Cultured neuronal networks monitored with microelectrode arrays (MEAs) have been used widely to evaluate pharmaceutical compounds for potential neurotoxic effects. A newer application of MEAs has been in the development of in vitro models of neurological disease. Here, we directly evaluated the utility of MEAs to recapitulate in vivo phenotypes of mature microRNA-128 (miR-128) deficiency, which causes fatal seizures in mice. We show that inhibition of miR-128 results in significantly increased neuronal activity in cultured neuronal networks derived from primary mouse cortical neurons. These results support the utility of MEAs in developing in vitro models of neuroexcitability disorders, such as epilepsy, and further suggest that MEAs provide an effective tool for the rapid identification of microRNAs that promote seizures when dysregulated. Epilepsy is a chronic disease that encompasses a broad spectrum of brain disorders characterized by recurrent and unprovoked seizures. It is the fourth most common neurological disorder, affecting people of all ages, and often includes additional debilitating neurological and cognitive consequences . While tMir128-2, one of the two genes that encode mature miR-128, in dopaminergic neurons developed fatal seizures within 3 mo of age (miRNAs are 20- to 23-nucleotide (nt) single-stranded RNAs that modulate post-transcriptional gene expression through imperfect base-pairing with target messenger-RNAs. miRNAs are thought to be particularly important in the complex gene regulation programs that occur in the brain . In facto of age .Here, we evaluated the utility of microelectrode array (MEA) technology to recapitulate in vivo excitability phenotypes due to inhibition of mature miR-128 in cultured cortical neuronal networks. We inhibited miR-128 expression in mouse cortical neurons by overexpressing a miRNA sponge, a competitive miRNA inhibitor, and recorded neuronal activity using an MEA, which captures the extracellular field potentials of electrically active cells. We identified significant increases in neuronal activity following miR-128 knockdown. Hence, in this study we describe an in vitro excitability phenotype due to miRNA regulation and establish a paradigm that can be used for effective identification of miRNAs that promote seizures when dysregulated.Supplemental Table S1). To control for effects of lentiviral transduction, we tested two controls that expressed either a nontargeting shRNA or a sponge containing sequences complementary to an artificial miRNA . Approximately 15% of neurons were transduced using the miR-128 sponge . There was no significant difference in cell viability between neuronal networks expressing a control sponge and networks expressing the miR-128 sponge . Knockdown efficiency was evaluated using a TaqMan expression assay specific to mature miR-128. The engagement of miRNAs with their targets can induce degradation of the miRNA via tailing and trimming . The small effects on MAPK3/1 pathway activation are likely due in part to the low transduction efficiency achieved (15%) .A miRNA sponge, delivered via lentiviral transduction, was used to inhibit miR-128 in primary dissociated cortical neurons from post-natal day 0 mice on the day of dissection (DIV0) . Fifteen minutes of recording data captures more than 10,000 spikes per well; when data from three wells or more are available, this length of recording provides information sufficient to make statistically sound decisions about neuronal activity patterns. MEA data provide a metric for cultured neuronal network activity and synchronicity via measurement of changes in spike, burst, and network events. Spike and burst rates provide a metric for the overall activity of the neuronal network, with more spikes and bursts corresponding to higher activity. Network events include synchronous network spikes and bursts in which 16 or more electrodes  capture activity simultaneously. While our data focused on spikes, bursts, and network events, several additional activity features were extracted from the data, including the number of active electrodes, mean firing rate (MFR) normalized to the number of active electrodes, burst rate and duration, network spike and burst rate, and percentage of total spikes in bursts and network events across the cultured neuronal network .We evaluated neuronal activity patterns following miR-128 knockdown in vitro. Fifteen minutes of activity data from control neuronal networks and networks expressing the miR-128 sponge was collected by MEA every day starting on DIV3 (P < 0.0001) . The effect of miR-128 knockdown on frequency of burst and synchronous network events was further investigated. The frequency of bursts and network events, including network level spikes and bursts, provides a readout of network level communication and activity. The miR-128 knockdown neurons showed a marked increase in the bursts per minute across active electrodes  , number of synchronous network bursts per second  , and number of network spikes  . Together, these data demonstrate that miR-128 inhibition increases neuronal activity.There was clear evidence of increased neuronal activity in the miR-128 knockdown neurons in comparison to control transduced cells. The MFR is considered to be an indicator of neuronal excitability and action potential frequency . TransduU test and combined P-value by Fisher's combined probability test, P = 1.5 × 10−5 and P = 6.3 × 10−7, respectively)  relative to controls. This indicates that miR-128 knockdown in cultured neuronal networks results in more numerous and longer lasting bursts, which occur in rapid succession with very short periods of inactivity between bursts , indicating that single firing events in knockdown cultures lead to bursts more often than in control networks. There were no differences between knockdown and control groups in the percentage of spikes in network events . Together, these data indicate that miR-128 knockdown cultures are hyperactive but are not more synchronous, which is consistent with the in vivo neuronal excitability of Mir128-2–deficient mice , yet we observe striking and reproducible network phenotypes, thus highlighting the importance and sensitivity of neuronal networks to the expression levels of miR-128.We also evaluated activity-independent features. Activity-independent features are those that take into account the activity of the network by normalizing by the firing rate and include the percentage of spikes occurring in bursts and network events. The percentage of spikes within bursts is a metric of network activity organization, whereas percentage of spikes in network events is a feature of network synchronicity. The percentage of spikes in bursts significantly increased in knockdown cultures . Other activity-dependent features were elevated but did not reach statistical significance. Furthermore, there was no significant difference between the MFR of wells transduced with a nontargeting shRNA control and untransduced cells . Synchronicity patterns of network events remained unchanged between groups, as noted in DIV0 experiments. Together, these results show that inhibition of miR-128 in mature culture also results in an excitability phenotype and is not attributed to an effect on network development.To investigate whether the effect of miR-128 inhibition on cultured neuronal network properties is an acute effect on functioning cultured neuronal networks, rather than an effect of their development, we inhibited miR-128 expression in mature cultured neuronal networks (DIV19) to compare with DIV0 experiments described above. Significant increases in MFR and burst rate were noted between DIV28 and DIV36 in comparison to controls . A nontargeting shRNA with a scrambled sequence was used as a control. Similar data were achieved using a control sponge based on the Cxcr4 gene sequence , which includes nine repeats of an artificial miRNA that is not complementary to any known miRNA and is also positioned downstream from the eGFP reporter gene in the pLCE vector.We designed a stably expressing lentivirus sponge to inhibit miR-128 according to a previously described protocol . The seqSupplemental Methods.Primary cortical neurons were dissociated from the brains of post-natal day 0 (P0) C57BL/6J wild-type mice and plated onto 12-well MEA plates (Axion Biosystems). All procedures involving mice were approved by the Division of Laboratory Animal Resources at Duke University and the International Animal Care and Use Committee at Columbia University. One to seven days before dissection, each 12-well plate was treated with polyethyleneimine and then washed four times with distilled water and allowed to dry overnight in a sterilized hood. On the day of dissection, the wells were coated with laminin and incubated at 37°C during the dissection. Laminin was removed by aspiration prior to seeding dissociated neurons onto the wells. P0 pups were isolated and immediately decapitated. Dissociated neurons from the entire cerebral cortex were used and cultured in Neurobasal-A media supplemented with B27. Media was 50% changed on DIV3 and every other day thereafter. For additional methods, including cell culture, miR-128 expression analysis by qRT-PCR, and immunoblotting technique, please see the Supplemental Methods). To analyze data over time, the values for each well between DIV8 and DIV16 were combined and a MWU test was performed. The labels on each well were then shuffled and permuted 10,000 times to create 10,000 data sets that were tested for significance using a MWU test. The P-value of the actual data set was then compared to the distribution of the P-values from the permuted data sets to determine whether differences between the groups were significantly different from random. For additional information pertaining to MEA data analysis and statistical testing, please see the Supplemental Methods.Spontaneous network activity was recorded for 15 min every day using the Axion Biosystems Maestro MEA at 37°C. We collected raw data and a spike list file, which contains the spike data for each recording. These files were used downstream to extract spike, burst, and network data, using code adapted from sjemea package created by Stephen Eglen . At the https://figshare.com/projects/Inhibition_of_microRNA-128_promotes_excitability_of_ cultured_cortical_neuronal_networks/15200). The following individual data sets are within the database, followed by their digital object identifiers:https://dx.doi.org/10.6084/m9.figshare.3543728.v2, https://dx.doi.org/10.6084/m9.figshare.3521726.v4, https://dx.doi.org/10.6084/m9.figshare.3521621.v3, https://dx.doi.org/10.6084/m9.figshare.3521435.v5, https://dx.doi.org/10.6084/m9.figshare.3521291.v4.Spikelist files generated during activity recordings: https://dx.doi.org/10.6084/m9.figshare.3543341.v2.The R code used to process the spikelist files: https://dx.doi.org/10.6084/m9.figshare.3543467.v1, https://dx.doi.org/10.6084/m9.figshare.3524531.v3, https://dx.doi.org/10.6084/m9.figshare.3524483.v1, https://dx.doi.org/10.6084/m9.figshare.3524447.v1, https://dx.doi.org/10.6084/m9.figshare.3524360.v2.Data files generated from processing spikelist files: The data from this study, including the data generated by the Axion Maestro system and the output of the data after running it through our software, as well as the analysis package (IGM.MEA_0.3.2.tar.gz) used to generate activity features discussed here, are publicly available on Figshare.com (


Cutibacterium (Propionibacterium) acnes, considered a part of the skin microbiota, is one of the most commonly isolated anaerobic bacteria from medical implants in contact with plasma. However, the precise interaction of C. acnes with blood cells and plasma proteins has not been fully elucidated. Herein, we have investigated the molecular interaction of C. acnes with platelets and plasma proteins. We report that the ability of C. acnes to aggregate platelets is dependent on phylotype, with a significantly lower ability amongst type IB isolates, and the interaction of specific donor-dependent plasma proteins (or concentrations thereof) with C. acnes. Pretreatment of C. acnes with plasma reduces the lag time before aggregation demonstrating that pre-deposition of plasma proteins on C. acnes is an important step in platelet aggregation. Using mass spectrometry we identified several plasma proteins deposited on C. acnes, including IgG, fibrinogen and complement factors. Inhibition of IgG, fibrinogen or complement decreased C. acnes-mediated platelet aggregation, demonstrating the importance of these plasma proteins for aggregation. The interaction of C. acnes and platelets was visualized using fluorescence microscopy, verifying the presence of IgG and fibrinogen as components of the aggregates, and co-localization of C. acnes and platelets in the aggregates. Here, we have demonstrated the ability of C. acnes to activate and aggregate platelets in a bacterium and donor-specific fashion, as well as added mechanistic insights into this interaction. Propionibacterium acnes is a predominant skin bacterium that primarily colonizes sebum rich areas, and is suggested to benefit our health [Propionibacteria, it has been proposed to rename P. acnes to Cutibacterium acnes [C. acnes subsp. acnes (type I), C. acnes subsp. defendens (type II), and C. acnes subsp. elongatum (type III) [C. acnes is associated with several diseases, including acne [C. acnes into sequence types by multi-locus and single-locus sequence typing, it has been demonstrated that specific types of C. acnes are associated with disease, while others are associated with health [Staphylococcus epidermidis and C. acnes [r health . Due to um acnes  and diviype III) . C. acneing acne , infectiing acne  and locaing acne . By furth health ,7–11. SeC. acnes , showingC. acnes .C. acnes associated with this pathogenesis [C. acnes is highly immunostimulatory [C. acnes has historically been used experimentally to trigger immune responses for tumor treatment in rodents [C. acnes had an adverse effect on circulating immune cells, generating thrombocytopenia and decreased fibrinogen levels in mice [C. acnes and the coagulation system, including platelets. However, no mechanism(s) to describe the thrombocytopenia or effect on the coagulation system has been given.Though not the main microorganism identified from infective endocarditis, there is an increasing number of reports of ogenesis . The impmulatory . Due to  rodents . During  in mice ,18, indiStreptococcus pyogenes [Staphylococcus aureus [Enterococcus faecalis [Aerococcus urinae [Platelets play a critical part in coagulation and immune response . These spyogenes , Staphyls aureus , Enterocfaecalis , and Aers urinae . Often, s urinae . This iss urinae . In addis urinae –28. In ts urinae , and rels urinae . On the s urinae , and by s urinae . On the s urinae ,34.C. acnes forms biofilms on medical implants and catheters in plasma rich environments, induce thrombocytopenia in mice, and being indicated as one of the foremost contaminants in platelet concentrates [C. acnes and platelets, and the downstream effects of such an interaction.In light of the demonstration that entrates –37, we bC. acnes strains are described elsewhere [C. acnes was routinely grown in Tryptic Soy Broth  supplemented with 1.5% glycerol, under anaerobic conditions  at 37°C.The isolated lsewhere ,38 and c3-citrate as an anticoagulant. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) was isolated through centrifugation . To obtain serum, blood was collected in empty tubes, and allowed to coagulate. Cells or blood clots were pelleted through centrifugation , and the supernatant saved. Sampling of blood from healthy volunteers giving informed consent has been approved by the regional Ethical Review Board in Lund, Sweden , and in accordance with the Declaration of Helsinki.Blood samples from healthy donors were collected with 0.1 M Na8 cfu/ml) were added while stirring. For dose-dependency experiments, bacteria at a concentration of 1x108 cfu/ml were serial diluted in PBS to generate two-fold dilutions while maintaining the same volume (e.g. 6x106-1x108 cfu/mL). PPP was used as a reference, and collagen I  as a positive control. The software AggroLink was used for all analyses. For certain experiments, pre-treatment of PRP with IdeS , anti-FcRγIIA , or prostaglandin E , was conducted. IdeS is a specific IgG protease with no other known activities in plasma or elsewhere [C. acnes, bacteria  was incubated with PPP, serum or heat inactivated serum  or PBS (control) and washed in PBS before being added to PRP.The platelet aggregation response to bacteria was investigated using a platelet aggregometer . PRP (450 μl) was added to pre-heated glass tubes placed in sample wells, and 50 μl bacteria suspension  was diluted in 30 μl HEPES buffer pH 7.4 and incubated with 5 μl bacteria  at room temperature for 25 minutes. ADP  or thrombin  were used as positive controls for platelet activation. Fluorescent antibodies  were added to detect the platelet population , surface-bound CD62P ), and platelet activation through presentation of an activation dependent epitope on GPIIb/IIIa . To determine the role of IgG and fibrinogen in platelet activation by C. acnes, PRP was pre-treated for 15 minutes at room temperature with AT10 (100 μg/ml) and ReoPro  prior to addition of bacteria and platelet antibodies. All samples were acquired and analyzed using a BD Accuri C6. The results are presented as median fluorescence intensity (MFI) fold increase in relation to the signal in the control sample with PRP and HEPES buffer alone for each experiment. Experiments examining platelet activation were not necessarily conducted concurrently.Flow cytometry was used to investigate the platelet activation in response to 8 cfu/ml final concentration) were incubated alone or with 450 μl PRP or PBS at 37°C while shaking (700 rpm) for 0, 25, and 120 minutes. The experiment was performed with and without the neutrophil antimicrobial peptide LL37 (100 μg/ml), as a control for bacterial killing. The mixture was either vortexed or sonicated  as described recently [Bacteria  were incubated with 50 μl plasma from different healthy donors for 15 minutes at room temperature. Bacteria were washed with PBS and incubated with FITC-labeled mouse anti-human IgG (1:50) . Bacteria pre-incubated in PBS (instead of plasma) were used to control for non-specific binding. Before analysis using flow cytometry, the samples were washed and resuspended in PBS.Bacteria , and the resulting supernatant dried. The samples were alkylated and reduced, before treatment with trypsin overnight at 37°C. The samples were analyzed on a Q Exactive Plus mass spectrometer  connected to an EASY-nLC 1000 ultra-high pressure liquid chromatography system . Peptides were loaded on an EASY-Spray column  at a constant pressure of 600 bars, and separated at a flow rate of 300 nl/min. Peptides were eluted with a linear gradient from 95–35% solvent A (0.1% formic acid in water) and 5–65% solvent B (0.1% formic acid in acetonitrile) over 60 min. The precursor ions were fragmented using high-energy collision induced dissociation (HCD) at normalized collision energy of 30. Acquired spectra were searched with X!Tandem [X!Tandem  against X!Tandem . A minimX!Tandem .C. acnes-induced platelet aggregates. C. acnes was added to PRP as previously described. For C. acnes labeling DAPI and Oregon Green were used as indicated. PBS, PPP and PRP alone were used as negative controls, and collagen I as a positive control for platelet aggregation. The samples were fixed in 4% paraformaldehyde for 45 minutes and washed in D-PBS supplemented with 5% BSA and 50 mM glycine. Primary antibodies were added  and incubated for 1 hour at room temperature, before washing in PBS + 5% BSA, and addition of secondary and/or labeled primary antibodies , continuing incubation for 1 hour. Finally, the samples were washed and re-suspended in PBS + 5% BSA and adsorbed on pre-coated poly-L-lysine coverslips for one hour before mounting to slides using DAPI ProLong Gold mounting medium . The samples were allowed to settle overnight and analyzed in a Nikon Eclipse Ti microscope.Fluorescence microscopy was used to visualize the C. acnes types and strains to induce platelet aggregation and activation. Mann Whitney was used to compare cfu/ml in the killing assay and to compare AS12 induced platelet aggregation and activation under different conditions. The co-localization of platelets and C. acnes in aggregates was calculated in NIS Elements , using Pearson's correlation. p < 0.05 was considered significant.Prism 7  was used for statistical analyses, using Kruskal-Wallis and Mann-Whitney. Kruskal-Wallis was used to compare the ability of the different C. acnes to activate and aggregate platelets has not been investigated. Using a skin isolate of C. acnes , and as the magnitude of aggregation, with the latter being reported as the percent of aggregation at the end of the experiment.Despite causing low-grade chronic inflammation in plasma rich environments, the ability of type II; ) we demoC. acnes at different concentrations with platelets and measured the aggregation. A C. acnes:platelet ratio of below 0.11 (e.g. ca 1 bacterium per 10 platelets) did not result in any measurable aggregation within 25 minutes, which was set as the upper time limit. However, higher ratio of bacteria:platelet (0.23–1.8) all resulted in aggregation within the set time frame. Higher doses of bacteria resulted in shorter lag times before aggregation (1) (Other studies have indicated that the bacterium:platelet ratio influences the ability of platelets to aggregate . Thereforegation . The aggtion (1) . By incrtion (1) . Rather,C. acnes has been divided into different types  based on reactivity against specific antibodies [C. acnes population into several different sequence types [C. acnes [Historically, tibodies . More dece types . The preC. acnes  to aggreStreptococcus mitis [C. acnes has been well documented, with a significant lower abundance in phylotype IB strains [C. acnes [A common feature for many bacteria is the presence of prophage regions in their genome. These prophages are bacterial viruses, but often also encode virulence factors, increasing the pathogenicity of their host . The preus mitis ,48. The  strains . HoweverC. acnes , did notC. acnes demonstrated distinct platelet aggregation responses, led us to investigate if also host differences (e.g. plasma proteins) would influence the platelet response. Four isolates of C. acnes, representing three phylotypes  with different abilities to trigger platelet aggregation in one donor, were used to induce platelet aggregation in PRP from three individual donors. The three donors responded to the C. acnes isolates with different lag times before aggregation (p = 0.036). There was also a marked interindividual difference in lag times between single isolates (e.g. AS12 and AD1) induced platelet aggregation after ~10 minutes in two donors, they only induced platelet aggregation after 20+ minutes (AD1), or failed to induce aggregation in a third donor . A similC. acnes with PRP and measured the bacterial survival at different time points representing the time of inoculation (t = 0 min), aggregation (t = 25 min), and prolonged interaction (t = 120 min). Aggregation was confirmed macroscopically for all samples at time points 25 and 120 min, indicating that the platelets had aggregated. Samples were gently sonicated to disrupt either bacterium:bacterium interactions, or platelet aggregates to accurately count live bacteria. Sonication resulted in similar levels of free bacteria as vortex treated samples, but was too gentle to induce bacterial lysis . While observed . Rather,observed . The chaC. acnes strain AS12 due to its high ability to induce aggregation. Pre-incubation of bacteria with plasma led to a significantly shorter lag-time as compared to non-treated bacteria (p = 0.03)[C. acnes aggregation of platelets.The observed differences in activation and aggregation between donors, and the relatively long lag-time before aggregation, could indicate deposition of plasma proteins on the bacterial surface, facilitating aggregation of the platelets. To investigate this, we used p = 0.03). To elucp = 0.03). Pre-incp = 0.03). To inve(p<0.001). Presencfragment), or by bC. acnes (p = 0.03), while not affecting activation by ADP or thrombin . However, the molecular mechanism(s) behind these phenotypic oddities have not been carefully elucidated. It is clear, based on our data, that the presence of IgG is but one critical factor necessary for the aggregation of platelets by C. acnes, but may not necessarily be the limiting factor in generating platelet aggregation, since both platelet-aggregating and non-aggregating strains of C. acnes had similar degrees of IgG opsonization. Even though C. acnes type IB is not as abundant as type IA on the skin, it is still a common skin bacterium. However, the abundance of type IA on the skin may explain why the immune system is more adapt in responding to those isolates through aggregation, constantly being stimulated by these strains [Type IB strains have earlier been demonstrated to have distinct properties as compared to type IA and II, in terms of carriage of bacteriophages , express strains ,62.C. acnes was recently shown to have specific fibrinogen-binding proteins [Staphylococcus aureus [Staphylococcus epidermidis [C. acnes aggregation of platelets seems more similar to that of Aerococcus urinae, recently demonstrated being able to cause aggregation of platelets [e.g. Streptococcus pyogenes), only demand a low number of bacteria to cause aggregation, occurring within 30–90 seconds [C. acnes and A. urinae [Many bacteria rely on the presence of IgG and fibrinogen for the activation and subsequent aggregation of platelets . C. acneproteins , and hasproteins , possibls aureus , and Sdrdermidis , being pdermidis . In manylatelets . Both balatelets . Other b seconds , as comp. urinae ; demonstDuring analysis of the donor-dependency in platelet responses, we found a discrepancy in the results regarding donor activation  and aggregation. However, all donors responded in either one or both the assays. The assays are only partly comparable because flow cytometry investigates an early stage platelet response and does not reflect aggregation, while aggregation in the Chrono Log only measures complete platelet aggregation and does not reflect the early platelet response. Further, during this analysis it became clear that there is a biological variance allowing for some variation in response to bacteria. Donor 3 was noticeably a weak initiator of platelet aggregation at two time-points, but initiated aggregation in two strains at the third occasion . Donor 1C. acnes; both on type II (AS12) and type IB (KPA171202). Using mass spectrometry we could further identify several other plasma proteins at the surface of C. acnes, eg. apolipoprotein A and plasminogen, currently not known to be involved in platelet aggregation. These proteins displayed a strain-specific binding. However, the biological relevance of this is currently unknown, making it an interesting future area of investigation. The lack of aggregation in several type IB isolates should thus not be due to a lack of plasma protein deposition on the surface, or reduced opsonization of the targets. A recent study highlighted the differences in surface and secreted proteomes of C. acnes types, and distinct immune responses towards these strains from PBMCs [C. acnes may also induce different responses from platelets, as seen in our study.Mass spectrometry data as well as fluorescence microscopy both support the presence of IgG, fibrinogen, and complement on the surface of om PBMCs , suggestC. acnes is also noteworthy. Complement is responsible for opsonization of bacteria, and as such should be identified on pathogens incubated with plasma. However, complement is also a mediator of activation/aggregation of platelets, usually in combination with IgG [A. urinae has demonstrated a dependence of complement, beyond their dependence on IgG and fibrinogen for platelet aggregation [C. acnes with heat-inactivated serum, and the accumulation of complement compounds on the bacterial surface after incubation with plasma, observed in this study suggests that complement plays a role also in platelet aggregation induced by C. acnes.The presence of complement on the surface of with IgG ,55. It hwith IgG . Other bregation . The proC. acnes leads to aggregation, the biological significance of this for the survival of C. acnes is debatable. Bacteria are supposedly contained by the platelets and delivered high concentrations of bacteriocidal substances from the activated platelets. Certain pathogens, including S. pyogenes have evolved means to survive this, and even take advantage of this containment for improved survivability in blood before releasement from the aggregate [C. acnes can induce aggregation of platelets without prior activation of the platelets, leading to an entrapment, but not killing, of the bacteria. Interestingly, even addition of high concentrations of LL-37 (250 μM) to the mixture did not affect the viability of the bacteria but was antibacterial in the absence of PRP. While LL-37 has a slightly lowered antibacterial activity in serum as compared to PBS, 20 μM LL-37 has been reported to still have a significant antibacterial effect [C. acnes can induce platelet aggregation for protection against serum proteins (e.g. LL-37), facilitating survival in this harsh environment. C. acnes is noticeably difficult for the immune system to handle, and has recently been suggested to survive intracellularly within macrophages and osteoblasts [C. acnes has been reported to withstand most of the effects of lysozyme, PMNs, monocytes, and their released contents, including MPOs [C. acnes to efficiently reduce free oxygen radicals through its secreted antioxidant RoxP remains to be elucidated [C. acnes is multifactorial, including several plasma proteins (IgG and fibrinogen) as well as possibly specific surface antigens, less commonly exposed (or expressed at a lower ratio) in type IB strains.Even though the platelet response towards ggregate . Seemingl effect . Thus, weoblasts ,71. Of ming MPOs . Whetherucidated . HoweverC. acnes will be in contact with platelets, it will mainly be in either a low infection dose , such that no platelet aggregation will be initiated, or as part of a biofilm. Initial experiments indicate that platelets will recognize a biofilm and aggregate upon contact with C. acnes biofilms, however more research is needed to further investigate this. Further, while not necessarily being a limitation, the partly activation-independent platelet aggregation mediated by C. acnes is rather unusual, since most platelet aggregations are initiated by an initial activation [C. acnes eliciting an aggregation in absence of activation remains to be elucidated. Thus, while providing evidence of a C. acnes-platelet interaction, being mediated by common platelet-activating proteins, leading to aggregation, it is clear that this particular interaction is more than meets the eye, and further research will be needed to investigate these interactions.The study has a few limitations that should be noted. Firstly, in most scenarios when tivation . There ativation . The mecS1 FigC. acnes (1x108 cfu/ml) and PBS , or C. acnes (1x108 cfu/ml) and prostaglandin E (1 μM). Aggregation was measured using a platelet aggregometer (ChronoLog) and analyzed using AggroLink.Platelet rich plasma (PRP) was incubated with collagen (5 μg/ml) as a positive control (A), and with (PDF)Click here for additional data file.S2 FigC. acnes (1x108 cfu/ml) and PBS . To PRP incubated with a high concentration of C. acnes (4x108 cfu/ml), PBS or collagen (5 μg/ml) was added after 16 minutes . The immediate drop is due to technical reasons when adding the reagents (e.g. PBS and collagen). Aggregation was measured using a platelet aggregometer (ChronoLog) and analyzed using AggroLink.Platelet rich plasma (PRP) was incubated with collagen (5 μg/ml) as a positive control , and with (PDF)Click here for additional data file.S3 FigC. acnes isolates AS12, AS13, AD1 and KPA171202 were incubated with platelets in plasma and aggregation measured using an aggregometer (Chronolog). Failure to induce aggregation (t>25 min) resulted in removal of that strain. Each dot in the figures represents one experiment . All experiments were performed three independent times and are presented as medians with range where appropriate.(PDF)Click here for additional data file.S4 FigRepresentative figure demonstrating the gating of a platelet population in platelet-rich plasma and the detection of PAC-1 and CD62P on the surface of the platelets in that population.(PDF)Click here for additional data file.S5 FigC. acnes and deposition of IgG detected through flow cytometry and reported as fold increase of median fluorescence intensity / platelet (anti-IgG FITC).Serum from three donors was incubated with 4 different strains of (PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Alphaproteobacteria  containing 4% (v/v) ethanol. Genomic DNA was extracted using phenol-chloroform extraction and ethanol precipitation. The quality of purified genomic DNA was tested by using NanoDrop 2000 UV–Vis spectrophotometer  and Qubit 2.0 fluorometer .Strains LMG 1590De novo assembly of the read sequences was carried out using the hierarchical genome assembly process workflow. The annotation of the sequences was carried out using a modified version of the Prokka annotation pipeline, which incorporated Prodigal 2.60, Aragorn, and RNAmmer 1.2 for the prediction of open reading frames, tRNAs, and rRNAs, respectively . Seemann, . Genome A. pasteurianus subsp. ascendens LMG 1590T) and CP015168-CP015171 (A. pasteurianus subsp. paradoxus LMG 1591T) in October 2016. The BioProject designations for strains LMG 1590T and LMG 1591T are PRJNA322127A. pasteurianus subsp. ascendens LMG 1590T and A. pasteurianus subsp. paradoxus LMG 1591T are available from the BCCM/LMG Bacteria Collection under accession numbers LMG 1590 and LMG 1591, respectively.The complete genome sequences have been deposited in GenBank under the accession numbers CP015164-CP015167 . A total of 3,163 genes were predicted from the genome of strain 1591T of which 3,088 genes were identified as CDSs. The genome sequences data are available in FASTA, annotated GenBank flat file, graphical, and ASN.1 formats.We obtained 218,360 raw reads covering a total of 1,387,777,653 bp with 228× genome coverage for strain LMG 1590A. pasteurianus strains, including LMG 1590T and LMG 1591T, and the previously sequenced A. pasteurianus species, a global alignment of genome sequences from 13 strains was performed using Mauve software  . The ADHs and ALDHs from the 13 strains were further aligned using Clustal Omega -alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are responsible for the oxidative metabolism of ethanol to produce acetic acid in AAB. These proteins generally consist of three subunits: a quinohemoprotein catalytic subunit, a triheme cytochrome c subunit, and a third subunit with unknown function. Sequence analysis of the catalytic subunits of the ADHs from strains LMG 1590A. pasteurianus subspecies were sequenced and assembled into one chromosome and three plasmids. Comparative genome and sequence analyses showed that rearrangements occurred in the A. pasteurianus strains and that the ADHs and ALDHs responsible for acetic acid production were highly conserved in these strains.In conclusion, the complete genomes of two BC performed the experiments. BJ and CJ analyzed the data and wrote the manuscript. GC, KK, JM, and S-HY helped in data analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer, FF, and handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.


Alternaria including Alternaria linariae (which includes A. solani and A. tomatophila), as well as A. alternata. In some instances, annual economic yield losses due to EB have been estimated at 79%. Alternaria are known only to reproduce asexually, but a highly-virulent isolate has the potential to overcome existing resistance genes. Currently, cultural practices and fungicide applications are employed for the management of EB due to the lack of strong resistant cultivars. Resistance sources have been identified in wild species of tomato; some breeding lines and cultivars with moderate resistance have been developed through conventional breeding methods. Polygenic inheritance of EB resistance, insufficient resistance in cultivated species and the association of EB resistance with undesirable horticultural traits have thwarted the effective breeding of EB resistance in tomato. Several quantitative trait loci (QTL) conferring EB resistance have been detected in the populations derived from different wild species including Solanum habrochaites, Solanum arcanum and S. pimpinellifolium, but none of them could be used in EB resistance breeding due to low individual QTL effects. Pyramiding of those QTLs would provide strong resistance. More research is needed to identify additional sources of useful resistance, to incorporate resistant QTLs into breeding lines through marker-assisted selection (MAS) and to develop resistant cultivars with desirable horticultural traits including high yielding potential and early maturity. This paper will review the current understanding of causal agents of EB of tomato, resistance genetics and breeding, problems associated with breeding and future prospects.Early blight (EB) is one of the dreadful diseases of tomato caused by several species of  Solanum lycopersicum Linnaeus), native to the Andean region of South America, is one of the most common horticultural crops and cultivated throughout the world. It can be grown in a wide range of climates from tropical to temperate; it also can be cultivated under cover conditions when outdoor temperatures are not favorable. Tomato is the world’s second most consumed vegetable after potato [Tomato , which have also been isolated from solanaceous plants and have been reported to cause EB-like symptoms in some situations [Alternaria species, several morphological, molecular and chemotaxonomic approaches have been applied. The taxonomy of  tomato” . More re tomato”  grouped ineariae . Large stuations . To diffA. solani and A. tomatophila are morphologically very similar. Measuring the size of conidia and beak length in various media showed that A. tomatophila was generally shorter and had a thinner body with a longer beak than A. solani. However, due to variation within the same species, it was not possible to draw definitive conclusions [A. solani isolates showed similar aggressiveness in both tomato and potato, while A. tomatophila was highly aggressive in tomato, but weaker in potato [clusions . Based on potato .A. solani from complex environments. Using the internal transcribed spacer (ITS) region, a PCR method was developed for the detection of Alternaria species from raw and processed tomato samples [AS1 and AS2) and cytochrome b were successfully used for the specific detection of A. solani from the closely-related Alternaria species including A. tomatophila in tomato and potato [Molecular techniques have been developed for detecting and distinguishing  samples . Furtherd potato ,13.A. solani. Amplified fragment length polymorphism (AFLP) analysis conducted on 112 A. solani isolates from Cuba and other countries around the world [Several studies support host speciation in he world  showed tAlternaria isolates from different hosts, mainly potato and tomato and a few from wild potato species in Russia, and compared them with representatives of A. solani and A. tomatophila collected in the USA [Alternaria major allergen (Alt a1), glyceraldehyde 3-phosphate dehydrogenase (gpd) and calmodulin revealed six, four and four single-nucleotide polymorphism (SNP) markers, respectively. Haplotype analysis showed two major groups representing A. solani and A. tomatophila [A. solani from the potato cluster had mating type locus 1 (MAT1) idiomorph MAT1-1, while 88% of A. tomatophila isolates had the MAT1-2 idiomorph.Similarly Gannibal, Orina, Mironenko and Levitin  analyzed the USA . Sequencatophila . FurtherAlternaria species, A. dauci, A. porri, A. solani and A. tomatophila [A. dauci, A. solani and A. tomatophila as three distinctive species with their own metabolite profiles. Furthermore, this study revealed that altersolanol A, altertoxin I and macrosporin are produced both in A. solani and A. tomatophila, but not in A. dauci. Another chemotaxonomic approach of proteins, using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) of 37 isolates showed three major clusters corresponding mainly to A. dauci, A. porri and A. solani/A. tomatophila, but was able to distinguish A. solani from A. tomatophila [Secondary metabolite profiling has been used to distinguish large-spored atophila . Metabolatophila .A solani, A. tomatophila and several other Alternaria species has been made publicly available [A. solani, recognizing that several others closely-related Alternaria species can cause EB of tomato.Recently, the genome sequence of vailable . Future A. solani reproduces asexually; a sexual stage of this fungus is unknown. The fungus overwinters in soil, plant debris, seed and alternate hosts in the form of either conidia or mycelia, which may serve as primary sources of inoculum , length of conidiophores, dimension of the spore body and vegetative compatibility. Morphological variations are often affected by environmental factors such as substrate, light intensity and temperature. Therefore, other variations should be taken into account while differentiating among species in addition to morphological variation. High variations among isolates have been detected at the molecular level using several molecular markers. In addition, A. solani also possesses extensive variation in terms of its toxin production. Several Alternaria species, which were difficult to distinguish by their morphological characteristics, have been classified based on their toxin production. Nonetheless, differentiation of A. solani based on toxin production is still not unanimous among researchers and scientists [Despite having only asexual reproduction, isolates of mutation . Morpholientists .A. solani can easily adapt to the changing environment and develop resistance to fungicides [A. solani known or confirmed so far, suggesting host non-specific recognition of the pathogen. This makes the studies of the pathology and genetics of EB more complex [Because of high genetic variation, ngicides . Furtherngicides . However complex . A. alternata has been reported to produce AAL toxin and causes Alternaria stem canker in tomato [PKS) ALT1 was involved in producing the AAL toxin biosynthesis. Additional toxins associated with Alternaria sp. infection have been reported such as Brefeldin, tentoxin, ACT toxin, AF-toxin, AK-toxin, ACTG-toxin, ACR-toxin, AM-toxin, AS-toxin and maculosin destruxin B [Fungal pathogens produce a number of enzymes and secondary metabolites that help to infect the host plant. The secondary metabolites may kill or activate the host cells and penetrate into the cells before invasion. n tomato . A polyktruxin B . Enzymestruxin B .A. solani. Many plant pathogenic fungi including Alternaria species produce phytotoxins. Based on selectivity, phytotoxins are classified as either host-specific toxins (HSTs) or non-host-specific toxins (NST). Alternaria species produce a variety of HSTs, and most are low molecular weight secondary metabolites. The HTSs are structurally diverse and some are involved in pathogenicity or virulence during host-plant interaction, as has been shown in A. alternata [To date, limited studies have been conducted to determine the molecular basis of infection and host responses to lternata ,28. A. solani produces various types of phytotoxic metabolites, including alternariol, altersolanol A, altertoxin, macrosporin and solanapyrone [Alternaria species revealed that A. solani, as well as A. dauci, A. porri and A. tomatophila strains produce common secondary metabolites including altersolanol A, altertoxin I and macrosporin, as well as unknown A. solani-specific secondary metabolites [napyrone ,29. Secoabolites . The rolA. solani. Solanapyrone is derived from a biosynthetic gene cluster consisting of six sol genes encoding a polyketide synthase, an O-methyltransferase, a dehydrogenase, a transcription factor, a flavin-dependent oxidase and cytochrome P450 [Sol5 encodes Diels-Alderase, which is involved in the last step of solanapyrone biosynthesis. Sol5 gene knock out mutants were unable to synthesize solanapyrone, but showed no change in virulence compared to the wild type. Thus, despite the strong phytotoxicity of solanapyrone, it is not required for pathogenicity by the fungus [Several forms of solanapyrone A, B and C are produced in ome P450 . Sol5 ene fungus .A. solani and proposed to be involved in phytopathogenicity [Extracellular proteases are key enzymes produced by fungi during the host-pathogen interaction and are involved in growth, development, survival and pathogenicity . The pregenicity ,33. HoweA. solani, plant chitinases produce strong antifungal effects, and transgenic expression of an endo-chitinase in potato provided disease resistance [Chitin is the major component of most fungal cell walls. Plants express chitinases during fungal infection and are recognized as pathogenicity-related (PR) proteins as part of the defense response against fungal attack . Againstsistance . Similarsistance .A. solani [A. solani possibly by activating PR protein synthesis [Microarray expression profiling has been used to investigate differential gene expression in susceptible and resistance tomato varieties in response to . solani . A numbeynthesis . HoweverA. solani is sensitive to quinone outside inhibitor, strobilurin and fungicides. However, resistance is being encountered. An amino acid substitution at position 129 in A. solani cytochrome b  is responsible for the decreased sensitivity [sitivity . A singlsitivity .A. solani, as well as several other key Alternaria species has been published [A. solani (BMP 0185) was predicted to contain 11,726 genes in a 32.9-Mb genome. In the coming years, comparative and functional genome analysis among Alternaria spp. will help shed new light on the molecular mechanism of disease caused by A. solani.Recently, a whole genome sequence for ublished . Based oA. solani appear in leaf (leaf blight or early blight), stem (collar rot or stem lesions) and fruit (fruit rot) of tomato plant [The symptoms of to plant ,39. Leafto plant . The disto plant . Older lto plant .Collar rot is typified by initial dark and sunken lesions on the stem, which later expand to form lens-shaped lesions with concentric rings similar to those on leaves. Lesions at the ground level girdle the stem in young seedlings, destroy the vascular system and form “collars” . Collar The infection on fruit causes dark, sunken, leathery and purple lesions on the stem-end. These lesions expand to a significant size and extend deep into the flesh of the fruit. Infected fruits mostly drop prematurely, and those reaching to maturity also become unmarketable .EB can be controlled by three measures: cultural practices, fungicide treatment and the use of resistant varieties. Cultural practices and fungicidal treatment are more common practices . CulturaSeveral types of fungicides have been developed for the control of EB, but fungicide treatment is not economically feasible, nor environmentally sound. Fungicides are first applied 1–2 days after transplantation and then require routine application at the interval of 7–10 days for effective control, thereby increasing production cost and environment pollution ,20. In NPseudomonas—P. fluorescens, P. aeruginosa, P. putida and P. cepacia—have resulted in less disease severity and higher fruit weights compared to control in tomato [P. gladioli B25 reduced EB disease by 60.2% by inducing systemic resistance in the host due to an enhanced level of phenol accumulation, peroxidase activity and PALase activity [Cinnamomum zeylanicum, Syzygium aromaticum, Ferula foetida, Inula racemosa, Hemidesmus indicus, Rubia cordifolia, Glycyrrhiza glabra and Saussurea lappa also showed antifungal activity against EB pathogen ranging from a medium to a high level [Trichoderma viride [T. harzianum [Bacillus subtilis also increased systemic resistance in tomato by inducing growth hormones and defense-related enzymes such as peroxidase, polyphenol oxidase and superoxide dismutase [The use of biological controls has also been documented in several research papers as an alternative method for the management of EB. The four species of n tomato . Similaractivity . Extractgh level . In addia viride  and T. harzianum  in the mismutase . Other aismutase .The association of the microbial community including mycorrhiza with plants’ growth and development has been known for a long time. These microbes produce different types of hormones including auxins and cytokinins and impact plant growth ,48,49. AUse of resistant varieties is the most provident, effective and sustainable control measure of EB of tomato. The genetic integration of effective EB resistance into cultivated tomato would reduce dependence on chemical inputs and have significant environmental, health and financial benefits.Disease screening plays a key role in the identification of resistant lines from diverse germplasm, which then can be used as a source of resistance in breeding programs. Proper disease screening methods are required in disease resistance breeding to get reliable and accurate data. Disease symptoms can be evaluated in the field, greenhouse and growth chamber.A. solani can be prepared in various culture media such as potato-dextrose agar (PDA) [2O (containing 0.01% Tween 20) and brushing the agar surface with a paintbrush [Inoculum of ar (PDA) , V-8 juiar (PDA) , lima bear (PDA)  and sporar (PDA) . Sporulaar (PDA) ,57. The intbrush . A sporeintbrush Field screening is the most common method of screening. It allows screening of a large number of populations at the same time under natural conditions and keeping track of disease severity in every phase of the plant life cycle . EB seveHowever, field screening has some disadvantages. It requires a large amount of inoculum, disease spreader rows and intensive labor for the evaluation of large populations. It is also difficult to maintain treatment uniformity in the field. The high disease pressure and confounding effects of other foliar diseases reduce the effectiveness of field screening . To avoiGreenhouse screening is useful in regions where field screening is difficult due to an unfavorable environment and confounding effects of other complex diseases. As the environment can be controlled in the greenhouse, it provides a conducive, uniform and repeatable environment. Unlike field evaluation, greenhouse screening allows several cycles of screening in a year . During Several studies have been conducted in greenhouses with different results. Greenhouse studies involving moderately-resistant and susceptible lines did not show a significant difference in their symptoms ,62, wherGreenhouse screening is affected by several factors including plant age, inoculum quality and quantity, inoculation technique and pre- and post-inoculation environmental conditions . As younA. solani and the evaluation of EB resistance along with other methods.The detached leaflet assay is performed in a growth chamber. Unlike field and greenhouse evaluation, it can only measure foliar resistance. A droplet of spore suspension is deposited on the upper surface of either punctured  or non-pS. lycopersicum) are mostly susceptible to early blight, and only a few resistant accessions are available. S. lycopersicum accession PI138630 has been used as a source of resistance in early EB-resistant breeding [S. lycopersicum that are not known to be used for EB resistance breeding in tomato such as S. lycopersicum f. sp. cerasiforme Accession PI406758 (highly EB resistant) [Cultivated tomatoes  .S. habrochaites, S. pimpinellifolium and S. peruvianum, and utilized to develop resistant lines in a tomato breeding program [S. habrochaites is the richest source of resistant genes among wild species of tomato, but these species are late maturing, indeterminate and low yielding. Some highly-resistant S. habrochaites accessions are: PI127827, PI126445, PI390513, PI390514, PI390516, PI390658, PI390660, PI390662, PI390663, LA2100 and LA2650 [S. habrochaites accession PI390662 was used to develop EB-resistant USDA Line 87B187 [1 hybrids of resistant accessions of S. habrochaites and the cultivated tomato showed a moderate to high degree of resistance, yet they also possessed other undesirable characteristics of S. habrochaites such as late maturity and small fruit size.Resistant sources have been identified in wild species  program . S. habrd LA2650 ,73. These 87B187  and PI12e 87B187 . The F1 S. pimpinellifolium is a close relative of cultivated tomato, hence, it can be easily crossed with cultivated tomato. It also possesses fewer undesirable traits compared to other wild species of tomato. Therefore, S. pimpinellifolium has been frequently used in tomato breeding programs. Several highly- and moderately-resistant S. pimpinellifolium accessions have been obtained in a field, greenhouse and growth chamber evaluation [S. pimpinellifolium Accession LA1921 and its BC4F4 progeny exhibited high resistance towards EB [aluation ,75. S. pwards EB .S. peruvianum, they have not been fully utilized in tomato breeding for EB resistance because of high genetic variation within each accession and the cross-incompatible nature of S. peruvianum with cultivated tomato [Although some resistant accessions PI129152, PI127829, PE33, LA1292, LA1365, LA1910, LA1983 and PI270435, have been identified in d tomato ,73 All tEB resistance is a complex trait that is quantitatively inherited and controlled by the additive or non-additive interaction of multiple genes and their interaction with the environment ,61. No sh2) of 0.26–0.38 [A. solani compared to susceptible lines. EB resistance was associated with enhanced expression of TPK1b (a defense-related gene), increased accumulation of H2O2, phenolic compounds and superoxide anion, a higher percentage of cross-linking and polyphenol activity, decreased DNA damage and less reduction in cell viability [A low to moderate heritability  ,87. TherThe development of molecular markers opens the possibility of solving the problems seen in traditional breeding of EB resistance. Molecular markers, such as restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs), cleaved amplified polymorphic sequences (CAPS), resistance gene analogues (RGAs), expressed sequenced tags (ESTs) and conserved orthologs sets (COS), have already been mapped onto all 12 tomato chromosomes and used for locating QTLs governing important agricultural traits . Such ma1 and BC1S1) resulting from a cross between susceptible S. lycopersicum (NC84173) and highly-resistant S. habrochaites (PI126445) [1 population before EB evaluations to avoid confounding effects of those factors on EB resistance. Nine significant QTLs on chromosomes 1, 2, 5, 9, 10, 11 and 12 and two minor QTLs on chromosomes 8 and 11 were detected in the BC1 population for EB resistance using the simple interval mapping method. The individual effects ranged from 8.4% (chromosome 5) to 21.9% (chromosome 1). Simple interval mapping analysis also detected thirteen significant QTLs in the BC1S1 population on chromosomes 1, 2, 3, 5, 8, 9, 10, 11 and 12 for EB resistance, with individual effects varying from 7.9% (chromosome 5) to 25.9% (chromosome 9). The six QTLs out of 11 in the BC1 population on chromosomes 1, 5, 9 (two QTLs), 10 and 11 and, again, the six QTLs out of 13 located on chromosomes 1, 5, 8 (two QTLs), 9 and 10 in the BC1S1 population were confirmed as independent QTLs using the composite interval mapping method [The first QTL mapping effort for EB resistance was performed on backcross populations (BCI126445) . In thisg method .1 population using a selective genotyping approach to detect and confirm QTLs governing EB resistance in PI 126445. They identified seven QTLs on chromosomes 3, 4, 5, 6, 8, 10 and 11; four of which, on chromosome 5, 8, 10 and 11, were also detected in the previous study of Foolad, Zhang, Khan, Nino-Liu and Lin [Zhang, Lin, Nino-Liu and Foolad  conducte and Lin , suggest2 and F3. F2 and F3 populations were obtained from a cross between EB-resistant S. arcanum (LA2157) and susceptible S. lycopersicum (Solentos) [Another study identified six QTLs governing EB resistance on chromosomes 1, 2, 5, 6, 7 and 9 that explained 7–16% of total phenotypic variance, using different environmental conditions and disease scoring methods in Folentos) . QTLs onolentos)  and Zhanolentos) .S. pimpinellifolium. After extensive screening of 300 accessions of S. pimpinellifolium, Accession LA2093 (previously designated as PSLP125) was selected for QTL mapping. LA2093 has good EB resistance plus other desirable characteristics [2, F3 and F4 populations derived from the cross of LA2093 and tomato breeding line NCEBR-1 were used for QTL mapping, which detected 10 QTLs conferring EB resistance on chromosomes 2, 3, 4, 5, 6, 7, 9 and 12 with an individual effect of 7.6–13.4% and a combined effect of 44% of total phenotypic variance [2 population of the same cross for QTL mapping through a selective genotyping approach, where nine EB QTLs were detected on chromosomes 1, 2, 3, 4, 5, 6 and 11 [QTL mapping for EB resistance has also been conducted in a population derived from eristics ,80,86. Fvariance . Out of 6 and 11 . Therefo7, F8, F9 and F10 generations of the same cross (LA2093 x NCEBR-1) [Again, five major QTLs for EB resistance were identified on chromosomes 2 (two QTLs), 5, 6 and 9, using recombinant inbred lines (RILs), i.e., the FNCEBR-1) . QTLs onNCEBR-1) . rolB gene of Agrobacterium rhizogenes. Babu, Jogaiah, Ito, Nagaraj and Tran [A. solani. Tomato plants pre-treated with PGPR showed improved resistance against early blight, which was associated with increased synthesis of antioxidant peroxidase (POX) and polyphenol oxidase (PPO) enzymes. This suggests the potential utility of POX and PPO genes to develop transgenic tomato lines with improved resistance against early blight [Several breeding efforts are ongoing to develop early blight-resistant varieties, but no commercial early blight-resistant variety has been reported yet . The lacand Tran  reportedy blight .To conclude, EB resistance is a complex trait, governed by multiple genes, and yet, no pathogen races have been reported. There is no single gene-for-gene model for EB resistance and no major resistance gene identified. Furthermore, EB resistance is affected by different factors including physiological maturity, plant type, growth habit and fruit load. More importantly, the existing EB resistance in cultivars is not enough, and still, growers depend on cultural practices and extensive use of fungicides for the control of EB . AlthougBreeding programs require stable QTLs having large additive effects and independent of epistatic interactions to be used in MAS. In the above studies, QTLs for EB resistance have been detected in almost all tomato chromosomes in different interspecific crosses. Among them, QTLs on chromosome 5 and 6 were reported in every study and have also been verified in a study by Foolad, Merk and Ashrafi . QTLs onS. pimpinellifolium, where there are limited marker polymorphisms [Additionally, narrow genetic diversity among cultivated tomatoes, limited polymorphic markers for intraspecific populations, extreme labor for markers like AFLP and RFLP and the unavailability of reliable PCR-based markers are major problems in molecular breeding of tomato. Most genetic maps were based on interspecific crosses of cultivated tomato and wild species because of the greater number of polymorphic markers, but most breeding populations are based on intraspecific crosses of cultivated tomato or with that of the closely-related species orphisms . The polorphisms .S. lycopersicum or between S. lycopersicum and closely-related species. The work in [S. pimpinellifolium. Identification of SNPs and their utilization to uncover genetic variation among elite cultivars provides new insights into tomato breeding for EB resistance. With the development of next generation sequencing technologies and the decreased cost of sequencing, highly polymorphic markers such as SNPs can now be incorporated into the tomato breeding program, which can detect the polymorphisms among closely-related individuals within  work in  identifi work in  identifi work in  identifiIn general, breeding for EB resistance in tomato without compromising other desirable horticultural traits is a demanding task. More research and investigations are needed to identify additional resistant sources, to validate, utilize and introduce detected QTLs in cultivated tomato species for the release of EB-resistant breeding lines and cultivars with high yield and early to mid-season maturity. S. habrochaites, S. pimpinellifolium and S. arcanum. QTLs on chromosome 5 and 6 were stably observed across generations and environments with the effect of 10–16% [S. habrochaites PI 126445 accession [S. arcanum LA2157 accession was also stable and showed a major effect on the stem lesion resistance, i.e., 35% of total phenotypic variance [QTLs have been identified in f 10–16%  and alsoccession ,93. The variance . HoweverAlternaria solani or A. alternate. Alternatively, there may be an enhanced level of resistance in tomato in the presence of certain microbes, which needs to be investigated. This is a completely new area of investigation. The new approach of crop improvement may be exploited to investigate if there are any microbial species that may suppress the growth of


Small non-coding RNAs, including miRNAs, and gene silencing mediated by RNA interference have been described in free-living and parasitic lineages of flatworms, but only few key factors of the small RNA pathways have been exhaustively investigated in a limited number of species. The availability of flatworm draft genomes and predicted proteomes allowed us to perform an extended survey of the genes involved in small non-coding RNA pathways in this phylum.Macrostomum lignano. We here described all the putative small RNA pathways present in both free living and parasitic flatworm lineages.Overall, findings show that the small non-coding RNA pathways are conserved in all the analyzed flatworm linages; however notable peculiarities were identified. While Piwi genes are amplified in free-living worms they are completely absent in all parasitic species. Remarkably all flatworms share a specific Argonaute family (FL-Ago) that has been independently amplified in different lineages. Other key factors such as Dicer are also duplicated, with Dicer-2 showing structural differences between trematodes, cestodes and free-living flatworms. Similarly, a very divergent GW182 Argonaute interacting protein was identified in all flatworm linages. Contrasting to this, genes involved in the amplification of the RNAi interfering signal were detected only in the ancestral free living species These findings highlight innovations specifically evolved in platyhelminths presumably associated with novel mechanisms of gene expression regulation mediated by small RNA pathways that differ to what has been classically described in model organisms. Understanding these phylum-specific innovations and the differences between free living and parasitic species might provide clues to adaptations to parasitism, and would be relevant for gene-silencing technology development for parasitic flatworms that infect hundreds of million people worldwide.The online version of this article (10.1186/s12862-017-1061-5) contains supplementary material, which is available to authorized users. Fasciola hepatica [Opisthorchis viverrini [Clonorchis sinensis [O. felineus [Moniezia expansa [Hymenolepis microstoma [Echinococcus granulosus [E. multilocularis [Taenia crassiceps [S. mansoni [F. hepatica [RNA interference (RNAi) is a reverse genetic tool that triggers post-transcriptional knock-down of a target mRNA by the introduction of complementary double-stranded RNA (dsRNA) molecules. It is almost the only available tool to study a gene function in helminths . In plathepatica –7, Opistiverrini , Clonorcsinensis , O. felifelineus  and in t expansa , Hymenolcrostoma , Echinocanulosus , E. multocularis  and Taenassiceps . While t mansoni –19 and Fhepatica . Is stilWhile the information on RNAi pathways in flatworms is fragmentary, several reports have detected the presence of microRNAs (miRNAs) in almost all the flatworm lineages –33. MicrM. lignano [The piRNA pathway is a small RNA mediated mechanism involved in the silencing of transposable elements in germline cells and hence, maintaining the genome stability . While c lignano , they ha lignano .Caenorhabditis elegans to conduct a bioinformatic search in flatworms with available transcriptomic/genomic data.Despite increasing evidence supporting the conservation of functional small RNA pathways in flatworms, they are still poorly described in most of the platyhelminth species. Reports comparing small RNA pathways among flatworms generally focus on the two main proteins, Dicer and Argonaute –42, whilC. elegans, we used a curated complete set of these factors as queries for comparisons .drsh-1 gene in all analyzed flatworms was observed, consistent with its central role in miRNA biogenesis  by ribonuclease III enzymes is a key step in the biogenesis of small non-coding RNAs. Dicer (Dcr) belongs to this group of ribonucleases and process dsRNA into 22-23 nt RNAs with two-nucleotide 3′ overhangs that are recognized by Argonaute proteins . Similartebrates . In C. efamilies . ExpecteD. melanogaster Dcr-1 and Dcr-2 proteins have evolved different roles, Dcr-1 processes pre-miRNA, i.e. miRNA precursor molecules, while Dcr-2 processes dsRNA in the RNAi pathway [In  pathway . Dcr-1 w pathway . Due to Arabidopsis thaliana Ago1; (2) Piwi-like proteins, closely related to D. melanogaster PIWI; (3) a specific group identified in C. elegans called group 3 [Argonaute proteins are essential for gene regulatory mechanisms mediated by small RNAs. The Argonaute (Ago) genes have been classified into four paralogous groups: (1) Argonaute-like (Ago-like) proteins, similar to  group 3 , 48, rep group 3 ; and (4) group 3  and clus group 3 .In agreement with previous reports, our exhaustive homology search among diverse flatworms showed multiple hits in all species ranging from 3 in cestodes to more than a dozen in free-living species. A more detailed phylogenetic analysis aided by structure determination allowed us to further classify these hits.S. mediterranea showed tree paralogues, in M. lignano thirteen paralogous sequences were detected  silencing, and hence, maintaining the genome stability in germline cells . This grterranea , 38 and japonica , and mor lignano . AccordiS. mansoni, F. hepatica and E. granulosus showed that FL-Agos were being expressed in several stages  pathway suppressing active TE in both the somatic and germline cells [While the presence of flatworm-specific Agos have been previously noticed in model species , 42, we ne cells . ConsideC. elegans [S. mansoni [C. elegans, Dcr-1 further process pre-miRNAs into a ~ 22 nt RNA duplex [D. melanogaster, however, Dcr-1 requires Loquacious (Loqs), a dsRNA-binding protein, for pre-miRNA processing [Pri-microRNAs are transcribed by RNApol II and processed into 60–100 nucleotides long pre-miRNA by the RNase III Drosha and its partner Pasha in a complex named microprocessor . We conf elegans . In vert elegans , 54. An  mansoni  and we dA duplex . In D. mocessing . Loqs orC.elegans, Ago1 in D. melanogaster) recognize short RNA duplexes, cleaving the passenger strand from the mature miRNA. The Ago protein in tandem with the miRNA and other associated factors form the miRNA Induced Silencing Complex (miRISC) that lead to the degradation of target mRNAs [Argonaute gene products  contributes to translation repression and ‘label’ the target mRNA for decay via deadenylation and decapping [C. elegans. This lack of common domain architecture suggests that AIN-1 and AIN-2 may not be members of the GW182 protein family, but rather represent functional analogs [GW182 is one of the most critical miRISC factors. In ecapping . Moreoveecapping . GW182 fecapping ). On the analogs .Fig. 4CoC. elegans, Gawky from D. melanogaster, and a GW182 family member recently reported in Crassostrea gigas [M. lignano using the C. gigas sequence, probably due to closer evolutionary distance between these species. The M. lignano sequence was predicted to contain UBA and RRM domains like the GW182 family member. Despite the unstructured nature of GW182 family members, outside UBA and RRM domains there are two short but well conserved motifs in the Ago-binding domain and the silencing domain  are clos CHUP-1 . It has r domain  suggestiC. elegans is SID-2. Interestingly, SID-2 is required for RNAi delivered by dsRNA soaking or feeding , but not for systemic RNAi. The protein is mainly expressed in the apical intestinal membrane where in coordination with SID-1 internalizes the interfering molecules [Other key transmembrane protein involved in dsRNA uptake in olecules . Even tholecules , 9, 16, olecules .C. elegans, the dsRNA is first recognized by the dsRNA-binding protein RDE-4 and presented to DCR for processing in the cytoplasm [D. melanogaster [, were searched. No proteins similar to RDE-4 or R2D2 were identified in flatworms, an intriguing observation considering its critical role in the generation of siRNAs [In ytoplasm . RDE-4 iytoplasm , 49, hennogaster , were sef siRNAs . HoweverIn addition to Dcr and Ago genes, we identified putative homologs of DRH-1, a DExH-box helicase protein associated to Dcr and XRN-1, a 5′-to-3′ exonuclase that degrades the mRNA targeted by the RISC complex  as query to search by tBLASTn on the flatworm genomes. Only M. lignano showed three genomic regions with high similarity (43.3% on average), two of them with annotated genes. These genes were used as bait to test the other flatworm genomes with no hits identified. Therefore, it is possible that Mut-7 homologs has been lost in the evolution of flatworms or a high evolution rate within the flatworm phylogeny made them undetectable by our method. RDE-2, on the other hand, has only been detected in C. elegans and other Caenorhabditis species [No homologs of Mut-7 and RDE-2 in flatworms were detected; these factors are involved in a protein complex in derstood . In addi sapiens .Therefor species , 71. We C. elegans, in addition to exo-siRNA, a endo-RNAi pathway triggered by endogenous dsRNAs derived from overlapping genes, long hairpins, and TE in the germline, has been reported [In reported , 73. Thereported . The supreported , 77, sugreported , 78.M. corti. In addition, in the Diphyllobothriidae order only D. latum seem to have lost this gene  domain, suggesting that RSD-3 may play a role in the traffic of vesicles during systemic RNAi [In pathways . This phpathways . In C.elmic RNAi .D. melanogaster. The demonstration of RNAi silencing by feeding in planaria [In flatworms, among all the factors of this pathway only homologous genes of RSD-3 were identified  exist, being expanded independently in the diverse linages Fig. . The relHere we used a strict homology search method against genomes that are in most cases in a draft stage. We did not rely only in current annotation  adding a whole genome homology search to confirm the putative absences. We are quite confident that this combined approach provide a good picture of the main aspects of small RNA pathways in flatworms. Other in silico approaches like PSI-BLAST search , a methoAdditionally, experimental approaches are needed to confirm some of the results reported here and to detect novel factors that could be developed uniquely in flatworms. Functional genomics utilizing RNAi , transgeIn any case, we believe that the results reported here are relevant not only to shine a light on the basic biology and gene regulation mechanisms in these organisms, but also to optimize the RNAi as functional genomic tool for parasitic worms, agents of neglected tropical diseases affecting hundreds of million people worldwide.C. elegans were identified from the literature . A local repository with the genomes and proteomes of 8 species of Trematodes, 12 species of Cestodes, 2 Monogeneans and 2 Turbelaria was generated (Table http://parasite.wormbase.org) and The Gyrodactylus salaris Genome Project (http://invitro.titan.uio.no/gyrodactylus/). Results were later corroborated with the new genomic annotations published in Wormbase Parasite release 8.Sixty-one proteins involved in small non-coding RNA pathways in C. elegans small RNA pathways factors were used to interrogate the proteomes of platyhelminthes using BLASTp [C. elegans proteome. Only the best reciprocal hits between flatworms and C. elegans were retained for further inspection.The protein sequences of all g BLASTp . All hitC. elegans. Alternatively, if a positive hit was retrieved in some flatworm species but not in others, these were used as queries for new genomic and proteomic reciprocal blast searches. This strategy helped to overcome the disparity on quality and completeness of genomic assemblies and/or gene annotation among the genomes deposited in Wormbase Parasite (http://parasite.wormbase.org/).When no homologous protein was identified or the retrieved gene was incomplete or fragmented, a genomic search using tBLASTn was conducted. A careful inspection of the resulting High-scoring Segment Pairs (HSP) aligned to the genomes were useful to extend gene annotation or to fully annotate putative novel genes. These were confirmed by reverse tBLASTn to C. elegans proteins, domains were predicted with HMMScan [C. elegans protein were considered as orthologs. However, we consider flatworm genes to be homologs when reverse BLAST produced a hit to a paralogous of the gene of C. elegans involved in the small non-coding RNA pathways, to avoid confusion these cases are explicitly indicated in the results section. In cases where protein domains were not detected with HMMScan, MEME suite [To confirm that the identified sequences from platyhelminthes were functional homologs of the  HMMScan , using aME suite  was usedC. elegans reference proteins and orthologous detected in other species. Alignments were visualized and edited when needed with BioEdit version 7.2.5 [Local alignment with MAFFT  was usedon 7.2.5 . The alion 7.2.5 , with ston 7.2.5 . The treon 7.2.5 .Additional file 1: Table S1.C. elegans small non-coding RNA pathway genes used for homology search. Table S2. Ribonuclease III genes identified in flatworms. Table S3. Argonaute genes identified in flatworms. Table S4. microRNA pathway genes detected in flatworms. Table S5. Exogenous RNA interference pathway genes detected in flatworms. Table S6. Endogenous RNA interference pathway genes detected in flatworms. Table S7. Amplification pathway genes detected in flatworms. Table S8. RNA interference spreading signal genes detected in flatworms. Table S9. Chromatin remodeling genes associated to RNA interference detected in flatworms. (XLSX 47 kb)Additional file 2: Figure S1.C. sinensis and O. viverrini genomes both paralogues are separated by less than 10 kb, while, in F. hepatica the intergenic region is almost 50 kb. Transcriptomic data of F. hepatica show that both genes are transcribed in several developmental stages. (TIFF 1513 kb)Dcr-2 and Dcr-3 genomic location and expression in Fasciolidae and Opisthorchiidae. In Additional file 3: Figure S2.S. mansoni, F. hepatica and E. granulosus. Transcriptomic data show that all FL-Agos are expressed in several developmental stages. Genes size was found to be variable ranging from around 4 kb in E. granulosus to 6.5 kb in S. mansoni and almost 40 kb in one of the F. hepatica genes. (TIFF 5708 kb)FL-Agos genomic location and expression in Additional file 4: Figure S3.Motifs detected in putative GW182 sequences of flatworms. Motifs detected in UBA and RRM domains are indicated in purple and lightblue boxes, respectively. The GW182 family conserved motif of AGO binding domain was also found in all flatworms (yellow box). Two additional motifs conserved only among flatworms were detected. The motif at the AGO binding domain (white box) is conserved in all flatworms, while, the other (black box) is rich in glutamine residues (Q) and is only conserved in trematodes and cestodes. Sequences of common motifs to all flatworm linages were aligned and residue conservation is indicated. Additionally, the number of GW repeats for each sequence are indicated. Species with parcial or no predicted gene model are not shown


Paragonimus species are human pathogens, but two of these, Paragonimus westermani and P. skrjabini, are responsible for the bulk of human disease. Despite their medical and economic significance, there is limited information on the gene content and expression of Paragonimus lung flukes.Paragonimiasis is an important and widespread neglected tropical disease. Fifteen P. westermani and P. skrjabini were studied with deep sequencing technology. Approximately 30 million reads per species were assembled into 21,586 and 25,825 unigenes for P. westermani and P. skrjabini, respectively. Many unigenes showed homology with sequences from other food-borne trematodes, but 1,217 high-confidence Paragonimus-specific unigenes were identified. Analyses indicated that both species have the potential for aerobic and anaerobic metabolism but not de novo fatty acid biosynthesis and that they may interact with host signaling pathways. Some 12,432 P. westermani and P. skrjabini unigenes showed a clear correspondence in bi-directional sequence similarity matches. The expression of shared unigenes was mostly well correlated, but differentially expressed unigenes were identified and shown to be enriched for functions related to proteolysis for P. westermani and microtubule based motility for P. skrjabini.The transcriptomes of adult P. westermani and P. skrjabini, inferred proteins, and extensive functional annotations generated for this project  represent a valuable resource for hypothesis driven research on these medically and economically important species.The assembled transcriptomes of The online version of this article (doi:10.1186/s13071-016-1785-x) contains supplementary material, which is available to authorized users. Paragonimus infection, and almost 300 million people are at risk of becoming infected . Rerameters . Scriptsrameters . AssemblThe complete, cleaned read sets were mapped to the corresponding filtered, high-quality transcript assemblies, and fragments per kilobase of exon per million mapped fragments (FPKM) were calculated for each unigene according to an RNA-Seq by expectation-maximization (RSEM) protocol using scripts included in the Trinity software package . UnigeneProtein sequences were predicted from transcripts using Prot4EST  based, iClonorchis sinensis (WormBase ParaSite BioProject PRJDA72781), Opisthorchis viverrini [Fasciola hepatica [Paragonimus kellicotti [-05. Sequences from Paragonimus species were excluded from NR prior to BLAST searches in order to facilitate identification of genus- and species-specific transcripts. The longest predicted protein isoform of each assembly unigene was also subjected to a reciprocal best BLAST match between the P. skrjabini and P. westermani transcripts with an e-value cut-off of 1e-05.Protein translations were compared to known proteins in NR (downloaded on 15 June 2015), iverrini , Fasciolhepatica  and Parallicotti  protein Predicted proteins were matched to conserved domains (InterPro) and gene ontology (GO) terms using InterProScan –27. AssoHomo sapiens (NCBI hs38) and the closest sequenced free-living relative, Schmidtea mediterranea (WormBase ParaSite Bioproject PRJNA12585), by BLASTP. Deduced Paragonimus proteins were considered putative “host mimics” when they shared at least 70 % sequence identity over at least 50 % of the length with the human ortholog but less than 50 % identity (if any) with the S. mediterranea ortholog.The longest isoform of each assembly unigene was compared to proteins from P-value cut-off of 0.01 [Functional enrichment of GO terms was calculated using FUNC with a  of 0.01 . In all P. westermani and P. skrjabini were sequenced, assembled de novo, and filtered to consider only high-confidence transcript sequences ; a majority of these also had close matches to non-Paragonimus sequences. Predicted proteins from 69.8 % and 60.6 % of P. westermani and P. skrjabini unigenes, respectively, had top matches to non-Paragonimus proteins in NR  compared to other FBTs  necessary to convert the initial substrate to the final product. Of 95 helminth-relevant KEGG modules , 35 wereP. westermani are thought to be facultative anaerobes with separate populations of mitochondria capable of either aerobic or anaerobic respiration [Paragonimus . This is consistent with the hypothesis that trematodes  are incynthesis , 24, 41.Paragonimus. They often play important roles in host parasite interaction [P. westermani and 750 P. skrjabini unigenes were found to contain classical signal peptides and no transmembrane domains. This suggests that they may be secreted from cells. Several GO terms related to proteolysis and redox regulation were enriched in the putative secreted unigenes in both species  than to the free-living, freshwater planarian platyhelminth, Schmidtea mediterranea  were identified by mass spectrometry. Some 205 of the immunoreactive P. kellicotti proteins have blast matches to proteins deduced from the transcriptomes of both P. westermani and P. skrjabini (Additional file Paragonimus diagnostic marker.In a previous study, proteins predicted from the  markers . ParagonP. westermani and P. skrjabini. Genetic conservation and diversification were assessed to characterize present and absent metabolic pathways. Like other FBTs [, these species appear capable of both aerobic or anaerobic metabolism, but not de novo fatty acid biosynthesis. For the most part, conserved unigenes were expressed to similar degree in both species. Genes upregulated in P. westermani were enriched for GO terms related to proteolysis while genes upregulated in P. skrjabini were enriched for GO terms related to microtubule based movement. Expressed orthologs of P. kellicotti serodiagnostic antigens were identified in both species, and should be explored in pan-Paragonimus diagnostic assays. We expect that the assembled transcriptomes and the accompanying functional annotations will be a valuable resource for future research, including ongoing genome sequencing projects [This study provides the first insights into gene content and expression in her FBTs , 24, 41,projects .