Paspalum genus have emerged as a versatile model allowing evolutionary, genetic, molecular, and developmental studies on apomixis as well as successful breeding applications. The rise of such an archetypal system progressed through integrative phases, which were essential to draw conclusions based on solid standards. Here, we review the steps adopted in Paspalum to establish the current body of knowledge on apomixis and provide model breeding programs for other agronomically important apomictic crops. In particular, we discuss the need for previous detailed cytoembryological and cytogenetic germplasm characterization; the establishment of sexual and apomictic materials of identical ploidy level; the development of segregating populations useful for inheritance analysis, positional mapping, and epigenetic control studies; the development of omics data resources; the identification of key molecular pathways via comparative gene expression studies; the accurate molecular characterization of genomic loci governing apomixis; the in-depth functional analysis of selected candidate genes in apomictic and model species; the successful building of a sexual/apomictic combined breeding scheme.In the past decades, the grasses of the  Apomixis   has longIn the last 30 years, coordinated international efforts have led to the elucidation of major molecular actors controlling apomictic development . The intPaspalum L. is one of the few genera in which sexual and asexual reproductive behaviors have been characterized side by side for more than 50 years. During this period, many approaches proved to be unsuccessful, while others offered significant advances. In this review, we aim at presenting the rationale supporting the work carried out in this particular genus, and, based on our previous experience, proposing a series of advisable steps that could help to explore the molecular control of the trait in other species, through genetic, genomic, and/or breeding approaches. We expect to favor the development of other research projects, in order to boost the investigation of this biologically amazing and complex field.While apomixis and sexuality frequently coexist within a species, sometimes within a single individual or even a single ovule (facultative apomicts), some genotypes reproduce mainly by apomixis (obligate apomicts) or only by sexuality . There is no possible way to carry out solid molecular comparisons of the sexual and apomictic developmental pathways in the absence of previous knowledge on the extent of apomictic reproduction in the particular species/genotype under study. Moreover, the cytological  and the embryological  backgrounds must be carefully examined in order to select the appropriate plant material/time frame to be compared.Paspalum species was settled at IBONE CONICET, Corrientes, Argentina [Polygonum type, typically composed of an egg apparatus (one egg cell and two synergid cells), a large binucleated central cell, and a mass of proliferating antipodal cells at the chalazal pole [Paspalum mature aposporous ESs usually contain an egg cell, one or two synergid cells, a binucleate central cell, and no antipodal cells [Hieracium-type apospory  was described in only two species: Paspalum secans Hitchc. and Chase [Paspalum simplex Morong [Taraxacum-type diplospory  was reported in three species: Paspalum commersonii Lam. (2n = 6x), Paspalum longifolium Roxb. (2n = 4x) and Paspalum conjugatum Berg. (2n = 4x) [Paspalum minus E. Fourn. [Paspalum scrobiculatum L. [To provide materials for research and breeding purposes, an extensive living collection of rgentina . Cytoembrgentina . Besidesrgentina . Cytoembzal pole . Meanwhizal pole . The Pasal cells ,18,19,20nd Chase  and Paspx Morong ,23. More2n = 4x) ,25. Fina. Fourn.  and Pasplatum L. . P. secans [P. conjugatum [P. minus [In polyploid apomictic individuals, microsporogenesis is characterized by a statistically significant increase of asynapsis/desynapsis, lagged chromosomes, chromosome bridges, and micronuclei . Such ab. secans , P. conjnjugatum , and P. P. minus . Paspalum species characterized so far are pseudogamous, i.e., they require the fertilization of the unreduced polar nuclei by a reduced male gamete to form the endosperm and a viable seed. The number of polar nuclei involved in pseudogamy is of practical importance in apomixis research, particularly when the mode of reproduction is determined by the flow cytometric seed screen (FCSS) method [Paspalum notatum show a 2:5 embryo:endosperm ratio (4x embryo and 4x + 4x + 2x = 10x endosperm). This method was successful during the last two decades for determining reproductive behaviors in individual or bulked seeds of several Paspalum species [Paspalum apomicts is the lack of deleterious response to deviations from the 2:1 maternal-to-paternal genomic ratio in the endosperm strictly required for sexual reproduction [All apomictic ) method . This me species ,36,37,38oduction . Typicaloduction . Paspalum species showed multiple cytotypes with variable ploidy levels. Out of them, 22 (81.48%) displayed sexuality at lower ploidy levels and apomixis at higher ones. The exceptions were Paspalum conspersum Schrad. ; Paspalum distichum L., P. secans (Itchc. & Chase), and Paspalum proliferum Arechav ; P. scrobiculatum L.  [Paspalum accessions allowed confirmation of the reproductive mode/ploidy level for several of the above-mentioned species and provided new information on the reproductive mode for 12 additional ones and one botanical variety [Paspalum entities belong to multiploid species of autoploid origin. Each multiploid contains a sexual self-sterile diploid cytotype and a series of aposporous apomictic autopolyploid cytotypes, usually from 3x to 6x, with tetraploids as the most common cytotype. The two main Paspalum species used as models for apomixis research, P. notatum and P. simplex, form agamic complexes made up of diploid sexual and autopolyploid apomictic individuals [Moreover, the study of the relationship between apomixis and ploidy is of importance when characterizing the biological materials to be used in apomixis research. For instance, according to the data reviewed by Ortiz et al. , 27  . Consequ variety . Most apividuals .Paspalum, the species that received most attention are P. simplex, P. notatum, Paspalum rufum, and Paspalum guenoarum. All of them grow as wild native populations in the argentine Litoral region, located within a limited radius around several local laboratories devoted to Paspalum apomixis research, which eases the collection and classification of novel germplasm. The genetic structure of some of these populations was carefully characterized, and sexual diploid individuals were proposed as the main genetic variability source, since they co-habit and hybridize with polyploid individuals [Paspalum species) are perennial and flourish during the warm season, are well represented in rangelands used for cattle production systems, and cultivated within and without the natural distribution region to be used as forage, turf, and cereal [Paspalum species are among the smallest within the Poaceae tribe, ranging from 0.550 pg in P. notatum to 0.900 pg in P. guenoarum [Once a significant number of materials were subjected to cytogenetic and cytoembryological examinations, the next step to be followed is the selection of those species/genotypes in which sexual and apomictic development will be analyzed from the genetic and molecular points of view. In the case of ividuals ,41,42. Ad cereal . Finallyuenoarum .P. notatum sexual diploid genotype and a newly formed sexual autotetraploid derivative revealed that at least 0.49% of the pre-meiotic floral transcripts changed their relative expression after an increment of ploidy [Paspalum flowers, as was revealed recently from RNAseq experiments [Chiefly among the points deserving consideration in the selection of the species for molecular analysis is the availability of natural or induced materials of identical ploidy and contrasting reproductive modes. These resources are essential for the subsequent establishment of useful populations segregating for apomixis, which will be used in inheritance analysis, genetic/epigenetic mapping, omics surveys, and breeding. Moreover, the comparison of sexual and apomictic developmental pathways using genotypes of different ploidy levels is directly not advised, simply because ploidy may affect expression levels independently of reproductive behavior at numerous loci ,44. Regaf ploidy . Accordieriments , ploidy-eriments . These eeriments , who selPaspalum, apomixis occurs mainly at polyploid levels and sexual counterparts of the same ploidy are not available in nature. Therefore, colchicine treatments were required to duplicate the chromosome content of sexual diploids [Paspalum plicatulum and P. notatum revealed that, immediately after polyploidization, genetic rearrangements affected 28–38% and 9.55% of the genome, respectively [Eragrostis curvula [P. notatum [In several species of diploids ,50,51,52diploids . Comparaectively ,55. More curvula  and P. n notatum . It shou notatum ,57.P. simplex [P. notatum [P. rufum [Finally, comparative transcriptomic and epigenetic surveys require the establishment of detailed timelines on reproductive development , to maximize the discovery of true differential representation results. Several reproductive calendars were established by correlating morphological features, e.g., ovary/ovule size and morphology, as well as mega- and microgametophyte developmental stages in  simplex , P. nota notatum , and P. P. rufum .In summary, as schematized in Understanding and exploiting the advantages apomixis represents for plant breeding requires detailed knowledge of the genetic and molecular bases that control both its inheritance and expressivity. Although genes governing specific reproductive steps have already been engineered into plants and artificial systems can mimic clonal reproduction ,63,64,65Paspalum, inheritance analyses were unfeasible until fully sexual tetraploid individuals were available [P. notatum [P. simplex [P. plicatulum [P. simplex and P. plicatulum were also used as female parents in interspecific crosses with different members of the Anachyris subgenus and the Plicatula group, respectively, widening the genetic studies to other species of the genus [In vailable . Indeed,vailable  could novailable . Towards notatum , P. simp simplex  and, moricatulum . These gicatulum ,53,67,68he genus ,70.1 and BC1P. notatum progenies showed that apospory is inherited as a simple dominant trait (Aaaa). However, distorted segregations were often observed in favor of sexuality (aaaa) with apomixis:sexuality ratios varying from 1:1 (expected) up to 1:9 [P. simplex, the segregation evidence indicates that apomixis is under the control of a single dominant allele, whose transmission is biased towards sexuality, while mapping reveals an association between the Apomixis Controlling Locus (ACL) and the telomeric region of the long arm of rice chromosome 12 [1 and F2 generations and three backcross populations derived from an artificial tetraploid sexual clone of P. plicatulum and an apomictic P. guenoarum cv. Rojas plant confirmed the previous genetic model, with a single Mendelian dominant factor with altered transmission (1 apo:1.6 sex) [P. simplex x Paspalum malacophyllum (1:5) [P. simplex x Paspalum procurrens (1:15.7) [P. notatum.Genetic analyses of Fp to 1:9 ,73,74,75osome 12 ,76. Inhe1.6 sex) . Interesum (1:5)  and P. s(1:15.7) . Moreove(1:15.7) . The lar(1:15.7)  is to daPaspalum showed a strict co-segregation between apospory, parthenogenesis, and unbalanced endosperm formation, suggesting that their relevant genetic determinants are located in the same low recombining chromosome area [All genetic analyses in ome area ,67,79. Tome area . P. notatum, the analysis of several segregating populations generated using an apomictic male progenitor revealed a high variability in the proportion of ovules bearing AESs among the hybrids [P. notatum, apospory expressivity can also show seasonal variation, reaching its maximum during peak flowering (summer) and decreasing in fall [Paspalum cromyorrhizon [P. simplex, as artificial wide-genome demethylation significantly reduced parthenogenesis but had no effect on apospory [Another central topic demanding special attention is the transmission of the apomixis expressivity capacity. Usually, apomictic individuals are classified as obligate when almost all ovules (>90%) show one or several aposporous embryo sacs (AESs), or facultative, when at least some ovules bear meiotic or mixed  female gametophytes . In P. n hybrids ,74. In m hybrids . Moreove hybrids . At leas in fall . A similorrhizon . Althougorrhizon . The aboorrhizon . Parthenapospory . Therefoapospory . Paspalum species form multiploid complexes composed of self-sterile sexual diploid and self-fertile pseudogamous apomictic polyploid cytotypes [P. notatum and P. rufum, including aposporous-like embryo sacs (AES-like) reported at the diploid level [P. notatum ACL was detected in the genome of 10 out of 57 diploid plants from a natural population . These results support the hypothesis that the factor/s controlling apomixis might occasionally occur in diploid individuals, but they remain silent until they are activated in response to hybridization and/or polyploidization.It is a well-known fact that many ytotypes . Howeverid level ,50,84; sid level ,52; artiPaspalum ACL, although quite simple in genetic terms, might be physically complex at the molecular level, as it was exposed by associating it to grass model species markers of defined map positions [Paspalum species  and a 6–10 Mb region of rice chromosome 12, as well as a segment of 10 Mb of rice chromosome 2 (for P. notatum only) [Anachyris subgenus show macrosynteny with the above-mentioned segment of rice chromosome 12 [P. notatum is a hybrid syntenic group resembling segments of chromosomes 12 and 2 of rice [The ositions ,77,85,86ositions ,83,86. Cosome 12 ,77, wherP. simplex bacterial artificial chromosomes (BACs) isolated with the aid of specific primers. Partial sequencing of one of them, corresponding to the ACL, revealed that although synteny at the marker level was conserved with respect to the rice genome, gene micro-collinearity was frequently interrupted with transposable elements and migrant genes from other rice chromosomes [P. simplex ACL is conserved among five reference grass species, being located at a telomeric position in chromosomes 12, 8, 3, and 4 of rice, Sorghum, Setaria, and Brachypodium, respectively, and in a more centromeric region of maize chromosome 1  sex-related genes were grouped by gene migration during speciation, (ii) a polyploidization event (through an intermediate triploid bridge) locally induced further small-scale rearrangements that, in turn, generated local sequence divergence, lack of chromosome pairing, and recombination blocking. This non-recombinant segment favored the accumulation of mutations, since they could not be discarded by meiosis. This kind of evolutionary and structural organization may have consequences on gene content and expression. Indeed, Polegri et al. [A deeper analysis of the ACL structure arose from the availability of apomixis-specific omosomes . More reomosomes  sequencemosome 1 . Based oi et al.  noticed i et al. ,90. Paspalum is that related to the Y-chromosome of dioecious plants, which originated from autosomal chromosomes by initial suppression of recombination in the regions containing the sex controlling genes and later on, by the migration of male determining genes [P. simplex is superimposed over fertilization-mediated embryo development by a mechanism controlled by DNA methylation. Furthermore, several P. simplex apomixis-linked genes expressed sense and antisense transcripts in reproductively committed cells, and showed a putative silencing effect of the apomixis-linked alleles on their sexual-specific counterparts [The expression of the ACL genes seems to have common features with operon-like gene clusters, which are defined as a set of two or more non-homologous functionally related genes that share a close genomic neighborhood . Operonsng genes . From a ng genes . This fing genes  showed tterparts ,90.Paspalum spp. is under the control of a single genomic region, the intrinsic characteristics of the ACL  hampers the identification of the trait’s key determinants through positional mapping strategies. In this scenario, a combination of transcriptomic and genetic/genomic information could provide hints on the primary or secondary role of DETs and be the method of choice for identifying genes and molecular routes involved in both activation and progression of the trait. In addition to the identification of key triggering factors, our knowledge on apomixis should be complemented by the understanding of the molecular pathways involved in subsequent developmental steps. Although genetic analyses suggest that apomixis in However, several concerns emerge for comparative transcriptomic analyses using apomictic/sexual systems: (i) the genetic nature of the materials, since comparisons usually involve polyploid, highly heterozygous individuals, which complicates the distinction between differential expression and genetic polymorphisms in PCR-based analyses as well as the classification of homologs  in RNAseq approaches, in particular if no genome sequence is available; (ii) the sample collection timing, because the rapid progression of the RNA landscape along with reproductive development might cause the frequent emergence of false DETs; (iii) the short- and long-term transcriptome responses after hybridization and polyploidization, since some of the materials used might have recently originated from colchicine treatments, while others emerged from natural whole genome duplication events in either ancient or relatively recent times . UnravelP. notatum since the early 2000s produced a large list of genes whose expression correlate with the occurrence of key reproductive features. An initial comparative examination of bulked RNAs from spikelets of sexual and apomictic F1 hybrids at anthesis was carried out by differential display and led to the identification of one transcript (ARP1) encoding an KSP consensus domain previously detected in several cdc 2-regulated cytoskeletal proteins [LORELEI family (GAP1) [TRIMETHYLGUANOSINE SYNTHASE1) [MAP3K YODA family (QUI-GON JINN [SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) [Transcriptomic surveys carried out in proteins . A seconproteins , includiy (GAP1) ,96, the NTHASE1) ,97; the GON JINN ,98 as weGON JINN ,99, someE (SERK) ,99,100, E (SERK) . FurtherE (SERK) ,103. P. simplex cv. Morong, the use of cDNA-AFLP on RNA extracted from flowers at several developmental stages, from premeiosis to 3–6 days after anthesis, rendered 202 DETs, the majority of which were present in apomictic florets only [P. simplex DETs belong to the same annotation classes of those reported for P. notatum, including extensins, YODA-like MAP3Ks, LRR-like proteins, transferase proteins, and retrotransposon proteins.In ets only . Near 20ets only . The remP. notatum spikelets by using the long-read Roche 454-FLX + technology [In the last decade, progress in DNA and RNA sequencing have provided biologists powerful tools to study and understand gene functions and interactions. However, the establishment of genomes/transcriptomes from genetically poorly characterized, polyploid, and highly heterozygous species might easily result in chimeric assemblies and/or fragmented transcripts ,105,106.chnology . Out of chnology . Since tP. notatum using Illumina sequencing of RNA samples extracted from leave and florets of 2x sexual, 4x sexual, and 4x apomictic genotypes. The database contains 114,306 reference transcripts, 536 of which correspond to genes possibly associated with apomixis. Interestingly, 89 differentially expressed transcripts mapped onto rice chromosome regions syntenic to the ACL [P. notatum, we generated Illumina TruSeq floral RNA libraries in triplicate, from sexual and apomictic materials collected at four different developmental stages, from premeiosis to anthesis (NCBI repository SRA accession: PRJNA511813). Data analysis  is on-going. While long-read Roche 454-FLX + sequencing produced genuine transcript assemblies, the derived quantitative expression comparisons lacked statistical accuracy . Recently, De Oliveira et al.  reported the ACL . MoreoveP. notatum sexual and apomictic floral transcriptomes (including siRNAs and miRNAs) and their possible target genes were characterized in detail [Finally, the short-sequence components of the n detail . A totaln detail , openingPaspalum, points out hypotheses as diverse as polyploidization and hybridization [Arabidopsis and rice, have already provided valuable information regarding the role of some candidate genes, their positioning into a comprehensive genetic network controlling apomixis remains elusive to date.As mentioned in previous sections, the mechanisms for establishing apomictic lineages from sexual ancestors and the evolutionary consequences of their emergence remain poorly understood and largely speculative. Mining the large body of knowledge gained in apomictic species, including dization ,109,110,dization ,112, oxidization , functiodization ,98,114, dization ,115, altdization , and hordization . AlthougPennisetum [Paspalum [Furthermore, apomictic plants usually have polyploid genomes, or possess aneuploid chromosome complements of highly heterozygous nature. Genetic approaches have revealed a singular genomic organization, characterized by a large, hemizygous and non-recombinant region of heterochromatic nature named Apomixis Controlling Region (ACR) in nnisetum  or ApomiPaspalum . Such gePaspalum ,118,119.Paspalum ,121, assBoechera retrofracta [E. curvula [Paspalum species and agamic complexes [Paspalum genomes are relatively small, as 2C values range from 0.5 to 6.5 pg [Paspalum ACL has revealed various extents of synteny with the subtelomeric part of the rice chromosome 12 long-arm [Paspalum species. Once assembled and annotated, we expect to molecularly resolve the ACL and determine its evolutionary trends; to re-analyze Paspalum transcriptomic resources, and to generate and compare epigenomes of sexual and apomictic plants. Finally, this effort will generate spillovers of great interest for forage grass breeding by providing critical genomic resources and allowing efficient molecular breeding, an important issue considering the need to mitigate both climatic change and anthropic pressures in economically important grassland and pasture agro-ecosystems.To date, a few genomes of sexual relatives of apomictic plants have been sequenced, including rofracta  and E. c curvula , providiomplexes ,46,108 mo 6.5 pg . Intereslong-arm ,88, and long-arm ,87. ReceAfter identifying candidate genes, the next rational step to advance apomixis research further consists of the establishment of functional analyses aimed at investigating their capacity to activate at least some steps of the trait. Those transcripts displaying differential regulation in apomictic/sexual backgrounds can be readily investigated through reverse genetic approaches, by using defective mutants or sense/antisense/RNAi transformants, since their down- or upregulation within the required genetic background would allegedly cause the emergence of apomixis-related phenotypes. Unfortunately, the absence of wide range germplasm banks for natural apomictic species forces either the prediction of orthology in model species or the adjustment of gene transformation techniques.Paspalum are as follows: (1) previous spatio-temporal and sense/antisense expression validation of candidate genes with a protocol allowing cell-specific expression detection and distinction of the expressed RNA strand, like in situ hybridization; and (2) generation of overexpression/downregulation transformants with an efficient transformation platform, or identification of mutant germplasm involving model species orthologs. While the first step  will provide information useful for planning modulation (up- vs. downregulation), selecting the required reproductive background  and guiding the promoter choice, the second one (mutant/transformant germplasm identification or generation) will facilitate the analysis of possible reproductive phenotype alterations. Based on these considerations, the steps to be followed when launching the functional characterization of an apomixis candidate gene in non-model apomictic/sexual systems like A point that deserves special attention is that, at least for many candidate genes, wide-range expression is associated with different functions in a multiplicity of organs/developmental stages, and only a subtle, timely, and cell-specific activity change might produce a deviation in the reproductive mode without causing collateral, sometimes detrimental, consequences. Therefore, when intending transformation, gene promoters of choice might be preferably time and organ/cell-type specific. Even if basic research could take advantages from additional information derived from constitutive promoter-based functional analysis, due to its potential to reveal the candidate function in numerous organs/tissues, the identification and effective use of cell-specific promoters is no doubt a bottleneck for apomixis-based breeding, which requires the modification of a restricted function set. If the project aims at generating a methodology to induce apomixis for breeding purposes, it should contemplate the identification, isolation, and/or validation of appropriate cell-specific promoters in the species of interest. On the contrary, when dealing with functional characterization of the gene, a constitutive promoter with verified expression in the ovule could be of choice, unless it causes unviable phenotypes due to deleterious side effects. Paspalum, and gradually optimized to analyze the spatio-temporal plus sense/antisense expression distribution within the ovule [In connection with this, in situ RNA hybridization protocols were developed for he ovule ,100,101.Paspalum genotypes. Such methodology is currently being used to produce Paspalum lines with up- or downregulated expression of apomixis candidates, which are later subjected to reproductive phenotype analyses. An example of the cytoembryological characterization of an antisense transformant line with downregulated expression of the apospory-inducer candidate gene QGJ is shown in Paspalum promoters identified from genome sequencing is currently under analysis . All these methods have allowed the functional characterization of three reproductive candidate genes  in species of Paspalum (see descriptions below), and several others are undergoing the same process.Regarding the development of protocols for modulating the expression of candidate genes in natural apomictic systems, Mancini et al.  considerORC3 (ORIGIN OF RECOGNITION COMPLEX 3), identified as PsORC3a, resulted genetically linked to apomixis in all Paspalum spp. for which segregating populations were available [P. simplex, ORC3 exists as three different copies, of which PsORC3a is a pseudogene specific for apomicts expressing an RNA transcript unlikely to be translated in a functional protein, whereas PsORC3b, probably coding for a highly conserved functional protein, together with PsORC3c, coding for a truncated protein, are common to both apomictic and sexual plants [PsORC3a is poorly and constitutively expressed at all developmental stages in apomictic flowers only. In situ analysis showed that, in apomictic plants, sense and antisense strands of PsORC3 are represented in cells and nuclei committed to reproduction , whereas both transcripts are silenced in the endosperm. Conversely, in sexual plants this gene is expressed as a sense transcript in the egg cell, polar nuclei, and endosperm, but not in the embryo [Arabidopsis and rice showed that ORC3 defective genotypes display normal gametophyte development, but endosperm/embryo arrest at early stages of development [PsORC3 (PsORC3a) could be involved in a relaxation of control mechanisms, which allow endosperm development even facing a maternal genome contribution excess [A particular copy of the gene vailable ,128. In l plants . PsORC3ae embryo . Reverseelopment . Based on excess . P. notatum Roche 454-FLX + floral transcriptomes were used to recover N46 full cDNA sequences and carry out a molecular phylogenetic analysis [YODA MAP3K family and renamed QUI-GON JINN (QGJ). At meiosis, in situ hybridization analysis revealed an altered pattern of expression in P. notatum apomictic plants, which could be analyzed at even more detail in apomictic Brachiaria brizantha [QGJ was strongly expressed at micropylar degenerating megaspores, in apomictic ones it showed activity within the enlarged meiocyte and the distal ovule nucellus, but was absent from the cell layer surrounding the meiocyte, from which apospory initials (AIs) originate. The effect of a QGJ diminished expression in an apomictic background was further investigated by producing RNAi lines [QGJ expression in an obligate apomictic background impaired the formation of AESs and, therefore, the expression of QGJ in distal nucellar cells is needed for aposporous development [QGJ and the ACL, but a long non-coding RNA partially related in sequence to QGJ (LNC_QGJ) cosegregated strictly with apomixis when mapped in a P. notatum segregating population [LNC_QGJ specific primers, which were conducted in several apomictic and sexual P. notatum individuals, showed that LNC_QGJ is expressed only in apomictic plants. The existence of a functional link between the particular QGJ expression pattern detected in apomictic plants and a putative regulatory long non-coding LNC_QGJ activity operating from the ACL should be further investigated [The transcriptomic surveys carried out by Laspina et al.  identifianalysis . N46 wasrizantha . While iAi lines . RelativAi lines . The proAi lines ), the coAi lines . The conelopment . Geneticpulation . Moreovestigated .TRIMETHYLGUANOSINE SYNTHASE 1 (TGS1) gene, which was named PN_TGS1-like [PN_TGS1-like showed expression levels positively correlated with sexuality rates. Moreover, it displayed contrasting in situ hybridization patterns in apomictic and sexual plant ovules from premeiosis to anthesis [PN_TGS1-like might be preventing the differentiation of apospory initials in sexual P. notatum plants [P. notatum genotype was transformed with a TGS1-like antisense construction under a constitutive promoter, to obtain lines with a reduced transcript representation [Paspalum unreduced megagametophyte morphology, including an egg cell, one-two synergids, two polar nuclei, and no antipodal cells). Moreover, around 9% of ovules showed a combination of meiotic and aposporous-like sacs. At early developmental stages, 32% of ovules displayed nucellar cells with prominent nuclei resembling apospory initials (AIs) surrounding the megaspore mother cell (MMC) or the MMC-derived meiotic products. Occasionally, immature binucleated (FG2) female gametophytes of the aposporous type  were detected [PN_TGS1-like might also be involved in either embryogenesis or endosperm development. These results suggest that PN_TGS1-like is a developmental repressor, whose expression in leaves blocks the formation of trichomes, while in ovules inhibit the onset of apospory initials and/or the progression of gametophytes. However, it does not influence parthenogenesis, even when it might play an unknown role during embryogenesis.After identification of an apomixis-associated DET (N69) homologous to a PRIP-interacting methyltransferase S-adenosyl domain protein by Laspina et al. , this caGS1-like . In facuanthesis , with him plants . Thereafentation . Antisendetected . Neitherdetected . MoreovePaspalum forage grasses, which have been selected and improved by classical and molecular methods over 80 years. The diversity present in the genus Paspalum for the modes of reproduction and ploidy levels is directly linked to adaptation to different environmental conditions and variation for a large group of traits of agronomic interest. Since the 1940s, near 30 apomictic cultivars belonging to the genus Paspalum have been released mainly for forage or turf [P. simplex [The identification of molecular markers (MM) cosegregating with apomixis, the generation of artificial sexual polyploids after colchicine duplication, the construction of transcriptome databases and, in a close future, genomic assemblies, the identification of genes controlling the reproductive mode, and the establishment of biolistic transformation platforms offer good and innovative prospects for harnessing reproductive and non-reproductive traits of interest in  or turf . All of  simplex . However simplex .Paspalum species form agamic complexes [P. notatum [P. simplex [P. notatum indicates that only a reduced fraction of the progeny (around 10%) inherits the full capacity to express apomixis [P. simplex [P. notatum [P. plicatulum and P. guenoarum [P. notatum identified as upright and fast-growing was recently released as a forage cultivar named Boyero UNNE, resulting from a collaborative research between the University of Florida and the National University of the North-East, Argentina [The majority of the omplexes . The relomplexes ,49,50,51 notatum ,73,74,75 simplex  and spec simplex . The expapomixis ,73,74 anapomixis  and the apomixis . There i simplex . In the  simplex . Neverth simplex  or by cr simplex . As a ge simplex . Heteros notatum  and for uenoarum ,135. An rgentina . Paspalum, particularly for the identification of true hybrids within progeny [P. notatum [P. simplex [Paspalum species, including the Plicatula group, makes the use of cytometric seed analysis an attractive screening technique for determining the reproductive mode [MMs have been used to monitor the process of hybridization between sexual and apomictic genotypes in  progeny ,74,131.  progeny . Moreove progeny ,85,137.  notatum  and P. s simplex . Moreove simplex . This is simplex . Althougive mode . Paspalum. Since the main objective of hybridization in apomictic species is to combine high levels of apomixis with heterosis for traits of agronomic interest, Marcón et al. [P. notatum. Crosses were made between pairs of sexual and apomictic parents with low, intermediate, and high genetic distances. As expected, higher genetic distances between parents increased the proportion of hybrids exhibiting heterosis for forage yield. Moreover, the same positive relationship was observed between genetic distances and the proportion of aposporous hybrids identified within the progeny. However, apospory expressivity was not significantly related to the genetic distance. These results suggest that random MMs may be successfully used to determine heterotic groups for forage yield in P. notatum, but also to predict a high occurrence of apomictic hybrids within segregating families. Furthermore, as part of a hybridization project in the Plicatula group, the genetic distances between an induced sexual genotype of P. plicatulum and 22 accessions belonging to 12 different species were determined using AFLP markers [MMs have also been used to identify heterotic groups in n et al.  used ISS markers . In thisP. notatum to increase the sexual tetraploid gene pool, as follows: (1) crossing the few available sexual tetraploid genotypes with a group of ecotypes collected across the Americas, and (2) polycrossing the resulting highly sexual hybrids to create the SSTP. This procedure allowed the transfer of the genetic diversity present among geographically and genetically distant ecotypes into a single sexual and cross-pollinated population [P. simplex or other species of the subgenus Anachyris.A synthetic sexual tetraploid population (SSTP) was established in pulation ,139. Morpulation . In thisBrachiaria spp. [P. notatum. Both methods proved to be efficient for improving forage yield in P. notatum. Although RSCA was expected to be more appropriate for exploiting heterosis since it was developed to accumulate heterotic effects over cycles, no difference was observed between the RPS and RSCA procedures for increasing forage yield [P. guenoarum. This approach identified a few sexual hybrids as possible progenitors to continue tapping into genetic diversity of apomictic individuals or to generate improved sexual populations.The availability of sexual tetraploid populations has allowed the application of breeding methods originally developed for cross-pollinated crops, such as maize or alfalfa, as was originally suggested for apomictic tetraploid ria spp. . Marcón ria spp.  evaluatege yield . A modifge yield . In thisP. notatum is represented in As was previously stated, molecular techniques may play an important role in the different phases of the selection procedures for generating superior apomictic hybrids. A theoretical scheme of RSCA developed for tetraploid P. notatum, since growth and forage production is markedly reduced during the winter in the subtropics. Once phenotypic and molecular data is available for selecting the best progeny, the apomictic genotypes may be separated using apomixis-linked markers. Superior apomictic hybrids usually need to go through a multiphase forage evaluation procedure [Random molecular markers may aid the identification of heterotic groups for the initial phase of the process  germplasm groundwork, including collection of variable materials, reproductive phenotyping, ploidy level assessment, genome content characterization, ploidy manipulation and reproductive calendar construction; (2) inheritance analysis, dense genetic mapping, and identification of markers cosegregating with apomixis; (3) genome sequencing; (4) transcriptome sequencing; (5) functional analysis in sexual and apomictic species; (6) traditional and molecular marker-assisted breeding to release élite apomictic hybrids. The molecular elucidation of the sexual–asexual transition would require the use of this methodological scheme in as many species as possible, in order to expose subtle variants that might have emerged during evolution and provide a comprehensive perspective of alternative pathways, disclosing optimal tools for harnessing apomixis into the breeding of major crops by genetic engineering. With this review, we hope to contribute to the design of the necessary experimental phases, which have been laid down in Paspalum after decades of effort, in a process that often involved trial and error. At best, the ordered management of the flowchart presented here could save both time and costs and stimulate apomixis research/breeding in other species of agronomic interest. From the


Petauroides volans) is a large, gliding eucalypt folivore. This nocturnal arboreal marsupial has a wide distribution across eastern Australia and is considered the sole extant member of the genus Petauroides. Differences in morphology have led to suggestions that the one accepted species is actually three. This would have substantial impacts on conservation management, particularly given a recent history of declining populations, coupled with extensive wildfires. Until now, genetic evidence to support multiple species has been lacking. For the first time, we used DArT sequencing on greater glider tissue samples from multiple regions and found evidence of three operational taxonomic units (OTUs) representing northern, central and southern groups. The three OTUs were also supported by our morphological data. These findings have important implications for greater glider management and highlight the role of genetics in helping to assess conservation status.The identification and classification of species are essential for effective conservation management. This year, Australia experienced a bushfire season of unprecedented severity, resulting in widespread habitat loss and mortality. As a result, there has been an increased focus on understanding genetic diversity and structure across the range of individual species to protect resilience in the face of climate change. The greater glider ( This is especially true in the wake of a natural disaster, when wildlife management decisions need to be made quickly and under challenging circumstances.Effective conservation management relies on accurate taxonomic classification and a robust understanding of the genetic structure of populations. A lack of knowledge about the genetic structure of species across their range can result in an inability to properly manage and protect species from extinction2 and directly or indirectly killed millions of native animals2. The impacts of fire on genetic diversity are nontrivial3, and the extent of the recent fires means that substantial portions of many species’ ranges were impacted2. As a result, the conservation status of a number of species are being revisited and the importance of protecting genetic diversity within species is becoming more widely recognised for its central role in conserving species’ resilience to anthropogenic climate change6.The catastrophic 2019–2020 bushfire season in Australia burnt over 97,000 kmPetauroides volans, (Kerr7) is a large, nocturnal gliding marsupial endemic to Australia  and resurrects the name P. armillatus, to apply to populations in mid-Queensland from the Eungella Range to just north of Townsville19. Petauroides armillatus was once considered a subspecies (P. volans armillatus) by Thomas in 192321 but subsequently synonymized within minor22. The multi-species taxonomy of Jackson19 and Jackson and Groves20 retains Petauroides volans as the most southerly distributed species from Bundaberg in Queensland (Qld) to Victoria (Vic), and includes two subspecies (P. volans incus and P. volans volans).Recently, JacksonP. volans, P. minor and P. armillatus are morphologically and genetically distinct at the level of species has been controversial because it lacks supporting published evidence and was referenced by Jackson and Groves20 from a personal communication with the late Dr Kenneth Aplin and a brief conference abstract by Arbogast et al.23. Neither of these sources have published data to support the distinctiveness of the proposed P. minor, P. armillatus, and P. volans. For the first time, we test Jackson and Groves’s20 three species designation using molecular sequence data, DArTseq24, and report the genetic population structure of greater gliders at multiple points across their natural range.The claim that the three taxa 25. Using linear models for unbalanced data with backward elimination method, we found that measurements for males and females in our dataset were not significantly different (p > 0.05) in seven out of eight attributes, with the exception that females had longer tails . We combined male and female data within locations for the remaining analyses. A principal components analysis (PCA) of morphological measurements broadly clustered into three groups with two samples showing some overlap , and Central and Southern greater gliders differed significantly in every attribute except ear length  and ear width  for any of the eight measured attributes . The raw data consisted of 78 individuals scored for SNP polymorphisms at 45,245 loci. Filtering, as outlined in Methods, resulted in only high-quality SNP loci being retained with high coverage across individuals and loci, ensuring completeness of the final dataset and minimising the amount of missing data. This yielded SNP calls for 11,317 loci for 75 individuals. Following this filtering, these data are considered to be highly reliable.Principal components analysis of this dataset revealed three distinct genetic groups corresponding to geographic locations of Taravale/Blackbraes (Northern), Bendoc and Wombat (Southern) and Redcliffe Vale/Museum  Fig. . Two ind27 identified four putative operational taxonomic units (OTUs) diagnosable on the basis of one or more fixed allelic differences—the Northern , and the two Southern, Bendoc and Wombat. Wombat (n = 6) and Bendoc (n = 9) were distinguished by only 2% (233) fixed differences in comparison with the other populations that differed between 11 and 30% (1373–4767). Test of significance demonstrated that the difference between Wombat and Bendoc was not significant . We therefore combined Wombat and Bendoc to yield a final three putative OTUs, representing North, Central, and Southern populations  and marginally higher than Central gliders . Northern gliders had the lowest heterozygosity (0.04), significantly lower also than Central gliders .Southern greater gliders had the highest expected heterozygosity (0.11), a measure of genetic diversity, substantially higher than Northern gliders  and the other a genotype consistent with a backcross of an F1 hybrid to the Northern group (T5)  had intermediate positions in the PCA falling between the Northern group and the Central group. Comparing these individuals with likelihood bins associated with parental genotypes, F1 hybrids, backcrosses of the F1 to the parentals, and F2 hybrids using New Hybridsn group T  representing Northern, Central and Southern groups. These broadly correspond with the species designations summarised by Jackson and Groves20 and provide the first genetic support for multiple greater glider species. The morphological measurement from the sampled greater gliders from the five study areas also align with these three OTUs. The Northern and Southern populations differed significantly in all morphological measurements, and this is in keeping with previously reported differences in the ear, body size, and pelage colouration of the currently recognised subspecies (P. volans minor and P. volans volans)16. There was some overlap between animals from the Central population in Redcliffe Vale and the other two groups in morphology, but the Central population still differed significantly in a number of measurements from the Northern and the Southern animals. We also found evidence of hybridisation between the Northern and Central groups and one of those hybrids had morphological measurements that were intermediate between those groups as well. As discussed by de Queiroz29, delimiting species is complicated by the numerous species concepts. The Genetic Species Concept recognises that species are genetically isolated but not reproductively isolated, allowing for hybridisation, which is likely to occur during the speciation process30.Greater gliders are currently classified as a single species, Petauroides volans has a long and complicated taxonomic history, which has been extensively reviewed by Maloney31 and Maloney and Harris32. The greater glider was first identified to western science by Kerr7, with further variability in morphology among collected individuals described by Collett18 who suggested a “variety” called Petauroides volans minor based on differences in skull size, body length and pelage colour. The greater glider was more formally described as one species by Oldfield Thomas21 with four subspecies 31. Petauroides v. armillatus was then synonymised with P. v. minor in 1934 by Iredale and Troughton who also changed the genus to Schoinobates resulting in three renamed subspecies S. v. minor, S. v. incanus, and S. v. volans (previously typicus)20. Petauroides was restored by McKay in 1982 who also synonymised P. volans incanus with P. v. volans in 198822. Based on personal communications with the late Dr Kennith Aplin and unpublished data in a conference presentation by Arbogast et al.23, Jackson and Groves20 recognised three species within Petauroides and two subspecies. Until now, there has been no published genetic evidence to support either multiple or single species designations, and P. volans has been legislated for the purpose of management and conservation as a single species14. Our results support a population structure consistent with the multi-species designation of Jackson and Groves20, with a Northern, Central and Southern species of greater glider. We are not able to resolve subspecies structure from our data.19, there are some notable differences. In particular, the Central OTU that corresponds with the proposed “central” species, P. armillatus, extends much farther south than anticipated based on the distribution described in Jackson19. Although we had a relatively small, non-continuous sample, it is also worth noting that we found no evidence of the Southern OTU that corresponds with P. volans in Queensland, despite the range proposed by Jackson19 extending to Bundaberg. There is no information provided by Jackson19 to explain how the distributions in that text were determined. Notably, Jackson’s distribution for P. volans did not agree with the range proposed by his source, Arbogast23, who does not extend the distribution of P. volans into Queensland, and that agrees with our data.While the three OTUs roughly align with the species designations described by Jackson33, and we have been cautious in interpreting our data in the context of existing work that defines the three species and draws support from the SNP data20. In particular, we have not attempted to describe the new taxa de novo using genetic data alone, but rather to examine the genetic data to see if it is consistent or inconsistent with the taxonomic concepts put forward by Jackson and Groves20 of three species. Our data are certainly inconsistent with the samples representing panmictic or close to panmictic populations, and in the view of the magnitude of the observed fixed differences and the very substantial counts of private alleles held in each of our three OTUs, the genetic divergences we observed are very unlikely to be explained by punctuated sampling along a cline. Additional surveys should be undertaken to obtain a more complete understanding of genetic structure across the range of greater gliders. While our data does not enable an exhaustive assessment of Petauroides systematics, it does provide the first genetic evidence to support multiple extant species in Petauroides, and this finding has important conservation implications.An important caveat is that the findings from this study arose in the process of conducting unrelated research to look at relationships between variations in physiology and climate at the northern and southern distribution of what was thought to be one species at the time . Given the distinct morphological differences between the northern and southern samples, we decided to use genetics to investigate the population structure. We did not comprehensively sample greater gliders throughout their range and no samples were obtained in New South Wales. Hypothesised species can represent arbitrary slices across geographic clines12. Population declines have been attributed to the cumulative effects of land clearing and climate change, including logging, lower than average rainfall and increased occurrence of intense fires36. This year, the most destructive wildfire season on record burned through substantial portions of greater glider habitat in south-eastern Australia. The extent of greater glider habitat impacted by fire and repercussions to populations are still under investigation. However, as a result of the recent widespread habitat destruction and the history of unresolved population declines, the conservation status of the currently recognised single species, P. volans, is being reassessed.Over the past decade, several studies have reported alarming declines in greater glider populations in the Blue Mountains, NSW and Central Highlands, Vic and localized extinctions in other areas, such as Booderee NP, NSWP. volans, further increasing conservation concern for this species and highlighting the lack of information about the other Petauroides spp. Ecological studies have focused primarily on populations in NSW and Vic with only a few published studies of P. minor16 and no prior studies of P. armillatus. The knowledge that there is now genetic support for multiple species, with distributions that are probably much smaller than the previously designated range of P. volans, should be a consideration in future conservation status decisions and management legislation.The division of the greater glider into multiple species reduces the presumed, widespread distribution of Petauroides latitudinal and longitudinal geographic range as part of a separate study to investigate relationships between animal physiology and climate  to detect greater glider eye shine. Individuals were then captured using a gas-powered, tranquilizer dart-gun  and darts specifically designed for mammals between 400 and 2000 g  containing 30–60 mg Zoletil 100 , depending on the estimated body mass of the animal. While still under sedation, captured individuals were weighed and sexed, and reproductive status was assessed. External measurements were taken with vernier callipers using external jaws to measure head length , head width , ear width (widest part of the ear when flattened), ear length , and knee to heal-hind limb (top of knee to base of the heel with limb flexed to ninety degrees). Body length  and tail length  were measured with a flexible tape measure. Each individual was marked with a PIT tag  implanted subcutaneously and a tissue sample was clipped from the margin of the ear for DNA analysis. All work involving live animals complied with animal ethics and relevant guidelines and regulations. The animal capture and tissue collection was approved by James Cook University .37. We then used a canonical variate analysis (CVA) in R package MASS38 to analyse regional group structure  in the multivariate data. To explore whether there were significant differences in measured traits between sexes and account for unequal sample sizes, we used linear models with backward elimination variable selection method and Tukey’s post-hoc multiple comparison test. Linear models included each morphological trait as the response variable and region , sex  and the interaction between region and sex. Insignificant variables were eliminated until only significant variables remained. Based on these results, sex was pooled and Tukey’s post-hoc multiple comparison test was used to compare measured morphological traits between regions. We also explored differences between sites with Tukey’s post-hoc multiple comparison test.Principal components analysis of the eight measured morphological traits for greater gliders from the five sites  was performed in R, using the “prcomp” function. The plot was generated using the ggplot2 package39, as described in detail by Kilian et al.40 and Georges et al.26. Essentially, DArTseq is an implementation of sequencing complexity-reduced representations41 and more recent applications on next generation sequencing platforms43. To achieve the most appropriate complexity reduction , four combinations of restriction enzymes  were evaluated and restriction enzyme combination of Pstl (recognition sequence 5′-CTGCAIG-3′) and Sphl (5′-GCATGIC-3′) was selected.DNA was extracted by Diversity Arrays Technologies  using a NucleoMag 96 Tissue Kit  coupled with NucleoMag SEP (Ref. 744900) to allow automated separation of high-quality DNA on a Freedom Evo robotic liquid handler . Tissue was first incubated overnight with proteinase K, adjusted in concentration depending on the tissue. Sequencing for SNP genotyping was done using DArTseq , which uses a combination of complexity reduction using restriction enzymes, implicit fragment size selection and next generation sequencing44 and the conditions applied are as described in Georges et al.26. After PCR, equimolar amounts of amplification products from each sample were pooled and applied to cBot (Illumina) bridge PCR for sequencing on the Illumina Hiseq 2500. The sequencing (single end) was run for 77 cycles to yield sequence tags of 20–69 bp after removing adaptors.Amplification using polymerase chain reaction (PCR)26. In particular, one third of samples were processed twice from DNA, using independent adaptors, to allelic calls as technical replicates, and scoring consistency (repeatability) was used as the main selection criterion for high quality/low error rate markers. The DArT analysis pipelines have been tested against hundreds of controlled crosses to verify Mendelian behaviour of the resultant SNPs as part of their commercial operations. The resultant data set contained the SNP genotypes and various associated metadata of which CloneiD (unique identity of the sequence tag for a locus), repAvg , CallRate  and SnpPosition (position in the sequence tag at which the defined SNP variant base occurs) are of particular relevance to our analyses.Sequences generated from each lane were processed using proprietary DArT Pty Ltd analytical pipelines as described by Georges et al.45) to facilitate processing with package dartR (version 1.8)46. We first removed all but one SNP from each sequence tag  and retained only those loci supported by a read depth between 5 × and 100 × (8895 loci removed). Three individuals were removed from the dataset owing to an exceptionally poor call rate of less than 0.5  and resultant monomorphic loci removed from the dataset. Loci with a repeatability less than 0.99 were removed (2380 loci) and finally, loci with a call rate of less than 0.95 were removed (9870 loci). We regard the data remaining after this additional filtering  as highly reliable.The SNP data and associated metadata were read into a genlight object ({adegenet}47) as implemented in the gl.pcoa and gl.pcoa.plot functions of dartR. A scree plot of eigenvalues guided the number of informative axes to examine48, taken in the context of the average percentage variation explained by the original variables (using the gl.pcoa.scree function in dartR).Genetic similarity among individuals, populations and colour morphs was visualized using ordination , and pairs of populations for which the count of fixed differences did not exceed the estimated false positive rate were also amalgamated. Counts of private alleles were obtained from gl.report.pa in dartR.Diagnosable units are populations or aggregations of populations that can be diagnosed by one or more fixed allelic differences. Individuals that were intermediate between two aggregations in the PCoA (T1 and T5) were removed as suspected hybrids or introgressed individuals and examined separately. A fixed difference analysis as implemented in dartR was undertaken, and pairs of populations with no fixed allelic differences were progressively amalgamated to yield a putative set of diagnosable operational taxonomic units (OTUs). Putative OTUs that were distinctive because of false positives (owing to sampling error) were identified using gl.fixed.diff with test = TRUE in dartR .28 without specifying parental source populations. Briefly, New Hybrids uses simulation to characterize likelihood bins for each of the parental populations, F1 hybrids generated by crossing the parentals, F2 hybrids, and backcrosses between the F1 hybrids and the parental populations. These bins are used to estimate the likelihood of an individual belonging to each bin, and these likelihoods are rendered and scaled to produce posterior probabilities of bin membership. Parameters were set as ThetaPrior = Jeffreys, PiPrior = Jeffreys, burnin = 10,000, sweeps = 10,000 and the default genotype frequency classes . New Hybrids gives a posterior probability of individual membership in each of the genotype frequency classes, allowing effective assignment of first generation hybrids (F1), second generation hybrids (F2) and backcrosses of F1 hybrids to the parental populations.The genotypes of suspected hybrids/introgressed individuals (T1 and T5) were examined using New HybridsTwo individuals with genotypes intermediate between Taravale/Blackbraes and Redcliffe Vale (T1 and T5) were found in the same cluster of trees in Taravale. Given their status and proximity, we wished to examine the possibility that they were in a parent–offspring relationship. We did this by assuming they were parent–offspring and examining the frequency of pedigree inconsistencies . Theoretically, the count of pedigree inconsistencies should be zero for true parent–offspring pairs, but errors in calling the SNPs generates false positives. To overcome this, we generated counts of pedigree inconsistencies for all pairs of individuals to generate a null expectation and identified putative pairs of parent–offspring as outliers using software to be included in the next release of dartR (gl.report.parent.offspring).Supplementary Information.


STAT3 mediates signalling downstream of cytokine and growth factor receptors where it acts as a transcription factor for its target genes, including oncogenes and cell survival regulating genes. STAT3 has been found to be persistently activated in many types of cancers, primarily through its tyrosine phosphorylation (Y705). Here, we show that constitutive STAT3 activation protects cells from cytotoxic drug responses of several drug classes. To find novel and potentially targetable STAT3 regulators we performed a kinase and phosphatase siRNA screen with cells expressing either a hyperactive STAT3 mutant or IL6-induced wild type STAT3. The screen identified cell division cycle 7-related protein kinase (CDC7), casein kinase 2, alpha 1 (CSNK2), discoidin domain-containing receptor 2 (DDR2), cyclin-dependent kinase 8 (CDK8), phosphatidylinositol 4-kinase 2-alpha (PI4KII), C-terminal Src kinase (CSK) and receptor-type tyrosine-protein phosphatase H (PTPRH) as potential STAT3 regulators. Using small molecule inhibitors targeting these proteins, we confirmed dose and time dependent inhibition of STAT3-mediated transcription, suggesting that inhibition of these kinases may provide strategies for dampening STAT3 activity in cancers. STAT3 is constitutively activated in many aggressive cancers where it promotes drug resistance, an inflammatory phenotype and poor clinical outcome –3. AberrSince STAT3 is frequently hyperactivated and is thought to mediate malignancy, it is considered as potential therapeutic target for several solid and haematological cancers –14. ThisHere we set out to test whether inhibition of other, previously unexplored druggable targets can achieve inhibition of STAT3 signalling. First, we showed that constitutively active STAT3 protects cells from cytotoxic drug responses of several drug classes including anti-mitotic inhibitors and inhibitors of apoptosis proteins. Second, using reporter cells expressing either STAT3 wild type (wt) or constitutively active mutant STAT3(Y640F) in an RNAi screen, we identified kinases whose knockdown resulted in altered STAT3-activity. Finally, we confirmed that small molecule inhibition of several of these kinases could block STAT3 activity. Inhibition of these kinases may therefore be alternative strategies to inhibit STAT3 in cancers and other pathological conditions.2 in a humidified incubator. HEK293sieSTAT3(Y640F) and HEK293sieSTAT3(wt) cells were generated as previously described Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: I Don't KnowReviewer #3: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The study authored by Parri et al reports on the identification of partially novel STAT3-regulators. The manuscript is very well written, the figures are clearly represented and overall the study is easy to read and to follow. Especially the description of the materials and methods section is very detailed. The manuscript shows a highly interesting and comprehensive data-set using a sophisticated screening assay  targeting more than 1000 genes and narrowing them down to 7 targets regulating STAT3 activity.There are still a few open questions:https://www.ncbi.nlm.nih.gov/books/NBK540958/). It would be very interesting to know, what the authors think that the reasons might be for the observed differences in cytotoxicity, while the viability stays unchanged when treating the cells with different chemicals and inhibitors .1. Celltiter-Glo and CellTox Green measure viability and cytotoxicity, respectively. It is thus anticipated and was recently nicely shown that these two assays should complement each other and basically show opposite result . And to make the story complete, please add the inhibitor treatment and STAT3-pY/pS-analysis on IL-6-treated STAT3-WT cells.In line 335, the authors mention that CDK8-i causes the strongest reduction of pY705 posphorylation, but Figure 5 actually shows that CDC7-i is equally potent.It is intriguing that serine kinase inhibition impacts on tyrosine-phosphorylation. It would be highly interesting to know the authors‘ thoughts about the reason for the reduced Y705-STAT3 phosphorylation upon serine-kinase inhibition.3. With regards to the observation, that some of the inhibitors showed a clear kinetic in the STAT3 reporter activity assays in STAT3-mutant cells, the authors conclude that this suggests a difference in protein phosphorylation turnover. Could this observation also be a result of constitutive STAT3 phosphorylation in the mutant cell line compared to de-novo-STAT3 activation upon IL-6 in WT cells? Or could this point towards an indirect effect of kinase-inhibition on STAT3 activity? It will be interesting to hear the author’s view on this.4. The reporter assays shown in Figure 4 suggest that in most cases, kinase inhibition has a more potent effect in STAT3-Y740F cells compared to IL-6-treated STAT3-WT cells. Please discuss these findings in the manuscript. These observations are particularly interesting, as Figure 1 shows that constitutively activated STAT3 protects cells from cell toxicity.5. The authors mention that the data in Figure 3 shows that only PI4KII inhibited STAT3 and STAT1 activity. To this reviewer, the effect of CDK8 KD looks very similar in this assay, and also CSK KD seems to have some (counterintuitive) effect on STAT1-activity. Please explain the rational why the authors do not mention these, and please add and/or discuss. Additionally, the authors claim that the KD generally caused stronger inhibition of WT STAT3 activity than mutant STAT3. It is not clear how the authors came to this conclusion. Please add the respective data to support this conclusion.6. In line, Figure 4 shows that the inhibition of CSNK2 and DDR2 have greater effects on STAT3-reporter activity in STAT3-Y640F mutant cells compared to STAT3-WT cells. Please discuss the discrepancy to the previous point.7. In lines 286-289 the authors mention that they see „stronger reduction“ or reporter activity in STAT3-Y740F cells than in STAT3-WT cells, but the major difference is not the reporter activity, but the kinetics of it. The authors may consider changing their wording.8. In the discussion in line 387 the authors mention that CDK8 has not been linked to STAT3 before, although afterwards they clearly discuss the previous findings by Bancerek et al. Likewise, also CSK has been previously (indirectly) linked to STAT3 phosphorylation.Minor points:1. Please explain the molecular details and functional consequences of the STAT3(Y640F) mutation in the manuscript .2. Please indicate whether the mycoplasma-tests were positive/negative.3. Please show the data mentioned in line 215 and 219 .4. Is there a reason, why the authors do not mention PI4KII and PTPRH in the abstract?5. The sentence in lines 273-275 is difficult to understand.6. For the purpose of consistency, please add the name of the CDC7 inhibitor in line 289.7. Please indicate in Figure 5, which samples have been analysed after 48 or 72 hours, respectively.8. In line 384 the authors mention that the six kinases and one phosphatase regulate EITHER STAT3-mutant OR STAT3-WT in HEK cells, but it should read AND/OR.9. In the legend of Figure S2: Please add „STAT3“- reporter activity... (line 618).10. In Figure 1C and 1C the axis of the GSK-461364 inhibior are not set to 100.11. The data in Figure 2D is displayed in a rather complicated manner. Maybe the authors find an easier way to plot their data.Reviewer #2: The supplemental files contain processed data, but should be labeled as to their content, and how data was normalized or processed. The supplemental files need legends and supplemental text. I did not see it?Reviewer #3: STAT3 mediates signalling downstream of cytokine and growth factor receptors where it acts as a transcription factor for its target genes, including oncogenes and cell survival regulating genes. STAT3 has been found to be persistently activated in many types of cancers, primarily through its tyrosine phosphorylation (Y705), and it is often taken as an target gene for the cancer therapeutic agents. In this paper, the authors find that constitutive STAT3 activation protects cells from cytotoxic drug responses of several drug classes. After performing a kinase and phosphatase siRNA screen with cells expressing either a hyperactive STAT3 mutant or IL6-induced wild type STAT3, the authors suggest that inhibiting the kinases  expressing cells after 48 or 72 hours, but had a strong inhibitory effect on STAT3(wt) Y705 phosphorylation already after 4 hours. Therefore, I suggest that the authors use figure 4S as the formal figure and discuss this phenomenon further.3. If the authors could draw a graph of these small molecular inhibitors on the targets of STAT3 signalling, it would be more helpful for the readers to understand the paper.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, AttachmentPONE-D-19-33342 JD.docxSubmitted filename: Click here for additional data file. 4 Mar 2020Reviewer #2? (comments sent as separate MS Word document without reference to Reviewer number)In these experiments, the authors have searched for previously unexplored druggable targets that inhibit STAT3 signalling, in cells with both WT and mutant STAT3. They show that constitutively active STAT3 protects cells from cytotoxic responses to severaldrug classes. They use transcription reporters in cells expressing STAT3 WT and STAT3(Y640F) in an RNAi screen, and identified kinases that altered activity. They use small molecule inhibition of several of kinase hits, which also blocked STAT3 activity, and conclude that targeting these kinases may therefore be an alternative strategy to inhibit STAT3 in cancers and other pathological conditions.Comments:The Introduction is informative with a nice rational, and its ends with a brief description of type of experiments. Previous work on drug sensitivity of STAT3 is referenced well. The experimental results are described in detail and look reasonable, but need a better take-away and what is the further work needed. Its is not clear what is novel in this study. In the discussion, they note that “in the siRNA screen three kinases and one phosphatase were identified , which have not previously been linked directly to STAT3”. A number of other hits were discussed as previously identified regulators of STAT3, which adds confidence to their iRNA screen. We feel that the novelty of our study lies in the comprehensive profiling of kinases and phosphatases that may regulate STAT3 activity. In this process (as mentioned by the reviewer), we identify both previously identified STAT3-regulalting proteins and ones that have not been linked to STAT3 activity. Importantly, we show that small molecule inhibitors of several of these targets can also induce STAT3 inhibition, opening up for follow-up studies in more complex models. To further emphasize these conclusions, we have adjusted the closing statement in the discussion (lines 427-435).However, drug screening does not appear to yield specificity for inhibition of activated STAT3(Y640F) compared to IL6 stimulated WT, nor for viability of STAT3(Y640F) cancer vs normal cells. The manuscript should be edited for clarity in this regard.We agree with the reviewer that siRNA and small molecule inhibition in general did not look significantly different between STAT3(wt) and STAT3(Y640F) expressing cells for the tested hit genes. This means that other than CSK, we did not identify clear STAT3 mutant-selective regulators. On the other hand, it means that the identified targets can be explored both in cancers with STAT3 mutations and in cancers with hyperactive wild-type STAT3. We have clarified this in the text at lines 226-232 and 375-379.Other comments:1. The Discussion is long and the 1st paragraph repeats previous ideas from the intro. The discussion has been shortened and we have tried to avoid repetition of ideas from the introduction.2. There too much description of the result in the Figure legends, which are in the Results section. Figure Legends should be concise, noting the method, and any specifics; the number biological and technical repeats and statistical significance and method. We have edited the figure legends accordingly.3. How many independent experiments and what do error bars represent?We have included this information to the figure legends.4. The Figure legends should all be together after the references in manuscripts. Figure legends have been moved to after the references. 5. Fig 2B “at least” is 2 words. This has been corrected.6. Fig 2A and B could be simply described in the methods, as they are repetitive and contain a lot of text.We have removed Fig 2A and 2B from the main figures.Reviewer #1: The study authored by Parri et al reports on the identification of partially novel STAT3-regulators. The manuscript is very well written, the figures are clearly represented and overall the study is easy to read and to follow. Especially the description of the materials and methods section is very detailed. The manuscript shows a highly interesting and comprehensive data-set using a sophisticated screening assay  targeting more than 1000 genes and narrowing them down to 7 targets regulating STAT3 activity.There are still a few open questions:https://www.ncbi.nlm.nih.gov/books/NBK540958/). It would be very interesting to know, what the authors think that the reasons might be for the observed differences in cytotoxicity, while the viability stays unchanged when treating the cells with different chemicals and inhibitors .1. Celltiter-Glo and CellTox Green measure viability and cytotoxicity, respectively. It is thus anticipated and was recently nicely shown that these two assays should complement each other and basically show opposite result (please see We have now addressed this point in the discussion (lines 330-333). We have previously described that in a strong viability response (with relatively fast-growing cells), it is often difficult to separate cytotoxic from cytostatic responses [1]. However, the viability inhibition curves in general in Fig 1 go a bit deeper in the case where greater cytotoxicity is induced. 2. As CDK8, CDC7 and CSNK2A1 represent serine/threonine kinases, it is puzzling why the authors chose to analyse the effect of the respective inhibitors on STAT3-Y705 phosphorylation, rather than STAT3-S727 phosphorylation. It would be advisable to add the effect of kinase inhibition on STAT3-S727 phosphorylation . And to make the story complete, please add the inhibitor treatment and STAT3-pY/pS-analysis on IL-6-treated STAT3-WT cells.This is an important point and we have therefore added data also on pS727 levels with all inhibitor treatments .In line 335, the authors mention that CDK8-i causes the strongest reduction of pY705 phosphorylation, but Figure 5 actually shows that CDC7-i is equally potent.It is intriguing that serine kinase inhibition impacts on tyrosine-phosphorylation. It would be highly interesting to know the authors‘ thoughts about the reason for the reduced Y705-STAT3 phosphorylation upon serine-kinase inhibition.We agree that CDC7 inhibition is similar in its potency to reduce pY705 levels to CDK8 inhibition and we have therefore have corrected this on lines 316-317. Our phospho-STAT3 western blotting data suggest that all hit genes likely are indirect regulators of STAT3 (not directly phosphorylating or dephosphorylating STAT3) and therefore assume that the signals that are affected by CDC7 or CDK8 inhibition ultimately lead to reduced phosphotyrosine levels through altered regulation of tyrosine kinases/phosphatases. We have addressed this in in the results and discussion (lines 316-322 and lines 372-380).3. With regards to the observation, that some of the inhibitors showed a clear kinetic in the STAT3 reporter activity assays in STAT3-mutant cells, the authors conclude that this suggests a difference in protein phosphorylation turnover. Could this observation also be a result of constitutive STAT3 phosphorylation in the mutant cell line compared to de-novo-STAT3 activation upon IL-6 in WT cells? Or could this point towards an indirect effect of kinase-inhibition on STAT3 activity? It will be interesting to hear the author’s view on this.We appreciate this point and agree that it is also possible that the difference between STAT3(wt) and STAT3(Y640F) expressing cells is due to the difference in assays. We have therefore addressed this in the manuscript on lines 254-264.4. The reporter assays shown in Figure 4 suggest that in most cases, kinase inhibition has a more potent effect in STAT3-Y740F cells compared to IL-6-treated STAT3-WT cells. Please discuss these findings in the manuscript. These observations are particularly interesting, as Figure 1 shows that constitutively activated STAT3 protects cells from cell toxicity.After reviewing the data, we find that only the DDR2 inhibitor regorafenib has a more potent effect in STAT3(Y640F) cells at later time points. Other treatments show highly similar effects in STAT3 mutant and wt cells (in the attached figure blue and green lines at 72h). At shorter time points (gray and red lines at 4h) the compounds are more effective in IL6-induced STAT3(wt)-cells. The figure below has data from manuscript Fig 4 and Fig S2 and clarifies this. We have added this to the manuscript to lines 254-263 and 279-280.In figure 4 we observed an error in the silmitasertib viability curve that has been corrected. Furthermore, we added data from one more biological replicate to the figure and edited the figure legend accordingly.5. The authors mention that the data in Figure 3 shows that only PI4KII inhibited STAT3 and STAT1 activity. To this reviewer, the effect of CDK8 KD looks very similar in this assay, and also CSK KD seems to have some (counterintuitive) effect on STAT1-activity. Please explain the rational why the authors do not mention these, and please add and/or discuss. We agree that the CDK8 KD is very similar in in STAT1 assay, but we could not conclude an effect by CDK8 KD on STAT1 transcriptional activity because of having tested only two siRNAs. We have clarified this in the text on lines 241-243. The counterintuitive CSK KD on effect to STAT3 vs STAT1 could be due to that SRC is activated by IFNγ and is involved in the activation of STAT3 but not STAT1 [2]. However, the effects we see on STAT1 transcriptional activity by CSK knockdown is not consistent enough across the tested siRNAs to draw a conclusion that it causes an inhibition rather than an activation. We have clarified this in the manuscript on lines 241-242 and in discussion lines 376-380.Additionally, the authors claim that the KD generally caused stronger inhibition of WT STAT3 activity than mutant STAT3. It is not clear how the authors came to this conclusion. Please add the respective data to support this conclusion.We observed stronger general inhibition of STAT3(wt) in the primary screen , but this observation didn’t carry through in the validation screen and the same difference was not observed in the final hits. Therefore, we have removed the claim from the manuscript (from figure legend 3).6. In line, Figure 4 shows that the inhibition of CSNK2 and DDR2 have greater effects on STAT3-reporter activity in STAT3-Y640F mutant cells compared to STAT3-WT cells. Please discuss the discrepancy to the previous point.The previous comment was about the siRNA screen as whole as mentioned earlier, we realized that this effect was not consistent through the follow-up screens and we therefore removed that statement. As discussed in point 4, in figure 4, although it may first look like several of the small molecule inhibitors were effective at inhibiting STAT3 reporter activity in STAT3 mutant cell than in STAT3 wt cells, only the DDR2 inhibitor has a clear selective effect. 7. In lines 286-289 the authors mention that they see „stronger reduction“ or reporter activity in STAT3-Y740F cells than in STAT3-WT cells, but the major difference is not the reporter activity, but the kinetics of it. The authors may consider changing their wording.It is true that the major difference is the kinetics of the two cell lines. We have changed the wording in the manuscript (lines 269-271).8. In the discussion in line 387 the authors mention that CDK8 has not been linked to STAT3 before, although afterwards they clearly discuss the previous findings by Bancerek et al. Likewise, also CSK has been previously (indirectly) linked to STAT3 phosphorylation.We agree that these were previous links that we had failed to report and the text has now been corrected (lines 374-375).Minor points:1. Please explain the molecular details and functional consequences of the STAT3(Y640F) mutation in the manuscript .The mutations in the SH2 domain, STAT3(Y640F) result in increased hydrophobicity of the STAT3 dimerization site (pY+3 pocket) that results in a more stable form of STAT3 dimer, constitutive Y705 phosphorylation, enhanced nuclear stability and increased transcriptional activity. The STAT3(Y640F) mutation does not have an effect on S727 phosphorylation [3]. We have clarified this in the manuscript (lines 45-49). 2. Please indicate whether the mycoplasma-tests were positive/negative.Mycoplasma tests were negative and we have clarified this in the manuscript (lines 87-88)3. Please show the data mentioned in line 215 and 219 .We have made supplementary figure using raw luciferase data addressing the data mentioned in lines 215 – 219  and the text is edited accordingly. Furthermore, we have labelled separately siSTAT3 and siDEATH in figure 2A where the positive control is siSTAT3 and assay control is siDEATH.4. Is there a reason, why the authors do not mention PI4KII and PTPRH in the abstract?The reason why we initially did not mention PI4KII and PTPRH in the abstract was that we did not have selective small molecule inhibitors against these proteins. We have included them now in the abstract.5. The sentence in lines 273-275 is difficult to understand.The sentence: “Generally, the reporter activity inhibition occurred more slowly in STAT3(Y640F) expressing cells than the STAT3(wt) cells, suggesting that the STAT3(Y640F) protein phosphorylation turnover is slower.” Has been edited to: “We observed that the reporter activity inhibition occurred more slowly in STAT3(Y640F) expressing cells than the STAT3(wt) cells, suggesting that the STAT3(Y640F) protein de-phosphorylation is slower.“ Lines 254-260.6. For the purpose of consistency, please add the name of the CDC7 inhibitor in line 289.We have corrected this to the text.7. Please indicate in Figure 5, which samples have been analysed after 48 or 72 hours, respectively.We have labelled the samples accordingly in figure 5. 8. In line 384 the authors mention that the six kinases and one phosphatase regulate EITHER STAT3-mutant OR STAT3-WT in HEK cells, but it should read AND/OR.The text has been edited according to the suggestion.9. In the legend of Figure S2: Please add „STAT3“- reporter activity... (line 618).We have edited the Fig S2 legend according to the suggestion.10. In Figure 1C and 1C the axis of the GSK-461364 inhibior are not set to 100.The GSK-461364 data had points outside the range of the ranges of the other data sets and we had therefore set a different scale on the y-axis for this inhibitor. As the reviewer suggests, it makes sense to have the graphs coherent and we have therefore now set the y-axis maximum to 125 for all the inhibitors in figure 1C and 1D.11. The data in Figure 2D is displayed in a rather complicated manner. Maybe the authors find an easier way to plot their data.Figure 2D  has been simplified.Reviewer #2: The supplemental files contain processed data, but should be labeled as to their content, and how data was normalized or processed. The supplemental files need legends and supplemental text. I did not see it?We have added supplemental legends and text explaining the data in supplemental files and corrected the labelling of the files. Reviewer #3: STAT3 mediates signalling downstream of cytokine and growth factor receptors where it acts as a transcription factor for its target genes, including oncogenes and cell survival regulating genes. STAT3 has been found to be persistently activated in many types of cancers, primarily through its tyrosine phosphorylation (Y705), and it is often taken as an target gene for the cancer therapeutic agents. In this paper, the authors find that constitutive STAT3 activation protects cells from cytotoxic drug responses of several drug classes. After performing a kinase and phosphatase siRNA screen with cells expressing either a hyperactive STAT3 mutant or IL6-induced wild type STAT3, the authors suggest that inhibiting the kinases . The used siRNAs are listed in supporting file S3. The bars are matched so that in the figures 3 A, B, C individual siRNAs are in same order, with exception with siCDK8 in Fig 3C where only the first two siRNAs are matched. The figure has been edited and the different siRNAs are labelled to clarify this. There are only two bars for CDK8 because we only had 2 siRNAs for this gene. 2. It is very interesting that 1μM ruxolitinib could not markedly reduce pY705 STAT3 level in STAT3(Y640F) expressing cells after 48 or 72 hours, but had a strong inhibitory effect on STAT3(wt) Y705 phosphorylation already after 4 hours. Therefore, I suggest that the authors use figure 4S as the formal figure and discuss this phenomenon further.The difference between the effects on the wild type vs. mutant STAT3 likely comes from that we are comparing de novo vs. persistent phosphorylation. In the STAT3(wt) expressing cells STAT3 is activated by the addition of IL6 after the ruxolitinib addition whereas in the STAT3(Y640F) the Y640F is already phosphorylated before adding ruxolitinib. Therefore, the effects on the wt phosphorylation are assessing the impact of directly blocking JAK-dependent phosphorylation of STAT3 (caused by IL6 stimulation). In the mutant case, we assess the impact on turnover of the phosphorylation (dephosphorylation followed by re-phosphorylation) of STAT3. Further enhancing the differential effect, The STAT3(Y640F) mutant has been described to cause a more stable dimer than the wild type protein, causing a slower rate of dephosphorylation. Since the pY705 activation by IL6 in STAT3(wt) cells is fairly fast and short-lived we used a 20-minute IL6 stimulation prior to lysing the cells for western blots and a 3-hour stimulation for the luciferase reporter readout. Ruxolitinib serves in this study as control for de novo vs. constitutive STAT3 activation. To clarify these differences between the wt and mutant-expressing cells, we have addressed this in the text on lines 45-49 and 254-263. 3. If the authors could draw a graph of these small molecular inhibitors on the targets of STAT3 signalling, it would be more helpful for the readers to understand the paper.We have added a summary figure 6 of the siRNA screen hits and inhibitors targeting them.References1. Gautam P, Karhinen L, Szwajda A, Jha SK, Yadav B, Aittokallio T, et al. Identification of selective cytotoxic and synthetic lethal drug responses in triple negative breast cancer cells. Molecular cancer. 2016;15(1):34. Epub 2016/05/12. doi: 10.1186/s12943-016-0517-3. PubMed PMID: 27165605; PubMed Central PMCID: PMCPMC4862054.2. Qing Y, Stark GR. Alternative activation of STAT1 and STAT3 in response to interferon-gamma. The Journal of biological chemistry. 2004;279(40):41679-85. Epub 2004/07/31. doi: 10.1074/jbc.M406413200. PubMed PMID: 15284232.3. Pilati C, Amessou M, Bihl MP, Balabaud C, Nhieu JT, Paradis V, et al. Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas. The Journal of experimental medicine. 2011;208(7):1359-66. doi: 10.1084/jem.20110283. PubMed PMID: 21690253; PubMed Central PMCID: PMC3135371.AttachmentResponse to reviewers.pdfSubmitted filename: Click here for additional data file. 10 Mar 2020Identification of novel regulators of STAT3 activityPONE-D-19-33342R1Dear Dr. Wennerberg,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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