Helicobacter pylori is well established as a causative agent for gastritis, peptic ulcer, and gastric cancer. Armed with various inimitable virulence factors, this Gram-negative bacterium is one of few microorganisms that is capable of circumventing the harsh environment of the stomach. The unique spiral structure, flagella, and outer membrane proteins accelerate H. pylori movement within the viscous gastric mucosal layers while facilitating its attachment to the epithelial cells. Furthermore, secretion of urease from H. pylori eases the acidic pH within the stomach, thus creating a niche for bacteria survival and replication. Upon gaining a foothold in the gastric epithelial lining, bacterial protein CagA is injected into host cells through a type IV secretion system (T4SS), which together with VacA, damage the gastric epithelial cells. H. pylori does not only establishes colonization in the stomach, but also manipulates the host immune system to permit long-term persistence. Prolonged H. pylori infection causes chronic inflammation that precedes gastric cancer. The current review provides a brief outlook on H. pylori survival tactics, bacterial-host interaction and their importance in therapeutic intervention as well as vaccine development. Helicobacter pylori is a Gram-negative bacterium that selectively colonizes the gastric epithelium. It is classified in the order of Campylobacterales and the family of Helicobacteraceae. The term “Helico-” is of Greek origin and means spiral or curved, representing the unique shape of H. pylori. In 1994, H. pylori was recognized as a Type I carcinogen by the World Health Organization (WHO) [H. pylori [Helicobacter pylori-mediated gastric cancers among infected population [on (WHO) . Infection (WHO) ,4. It ha. pylori , and tha. pylori . Approxi. pylori . A recenpulation .H. pylori. The infection is usually acquired during childhood and can persist to adulthood when left untreated [H. pylori was reported in Africa (70.1%), followed by South America (69.4%) and Western Asia (66.6%), whereas the lowest prevalence was observed in Oceania (24.4%), Western Europe (34.3%), and Northern America (37.1%). These data denote lower socioeconomic status and poor hygiene practices as the risk factors for H. pylori infection [It is estimated that 50% of the world’s population is infected with ntreated . In a syntreated , the hignfection . Currentnfection ,13.H. pylori is one of the most successful human pathogens and has coexisted and adapted to humans for at least 50,000 years. This review discusses the unique bacterial characteristics that confer its successful survival within the stomach lumen. The unique helical shape, ability to produce urease, flagella movement, and outer membrane proteins (OMPs) are among the contributing factors [H. pylori attaches firmly to host cells via various OMPs and produces a permanent colonizing strain to prevent detachment from the peristaltic movement of the bowels. Then, it induces gastric mucosal damage via its virulence proteins, CagA and VacA. Damage to the gastric epithelial cells following bacteria colonization induces chronic inflammatory reaction, leading to gastritis, peptic ulcer, or gastric carcinoma. The following sections discuss the key features of H. pylori that determine a successful colonization of the gastric mucosa  and flagellar hook  have been reported to cause the formation of truncated flagella or result in aflagellated strains with reduced colonizing abilities [flgB (encoding rod protein), flgE, flgK and flgL (both encoding hook-filament junction proteins), fliK (encoding hook length control protein), and flaB and flag (encoding filament protein) [us layer . It has istalsis . In addir sheath . The expbilities . Moreoveprotein) .H. pylori, which is twice the amount compared to E. coli [Helicobacter outer membrane porin (Hop) family. Among the well-studied Hop proteins are HopS and HopT , which facilitate the attachment of bacteria to host cells via binding with the Lewis b (Leb)-histo-blood group antigen [H. pylori SS1 mutant caused a more severe inflammation in gerbils [H. pylori OMP HopH/OipA can suppress dendritic cell maturation and interleukin-10 (IL-10) secretion, dampening immune activation [The outer membrane proteins (OMPs) provide a barrier for the bacterium to resist its external environment. Furthermore, OMPs have been shown to be an important component for bacteria attachment to host cells. A total of 4% of the bacterial genome encodes for a diverse family of OMPs in  E. coli ,21. The  antigen . HopP  and carbon dioxide (CO2). Urease is typically found on the bacterial surface or in the cytoplasm that is released upon bacteria lysis. A urease-negative H. pylori strain showed impaired colonizing ability and was unable to be isolated from gnotobiotic piglets following 3 days of infection [H. pylori [4 produced function as a buffer, it is also known to be a toxic substance that causes damage to the host cells [H. pylori also limits its ecological niche to the stomach, where optimal proton motive force can only be maintained in pH 3.5 to 8.4 in vitro to drive adenosine triphosphate (ATP) generation essential for its survival [Several genes in the genome of nfection . Additio. pylori . Urea trst cells ,31. Intesurvival . Besidessurvival . Furthersurvival .H. pylori to modulate host immunity is acquired through its long course of coevolution with humans. Among these survival tactics is the synthesis of bacterial components of low immunogenicity. For example, lipopolysaccharide (LPS) and flagellin derived from H. pylori have been reported to be weakly immunogenic [The ability of unogenic ,36. Actiunogenic ,38. ConsH. pylori exploits Mincle and Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin (DC-SIGN) in macrophages and dendritic cells to induce anti-inflammatory IL-10 secretion, and suppress both proinflammatory IL-12 and IL-6 production [H. pylori has been found to attenuate macrophage proliferation in an in vitro setting [H. pylori induces the recruitment of dendritic cells that produce transforming growth factor-β (TGF-β) and IL-10. This skews the T cells’ response towards a more regulatory phenotype [H. pylori and IL-22 induce metalloprotease-10 (MMP-10) expression in gastric epithelial cells, which enhances bacterial colonization and associated pathology via reduction in antibacterial protein Reg3a production, decrement of tight junction proteins including E-cadherin and ZO-1, and enhancement of inflammation indicated by the influx of CD8+ T cells [Furthermore, oduction . Additio setting , resist  setting ,42, ther setting . Apart fhenotype . It was henotype ,46. More T cells .H. pylori, it is encoded by the 40 kb genetic locus of cytotoxin-associated gene pathogenicity island (cagPAI), a portion of the chromosome that possesses different CG content and is usually acquired through horizontal transfer. It has a 41 nm-long core structure consisting of different bacterial proteins including CagM, CagT, Cag3, CagX, and CagY, which protrude from the bacterial surface into the host cell. CagX and CagY are orthologous bacterial components that form the putative channel of the T4SS [5β1 integrin on the epithelial cell [The type IV secretory system (T4SS) is a large transporter complex expressed on the surface of Gram-negative bacteria and archaea to facilitate the transportation of proteins and DNA into host cell in a contact-dependent manner . In H. pthe T4SS . CagL isial cell . It alsoial cell . Additioial cell . A functial cell .H. pylori. Due to its importance in the outcome of infection, classification based on the presence of the CagA-encoding cagPAI results in the identification of cytotoxin-associated gene A (CagA) positive, Cag+, or negative, Cag− H. pylori strains [Cag+ H. pylori was detected among 33.3% of patients with gastritis, 68.7% of peptic ulcer cases, and 50% of gastric carcinoma cases [H. pylori prevalence among patients with duodenal ulcers (20.8%) and gastric cancer (16.6%) found Cag+ strains among 78% and 85% of the isolated H. pylori strains from respective diseases [Cag+ H. pylori, two different variants of CagA have been described based on binding affinity to SHP2, namely, the East Asian CagA, with stronger binding affinity and higher pathogenicity, and the Western CagA, with lower binding affinity that makes it less virulent than the former [CagA is a hydrophilic, surface-exposed protein that may be present or absent in  strains . In Egypma cases . Meanwhidiseases . With hie former .Within the cell, CagA is phosphorylated at the C-terminal EPIYA motif by host c-SRC and c-ABL kinases. It then binds to the SH2 domain to activate a series of oncogenic signaling processes . This acH. pylori to induce the formation of large cytoplasmic vacuoles in host cells. It has two functional domains that are linked together via a hydrophilic loop, namely the p58 domain, which allows host cell binding, and the N-terminal p33 domain, which is responsible for vacuole formation [cagA, the vacA gene is highly polymorphic across different H. pylori strains and contributes to variable disease outcomes. There are two different families within each vacA sequence: s1 and s2 within signal sequences, i1 and i2 in intermediate regions, and m1 and m2 in middle-region sequences. Type s1/m1 strain produces higher levels of cytotoxic and inflammatory activity in the gastroduodenal region [VacA is a toxin secreted by ormation ,63. Simil region ,65.H. pylori from the underlying mucosa [Structurally, VacA is heat-labile, protease-sensitive and assume an oligomeric structure that dissociates upon acid or alkaline exposure. This process activates the toxin and potentiates its binding to host cell surface receptor-type protein tyrosine phosphatase β . The toxg mucosa .During carcinogenesis, VacA causes a decrease in trans-epithelial resistance and a concomitant increase in paracellular permeability ,71,72. IH. pylori infection decreases specific CD8+ cytotoxic T cell and CD4+ helper T cell activities, leading to depressed local immune response [1/S phase through abrogation of nuclear factor of activated T cells (NFAT) translocation, thus effectively suppressing the production of T cell growth factor IL-2 [VacA-induced vacuolization causes a marked decrease in antigen proteolysis among antigen-presenting cells, a process that is required to generate peptide epitopes. The decrease in proteolytic activity reduces peptide presentation, consequently inhibiting the stimulation of T cells . There hresponse ,77. In ator IL-2 .H. pylori infection causes a typical series of events that eventually leads to gastric cancer in a progression cascade proposed by Correa, Haenszel [H. pylori infection. This is followed shortly by acute gastritis that slowly becomes chronic gastritis. Chronic gastritis worsens, which leads to atrophic gastritis. The loss of gastric glandular cells causes increased stomach pH. Altered conditions in the stomach together with other genetic and environmental factors contribute to the development of intestinal metaplasia, then dysplasia, and finally gastric cancer. Because of the lack of specific symptoms during the early stages of gastric cancer, patients are usually diagnosed only after the cancer has invaded the muscularis propria. This may be one of the contributing factors as to why the 5-year survival rate for gastric cancer is less than 15% in the United States [Haenszel . The casd States .H. pylori elimination because of the bacterium’s various immune evasion strategies. Hence, the recruitment of these cells results in epithelial damage instead of the expected eradication. The persistent presence of H. pylori thus leads to both the chronic proinflammatory response and cellular damage that contribute to the development of cancer. H. pylori in the transformation of an uninfected healthy gastric epithelium to the state of gastric cancer.At early timepoints, most infected patients are unaware of the bacterium’s colonization because of its asymptomatic manifestations. The initial response to the infection is marked by an increase in highly inflammatory immune cells, which are unfortunately ineffective at Helicobacter pylori as a causative agent for gastritis and gastric cancer has geared the direction of therapeutic strategy towards antimicrobial regimens. However, attempts to achieve a complete elimination of the bacteria using the devised proton-pump inhibitors-based triple therapy has proven to be challenging, partly because of increasing antibiotic resistance [H. pylori virulence factors during its pathogenesis has not significantly altered the management of the disease from the decades-old antibiotic regimen. To date, gastric cancer is still the fifth most common and fourth most deadly cancer worldwide [H. pylori is crucial for the rational development of a better curative method. The discovery of sistance . Furtherorldwide , with thorldwide ,83,84. HH. pylori virulence and interaction with host cells have been explored, albeit with little translation into the clinical setting. Among these targets, drugs against H. pylori urease production have been popularly sought after, as it contributed significantly to bacterial survival advantage and immune activation [in silico genome-scale protein interaction network was constructed, potentiating the identification of therapeutic targets [H. pylori-host interaction has also seen major advancement, especially in the alleviation of chronic inflammation. It has been identified that H. pylori induces expression of an inflammatory protein, podoplanin, via LPS to induce strong proinflammatory IL-1β secretion from macrophages [H. pylori chronic infection. Other host molecules that have been identified include heparanase, an enzyme that has been exploited to facilitate immune cell recruitment and inflammation during infection [H. pylori during the initiation of gastric cancer has also been suggested. These pathways include COX-2/Wnt/beta-catenin/VEGF, TLR2/TLR9/COX-2, COX2-PGE2, and NF-κB/COX-2, as well as EPHA2, MMPs, and the miR-543/SIRT1 axis [Various alternatives targeting tivation . Furthertivation . In a re targets . Targetirophages . Podoplarophages . Hence, nfection . Inhibitnfection . Other tRT1 axis .H. pylori infection, especially in developing countries. Most of the existing vaccine candidates are made up of purified or recombinant components of H. pylori antigens with an adjuvant [H. pylori adhesion A subunit (HpaA)) and other bacterial factors , FlaA) [An effective vaccine has been in active search with the hope of reducing severity and prevalence of adjuvant . For insadjuvant . This as), FlaA) .H. pylori continues to be relevant and important as our knowledge about this gut pathogen seems to continually expand. As discussed above, H. pylori can persist in the human stomach through establish of founder colonies, pH neutralization, gastric epithelial cell disruption, and host immune modulation. Exploration of the virulence targets may serve to accelerate vaccine research and provide better treatment alternatives to complement the existing antibiotic therapy.In summary, understanding the virulence and pathogenesis of


Staphylococcus aureus was shown to contribute to disease progression. Only little is known about the complex microbiome patterns involved in CTCL and how microbial shifts might impact this malignancy. Nevertheless, first hints indicate that the microbiome might at least in part explain transcriptional heterogeneity and that microbial approaches could serve in diagnosis and prognosis. Shaping the microbiome could be a treatment option to maintain stable disease. Here, we review current knowledge of transcriptional heterogeneity of and microbial influences on CTCL. We discuss potential benefits of microbial applications and microbial directed therapies to aid patients with CTCL burden.Cutaneous T-Cell Lymphomas (CTCL) presents with substantial clinical variability and transcriptional heterogeneity. In the recent years, several studies paved the way to elucidate aetiology and pathogenesis of CTCL using sequencing methods. Several T-cell subtypes were suggested as the source of disease thereby explaining clinical and transcriptional heterogeneity of CTCL entities. Several differentially expressed pathways could explain disease progression. However, exogenous triggers in the skin microenvironment also seem to affect CTCL status. Especially  Staphylococcus aureus can aggravate the progression of CTCL have been investigated [Cutaneous T-cell lymphomas (CTCL) are a group of skin homing neoplastic malignancies comprising approximately 10% of total non-Hodgkin lymphomas (NHL) with Mycosis fungoides (MF) as the most common entity ,3,4. Onestigated . In genestigated ,19. Regastigated ,21. GenoIn this review, we discuss current knowledge of transcriptional heterogeneity and the microbiome of CTCL. We will first show that CTCL seems to arise from malignant cells in the blood, leading to intra-patient heterogeneity. We also indicate first evidence of microbial implications on disease progression in CTCL and how differential colonization could lead to inter-patient heterogeneity. Then, we outline transcriptional heterogeneity on the inter- and intra-patient level and underline aspects that could point to microbial influence. Next, we summarize current knowledge of the microbiome in CTCL. Finally, we discuss the interplay between the microbiome and transcriptome of CTCL and suggest possible microbial applications in CTCL.+ tissue resident effector memory phenotype [In the majority of cases the malignant T-cell exhibits a CD4henotype . The malhenotype . It is bhenotype . With onhenotype . Moreovehenotype ,26. Henchenotype ,25,26,27Staphylococcus aureus (S. aureus) infections in a subset of CTCL patients [S. aureus [S. aureus in CTCL pathogenesis and its association with TCR Vβ in more detail.High-throughput sequencing of the TCR-beta gene showed that MF patients with a tumour clone frequency (relative abundance of T-cell clones) in lesions less than 25% have a good prognosis for overall and progression-free survival, while this is inverse for tumour clone frequencies over 25%. However, such associations were not seen in patients with Sézary syndrome (SS), the leukemic variant of CTCL . Interespatients ,29. Thispatients . Because. aureus  and colo. aureus ,32, thisA series of studies profiled the mutational landscape of CTCL ,34,35,36TOX, FYB, LEF1, CCR4, ITK, EED, POU2AF, IL-26, STAT5, BLK, GTSF1 and PSORS1C2, as these genes were already characterized in previous investigations using quantitative RT-PCR [On the single gene level, however, 75 transcripts were found to be upregulated across different CTCL stages, including inflammation-mediating genes like STAT and CD70. The authors underlined differential expression of e RT-PCR .TOX and FYB were upregulated. In early disease stages that are about to progress, TOX, FYB and other genes were overexpressed as compared to samples with an indolent course [TOX is not an exclusive CTCL marker, since it has been shown to be upregulated in primary cutaneous B-cell lymphomas and benign dermatoses as well [TOX expression in CTCL was several magnitudes higher than in benign dermatoses, climaxing in most advanced stages [Focusing on advanced disease stages, T-cell related genes like t course ,9,11,39.t course . TOX is  as well ,41. Howed stages ,43, but d stages . A follod stages , althoug+ T-cells, regulatory T-cells (Tregs), dendritic cells (DCs), macrophages and mast cells. The infiltrative proportion decreases with disease progression and varies between patients [With the advent of single cell RNA sequencing (scRNA-seq), more detailed insights into the behaviour of single cells and their states within a specific context have emerged. This can lead to improved onco-biological knowledge leading to better treatment and precision medicine. Therefore, scRNA-seq might be superior to bulk RNA-seq, although the latter still provides less noisy and more precise data ,46,47,48patients ,50. Hencpatients ,46. Fivepatients ,52 with patients  and two patients ,54. Gaydpatients  sequencepatients . NoteworTOX-positive cells and furthermore showed similar characteristics as the lymphocyte-tumour-clusters described above [TOX might potentially serve as a diagnostic and prognostic marker [+ and CD8+ TILs [Focusing only on actively proliferating lymphocytes of the five patients investigated revealed a shared panel of 17 genes that are overexpressed compared to healthy control individuals . Among oed above . Hence, c marker ,12. Althc marker . ConcernRindler et al.  showed iTo further investigate lineage relationships between malignant cells of the three body compartments, the authors used trajectory analysis. This computational method orders cells along branches based on similarities in their expression profile. As a result, lineage trees with branch points inferring cell fate are compiled. Mostly the resulting tree is 2-branched with a starting pre-branch (or root) ,57. UsinFOXP3 (Tregs) positive cells to either GATA3 (T helper 2 cells) or IKZF2 (Tregs) positive cells, which may depend on the tumour micromilieu [Likewise, investigation by Borcherding and co-workers on Sézary syndrome revealed substantial heterogeneity within malignant T-cells of a single patient . Overexpromilieu . T helperomilieu . Since pBoth studies by Rindler et al.  and BorcS100A4, S100A10, IL7R, CCR7, and CXCR4). Three of which comprise known functions in growth and migration of malignant T-cells  might cleave LL-37 into inactive fragments [Further indication for the influencing effect of AMPs on CTCL progression is added by the discovery of geographic patterns of CTCL cases. They indicate a link between sunlight exposure leading to vitamin D expression which in turn upregulates AMPs: Demographic data from CTCL patients in Texas, USA, shows that several communities had five to twenty times higher incidences than the expected rate for the population . On the ragments .In summary, CTCL presents with a dysfunctional skin barrier and reduced AMP production . This results in an enhanced skin permeability leading to greater susceptibility for skin infections ,69.Staphylococcus aureus on CTCL lesions has been reported [S. aureus have been studied [Many common skin diseases are associated with changes in the microbiota, which is termed dysbiosis . While treported  and will studied . An earl studied . However studied . The vas studied . There a studied ,88. The  studied .Serratia spp. and Pseudomonas spp., that affect the skin in nosocomial infections and immunocompromised patients [Staphylococci on CTCL lesions using 16S sequencing. Moreover, the phylogenetic diversity was decreased, indicating a distinct microbiome as compared to healthy volunteers and psoriasis patients [S. aureus also showed no differences whereas other commensal staphylococcal species trended higher in MF but not in SS. Nonetheless, principal coordinate analysis showed separation of samples from healthy volunteers and advanced stage CTCL. This may be driven by overrepresentation of two corynebacterial species and decreased abundances of two cutibacterial species. Bacterial shifts, furthermore, seem to correlate with disease stage  that can act as so-called superantigens [Investigations show that CTCL lesions are often colonized by S. aureus . This baantigens . Superanantigens . In a stantigens . The staantigens . Moreoveantigens . Howeverantigens . Hence, antigens  that difantigens . Taken tantigens , loss ofantigens  during Cantigens  and (ii)antigens  that canantigens  and withantigens .In Vitro, SEs can stimulate CTCL disease activity via cell-cell contact of malignant and benign T-cells , which mS. aureus on the progression of CTCL.In conclusion, these results hold compelling evidence for the role of S. aureus is also a member of the physiological skin flora [S. aureus strains dispose of different SEs [S. aureus, but also the specific strain may be responsible for disease stimulating actions. Since TSST-1 was associated with the specific TCR Vβ family TRBV20 [Because in flora , specifiin flora  and diffrent SEs . While srent SEs . Strain-rent SEs ,111. Heny TRBV20 ,29, but y TRBV20 , these cS. aureus after antibiotic treatment over several months was linked to a decline in disease severity in 2 patients [S. aureus positive after administering oral and topical (nares) antibiotic agents over a course of several months. Response to treatment was observed over all disease stages [Because hospitalized CTCL patients suffer from recurrent staphylococcal sepsis, systemic antibiotic treatment is often applied. However, as a side benefit, rapid clinical improvement of CTCL burden in some patients has been observed ,68. In apatients . Anothere stages .S. aureus colonization after antibiotic treatment is linked to a decrease in malignant T-cells and normalization of several markers and pathways typically upregulated in CTCL: Systemic antibiotics were applied to six advanced stage MF patients and two SS patients over 24 days, who did not respond to standard treatment. Eradication of S. aureus resulted in significant disease improvement and a decrease in malignant T-cells depicted as the relative frequency of the most dominant TCR-beta clone. Microarray gene expression analysis revealed normalization of markers for cell proliferation, inflammatory response, neoplasia and the Stat3 and IL-2 pathways compared to healthy controls, even one month after cessation of antibiotic treatment. In isolated primary malignant cells, Staphylococcal enterotoxin A (SEA) led to an activation of Stat3 and IL2-Receptor, while antibiotics showed no effect. Together these findings strongly suggest that S. aureus directly or indirectly stimulate CTCL tumour cells, and that antibiotic mediated disease relief is depended [S. aureus re-emerges quickly in most patients after termination of systemic antibiotic application. This finding emphasizes the clinical need for appropriate management of CTCL infections. Lifelong antibiotic therapy is not feasible due to antibiotic side-effects and the risk of new antibiotic resistances [S. aureus are methicillin-resistant S. aureus (MRSA) [Lindahl et al.  showed tdepended . Unfortuistances . One this (MRSA) , hence ns (MRSA)  reporteds (MRSA) .S. aureus can highly influence disease course and eradication of this pathogen may result in profound clinical improvements [S. aureus seems to foster expansion of a specific TCR Vβ family [S. aureus constitutes only a minor part of the complex microbiome on human skin with many other microbes orchestrating human health and disease [S. aureus could lead to a homogenization of transcriptional response, observed transcriptional heterogeneity might at least in part also be attributed to differential microbial colonization between CTCL lesions and between CTCL patients. Of course, it could also be speculated that transcriptional heterogeneity of CTCL itself leads to the observed microbiome patterns. Thus, there could also be a reciprocal influence of the microbiome and transcriptome in CTCL. Studies investigating mechanistic relationships are needed to clarify this “chicken-and-egg” situation.CTCL presents with substantial clinical and transcriptional heterogeneity that might originate from adaptive and functional plasticity of malignant T-cells ,10,53,54ovements ,113. CTCovements . On the ovements ,51,52,54β family , thereby disease , other m disease ,21. Sinc disease ,32,96, tS. aureus in AD patients was successfully tested in a phase 1 clinical trial presumably leading to improvement in eczema severity [There is no curative therapy available until today and optimal treatment remains to be elucidated . Early sseverity . Howeverseverity ,123,124.severity ,30, micrseverity ,125. Fur


Colletotrichum gloeosporioides is a devastating plant disease with an extensive impact on plant productivity. The process of colonization and disease progression of C. gloeosporioides has been studied in a number of angiosperm crops. To better understand the evolution of the plant response to pathogens, the study of this complex interaction has been extended to bryophytes. The model moss Physcomitrium patens Hedw. B&S (former Physcomitrella patens) is sensitive to known bacterial and fungal phytopathogens, including C. gloeosporioides, which cause infection and cell death. P. patens responses to these microorganisms resemble that of the angiosperms. However, the molecular events during the interaction of P. patens and C. gloeosporioides have not been explored. In this work, we present a comprehensive approach using microscopy, phenomics and RNA-seq analysis to explore the defense response of P. patens to C. gloeosporioides. Microscopy analysis showed that appressoria are already formed at 24 h after inoculation (hai) and tissue colonization and cell death occur at 24 hai and is massive at 48 hai. Consequently, the phenomics analysis showed progressing browning of moss tissues and impaired photosynthesis from 24 to 48 hai. The transcriptomic analysis revealed that more than 1200 P. patens genes were differentially expressed in response to Colletotrichum infection. The analysis of differentially expressed gene function showed that the C. gloeosporioides infection led to a transcription reprogramming in P. patens that upregulated the genes related to pathogen recognition, secondary metabolism, cell wall reinforcement and regulation of gene expression. In accordance with the observed phenomics results, some photosynthesis and chloroplast-related genes were repressed, indicating that, under attack, P. patens changes its transcription from primary metabolism to defend itself from the pathogen.Anthracnose caused by the hemibiotroph fungus  Colletotrichum gloeosporioides is a hemibiotrophic ascomycete fungus that, together with other members of the genus Colletotrichum , produces anthracnose in many plants [C. gloeosporioides is considered the main causal agent of anthracnose, infecting more than 470 plant species [y plants ,2. The s species ,4,5,6 an species . The inc species .C. gloeosporioides attack has been studied in vascular plants. Dual RNA-sequencing profiles during the interaction of C. gloeosporioides with tomato fruits (Solanum lycopersicum L.) and strawberry leaves (Fragaria ananassa Duch) showed that this fungus induced in host plant cells a salicylic acid-dependent hypersensitive response (HR), autophagy, MAPKKK, phenylalanine and flavonoid biosynthesis, and plant cell wall modification [C. gloeosporioides-inoculated tea leaves  revealed that the main metabolic processes upregulated by miRNA during the fungus–plant interaction were related to receptor kinases, ROS scavenging, auxin and salicylic acid-mediated pathways [C. gloeosporioides possess efficient mechanisms to colonize the host and has specific effector proteins that disrupt host response mechanisms.Plants under biotic stress activate defense responses mediated by cell surface-localized pattern recognition receptors (PRRs) and intracellular disease resistance proteins, which detect pathogen-associated molecular patterns (PAMPs) and effectors, respectively, leading to pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) ,8. Defenfication ,10. In apathways . DespiteMarchantia polymorpha L. is colonized by fungal endophytes [Physcomitrium patens Hedw. B&S (formerly Physcomitrella patens) and Funaria hygrometrica Hedw. by fungi , oomycetes  or bacteria  have been previously documented [B. cinerea is a necrotrophic ascomycete that infects Polytrichum juniperinum Hedw. in nature [P. patens in laboratory conditions [P. patens towards pathogens were recently reviewed [P. patens [P. patens is one of the non-vascular plant models mostly used in plant biology studies; thus, an analysis of its defense mechanisms against pathogens allows a better understanding of the evolution of plant defense mechanisms from mosses to seed plants.Plant interactions with fungi are ubiquitous and are registered in fossil records ,13. The dophytes . The infcumented ,19,20,21n nature  and P. pnditions . The defreviewed . Several. patens . P. pateC. gloeosporioides) on bryophytes [P. patens by microscopy, phenomics and RNA-sequencing. Studies of fungal–bryophyte interactions at the genomic scale could reveal the molecular mechanisms and ecosystem functioning effects of detrimental or endophytic fungi on metabolite production, nutrient acquisition, plant–pathogen crosstalk and resistance, and abiotic stress alleviation. It could also provide insights into the coevolution mechanisms of pathogens with bryophytes and the fungal infection strategies different from those described in vascular plants.Although a vast knowledge is available for angiosperm–pathogen interactions, few omics studies have analyzed the effects of widely distributed pathogen fungi , turning the protonema brown colored and with a macerated appearance. Fungal colonization progressed successfully over time (24 and 48 hai), and the most prominent symptoms were observed at 48 hai , strawberry (Fragaria ananassa Duch.) and mango (Mangifera indica L.) [P. patens. Because C. gloeosporioides directly penetrates the host cell walls, their molecular composition is extensively modified and strengthened by lignification in flowering plants or accumulation of callose and phenolic compounds in non-vascular plants [P. patens cells to reinforce its cell walls as a defense mechanism against fungal infection [Phytophthora capsici and the necrotrophic fungus B. cinerea, provoke browning of chloroplasts and its migration towards the active infection site and cytoplasmatic shrinkage [P. patens attempted to control C. gloeosporioides infection, as was previously reported [Appressorium formation during the early stage of nt cells . This stdica L.) ,28,29, ar plants . The posnfection ,23,24. Chrinkage . This rehrinkage ,32. Morereported .P. patens colonized with other pathogens, such as A. alternata, B. cinerea, F. avenaceum, P. debaryanum and P. irregulare [C. gloeosporioides) [M. polymorpha exposed to polycyclic aromatic hydrocarbons [All these findings are consistent with those observed in regulare ,19,30. Srioides) ,35,36; hocarbons . Thus, tP. patens by C. gloeosporioides. A color segmentation analysis was performed to quantify the C. gloeosporioides infection in P. patens , estimated using the chlorophyll a fluorescence values, has been widely used as an indirect measure of stress in plants [Fv/Fm between the controls and infected mosses  susceptible to the fungus Zymoseptoria tritici, where Fv/Fm decreases up to 40% in infected plants [Euterpe oleracea Mart.) infected with Colletotrichum spp. showed a decrease of 13% in Fv/Fm [The quantum yield of photosynthesis , reflecting the role of salicylic acid in moss defense responses to B. cinerea [P. patens colonies exposed to salicylic acid show higher resistance to the infection by P. wasabiae [P. irregulare, also turns on a salicylic acid-like response in the moss Amblystegium serpens Hedw. [M. polymorpha promotes the necrotrophic infection by the fungus Irpex lacteus [M. polymorpha immunity against I. lacteus [P. patens also increases its resistance to infection by addition of jasmonic acid at the medium culture , potato (Solanum tuberosum L.) and wheat (T. aestivum L.), where the accumulation of salicylic acid resulted in greater expression of plant defense genes [Moss colonies grown on media supplemented with salicylic acid and jasmonic acid showed less infection levels, reflected in lower cell damage I. This rc stress ,50. For  cinerea . Moreovewasabiae . Other pns Hedw. . Contrar lacteus . This is lacteus . Interes culture I. Althoumponents . Jasmonas plants . The majse genes ,56,57. Ise genes .P. patens, including the nuclear, chloroplast and mitochondrial genomes. A total of 93.7% of moss reads were successfully mapped to the genomes of C. gloeosporioides vs. the control (non-infected moss)); (ii) time effect ; (iii) 8 hai vs. non-infected mosses; (iv) 24 hai vs. non-infected mosses; and (v) 24 hai vs. 8 hai (C. gloeosporioides (treatment–infection–effect) denoted the bigger transcriptional reprogramming in P. patens.For differential gene expression analysis, the time effect (24 hai vs. 8 hai) and the treatment effect (infected moss vs. non-infected moss) were simultaneously evaluated E,F. A sts. 8 hai G,H. The P. patens by C. gloeosporioides infection. The moss upregulated and downregulated 837 and 403 genes, respectively   24 hai vs. 8 hai was not informative about the moss defense response during fungal infection since 363 genes were differentially expressed and 112 of them were exclusive of this comparison G,H, whicn total) . Eleven n total) G,H. In tP. patens defense response to fungal infection [P. patens by C. gloeosporioides colonization. Transcriptional reprogramming has been previously reported for many plants during fungal infections [Gene ontology (GO) enrichment analysis showed that the most enriched metabolic processes (fold enrichment > 25) were the cinnamic acid pathway, erythrose 4-phosphate/phosphoenolpyruvate (E4P/PEP) family amino acid pathway, L-phenylalanine and chorismate metabolic process A. All thnfection . Defensenfection . Finallyfections ,61,62.B. cinerea, F. oxysporium, Mycosphaerella oxysporium and Pseudomonas syringae [Among the biological processes, several metabolic pathways related to photosynthesis, chloroplast functioning and starch biosynthesis were downregulated A. The prsyringae ,66.P. patens during C. gloeosporioides interaction. While chs, chi, cad and 4cl increased 4–8-fold their expression, pal genes were strongly upregulated. Interestingly, 10 different pal genes were upregulated, some of them until 330-fold  has been described as a cell wall fortification response in this moss [pal and chs genes, encoding the enzymes involved in the early steps of lignan biosynthesis [P. patens fungal infection , arabinogalactan-proteins and, to a lesser extent, pectin  [cals genes are involved in the callose synthesis, a polymer of β-1,3-D-glucan. Callose accumulates in plants cell walls in responses to different stresses, including pathogen attack [P. patens in the presence of pathogens has been previously reported [Several genes related to cell wall biogenesis and modification were also overexpressed, including those encoding for callose synthase (CALS), cellulose synthase (CSLD), protein pectic arabinogalactan synthesis-related (PAGR) and β-mannosyl transferase (CSLA), among others B. The ceturonan) . Therefon attack . Local dreported ,79. Papireported .Oryza sativa L. [gals genes participate in the biosynthesis of β-1,4-galactan, which is one of the main side chains of galacturonans, the abundant polysaccharides in plant cell walls that are the major component of pectin [P. patens during C. gloeosporioides attack.Other genes that were overexpressed are those coding trichome birefringent protein (TBL) and galactan enzyme β-1,4-galactosyltransferase (GALS). TBL is a protein located in the Golgi complex and is responsible for acetylating polymers present in the cell wall. This protein has been related to resistance to rice blight disease in ativa L. . On the f pectin . The higC. gloeosporioides  and transcriptional corepressor (TPR1) (Topless Related 1), respectively, were the most upregulated genes (logFC > 6) related to defense response in P. patens during infection by C. gloeosporioides  [snc genes were upregulated in P. patens infected by C. gloeosporioides (P. patens) [Plants respond to pathogen infection by reprogramming the transcription of defense-related genes involved in signaling, resistance to diseases and tolerance to different stressors . We founorioides B. Two georioides B. ERG isffectors . The trae grisea . On the n plants . During nd dnd2) . We alsoorioides B. SNC1,  patens) . Salicyl patens) . Salicyl patens) .P. patens infected by C. gloeosporioides  and auxin 1-napthaleneacetic acid (NAA) but downregulated by ethylene and auxin inhibitors [tao expression is associated with ethylene and auxin levels in Arabidopsis H&H, suggesting that TAO1 could be involved in hormone-dependent defense mechanisms. nramp genes participate in the ethylene-defense response and were also upregulated . EDR2 is a negative regulator of the salicylic acid-dependent response [P. patens to infection by C. gloeosporioides show that hormone-dependent responses play a pivotal role in moss immunity to overcome pathogens.Genes coding for TAO proteins (target of AvrB operation) were also upregulated in orioides B. TAO1, n growth . The actynthesis . It alsohibitors . These regulated B. NRAMP d plants . On the t stress . In addiresponse . These tdir1  among genes coding LRRs. TRAF1A is a TNF receptor-associated factor that regulates the plant immune response, mediating the renewal of the SNC1 nuclear-binding domain. The activation of traf prevents the hyperaccumulation of SNC1 that could cause autoimmune plant phenotypes [Membrane receptor protein kinases containing leucine-rich repeat domains (LRRs) constitute the first line of defense in plants and are key regulators of plant growth and development . LRRs recriptome , from whcriptome C. The trenotypes .P. patens does not have fls2 genes [S. lycopersicum L., BAK1 has been reported as an essential coreceptor for immune response activation mediated by receptor-like protein Ve1 and Cf receptors, which recognize pathogen effectors [A. thaliana H&H, the function of some LRR receptors  was also compromised in bak1 mutant plants, corroborating the role of bak1 genes to install defense response in plants [lrrc-pk and elr genes, encoding serine/threonine kinases containing LRR domains, were also differentially expressed (logFC > 2). These receptors participate in the recognition of PAMPs in the plant–microbe interaction [A. thaliana H&H and S. lycopersicum L. and it has been shown that ELR1 expression reduces the growth of P. infestans in infected plants [A gene encoding BAK1, a dual specificity kinase acting on both serine/threonine- and tyrosine-containing substrates, was also upregulated. BAK1 forms a protein complex with BIR1 (brassinosteroid insensitive receptor) and FLS2 (flagellin sensing 2 receptor) . In thiss2 genes . BAK1 iss2 genes . In S. lffectors . In A. tn plants . The lrreraction . Particud plants . The rolP. patens transcriptome, we identified an overexpressed gene (lrx) that encodes a moss extensin. These proteins play a key role in plant development as regulators of cell wall expansion, or as chains that connect the cell wall with the plasma membrane [gso gene, involved in plant development [P. patens, the processes of development and plant growth were negatively regulated.Extensins are another important class of plant cell wall proteins with LRR domains. In the membrane . Howeverelopment , was negP. patens transcriptome, several genes related to ROS metabolism were differentially expressed, including glox, prx and sod . This enzyme is involved in regulating the H2O2 levels to alleviate damage by oxidative stress [sod genes are relevant, and we found one overexpressed sod gene  in tobacco during abiotic stress [ROS play a central role in plant signaling and immune response, since they have a direct antimicrobial effect and, most importantly, act as local and systemic signaling molecules . In the  and sod C. Aldehysod , for example, was overexpressed. As previously discussed, this kinase has been related with salicylic acid-mediated resistance, but also it has been associated to differential expression of defense genes and ROS synthesis [2+-dependent potassium channel was also upregulated (logFC = 3.18). Potassium is an important molecule for the defense response since it regulates ROS levels and increases the phenols concentration and synthesis of defense compounds [Cell signaling pathways are vital in defense responses in plants. Among them, Caesponses . Several signals . Among tentified C. The Caynthesis . A sequeompounds .erf-rap2.11 was overexpressed . This is consistent with the result obtained in this work since WRKY-like transcription factors were not upregulated in P. patens at the early times (8 and 24 hai). In contrast, the transcriptome of P. patens infection with B. cinerea showed upregulation of several WRKY-like transcription factors from 8 hai [B. cinerea compared to C. gloeosporioides.No differentially expressed genes encoding WRKY-like transcription factors were found. These transcription factors belong to one of the largest families of transcriptional regulators in plants and are key regulators of plant immunity . The uprresponse ,10. Howees 8 and  hai. In rbc genes that encode enzyme ribulose bisphosphate carboxylase (RuBisCO), which fixes CO2 in the first step of the Calvin Cycle, were downregulated [psa), Photosystems II complex proteins , and other genes relevant for chloroplast metabolism, such as those related to the electron transfer across the thylakoid membrane (ndhN), chlorophyll biosynthesis (cao) and chloroplast envelope membrane proteins (cemA), were also downregulated during the moss–fungus interaction. Chloroplasts are an important source of ROS and salicylic acid during the defense response [Finally, we found that every differentially expressed gene related to photosynthesis or chloroplasts was downregulated C. Importegulated . Genes eresponse . The negAs evidenced, plant defense responses against pathogens are mediated by the activation and repression of a large set of mRNAs. However, nothing is known about the role of non-coding RNAs  in the bryophyte–fungus interaction. For example, plant miRNAs can regulate the expression of pathogenesis-related genes during fungal infection ,132. TheC. gloeosporioides isolated from orange fruits [5 conidia/mL spore suspension was prepared using milliQ water. Three milliliters of this solution were sprayed over 21-days-old moss P. patens (Gransden wild type) colonies grown on Hoagland solid media  at 22 °C and with a 16 h light/8 h dark regime under 60–80 μmol photons m−2 s−1 white light. Control (non-inoculated) moss cultures were implemented and sprayed with sterile milliQ water.e fruits  was growC. gloeosporioides colonization, respectively. In addition, 48-h-old (late stage of infection) co-cultures were included for microscopic and phenomic characterization. All experiments were performed in triplicate using three independent biological replicates.For microscopic, phenomic and transcriptomic studies, the fungal–moss co-cultures were analyzed at 8 h and 24 hai, time points corresponding to the early (spore germination) and active (moss cell infection) stage of C. gloeosporioides infection, moss samples were stained with 0.1% solophenyl flavine 7GFE 500 [Moss samples were stained with 0.01% safranin O solution for 15 min, rinsed twice with distilled water and observed on a Leica DM500 microscope, . To demonstrate 7GFE 500  and obseP. patens’ colony images were acquired using a semi-automatized imaging acquisition system ScanalyzerPL  equipped with a high-resolution (1628 × 1236 pixels) camera . Moss images were obtained using RGB-LED within the visible spectrum (400–700 nm). The top view of each moss colony, as well as 360° lateral views, were photographed. Tissue pixel area was determined to estimate the digital biomass and senescence was evaluated using the color segmentation profile, which was calculated using the LemnaGrid software . This computer algorithm segments and classifies the total canopy into values that correspond to the percentage of colors, taking the optimal condition criterion (green), senescent (yellow) and necrotic (brown).P. patens’ colonies were adapted to dark conditions for 20 min. Fluorescence maps of the moss colonies were obtained by stimulating the maximum quantum yield of Photosystem II, expressed as Fv/Fm . The PSI Open FluorCam system is equipped with four LED panels  divided into two sensor pairs, which measure the initial fluorescence state (Fo) and the maximum fluorescence state (Fm). Additionally, a CCD camera with a 512 × 512-pixel resolution (12-bit dynamic) was used to obtain the color scale images, which were used to determine the Fv/Fm values (0.1–1.0).Chlorophyll fluorescence images were captured from moss colonies using a PSI Open FluorCam FC 800-O system . Previously, P. patens’ colonies were incubated during 24 h in the presence of salicylic acid or jasmonic acid, and after that period tissues were inoculated with C. gloeosporioides as previously described, and incubated for 72 h to evaluate cell damage using Evans Blue staining [Three-week-old moss colonies were transferred to Hoagland solid media containing salicylic acid  at 0.1 mM and 0.5 mM, or jasmonic acid (Sigma) at 50 µM final concentrations. staining . Brieflyp < 0.01. The data analysis software GraphPad Prism version 7.03 (GraphPad Software) was used.A one-way analysis of variance (ANOVA) test was conducted to determine any statistically significant differences. Significance levels were always expressed as a value of 20 > 97.5 %.As mentioned before, 8- and 24-h-old co-cultures were recovered, frozen using liquid nitrogen and pulverized. Total RNA was extracted using the RNeasy Plant Mini Kit . RNA quality control, library preparation and sequencing were performed by Macrogen Inc. . Briefly, RNA integrity number (RIN > 9) was checked using an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies) and cDNA libraries were prepared for paired-end sequencing using 1 μg of RNA and the TruSeq Stranded Total RNA Plant LT Sample Prep. Finally, sequencing was performed on Illumina platform  to generate paired-end 150 bp reads, obtaining ~50 M reads per sample with Q20. Then the RSubread package version 3 [P. patens . In addition, the sequence alignment was also performed using P. patens available genomes of chloroplasts (NC_005087.1), mitochondria (NC_007945.1) and ribosomal RNA sequences .The quality of the obtained sequences was checked using the FastQC software v0.10.1 . Adaptorersion 3  was usedTo perform the differential expression analysis, genes with a low number of read counts (CPM < 0.5) were filtered, aiming to increase the statistical strength of the prediction of differentially expressed genes . Expresshttps://www.arabidopsis.org/tools/go_term_enrichment.jsp, accessed on 15 April 2020), using the Panther classification system [P. patens database. To determine the enrichment rate, the Fischer’s Exact Test method was used. GO terms with an FDR < 0.05 were considered in this analysis. To summarize the long list of GO terms, redundant GO terms were removed with REViGO  [Gene Ontology (GO) enrichment was conducted following the protocol described by . GO termn system  on the Pntology) .P. patens during infection with the fungal pathogen C. gloeosporioides, which we depict in P. patens is susceptible to infection with C. gloeosporioides, once the pathogen is recognized by receptors, a defense response is activated, evidenced by the induction of genes involved in MAPK signaling cascades, ROS production and homeostasis, calcium signaling, an HR-like response, salicylic acid-dependent signaling, cell wall reinforcement and genes involved in different defense mechanisms. The defense response developed by P. patens coincides in several aspects with those observed in vascular plants. During the transcriptional reprogramming that occurs in P. patens during C. gloeosporioides colonization, several genes related to photosynthesis and chloroplast functioning are downregulated, indicating that the defense response and secondary metabolism processes are favored in moss tissues over metabolic functions of primary metabolism.In this study, we present a comprehensive description of the cellular and molecular response of the bryophyte In conclusion, this study extends our knowledge of moss defense responses against pathogens and highlights the important molecular players during the evolution of land plant defense against biotic stress.


Snake venoms represent a danger to human health, but also a gold mine of bioactive proteins that can be harnessed for drug discovery purposes. The evolution of snakes and their venom has been studied for decades, particularly via traditional morphological and basic genetic methods alongside venom proteomics. However, while the field of genomics has matured rapidly over the past 2 decades, owing to the development of next-generation sequencing technologies, snake genomics remains in its infancy. Here, we provide an overview of the state of the art in snake genomics and discuss its potential implications for studying venom evolution and toxinology. On the basis of current knowledge, gene duplication and positive selection are key mechanisms in the neofunctionalization of snake venom proteins. This makes snake venoms important evolutionary drivers that explain the remarkable venom diversification and adaptive variation observed in these reptiles. Gene duplication and neofunctionalization have also generated a large number of repeat sequences in snake genomes that pose a significant challenge to DNA sequencing, resulting in the need for substantial computational resources and longer sequencing read length for high-quality genome assembly. Fortunately, owing to constantly improving sequencing technologies and computational tools, we are now able to explore the molecular mechanisms of snake venom evolution in unprecedented detail. Such novel insights have the potential to affect the design and development of antivenoms and possibly other drugs, as well as provide new fundamental knowledge on snake biology and evolution. Snakes (Squamata: Serpentes) represent a monophyletic lineage, comprising ∼3,600 extant species found in all continents, except Antarctica ,2. From Python bivittatus), the red-tailed boa (Boa constrictor constrictor), and the king cobra (Ophiophagus hannah)  and Nanopore) and short-read platforms (Illumina), as well as Chicago, Hi-C, and optical mapping in the case of N. naja ..C. pyrrhHomo sapiens genome (2.90 Gb) is ∼2 times larger than the T. sirtalis genome (1.42 Gb), the number of genes is similar between the two  .,22.C. vimosome 7 . Phyloges genome . Therefos genome .N. naja, where as many as 16 toxin gene families are located on macrochromosomes ), indicating a more neutral nucleotide substitution and the maintenance of similarity between gene copies [Crotalus cerastes), data showed evidence of selection being stabilized, which supports that the maintainance of a generalist phenotype is favoured [Determining and unraveling the driving factors behind the dynamic evolutionary processes in snake venom gene families has garnered the interest of scientists for decades—a quest that could only benefit from increasing efforts in WGS of venomous snakes. Positive selection appears to be the force behind the evolution of genes involved in predator-prey arms races , and it the gene . In P. f± 0.062) , suggestr copies . Similarse genes . Converse copies . On the favoured . It mustfavoured ), relatifavoured , 134. ThP. mucrosquamatus, where dominant toxin-encoding genes displayed relaxed selective constraints for deleterious mutations despite statistically significant rates of positive selection [Crotalus adamanteus) and the South American rattlesnake  is significantly more correlated with differences in number of duplication-derived gene copies between populations than with adaptive divergence in the sequences themselves [New -omics tools and methods are rapidly advancing our knowledge of the mechanisms behind venom evolution . In partelection . Furtheremselves ,143.eN, namely, the number of reproductive individuals that actually produce offspring) [eN greatly contributes to sequence variation, as the fate of a favourable mutation spreading is controlled by eN and the strength of selection [Sistrurus catenatus), a threatened species whose range consists of several scattered populations largely isolated from each other. Although the influence of genetic drift on venom evolution in this species is currently weak, it is likely to increase dramatically over time once the impact of drift is augmented due to the low eN found in most populations [The strength at which genetic drift acts on the genome is inversely proportional to effective population size (fspring) . Ne greaelection ,145. A pulations . Thus, cde novo genome assembly is the high content of repeat sequences in snake genomes. Some of these challenges might be adequately addressed by using third-generation sequencing technology. As the costs and error rates of this and other approaches decrease, they are certain to be used more widely in snake genome research. In turn, the assembly of more venomous snake genomes will allow us to explore adaptation and venom evolution at all phylogenetic levels, bringing a new perspective to the study of snake genomes and venoms.WGS is a revolutionary advance in genetic research that has only recently been applied to the fields of herpetology and toxinology. Nonetheless, sequencing of complete snake genomes has already shed light on the evolutionary history of toxin-encoding genes as well as their expression patterns in the venom gland. In the future, WGS may be harnessed to obtain a better understanding of the molecular mechanisms involved in snake evolution ,103, finNot applicable.2: phospholipase A2; SSR: simple sequence repeat; SVMP: snake venom metalloproteinase; SVSP: snake venom serine proteinase; TE: transposable element; WGS: whole-genome sequencing.3FTx: 3-finger toxin; bp: base pairs; BUSCO: Benchmarking Universal Single-Copy Orthologues; CRISP: cysteine-rich secretory protein; CTLP: C-type lectin-like protein; DoC: depth of coverage; Gb: gigabase pairs; kb: kilobase pairs; LAAO: L-amino acid oxidase; LINE: long interspersed element; NCBI: National Center for Biotechnology Information; NGS: next-generation sequencing; PLAW.R., W.Z., and S.L. are employees at the BGI. The authors declare that they have no other competing interests.This research was supported by the Beijing Genomics Institute and the Technical University of Denmark. M.E.A. is funded by the Independent Research Fund Denmark (7027–00147B). C.K. is funded by Innovation Fund Denmark (9065–00007B). T.P.J. is funded under Marie Sklodowska-Curie grant agreement No. 713683 (COFUNDfellowsDTU).A.H.L. and S.L. conceived the project. A.H.L., W.R., K.K., T.P.J., C.K., C.T.W., W.Z., S.G., L.S., M.M.D., B.J.M., E.R.T., and M.E.A. structured the draft and provided final editing. A.H.L., K.K., T.P.J., and W.R. coordinated and drafted the manuscript and implemented comments provided by all authors. All authors contributed critically to the scientific content. All authors read and approved the final manuscript.giac024_GIGA-D-21-00410_Original_SubmissionClick here for additional data file.giac024_GIGA-D-21-00410_Revision_1Click here for additional data file.giac024_Response_to_Reviewer_Comments_Revision_1Click here for additional data file.giac024_Reviewer_1_Report_Original_SubmissionBlair Perry -- 1/18/2022 ReviewedClick here for additional data file.giac024_Reviewer_2_Report_Original_SubmissionMatthew Holding -- 1/18/2022 ReviewedClick here for additional data file.


Pseudomonas aeruginosa, the rhamnolipids-producer, is one of dominant bacteria in oil reservoirs. Although P. aeruginosa strains are facultative bacteria, the anaerobic biosynthesis mechanism of rhamnolipids is unclear. Considering the oxygen scarcity within oil reservoirs, revealing the anaerobic biosynthesis mechanism of rhamnolipids are significant for improving the in-situ production of rhamnolipids in oil reservoirs to enhance oil recovery.Pseudomonasaeruginosa SG anaerobically produced rhamnolipids using glycerol rather than glucose as carbon sources. Two possible hypotheses on anaerobic biosynthesis of rhamnolipids were proposed, the new anaerobic biosynthetic pathway (hypothesis 1) and the highly anaerobic expression of key genes (hypothesis 2). Knockout strain SGrmlB failed to anaerobically produce rhamnolipids using glycerol. Comparative transcriptomics analysis results revealed that glucose inhibited the anaerobic expression of genes rmlBDAC, fabABG, rhlABRI, rhlC and lasI. Using glycerol as carbon source, the anaerobic expression of key genes in P. aeruginosa SG was significantly up-regulated. The anaerobic biosynthetic pathway of rhamnolipids in P. aeruginosa SG were confirmed, involving the gluconeogenesis from glycerol, the biosynthesis of dTDP-l-rhamnose and -hydroxy fatty acids, and the rhamnosyl transfer process. The engineered strain P. aeruginosa PrhlAB constructed in previous work enhanced 9.67% of oil recovery higher than the wild-type strain P. aeruginosa SG enhancing 8.33% of oil recovery.P. aeruginosa SG to anaerobically biosynthesize rhamnolipids. The genes, rmlBDAC, fabABG, rhlABRI, rhlC and lasI, are key genes for anaerobic biosynthesis of rhamnolipid by P. aeruginosa. Improving the anaerobic production of rhamnolipids better enhanced oil recovery in core flooding test. This study fills the gaps in the anaerobic biosynthesis mechanism of rhamnolipids. Results are significant for the metabolic engineering of P. aeruginosa to enhance anaerobic production of rhamnolipids.The highly anaerobic expression of key genes enables  At present, the demand for oil in economic development continues to increase, while the recoverable oil reserves in oil reservoirs are decreasing, and it is more difficult to find new oil and gas resources. It is of great strategic significance to enhance the oil recovery of existing oil fields through scientific research and innovation. Microbial oil recovery technology (MEOR) is an economical, effective and environmentally friendly oil recovery technology . It is f2Pseudomonas sp. and Burkholderia sp.  was injected into rock core. The medium contained 40g/l glycerol, 4g/l of NaNO3, 3g/l of KH2PO4, 4g/l of K2HPO43H2O, 0.5g/l of KCl, 0.5g/l of NaCl, 0.2g/l of CaCl22H2O. Then the rock core was incubated at 39C for 8days. During the core flooding process, the volumes of displaced oil (ml) and displaced water (ml) were recorded. Enhanced oil recovery efficiency (%) was calculated.Enhanced oil recovery by in-situ anaerobic production of rhamnolipids was evaluated by core flooding test. The experimental protocol of core flooding test was referred to the previous study . The enginosa SG . The rocP. aeruginosa SG decreased the culture surface tension to 27.0mN/m and 27.3mN/m using both glucose and glycerol as carbon sources, respectively. The oil spreading circles diameters of the aerobic culture were 482mm and 565mm when P. aeruginosa SG using glucose and glycerol as carbon sources, respectively. The surface activity and oil spreading activity indicate that P. aeruginosa SG produced biosurfactants [P. aeruginosa was rhamnolipids [P. aeruginosa SG produced rhamnolipids using both glucose and glycerol as carbon sources.Under aerobic conditions, strain factants , 18. Thenolipids , 15. Und600), surface tension and oil spreading circles diameters were shown in Fig.600) point of view, strain P. aeruginosa SG grew well under anaerobic conditions using both glucose and glycerol as carbon sources  cultured P. aeruginosa using 14C-labeled glycerol--14C and glycerol--14C as carbon sources. They found that the C6 skeleton of rhamnose was composed of two 14C-labeled C3 units that were derived from two molecules glycerol (14C-labeled C3 unit) without carbon chain rearrangement [P. aeruginosa, the rhamnolipids biosynthetic pathway is same under both aerobic and anaerobic conditions, but the anaerobic expression of key genes for rhamnolipids synthesis is inhibited using glucose as carbon source, and these key genes can be highly expressed under anaerobic conditions using glycerol as carbon source. Previous studies compared the gene expression differences of P. aeruginosa using glucose as carbon source under aerobic and anaerobic conditions, and the results showed that the expressions of rhlR, rhlI and rhlAB genes related to rhamnolipids synthesis were inhibited under anaerobic conditions using glucose [Here, two possible hypotheses on anaerobic biosynthesis of rhamnolipid by angement . Hypothe glucose 30.Fig. 2rmlBDAC operon can identify whether the rmlBDAC genes involved in the anaerobic biosynthesis of rhamnolipid using glycerol. If the knockout strain SGrmlB can anaerobically produce rhamnolipids using glycerol as carbon source, there is a new biosynthetic pathway of dTDP-l-rhamnose under anaerobic conditions (hypothesis 1). If not, the rmlBDAC genes control the anaerobic biosynthesis of dTDP-rhamnose, and the expression of rmlBDAC genes and other key genes are inhibited under anaerobic conditions (hypothesis 2). The transcriptomics results can provide the related information.From the two hypotheses point of view, the anaerobic synthesis of dTDP-rhamnose is one key metabolic pathway for rhamnolipids biosynthesis under anaerobic conditions. Knocking out the rmlBDAC operon genes control the biosynthesis of dTDP-rhamnose, and -hydroxy fatty acids are derived from the de novo synthesis of fatty acids [P. aeruginosa. Therefore, anaerobic synthesis of dTDP-rhamnose is one key metabolic pathway for rhamnolipid biosynthesis under anaerobic conditions.dTDP-Rhamnose and -hydroxy fatty acids are two required precursors for rhamnolipids biosynthesis. The ty acids . The de l-rhamnose for rhamnolipids synthesis using glycerol under anaerobic conditions. If not, the rmlBDAC operon genes control the anaerobic biosynthesis of dTDP-rhamnose then for rhamnolipids synthesis using glycerol under anaerobic conditions. As shown in Fig.600=2.18 and 2.33. The wild-type strain SG can anaerobically produce rhamnolipids using glycerol as carbon source, reducing surface tension to 29.8mN/m and forming oil spreading circle with diameter of 22.0mm. Although glycerol used as carbon source, the knockout strain SGrmlB can not anaerobically produce rhamnolipids, reducing surface tension to 48.9mN/m and forming oil spreading circle with diameter of 9.3mm. Results indicated that the rmlBDAC operon genes involved in the anaerobic biosynthesis of rhamnolipids using glycerol.The anaerobic cell growth and anaerobic rhamnolipids production of wild-type strain SG and the knockout strain SGrmlB were comparatively determined. If the knockout strain SGrmlB can anaerobically produce rhamnolipids using glycerol as carbon source, there is a new biosynthetic pathway of dTDP-P. aeruginosa PAO1 (NC_002516). The RNA sequencing data have been deposited in NCBIs Sequence Read Archive (SRA) with Accession Number PRJNA670563 (https://dataview.ncbi.nlm.nih.gov/object/PRJNA670563?reviewer=q57rp98cvqi8nvid6dv8urgint). FPKM values of the mapped genes were calculated for analysis of the gene expression level. The FPKM value of one was used as the threshold to judge whether the gene was expressed or not. The correlation of gene expression levels between samples is an important indicator to test the reliability of experiments and the reasonableness of sample selection [2) is to 1, the higher the similarity of expression patterns between samples is and the less different genes among samples. In this study, Pearson correlation coefficient squared (R2) between samples in GluAn and samples in GlyAn were all greater than 0.92, which indicated the ideal sampling and experimental conditions.To understand why glycerol can be used for anaerobically synthesizing rhamnolipids by strain SG while glucose cannot, RNA sequencing (RNA-seq) was performed on strain SG using glucose and glycerol as carbon source under anaerobic conditions, respectively. The RNA-seq data were shown in Table election . The cloP. aeruginosa SG using glycerol and glucose as carbon sources under anaerobic conditions. There were 549 up regulated genes and 423 down regulated genes in GlyAn samples vs GluAn samples. As shown in Fig.According to the readCount data obtained from gene expression level analysis, a total of 972 significantly differentially expressed genes with log2(FoldChange) value>1 and q-value<0.05 were identified between strain l-rhamnose and -hydroxy fatty acids, and the rhamnosyl transfer process pathways were picked out from the differentially expressed genes between GlyAn samples and GluAn samples. The precursor, dTDP-l-rhamnose, provides the glycosyl part for the biosynthesis of rhamnolipids. It can be initiated by the gluconeogenesis pathway or the EntnerDoudoroff pathway, and the algC gene and rmlBDAC operon genes control the dTDP-l-rhamnose biosynthesis [l-rhamnose and -hydroxy fatty acids, were assembled to mono-rhamnolipids and di-rhamnolipids catalyzed by rhamnotransferase I (RhlB) and rhamnotransferase II (RhlC), respectively [rhlB and rhlC, respectively. The genes lasRI and rhlRI in quorum-sensing pathways are responsible for activating the expression of genes, rhlAB and rhlC [rmlBDAC, rhlAB, rhlC, fabA, fabG and rhlRI, lasI, were significantly up-regulated or activated when using glycerol as carbon source. Glycerol metabolism up-regulated the expression of genes in Quorum-sensing system, which promote the expression of genes rmlBDAC, fabA, fabG, rhlAB and rhlC. The anaerobic high expression of key genes is significant for P. aeruginosa producing rhamnolipids under anaerobic conditions.As listed in Table ynthesis , 33. Theynthesis , 33. Theectively . Rhamnotand rhlC , 35. Undl-rhamnose was also controlled by rmlBDAC operon genes under anaerobic conditions. The gene expression results showed that the related genes for rhamnolipids biosynthesis were highly expressed under anaerobic conditions using glycerol. The genes, rmlBDAC, rhlAB, rhlC, fabA, fabG and rhlRI, lasI, were significantly up-regulated expression using glycerol as carbon source, compared to using glucose under anaerobic conditions.In this study, the knockout strain SGrmlB could not anaerobically synthesize rhamnolipids even using glycerol as carbon source. Results showed that the pathways to metabolize glycerol to synthesize dTDP-P. aeruginosa was revealed at the gene level using glycerol as carbon source. The anaerobic biosynthetic pathway of rhamnolipids in P. aeruginosa SG were confirmed, involving the gluconeogenesis from glycerol, the biosynthesis of dTDP-l-rhamnose and -hydroxy fatty acids, and the rhamnosyl transfer process, which is consistent with the aerobic biosynthetic pathways in model strain P. aeruginosa PAO1. The key genes involved in anaerobic biosynthesis of rhamnolipids are rmlBDAC, rhlABRI, rhlC, fabA, fabG and lasI. Glycerol metabolism up-regulated the expression of genes in Quorum-sensing system (rhlRI and lasI), which promote the expression of genes rmlBDAC, rhlAB and rhlC, thus enable P. aeruginosa to anaerobically produce rhamnolipids using glycerol.Based on the above results, Hypothesis 2 is more convincing . Compared with anaerobic conditions, aerobic conditions are conducive to cell synthesis of reducing power, which promotes fatty acid synthesis and ultimately enhances rhamnolipids synthesis. Increasing the reducing power level in P. aeruginosa cells will also enhance the production of rhamnolipids under anaerobic conditions. Results of this study would be significant for the metabolic engineering of P. aeruginosa to enhance anaerobic production of rhamnolipids.bacteria , but theynthesis , 34, 35.ynthesis , 13. Rhaynthesis , 36. TheP. aeruginosa can anaerobically produce rhamnolipid using glycerol rather than glucose. Two possible hypotheses on anaerobic biosynthesis of rhamnolipid using glycerol were proposed. The rmlBDAC operon genes involved in the anaerobic biosynthesis of rhamnolipid using glycerol. The expression of key genes was inhibited under anaerobic conditions when P. aeruginosa using glucose. The highly anaerobic expression of key genes enables P. aeruginosa to anaerobically biosynthesize rhamnolipids using glycerol. The anaerobic biosynthetic pathway of rhamnolipid using glycerol was confirmed. Improving the anaerobic production of rhamnolipids better enhanced oil recovery. This study fills the gaps in the anaerobic biosynthesis mechanism of rhamnolipids.


Campylobacter jejuni (C. jejuni) is a common cause of mostly self-limiting enterocolitis. Although rare, myocarditis has been increasingly documented as a complication following campylobacteriosis. Such cases have occurred predominantly in younger males and involved a single causative species, namely C. jejuni. Case report: We report herein a case of myocarditis complicating gastroenteritis in a 23-year-old immunocompetent patient, caused by this bacterium with a favorable outcome. Cardiac magnetic resonance imagining was useful in establishing an early diagnosis. Conclusions: Myocarditis should be considered in younger patients presenting with chest pain and plasmatic troponin elevations. The occurrence of myocarditis complicating C. jejuni is reviewed.Background:  Campylobacter spp. are among the most common bacterial causes of gastroenteritis [Campylobacter genus encompasses many species, among which C. jejuni, C. coli and C. fetus are the main human pathogens [C. jejuni are a well-recognized but rare cause of myocarditis and perimyocarditis [C. jejuni, occurring in an immunocompetent patient. The diagnosis was based on clinical, biological and imaging findings.nteritis . Campyloathogens . Typicalcarditis ,3,4,5,6.3  on cell blood count, with elevated C-reactive protein of 130 mg/dL , serum electrolytes, creatinine and liver enzymes within normal limits. Maximum Troponins and creatine kinase MB (CK-MB) were elevated to 678 ng/L  and 54 ng/mL . The patient was admitted to the cardiac department for further workup. He was managed symptomatically with analgesics, anti-reflux and fluids. Transthoracic echocardiography (TTE) revealed a preserved ejection fraction (EF) of 55% with normal wall motions, no valvular dysfunction, normal pulmonary pressure and no pericardial effusion. His risk of coronary artery disease (CAD) was low; moreover, clinical, biological and echocardiographic presentation summed the hypothesis of an acute myocarditis. A large etiological workup, including repeated peripheral blood culture, Mycoplasma pneumoniae, Chlamydia pneumoniae, Coxiella burnetii, Borrelia burgdorferi, Leptospira spp., Rickettsia spp. and Brucella spp., was conducted. The serologies, such as urinary Legionella pneumophila antigen, were negative. Moreover, serologies of RNA viruses  and DNA viruses  were negative. Autoimmune assessment, including antinuclear, anti-neutrophil cytoplasmic antibodies, systemic sclerosis and autoimmune myopathies specific antibodies and converting enzyme assay, were negative. The patient denied any recent drug intake. A Gram stain of specimen stool collected showed multiple curved and spiral Gram-negative rods. Biochemical tests indicated an oxidase, catalase and hippurate negative and indoxyl acetate-positive bacterial species, corresponding to C. jejuni. Stool cultures confirmed the diagnosis of C. jejuni sensitive to macrolides (Azithromycin/Roxithromycin/Clarythromycin) and flouroquinolones (Ciprofloxacin). Continuous telemetry monitoring showed some runs of non-sustained ventricular tachycardia (NSVT). Oral bisoprolol 2.5 mg twice daily was started for that, and oral 1 g of Azithromycin was administered. He remained clinically stable over the rest of the hospital course, and the diarrhea was progressively resolved. The patient remained stable, and we could perform cardiovascular magnetic resonance (CMR) imaging. Triple inversion-recovery black-blood T2-weighted STIR sequences showed focal areas of hypersignal in the subepicardium of the posterolateral left ventricular (LV) wall, indicative of myocardial edema  symptoms of nausea, vomiting, 4-day watery diarrhea with transient episode of fever 38.7 °C). At admission, physical examination showed an impaired general status. He was afebrile (37.2 °C), respiratory rate 20 per min., cardiac assessment showed blood pressure of 140/70 mmHg, regular tachycardia of 100 beats per minute and normal cardiac murmur. Pulmonary examination showed a respiratory rate of 16 cycles per min, and auscultation was normal. He denied abdominal pain, and palpation showed no tenderness and no liver or spleen enlargement. He reported generalized myalgia but rheumatological evaluation did not show arthritis or productive myalgia. The electrocardiogram (ECG) showed left axis deviation with regular sinusal tachycardia. Routine laboratory showed leukocytosis 13 G/L , neutrophilia 8.2 G/L  and lymphopenia 600/mmal edema . In addial edema . Inversial edema . The fin °C. At aC. jejuni infection in a young male patient. Myocarditis may have different patterns of clinical presentation, ranging from mild symptoms of chest pain and tightness associated with transient ECG changes, as illustrated in the current case, to life-threatening cardiogenic shock and ventricular arrhythmia. Thus, it is of high importance to evoke this diagnosis and carry out appropriate investigation to identify its cause [C. jejuni-related myocarditis have been reported [This is a rare case of an acute myocarditis related to ts cause . The diats cause . TTE musts cause . TTE cants cause . In the reported . CMR imareported . The proAn etiological infectious workup was performed, according to the European cardiac recommendation. The viral serology is of limited utility in the diagnosis of myocarditis because it does not imply myocardial infection but rather the interaction of the peripheral immune system with infectious agent. In the North American series of myocarditis, molecular techniques, mainly (reverse transcriptase) RT-PCR amplification, showed genomes of enterovirus, adenovirus, influenza viruses, human herpes virus-6 (HHV-6), Epstein-Barr-virus, cytomegalovirus, hepatitis C virus and parvovirus B19 in the myocardium of patients with myocarditis . This apThe recent scientific statement on EMB gave the highest levels of recommendation in life-threatening clinical presentations. However, the diagnostic, prognostic and therapeutic value of EMB was based on the Dallas histopathologic criteria and did not include immunohistochemistry and viral genome analysis .Further studies are needed to establish the real link between the viral agents and their cardio tropism. The place of RT-PCR amplification in the myocardium is to be determined.Campylobacter spp. have an uncommon but increasingly recognized link with myocarditis, as supported by a growing number of case reports and reviews of literature [C. fetus is less common as a human pathogen [C. jejuni and development of other immune-mediated diseases , auto immune mechanism seems not to be the dominant mechanism in the myocardial lesions due to C. jejuni [terature ,5,6. C. pathogen  but knowpathogen . The put. jejuni . The pat. jejuni .


Phalaenopsis as well as biotic and abiotic stress-treated Phalaenopsis. The database includes floral transcriptomic sequences from 10 orchid species across all the five subfamilies of Orchidaceae.The Orchid family is the largest families of the monocotyledons and an economically important ornamental plant worldwide. Given the pivotal role of this plant to humans, botanical researchers and breeding communities should have access to valuable genomic and transcriptomic information of this plant. Previously, we established OrchidBase, which contains expressed sequence tags (ESTs) from different tissues and developmental stages of Apostasia shenzhenica, Dendrobium catenatum, and Phalaenopsis equestris were de novo assembled and analyzed. These datasets were used to develop OrchidBase 4.0, including genomic and transcriptomic data for these three orchid species. OrchidBase 4.0 offers information for gene annotation, gene expression with fragments per kilobase of transcript per millions mapped reads (FPKM), KEGG pathways and BLAST search. In addition, assembled genome sequences and location of genes and miRNAs could be visualized by the genome browser. The online resources in OrchidBase 4.0 can be accessed by browsing or using BLAST. Users can also download the assembled scaffold sequences and the predicted gene and protein sequences of these three orchid species.Recently, the whole-genome sequences of http://orchidbase.itps.ncku.edu.tw/OrchidBase 4.0 is the first database that contain the whole-genome sequences and annotations of multiple orchid species. OrchidBase 4.0 is available at  The Orchid family is the largest families of the monocotyledons and an economically important ornamental plant worldwide. The orchid is a valuable evolutionary model organism with an unparalleled diversity of innovative vegetative, floral and ecological features. They have colonized successfully almost all habitats on earth. The reasons of the orchid’s dramatic diversification have been associated to the specific interaction between the orchid flower and pollinators , rapid aPhalaenopsis and their hybrids are important for the orchid breeding and the availability of horticultural mutants, the Phalaenopsis plants are often chosen for the orchid development study [Phalaenopsis are found throughout the islands of the Pacific Ocean and the tropical Asia. Phalaenopsis equestris and Phalaenopsis aphrodite subspecies formosana, two native species in Taiwan, are often chosen as parents for breeding commercial cultivars. P. equestris has several beneficial traits such as branches with abundant colorful flowers and numerous spikes. P. equestris is a diploid plant and the estimated haploid genome size 1.6 Gb, which is relatively small in Phalaenopsis [P. equestris has 38 chromosomes that are small and uniform in size (< 2 μm). The fundamental studies and genomic sequences availability have laid the basis for P. equestris to be the first whole-genome sequenced orchid plant [Dendrobium is the third largest genus of Orchidaceae. Dendrobium is a fascinating group of orchids because of their diverse floral architectures, fleshy stems, and synthesis of many kinds of polysaccharides [Dendrobium catenatum contains various kinds of polysaccharides. Many of these polysaccharides have medicinal applications, such as immuno-enhancing, anti-inflammatory, antioxidant and anti-glycation activities [Apostasia shenzhenica is a representative of one of two genera, Apostasia and Neuwiedia, that form a sister clade to the rest of the Orchidaceae. Apostasioideae possess several morphologically unique characteristics different from other orchids. The most remarkable one is their floral morphology. Apostasia shows an undifferentiated labellum at the adaxial side of second floral whorl and relatively simple gynostemium at the center of the flower [Containing more than 900 genera and 27,000 species , the Orcnt study –11. Specaenopsis , 13. P. charides . The fletivities . Apostase flower .Fig. 1PP. equestris, P. bellina and P. aphrodite subsp. Formosana) [Neuwiedia malipoensis and Apostasia shenzhenica (Apostasioideae); Paphiopedilum armeniacum and Cypripedium singchii (Cypripedioideae); Cymbidium sinense and Phalaenopsis equestris (Epidendroideae); Hemipilia forrestii and Habenaria delavayi (Orchidoideae); Vanilla shenzhenica and Galeola faberi (Vanilloideae) [Previously, we established OrchidBase to accommodate and manage the transcriptome sequences in 11 cDNA libraries, generated from various tissues, including inflorescence and flower buds, vegetative tissue, leaf, developing seed, protocorm, cold-treated and pathogen-inoculated plantlets of the three orchid species (rmosana) . The secloideae) .P. equestris was sequenced via a whole-genome shotgun strategy. Its genome size is estimated to be 1.16 Gb, which contains 29,431 predicted protein-coding genes [P. equestris was used to construct OrchidBase 3.0 [D. catenatum was sequenced by Illumina HiSeq 2000 platform [A. shenzhenica was whole genome sequenced by using several different approaches including PacBio, Illumina, and 10X genomics technologies [P. equestris and D. catenatum assembled genome was respectively improved by adding sequence reads generated by both PacBio and 10X genomics technologies. Owing to OrchidBase 3.0 contained the limited old version information of Phalaenopsis genome published in 2015, we update the new version genome of P. equestris, and added genomic information of two other orchid species, D. catenatum and A. shenzhenica, in OrchidBase 4.0. Useful annotation information and easy-to-use web interfaces are provided in OrchidBase 4.0 to access comprehensive sequence data.In year 2015, the genome of ng genes . This spBase 3.0 . In yearplatform . In yearOrchidBase 4.0 is composed of a SQL server database server, a windows application, and a web interface. For storing and managing collected orchid genome sequence information and the annotation data, the SQL Server 2012 system is adopted. The windows application executes sequence analysis, and the C# programs and Perl scripts are applied to parse orchid genome data and construct the database. Several existing tools were used for improving database coverage, system performance, and the user interface. The web interface is constructed using HTML and the Microsoft. NET (framework 4.62). The OrchidBase 4.0 was developed based on Model-View-Controller (MVC) architecture principles by using the ASP.NET MVC 4 framework , 22.Figure Phalaenopsis equestris (BioProject PRJNA192198 and PRJNA389183) were downloaded from the NCBI database. The Bioproject PRJNA262478 containing raw data and whole genome-assembled scaffold sequences of Dendrobium catenatum were also downloaded. Genome sequences and whole-genome assembly of Apstasia shenzhenica included in BioProject PRJNA310678 were fetched. Statistics of these three orchid genomes is shown in Table The raw data and whole genome-assembled scaffold sequences of Based on these datasets, predicted protein-coding genes and translated amino acid sequences were annotated by combining homology-based prediction, de novo gene prediction, and RNA sequence-aided prediction . Each prThe transcriptomics data were downloaded from BioProjects PRJNA288388, PRJNA304321, and PRJNA348403. For providing expression profile of each orchid gene, all RNA-seq reads were mapped to the predicted genes and counted FPKM values for each gene in the various tissues and different developmental stages. All of this biological information has been integrated into the OrchidBase 4.0.A. shenzhenica, D. catenatum, and P. equestris genome information in OrchidBase 4.0 can be searched to acquire the assembled scaffolds and predicted gene information. Through the web interface, the three orchid genome information contained in OrchidBase 4.0 could be freely accessed. The information can be accessed via the “Orchid Genome” icon  terms, Interpro, Swissprot, and TrEMBO classification Fig. . Users cThe list of annotated genes with the FPKM value can be explored in the “Gene expression” page Fig. . Users cThe “Metabolism pathway” page provides information for the selected genes involved in the KEGG pathways Fig. . Users cTo help users perform sequence alignment, OrchidBase 4.0 provides a graphic user interface for users to run BLAST Fig. . The assThe addition and integration of orchid whole genomic sequences with detailed annotation information and easy-to-use web interfaces in OrchidBase 4.0 allow users to efficiently find target genes, such as floral development-related genes , floral Project name: OrchidBase 4.0.http://orchidbase.itps.ncku.edu.tw/Project home page: Operating system(s): Microsoft Windows Server 2016 Standard.Programming language: ASP.NET and Visual C#.Other requirements: none required.License: none required.Any restrictions to use by non-academics: no restriction.