature, clots’ morphological and structural differences may influence cell homing and clot interaction with the surrounding environment. Certainly, more detailed studies on PRP-clots as scaffolds will be required. Three-dimensional structural tests on fiber orientation, pore dimensions, and mechanical properties are only some variables that need more attention. Even if no clinical conclusions can ever be drawn from an in vitro study, a scaffold with low mechanical properties can be considered a good bone substitute only for clinical situations that did not require esthetic and functional outcomes and that require low time of permanence such as the filling of wide pneumatized sinus lift . An in vThe role of LPA should also be evaluated further in future research, as it constitutes a promising PRP research topic. Moreover, as the presence of LPA in PRP had never been highlighted before, in the same way, further research could lead to the discovery of other new growth factors correlated to PRP.Our results, in spite of the evident need for further confirmations and research, represent an important step for an improved understanding of PRP use for bone regeneration and for its more appropriate clinical use.


Plasmodium dormant liver hypnozoites compromise malaria eradication efforts. New radical cure drugs are urgently needed, yet the vast gap in knowledge of hypnozoite biology impedes drug discovery. We previously unraveled the transcriptome of 6 to 7 day-old P. cynomolgi liver stages, highlighting pathways associated with hypnozoite dormancy . We now extend these findings by transcriptome profiling of 9 to 10 day-old liver stage parasites, thus revealing for the first time the maturation of the dormant stage over time. Although progression of dormancy leads to a 10-fold decrease in transcription and expression of only 840 genes, including genes associated with housekeeping functions, we show that pathways involved in quiescence, energy metabolism and maintenance of genome integrity remain the prevalent pathways active in mature hypnozoites.Relapses of  Plasmodium vivax malaria puts 35% of the world’s population at risk of disease  inhibitor  . Although established hypnozoites continue to express a subset of genes, a marker gene which specifically distinguishes hypnozoites from schizonts was not detected. Genes and pathways associated with quiescence, energy metabolism and maintenance of genome integrity remain prevalently expressed in mature hypnozoites, indicating that the hypnozoite stage may be defined by a network of regulatory factors cooperating in the maintenance of genome stability and in the epigenetic control of gene expression. disease . A largenfection . This ashe liver , there ihe liver . For thinhibitor , was shonhibitor . This inarasites , in vitr culture , cell-so stages)  by transP. cynomolgi M strain sporozoite infection. Mean fluorescence intensities, a measure that depends on cell size and fluorescence intensity, slightly increased from day 6 to day 9 in the GFPlow samples, illustrating a small increase in the volume of hypnozoites over time  and liver schizonts (GFPhigh) at later time points, 9 and 10 days after ver time . A more ver time . Microscver time . The GFPmsp1 was positive for schizonts at day 10, while no transcripts were detected in hypnozoites at day 10  liver schizonts express a similar number of genes as those observed at days 6/7 . However at day 9 genes are expressed at higher levels  . Most of day 6/7  . This is also reflected in the significant 10-fold decrease of gene expression levels in hypnozoites . We observed that only 18% of genes expressed in days 6/7 hypnozoites (628 out of 3462 genes with FPKM ≥1) are as well expressed at day 9 (628 out of 840 genes with FPKM ≥1). This transcriptional decrease is even more pronounced for highly expressed genes where only 2.4% of genes expressed at day 6/7 (21 out of 870 genes with FPKM ≥10) show high transcriptional activity also at day 9 (21 out of 30 genes with FPKM ≥10) . The smaP. vivax hypnozoite transcriptome  with high transcript abundance in P. vivax hypnozoites  revealed that cytoadherence is the only function with expression of less than 70% of the genes  and, for each group, the mean percentage of genes active in the pathways was calculated for hypnozoites at day 6/7 and day 9. This revealed lower transcription levels of genes involved in transcription, translation, replication and merozoite invasion in the day 9 hypnozoites. Functions such as chromatin structure and energy metabolism were less represented in hypnozoites at day 9, however, still showed a moderate expression level , chaperones and protein structure modifications (e.g. HSP70 cycle), mitochondrial respiration and redox metabolism showed either upregulation or sustained expression in day 9 hypnozoites, suggesting a key role in the maintenance of the quiescent state . Moreoverculosis .To conclude, our data show that gene expression levels of housekeeping pathways in later stage, day 9 hypnozoites are dampened. Only pathways previously associated with quiescence and required for energy metabolism and maintenance of chromosome integrity remain expressed in hypnozoites.up-regulated in infective sporozoites gene 4 (UIS4) and the exported protein-1 (EXP-1), located at the site of the parasitophorous vacuole membrane (PVM), have indeed been shown to interact with host cell proteins in rodent malaria models  chaperone cycle to be highly active in late stage hypnozoites. These data were validated by RNA-FISH and immunofluorescence assays. Intriguingly, in Toxoplasma expression of hsp70 is induced during bradyzoite differentiation and its inhibition can suppress bradyzoite development in vitro  (PcyM_0702400) is expressed in hypnozoites (3.5 FPKM at day 6/7 and 2.2 FPKM at day 9). It is hence tempting to speculate that this protein might play a role in chromosome condensation during dormancy. Moreover, we find Alba genes transcribed in hypnozoites. Members of the Alba family from protozoan parasites bind to both DNA and RNA and are implicated in transcriptional regulation, chromatin packaging, and translational control, as well as cellular differentiation and developmental processes  were suitable for the aims of this project. The research protocol was approved by the local independent ethical committee conform Dutch law . Details are described by Nonhuman primates were used because no other models  values of GFPlow and GFPhigh samples were calculated using four separate recordings of 1 million cells per experiment .Procedures for liver cell isolation, liver stage culture and cell sorting were essentially as described previously . BrieflyE. coli, purified using a Ni-IMAC column followed by gel-filtration and used to immunize rats .PcyM_0602100 (UIS4) aa20-166 and PcyM_0924700 (EXP-1) aa19-130 were expressed in IFA staining of methanol fixed liver stage parasites was carried out as before . AlternaP. cynomolgi liver stage cultures in 96-well collagen-coated CellCarrier plates (PerkinElmer) were washed once in PBS and fixed in 4% paraformaldehyde (Affymetrix) at room temperature for 30 min. Cultures were then washed, dehydrated and stored in 100% ethanol at −20°C until RNA-FISH was performed using a Tyramide Signal Amplification (TSA) based assay from Advanced Cell Diagnostics (RNAscope Multiplex Fluorescent Assay v2), essentially according to the manufacturer’s instructions. Following rehydration protease digestions were performed using pretreatment solution three from the kit at 1:10 dilution for 20 min. at room temperature. Hybridizations were 2 hr at 40°C. Probes used were directed against P. cynomolgi hsp70 , PTEX150 (PcyM_1315200 targeting 871–2360 of XM_004224250.1), Alba1  and MSP1 (PcyM_0731200 targeting 147–1127 of XM_004221774.1). After hybridization, TSA amplification steps were performed as described by the manufacturer. Following DAPI staining, cells were kept in PBS for imaging. Images were acquired with a Leica DMI6000B inverted fluorescence microscope equipped with a DFC365FX camera using a HC PL APO 63x/1.40–0.60 oil objective.For RNA-FISH, Total RNA was isolated using the Direct-zol RNA MiniPrep Kit (Zymo Research) including on-column DNase digestion according to the manufacturer’s instructions. The quality of the RNA samples was assessed with the High Sensitivity RNA kit using the TapeStation 4200 instrument (Agilent Technologies).RNA samples were processed using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech) to generate high-quality cDNA. The results obtained were evaluated with the High Sensitivity DNA kit using the Bioanalyzer 2100 instrument (Agilent Technologies). The cDNA samples were then sheared to 200–500 bp length using a Covaris E220 instrument (Covaris) and subsequently processed with the Low Input Library Prep Kit v2 (Clontech) to generate sequencing libraries. The quality of the libraries was assessed with the D1000 TapeStation kit (Agilent Technologies). RNA-seq cDNA libraries were sequenced in paired-end mode, 2 × 76-base-pair (bp), using the Illumina HiSeq2500 platform. Read quality was assessed by running FastQC (version 0.10) on the FASTQ files. Sequencing reads showed high quality, with a mean Phred score higher than 30 for all base positions. The obtained 76-bp paired-end reads were used for the alignment to the reference genomes and used for the gene expression quantification with the Exon Quantification Pipeline (EQP)  as descrP. cynomolgi gene, we determined the number of sequencing reads aligned to genes and computed gene expression values as the number of Fragments per Kilobase per Million fragments mapped (FPKM). For the normalization of the schizont expression values, we applied the previously developed method of normalization against the total number of host reads  to demonstrate protein expression of the genes transcribed during previously reported biomarkers for day 6/7 hypnozoites that were not found at day 9; (2) to validate some pathways revealed at the transcriptomic level.As you can see from the comments included below, each reviewer expressed positive views regarding the organization and the performance of the experiments as well as the robustness of your results on transcriptomics during the development of Reviewer #1:P. cynomolgi liver stage hypnozoites and liver stage schizonts at day 9 and day 10 post infection. The parasite material was derived from in vitro-cultured hepatocyte infections that were subject to cell sorting for enrichment of parasite-infected hepatocytes. The work is an extension of data recently published by the same group in eLife , which described hypnozoite and schizont transcriptomes from parasites at days 6/7 post infection.The paper by Bertschi et al. describes the transcriptomes of The experimental work is technically sound, and the data analysis is reasonably well done. The main conclusion drawn by the authors is that more 'mature' day 9 hypnozoites exhibit similar gene expression patterns when compared to day 6/7 hypnozoites but the former shows a further decrease of transcription.While the work is of value and a descriptive and incremental step forward towards the delineation of liver stage gene expression profiles and gene expression in hypnozoites, I cannot see that it provides a significant step towards an understanding of the molecular mechanisms of dormancy and persistence of hypnozoites. It is entirely possible that the answer to the question "what regulates dormancy" will not be revealed by further transcriptome analysis beyond what has been recently published  and this manuscript further confirms this problem. It might be time that the focus is shifted towards potential posttranscriptional or posttranslational mechanisms that might regulate dormancy.Reviewer #2:P. cynomolgi liver stage parasites, including the maturation of its dormant stage hypnozoite in their manuscript, titled: "Transcriptomic analysis reveals malaria hypnozoite maturation." Data analysis shows that the progression of parasite dormancy leads to a 10-fold decrease in transcription with only 840 transcribed genes. Expression analysis shows that pathways involved in quiescence, energy metabolism and maintenance of genome integrity remain the prevalent pathways active in mature hypnozoites. The manuscript is well written. The data generated are robust, well validated and further confirm the importance of epigenetic control in parasite development. I would, however, like to stress that while the described work has been thoroughly performed, the work appears narrow and incremental to previous published datasets. Additional experiments targeting/validating the identified pathways as essential for hypnozoite persistence would hold more value. Is there anyway the team can test the inhibitory activity of drugs/molecules targeting histone-modifying enzymes such as histone acetyltransferases (HATs), histone methyltransferases (HMT) or histone chaperones against hypnozoite maturation and survival? This would significantly improve the relevance of this manuscript.Bertschi and colleagues unravel the transcriptome of 9 to 10-day-old Reviewer #3:in vitro experiments using non-human malaria, the authors point out several consistencies of these results with P. vivax studies and these results also corroborate the prophylactic but not radical curative activity of PI4K. The manuscript is well organized, and the conclusions are supported by the design and analysis of their experiments. I would recommend with work for publication.Although the sample size is small, this manuscript shows convincingly that as hypnozoites mature from day 6/7 to day 9, transcriptional activity is massively reduced. This is in stark contrast to the increased transcriptional activity at the same timepoints during schizont development. There is a strong rationale for doing this work because in order for therapeutics to provide effective radical cure, they must target processes in even the most dormant of hypnozoites. Although the inferences of this study are limited to eLife. Your plan has been considered by a Senior Editor, Reviewing Editor and three reviewers, and we are prepared to consider a revised submission.Thank you for choosing to send your work entitled "Transcriptomic analysis reveals malaria hypnozoite maturation" for consideration at We will not request any additional experiments, but we will suggest that the authors do respond to the other comments posted by the reviewers – these are mainly editorial in nature – and to revise the title of the manuscript. [Editors’ note: what follows is the authors’ plan to address the revisions.]As you can see from the comments included below, each reviewer expressed positive views regarding the organization and the performance of the experiments as well as the robustness of your results on transcriptomics during the development of P. cynomolgi. While they considered the information to be of value and worthy of publication, they also commented on the small incremental step that your data provide and thus questioned the advancement towards understanding dormancy of hypnozoites. To substantially enhance your story, they suggested performing additional experiments: (1) to demonstrate protein expression of the genes transcribed during previously reported biomarkers for day 6/7 hypnozoites that were not found at day 9;This is an interesting suggestion; however, the analysis of the day 6/7 transcriptome data unfortunately did not detect any hypnozoite-specific biomarkers. This means that we do not have any hypnozoite biomarkers we could select for the proposed experiment at day 9.In the previous manuscript, we indicated a dozen of transcripts upregulated in the hypnozoites versus schizonts, which correspond to genes with low expression levels in the hypnozoites and very-low expression levels in the schizonts.Thefollow-up validation of the most upregulated gene in hypnozoites, a member of the ETRAMP family, could not confirm protein expression in sporozoites, hypnozoites or schizonts during liver stage development; this appeared to be due to a different alternative splicing (intron retention events) occurring in these stages, resulting in the presence of premature stop codons in the mRNA sequence and hence in the absence of protein expression at days 6 and 7.Thus, the ETRAMP protein would not qualify as a hypnozoite-specific marker to be validated at day 9.Selecting the other low-level transcribed genes from the day 6/7 hypnozoite transcriptome, raising antibodies to recombinant proteins , preparing new EEF cultures and probing those with the antibodies could easily take 6 months and would require another monkey infection.In addition, we understand that the reviewer raised this point merely as an interesting route to follow, beyond the scope of our current work, and they do not seem to suggest additional experiments.(2) to validate some pathways revealed at the transcriptomic level.Reviewer 2 suggests evaluating the effect of compounds targeting histone-modifying enzymes. This is also an interesting suggestion but would require a massive number of studies that we feel are beyond the scope of this paper. In fact, we would expect the effects of these compounds to be pleiotropic by definition and to lead to the generation of data that may be very difficult to interpret and would require substantial validation even if we see an effect. To more effectively test this type of compounds we would have to develop new types of assays where we can follow individual parasites over time. A pilot experiment with a limited set of inhibitors may likely take at least 6 months, without any guaranteed success. For this reason, we would prefer to consider the drug screening as a new chapter compared to the proposed “research advance” article that builds on and complements our recent paper of malaria liver stage transcriptomics.[Editors’ notes: the authors’ response after being formally invited to submit a revised submission follows.]We will not request any additional experiments, but we will suggest that the authors do respond to the other comments posted by the reviewers – these are mainly editorial in nature – and to revise the title of the manuscript.We carefully addressed the concerns of the referees and give detailed answers to the different points below. In addition, we revised the title of our manuscript as follows:“Transcriptomic analysis reveals reduced transcriptional activity in malaria hypnozoites during progression into dormancy”.Reviewer #1:in vitro-cultured hepatocyte infections that were subject to cell sorting for enrichment of parasite-infected hepatocytes. The work is an extension of data recently published by the same group in eLife , which described hypnozoite and schizont transcriptomes from parasites at days 6/7 post infection.The paper by Bertschi et al. describes the transcriptomes of P. cynomolgi liver stage hypnozoites and liver stage schizonts at day 9 and day 10 post infection. The parasite material was derived from The experimental work is technically sound, and the data analysis is reasonably well done. The main conclusion drawn by the authors is that more 'mature' day 9 hypnozoites exhibit similar gene expression patterns when compared to day 6/7 hypnozoites but the former shows a further decrease of transcription.While the work is of value and a descriptive and incremental step forward towards the delineation of liver stage gene expression profiles and gene expression in hypnozoites, I cannot see that it provides a significant step towards an understanding of the molecular mechanisms of dormancy and persistence of hypnozoites. It is entirely possible that the answer to the question "what regulates dormancy" will not be revealed by further transcriptome analysis beyond what has been recently published  and this manuscript further confirms this problem. It might be time that the focus is shifted towards potential posttranscriptional or posttranslational mechanisms that might regulate dormancy.We thank the reviewer for this thorough review and agree with the need to shift the focus of future studies towards posttranscriptional or posttranslational mechanisms that might regulate dormancy in the hypnozoite.Reviewer #2:Bertschi and colleagues unravel the transcriptome of 9 to 10-day-old P. cynomolgi liver stage parasites, including the maturation of its dormant stage hypnozoite in their manuscript, titled: "Transcriptomic analysis reveals malaria hypnozoite maturation." Data analysis shows that the progression of parasite dormancy leads to a 10-fold decrease in transcription with only 840 transcribed genes. Expression analysis shows that pathways involved in quiescence, energy metabolism and maintenance of genome integrity remain the prevalent pathways active in mature hypnozoites. The manuscript is well written. The data generated are robust, well validated and further confirm the importance of epigenetic control in parasite development. I would, however, like to stress that while the described work has been thoroughly performed, the work appears narrow and incremental to previous published data sets. Additional experiments targeting/validating the identified pathways as essential for hypnozoite persistence would hold more value. Is there anyway the team can test the inhibitory activity of drugs/molecules targeting histone-modifying enzymes such as histone acetyltransferases (HATs), histone methyltransferases (HMT) or histone chaperones against hypnozoite maturation and survival? This would significantly improve the relevance of this manuscript.We thank the reviewer for this thorough review and for the suggestion to perform additional drug screening with drugs targeting histone-modifying enzymes. In our revision plan, we have clearly addressed the main challenges related to the execution of these follow up experiments.


GTPase signal transduction pathways control cellular decision making by integrating multiple cellular events into a single signal. The Mitotic Exit Network (MEN), a Ras-like GTPase signaling pathway, integrates spatial and temporal cues to ensure that cytokinesis only occurs after the genome has partitioned between mother and daughter cells during anaphase. Here we show that signal integration does not occur at a single step of the pathway. Rather, sequential components of the pathway are controlled in series by different signals. The spatial signal, nuclear position, regulates the MEN GTPase Tem1. The temporal signal, commencement of anaphase, is mediated by mitotic cyclin-dependent kinase (CDK) phosphorylation of the GTPase’s downstream kinases. We propose that integrating multiple signals through sequential steps in the GTPase pathway represents a generalizable principle in GTPase signaling and explains why intracellular signal transmission is a multi-step process. Serial signal integration rather than signal amplification makes multi-step signal transduction necessary. Accurate chromosome segregation is essential for genome maintenance and viability of the organism. Central to this accuracy is the integration of temporal and spatial signals by the cell cycle machinery. Deciphering the molecular mechanisms that bring about signal integration is thus critical to understanding how organisms are built.In eukaryotes, a family of protein kinases, known as cyclin-dependent kinases (CDKs) drive progression through the cell cycle. G1 CDKs promote cell cycle entry, S phase CDKs initiate DNA replication, and mitotic CDKs drive entry into mitosis  network releases Cdc14 from its inhibitor in the nucleolus upon anaphase entry . The FEAThe FEAR network is comprised of some of the same proteins that control chromosome segregation at the onset of anaphase . SeparasThe Mitotic Exit Network (MEN) is activated during anaphase and promotes the sustained release of Cdc14 from the nucleolus. Released Cdc14 then triggers complete CDK inactivation and hence exit from mitosis. The MEN is essential. Cells lacking MEN function arrest in late anaphase failing to exit from mitosis . The sigOne function of the MEN is to ensure that sustained Cdc14 activation only occurs when the nucleus moves into the bud during anaphase. Asymmetric localization of MEN activators and inhibitors causes the MEN to react to spindle position. Kin4, which activates the Tem1 GTPase activating protein (GAP) complex Bub2-Bfa1 and hence maintains Tem1 in the GDP bound form  localizeWhile an appealing model for how cells sense spindle position, this zone model of MEN activation fails to explain how mitotic exit is coordinated with cell cycle progression. It has previously been observed that when cells experience a prolonged metaphase arrest the nucleus moves into the bud , yet theThe MEN promotes exit from mitosis. This activity of the MEN is confined to anaphase. During this cell cycle stage movement of a SPB into the bud leads to the association of the MEN kinase Cdc15 with SPBs where it phosphorylates the scaffold Nud1 . This prA recent study reported that the MEN kinases Cdc15 and Dbf2-Mob1 are active prior to anaphase and prior to a SPB moving into the bud, but that this activity cannot be measured by conventional kinase activity assays . This mecdc15-2) or an analog sensitive (cdc15-as1) allele of the MEN kinase CDC15. Kar9 association with astral microtubules is asymmetric. Kar9 preferentially binds to microtubules that emanate from the old SPB, that is destined to move into the bud during anaphase  arrest in metaphase. During the arrest, 97% of cells move part or the entirety of the undivided nucleus into the bud. To determine whether MEN activation occurs in cdc23-1 cells in which the nucleus translocated into the bud, we analyzed Mob1 localization. In wild-type cells, Mob1 associates with SPBs when the MEN is activated during anaphase   . To prevll cycle .mcd1-1 mad1Δ control cells Nud1 T78 phosphorylation occurred during anaphase. Mitotic exit followed as judged by the disassembly of the anaphase spindle. In cells depleted for Cdc20, T78 phosphorylation was greatly delayed and diminished, irrespective of whether or not spindle elongation occurred  caused a 15 min delay in Nud1T78 phosphorylation and anaphase spindle break-down (esp1-1) and in two other FEAR network mutants: slk19Δ and spo12Δ bns1Δ . We found that overexpression of Clb2dBΔ prevented MEN activation during anaphase as judged by reduced Mob1 association with SPBs . Exit fr to SPBs . These dcdc20-1 or cdc23-1 mutations. To lower mitotic CDK activity we introduced the temperature sensitive clb2-VI allele into these strains and examined exit from mitosis. To measure exit from mitosis we analyzed Cdc3 localization. Cdc3 is a component of the septin ring that dissociates from the structure in response to mitotic CDK inactivation during exit from mitosis . Release of NLSCdc14 from the nucleus into the cytoplasm depended on MEN activity, validating this novel MEN assay , we also observed translocation of NLSCdc14 from the nucleus into the cytoplasm . CDC13 is required for telomere function. In its absence large amounts of single stranded DNA accumulate at telomeres, which causes activation of the DNA damage checkpoint  prevents their phosphorylation by mitotic CDKs.Previous work had established that the MEN kinase Cdc15 has 7 CDK phosphorylation sites ; Mob1 hacdc15-7A and mob1-2A mutants to exit from mitosis in a cdc20-1 metaphase arrest. We found that 21% of cdc15-7A cdc20-1 double mutants were able to exit from mitosis  a SPB enters the bud and (2) mitotic CDK activity has decreased. We propose that regulating the MEN signaling cascade in series decreases susceptibility to noise; a downstream dependence on CDK inactivation insulates the MEN against spindle movement prior to anaphase. Our work also provides an explanation for why signaling pathways are multi-step processes. Signal integration rather than signal amplification may be the reason for why signal transduction pathways are multi-step processes.Positive feedback loops, such as the mutual regulation between Cdc14 and the MEN  as well Observations of MEN activation prior to anaphase clarify the importance of the feedback loops governing MEN activity. When a SPB moves into the bud during metaphase a slight, but appreciable MEN activation occurs. Small amounts of Mob1 localize to the SPBs, yet signaling strength (as judged by Mob1 association with SPBs) is weak and inconsistent, never leading to release of Cdc14 from the nucleolus. This emphasizes the importance of the FEAR network and mitotic cyclin degradation at the metaphase – anaphase transition. Without the FEAR network, the Cdc14 positive feedback loop is disrupted, slowing MEN activation by 20 min. Likewise, without APC/C-Cdc20 degradation of Clb2 and Clb5 at the metaphase – anaphase transition, the double-negative feedback loop between mitotic CDKs and the MEN is too heavily weighted towards CDK activity, preventing MEN activation.The initial characterization of MEN regulation focused on the MEN GTPase Tem1. Tem1 was found to make the MEN responsive to spindle position and DNA damage . Our worcdc15-7A mob1-2A double mutant indicates that control of the MEN kinases protects cells from spindle movements into the bud such as occur when cells arrest in metaphase for prolonged periods of time. It is worth emphasizing that movement of the nucleus into the bud during metaphase arrests is not merely a byproduct of cell cycle arrests brought about by mutations in genes critical for anaphase entry. DNA damage or microtubule – kinetochore attachment defects can arrest cells in metaphase for hours. Indeed, our live cell analyses show that wild-type cells arrest in metaphase at an appreciable frequency presumably due to light-induced DNA damage. In 67% of these arrested cells the spindle moves in the bud. Our data further suggest that even transient excursions of the spindle into the bud during metaphase could lead to inappropriate Tem1 activation. 15% of cdc15-7A mob1-2A double mutants exited mitosis during the metaphase arrest without obvious and hence inferred transient movement of the nucleus into the bud. However, we cannot exclude the possibility that the mitotic exit observed in these 15% of cells is due to partial loss of spindle position sensing. In either case our findings indicate that inhibition of the MEN kinase cascade by mitotic CDKs prevents inappropriate mitotic exit prior to anaphase.The phenotype of the cdc15-7A mob1-2A double mutant was similar to that of the clb2-VI mutant, it is possible that mitotic CDKs regulate additional MEN components. Mass spectrometry studies have detected phosphorylation consistent with modification by CDKs on many MEN components and regulators including Bfa1, Cdc5, Cdc14, Lte1, Nud1, Tem1, and Dbf2 (Although the phenotype of the and Dbf2 . The MENThe MEN is a member of the Hippo pathway family (Reviewed in S. cerevisiae strains in this study are derivatives of W303 (A2587) and are listed in All Plasmids used in this study are listed in z sections 1 μm apart were acquired and maximally projected.The growth conditions for live cell imaging are described in the figure legends. Cells were imaged on agarose pads  medium containing 2% glucose, unless otherwise noted) affixed to a glass slide and covered with a coverslip. The microscope platform was a DeltaVision Elite  consisting of an InsightSSI solid state light source, an UltimateFocus hardware autofocus system and a model IX-71, Olympus microscope controlled by SoftWoRx software. A 60X Plan APO 1.42NA objective and CoolSNAP HQ2 camera were used for image acquisition. 6 cdc20-1 which never exit mitosis; Cdc14 translocation into the cytoplasm was quantified by manually segmenting the cell and measuring the coefficient of variation (CV = stdev/mean) of intensities within the cell as previously described (Image processing was performed using Volocity (PerkinElmer) software. Twenty non-mitotic cells that subsequently move at least one SPB into the bud , reachedescribed , normaliNud1-3V5 immunoprecipitation, Nud1pT78 detection, and Nud1-3V5 detection were performed as previously described  with theDbf2 kinase assays were performed as previously described  with theIndirect in situ immunofluorescence microscopy to detect Tub1 was performed as previously described  using a Primary antibody and dilution are listed under individual experimental procedures. Secondary anti-mouse and anti-rabbit antibodies were used at a 1:5000 dilution (GE). 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 has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Anna Akhmanova as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Yves Barral (Reviewer #1).Thank you for submitting your article "The Mitotic Exit Network integrates temporal and spatial signals by distributing regulation across multiple components" 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:Campbell et al. investigate the nature of the signals that trigger mitotic exit in yeast. Using a large panel of imaging and biochemical readouts, they show that mitotic exit requires the convergence of two independent signals, (1) the proper entry of a spindle pole into the bud and (2) the activation of APC, which lowers Cdk1 activity. Their conclusion is that all stages of the MEN are responsive to regulation with a spatial control on Bfa/Bub2 and CDK control of Cdc15 and Dbf2 combining to function as an AND gate to provide greater fidelity of MEN activation.Essential revisions:cdc13 mutant cells is considered, which might be a very specific case. The data should be confirmed with other methods for damaging the DNA and more rigorous analyses, for example: on Pds1 degradation in the different mutants.Three major issues should be addressed in the revision. The first is that the studies on DNA damage should be extended since currently only the case of The second difficulty of the paper is that it uses a flurry of different assays, many of which are used for the first time without describing and validating them thoroughly, and without using them consistently throughout the paper. For example, the assay with Cdc14-NLS is novel, is used only for late parts of the paper, and is not truly validated. In contrast, more direct and better established assays for mitotic exit, such as actomyosin ring contraction, are not used and it is unclear why. More consistent assays should be used throughout the paper.The third issue is that the paper requires substantial rewriting since it confuses mitotic exit and the mitotic exit network. The only readout of the network that the authors consider is the late one, namely mitotic exit. Early readouts that would be highly relevant to monitor the activation of the MEN, such as Hmt1 phosphorylation , Kar9 localization, SPB differentiation and the positioning of the metaphase spindle , phosphorylation of the kinesin Kip2  are omitted. Therefore, many conclusions about the activation of the MEN are not assayed directly, only the activation of mitotic exit by the MEN. In this respect, the published literature concerning the early functions of the MEN should be properly cited; indeed some of the findings presented in the paper have been predicted by others . Essential revisions:Three major issues should be addressed in the revision. The first is that the studies on DNA damage should be extended since currently only the case of cdc13 mutant cells is considered, which might be a very specific case. The data should be confirmed with other methods for damaging the DNA and more rigorous analyses, e.g.: on Pds1 degradation in the different mutants.cdc9-1 (DNA ligase) and hydroxyurea (nucleotide depletion). We obtained the same results as with the cdc13-1 mutant. These new data are shown in Figure 5G. Examining Pds1 levels in this experiment does not provide any additional information. Cells do not exit from mitosis when lowering CDK activity but remain arrested in metaphase. Hence, Pds1 degradation has not occurred.The study of DNA damage has been expanded to include a second mutant, The second difficulty of the paper is that it uses a flurry of different assays, many of which are used for the first time without describing and validating them thoroughly, and without using them consistently throughout the paper. For example, the assay with Cdc14-NLS is novel, is used only for late parts of the paper, and is not truly validated. In contrast, more direct and better established assays for mitotic exit, such as actomyosin ring contraction, are not used and it is unclear why. More consistent assays should be used throughout the paper.and mitotic exit. We would like to point out that we observe a remarkable consistency between assays for MEN activity  and procedures measuring exit from mitosis . However, we acknowledge that this can make the manuscript difficult to follow. Thus, we have edited the text and figures to draw attention to how the various measures of MEN activity correlate with measures of mitotic exit. Every biochemical assay is accompanied by tubulin immuno-fluorescence to assess anaphase spindle disassembly and hence exit from mitosis. To the analysis of Mob1 and Cdc14 localization we have added analyses assessing the formation of new buds, which is yet another assay to assess exit from mitosis. Thus, every experiment provides a measure of MEN activity and exit from mitosis.We believe that the strength of our manuscript is that we assay both MEN activity NLS nuclear export assay because we required an assay to measure both mitotic exit and MEN activation in the same cell.This was not possible with existing techniques. We would like to emphasize that we did validate the Cdc14NLS assay, demonstrating that it is responsive to modulating MEN but not FEAR network activity. These validations are shown in Figure 5—figure supplement 1. We expanded the description of these validations in the text.We developed the Cdc14The third issue is that the paper requires substantial rewriting since it confuses mitotic exit and the mitotic exit network. The only readout of the network that the authors consider is the late one, namely mitotic exit. Early readouts that would be highly relevant to monitor the activation of the MEN, such as Hmt1 phosphorylation , Kar9 localization, SPB differentiation and the positioning of the metaphase spindle , phosphorylation of the kinesin Kip2  are omitted. Therefore, many conclusions about the activation of the MEN are not assayed directly, only the activation of mitotic exit by the MEN. In this respect, the published literature concerning the early functions of the MEN should be properly cited; indeed some of the findings presented in the paper have been predicted by others .The Barral lab previously reported that low levels of MEN activity control Kar9 localization during metaphase and spindle pole inheritance. In our manuscript, we specifically analyzed MEN’s anaphase function that promotes Cdc14 release from the nucleolus and exit from mitosis. As requested by the reviewers we have now analyzed Kar9 localization and spindle pole inheritance in MEN mutants. This analysis is shown in new Figure 1—figure supplement 1. Unfortunately, we do not observe this previously reported metaphase function of the MEN. Neither Kar9 localization nor SPB inheritance was altered in cells lacking MEN function. We cannot explain this discrepancy besides to point to a difference in strain background. Given that we have no evidence for MEN activity during metaphase, we have focused our analyses on the well-established role of the MEN in anaphase. All this is described in a new section entitled “MEN activity is confined to anaphase”. The data are presented in new Figure 1—figure supplement 1.