Klebsiella. The whole-genome sequences will aid in comparative genomic studies of ISS Klebsiella strains with Earth counterparts, to gain insight into their adaptation to space conditions.Isolated across four locations aboard the International Space Station (ISS), 10 bacterial strains were compared using whole-genome sequencing analysis and were phylogenetically identified as  Klebsiella. The whole-genome sequences will aid in comparative genomic studies of ISS Klebsiella strains with Earth counterparts to gain insight into their adaptation to space conditions.Isolated across four locations aboard the International Space Station (ISS), 10 bacterial strains were compared using whole-genome sequencing analysis and were phylogenetically identified as  Klebsiella was discovered by Carl Friedlander in 1882 from the lungs of patients who had died of pneumonia   , 6. Sincon (ISS) , the strbapenems , 4, 7. TKlebsiella species, including K. aerogenes (n = 1), K. pneumoniae (n = 1), and K. quasipneumoniae (n = 8), were isolated from various locations on ISS environmental surfaces  using the EZBioCloud calculator . Default parameters were used for all software.Genomes were sequenced using the Illumina  Nextera Flex protocol for library preparation, and a NovaSeq 6000 S4 flow cell  was used for paired-end sequencing. The quality was assessed with FastQC v0.11.7) . Adapter.7 . Adape length . The GC PRJNA635942, PRJNA640688, and PRJNA640693. Whole-genome sequencing data have also been deposited in NASA GeneLab . The versions described in this paper are the first versions.This whole-genome sequencing project has been deposited in GenBank, and the GenBank and raw read accession numbers are given in


Iron is an essential element required to support the health of organisms. This element is critical for regulating the activities of cellular enzymes including those involved in cellular metabolism and DNA replication. Mechanisms that underlie the tight control of iron levels are crucial in mediating the interaction between microorganisms and their host and hence, the spread of infection. Microorganisms including viruses, bacteria, and fungi have differing iron acquisition/utilization mechanisms to support their ability to acquire/use iron . These pathways of iron uptake are associated with promoting their growth and virulence and consequently, their pathogenicity. Thus, controlling microorganismal survival by limiting iron availability may prove feasible through the use of agents targeting their iron uptake pathways and/or use of iron chelators as a means to hinder development of infections. This review will serve to assimilate findings regarding iron and the pathogenicity of specific microorganisms, and furthermore, find whether treating infections mediated by such organisms via iron chelation approaches may have potential clinical benefit. Iron is a key element needed to support fundamental cellular processes including oxygen transport, DNA replication, transcription, and metabolic processes in many living organisms ,2. It isDietary iron in the host can be obtained in the form of heme from various sources including red meat, seafood, and poultry . This heIn an adult human, hemoglobin from red blood cells (RBCs) contains approximately two-thirds of the total iron present in the body (~3–4 g) . Not onlThe specific mechanisms that support the ability of microorganisms such as viruses, microbes, and fungi to uptake iron from sources in the host are discussed in this review, along with the effect of iron chelators which may potentially antagonize their growth and virulence.It is well established that deregulated iron control can lead to detrimental effects on survival ,2. Since3+-bound complex (TBI)), facilitates the transport of iron into cells via the transferrin receptor (CD71) [3+ (ferric) to the Fe2+ (ferrous) form [Transferrin, a carrier glycoprotein which binds to iron (as Fer (CD71) . Cellulaus) form . Once reus) form .The divalent metal transporter ZIP8 (Solute carrier family 39 member 8 (SLC39A8)) is one way through which NTBI can enter cells . AnotherThe imported iron (from either NTBI or TBI) can either 1) be stored in a complex with ferritin (FTN), 2) be added to the labile iron pool (LIP), (3) be exported extracellularly via ferroportin (FPN), or (4) be integrated within key enzymes involved in regulating cellular metabolic processes . Extrace be store be addedIron-binding proteins in the host plasma  have the ability to sequester iron from various sources; their iron binding capacity can contribute to reducing the availability of extracellular iron that may support the growth and virulence of microorganisms within this environment . WithholStreptomyces pilosus) and Desferrithiocin ((DFT), a tridentate siderophore, produced by Streptomyces antibioticus) [In multiple diseases, iron chelation has been explored as a therapeutic regimen to reduce iron levels to promote health . Efficacioticus) ,29,30. Mioticus) ,32. Mammioticus) ,34. Not ioticus) ,34.Yersinia and Klebsiella infections may develop [A subset of FDA-approved iron chelators includes DFO, DFRA, and DFP  and thes develop . DFP, ad develop . DFRA, a develop . Further develop .In this review, we summarize findings involving the application of iron chelators primarily in the context of infectious diseases (see Overview—Viruses have adapted themselves to use host cell components to propagate. In contrast to Hepatitis C Virus (HCV) and Human Immunodeficiency Virus (HIV) which utilize SR-B1 and CD4 receptor, respectively, for host cell entry, New World Arenaviruses enter host cells by binding to transferrin receptors, a host cell receptor also responsible for iron uptake ,44. To sFlaviviridae family and is a single-stranded positive sense virus with a diameter of 50–80 nm [Viral Genome and Structure—Hepatitis C Virus is a member of the 50–80 nm . Its gen50–80 nm  and asse50–80 nm .Health Implications and Current Treatments—HCV infections are associated with inflammation potentially leading to liver fibrosis, cirrhosis, and hepatocellular carcinoma . AlthougIron Contribution to HCV Infections—In liver-biopsy specimens from infected HCV patients, a positive correlation between hepatic iron content and HCV was reported . Efforts4 for two days was found to increase the replicative capacity of HIV  in hepatocytes [In in vitro studies, supplementation with FeSOatocytes . Furtheratocytes  and La patocytes ,62,63. Watocytes .Although it has been suggested that therapies that reduce iron levels could be utilized as adjuvant to existing HCV therapies , additioRetroviridae family and is a single stranded RNA virus with a diameter of 100 nm [Gag, codes for the core structural proteins  and the matrix (p17), (2) Pol, codes for viral replication enzymes including reverse transcriptase, integrase, and protease, (3) Vif, encodes a protein that promotes infectivity of viral progeny, (4) Vpr, codes for a protein that causes cell cycle arrest, (5) Vpu, codes for a protein involved in the release of the viral particle, (6) Tat, codes for a protein which is involved in HIV gene expression, (7) Rev, codes for a protein that allows the export of the RNA from the nucleus into the cytoplasm, (8) Env, encodes the glycoproteins in the envelope (gp120 and gp41), and (9) Nef, codes for a protein that can modulate signaling and promote viral budding [Viral Genome and Structure—Human immunodeficiency virus is a member of the f 100 nm . HIV con budding . The vir budding ,66.Health Implications and Current Treatments—HIV can lead to acquired immunodeficiency syndrome (AIDS), a terminal stage of HIV-infections, in which patients are afflicted by opportunistic infections and cancer . There iHFE gene); specifically, a major reduction in viral particles was noted following an extensive 18-month phlebotomy period [Iron Contribution to HIV Infections—In patients with HIV infections, excessive iron content in their serum , tissuesy period .gag) and reverse transcriptase activity in the cell supernatants [At a cellular level as determined from in vitro studies, the addition of exogenous iron or modulation of key modulators of host iron metabolic pathways could alter HIV-1 replication and transcription. Specifically, overexpression of FPN1, the iron export receptor, decreased transcription of HIV-1 in HEK293 cells . In contrnatants ; these crnatants .The mechanisms underlying these iron-associated cellular effects on HIV-1 transcription were elucidated to be mediated through NF-kB (a transcription factor that plays a role in regulating multiple cellular activities), which binds to the long-terminal repeat sequence of HIV  on NF-kB response elements . This paStreptomyces verticillus which forms iron complexes generating ROS that leads to base modifications in the viral DNA) could also reduce the replicative capacity of HIV without affecting cellular viability in PBL ; whether the effects of BLM is due to iron-chelation activity is unclear [3H-thymidine) of peripheral blood lymphocytes [Inhibition of the iron-dependent DNA replication enzyme, RNR, with iron chelators (DFRA and DFP) in lymphocytes could also alter HIV viral replication ,85. Bleophocytes .Tat gene by reducing CDK2 and CDK9 kinase activity on specific target proteins including cyclin T1 and the C-terminal domain of RNA polymerase II in multiple cell types including HeLa-CD4-LTR-β-gal cells, 293T cells, and CEM cells [In vitro cell studies were performed to identify changes in the expression and activities of cell cycle mediators by iron chelators in HIV-1 infected cells. Specifically, the iron chelators DFO and 311  reduced HIV-1 viral transcription by modulating protein expression of cyclin-dependent kinases  in human lymphoid CEM cells . MoreoveEM cells . This fiEM cells . Other nEM cells .Although there appears to be an abundance of data supporting the use of iron chelators in HIV-1 infected cell lines, additional research is needed to enable the use of iron chelators in clinical treatment strategies.Physical Characteristics of Gram-negative and Gram-positive Bacteria with Relevance to Iron Uptake—It is well established that iron acquisition is critical in promoting the growth and virulence of numerous pathogenic bacteria . Since aPseudomonas aeruginosa, which we discuss in greater detail below [Iron Uptake Pathway in Gram-negative Bacteria—Greater than 30 outer membrane protein (OMP) heme receptors (involved in transporting heme intracellularly) have been characterized across a wide variety of Gram-negative microbes . Some spil below . Anotheril below . The feril below .Corynebacterium diphtheriae, a well-studied Gram-negative microbe [Streptococcuspyogenes, another well-studied Gram-negative microbe, involves the Shr protein (which binds heme), the streptococcal heme-binding protein Shp , SiaA (which is the heme-binding lipoprotein), SiaB (membrane permease), and SiaC (ATPase) [Staphylococcus Aureus, has been identified which uses hemolysins in its pursuit to acquire bound heme [Iron Uptake Pathway in Gram-positive Bacteria—In contrast to Gram-negative microbes, far fewer details have been uncovered with respect to the mechanisms underlying heme uptake in Gram-positive microbes. However, the HemT-like lipoprotein, HmuT, has been identified to participate in this process, specifically in  microbe . The com(ATPase) ,39. A mound heme .Pseudomonas aeruginosa, a multi-drug resistant pathogen, has a genome size of 5.5–7 Mbp with the capacity to express genes underlying resistant phenotypes [P. aeruginosa is mediated by structural components including flagellum and pili as well as cell surface glycolipids and lectins that are involved in bacterial movement and adhesion to host cells [P. aeruginosa is capable of injecting cytotoxins into host cells [Bacterial Features—enotypes . Togetheenotypes . The patst cells . Furtherst cells . In addist cells .P. aeruginosa growth properties [P. aeruginosa [P. aeruginosa injected intraperitoneally in Swiss–Webster mice resulted in increased virulence [P. aeruginosa deficient in Pvd or Pch/Pvd inoculated intranasally, elicited reduced growth in the pulmonary tissue coinciding with decreased virulence [Contribution of Iron—Several iron uptake mechanisms are involved in mediating operties . The pyoruginosa ,107 usinruginosa . The addirulence . Using iirulence .P. aeruginosa, the rate of repair is diminished in multiple animal models  [P. aeruginosa was applied), transcriptional profiling identified 7 out of 136 differentially expressed genes that were involved in pyochelin biosynthesis, including the pyochelin receptor fptA, pchH, pchG, pchE, pchD, pchB, and pchC (the biosynthesis of Pch requires the iron-regulated pchDCBA operon) [P. aeruginosa include the citrate-mediated Fe3+ uptake pathways that engages FecA, an outer membrane ferric citrate receptor, the FeoB transporter, and the PcoA, periplasmic ferroxidase [phuSTUVW operon (a gene cluster encoding an outer membrane receptor and specific ABC transporters for heme and hemoglobin uptake) [In wounds infected with  models) . Specifi operon) . In addi operon) . These f operon) . Other proxidase . The pro uptake) .P. aeruginosa infections in lungs are frequent in patients afflicted with cystic fibrosis [P. aeruginosa infection. Neutrophil numbers, pro-inflammatory cytokines, and microbial numbers were reduced with bLF treatment [In addition to wound infections, fibrosis . In an ereatment . Althougreatment , it is uLeptospermum scoparium (manuka bush), was discovered to elicit anti-microbial activity against multiple pathogens [P. aeruginosa, Escherichia coli, and S. aureus [P. aeruginosa, which could potentially be utilized as a strategy to overcome antibiotic resistance.Manuka honey, produced from the nectar of athogens . Specifi. aureus . Althoug. aureus  and the . aureus , it remaP. gingivalis and Prevotella intermedia are mutualistic in terms of heme acquisition, as described in detail below. P. gingivalis (Strain W83) is a Gram-negative oral bacteria with a genome size of 2,343,479 bp. P. intermedia is described as a anaerobe that is Gram-negative with a genome size (OMA14 strain) which is represented by two circular chromosomes of 2,280,262 and 867,855 bp, respectively [Bacterial features—The oral microbiome can be composed of up to 700 species; an imbalance of these species could lead to the development of periodontitis . Specifiectively . Genome ectively ,120. Witectively ; this exectively .P. gingivalis is deficient in siderophores [P. gingivalis contains proteins that physically interact with hemoglobin  and heme  in gingival crevicular fluid [rgpA (which codes for gingipain that cleaves arginyl peptide bonds), hagA (which codes for hemagglutinin A), and kgp (which codes for gingipain that cleaves lysyl peptide bonds) [P. gingivalis and P. intermedia involves the process of heme acquisition; specifically, this involves the HmuY protein in P. gingivalis and the proteolytic activity of P. intermedia [P. intermedia, which increases free hemoglobin, is thus proposed to provide an optimal growth environment for P. gingivalis [P. intermedia), supports iron (III) protoporphyrin IX generation via hmuY from P. gingivalis [Contribution of iron—Since rophores ,124 as wrophores ,126,127,rophores . This barophores . With rear fluid ,130,131.e bonds) . One muttermedia . The hemngivalis ,134. In ngivalis ,135.P. gingivalis could be effectively inhibited in its growth rate and adhesiveness by the iron chelator, DFO, by reducing its ability to accumulate hemin, Fe3+-protoporphyrin IX, a virulence factor [P. intermedia, another Gram-negative microbe present in periodontal lesions [F. nucleatum [Subgingival plaque e factor . The eff lesions . DFRA co lesions . A bluebucleatum . The micucleatum .Over the past two decades, there has been a rise in antibiotic-resistant infections, which could be attributed to the overuse of antibiotics (with evidence of country dependency ) and antStreptococcus pneumoniae, a pathogen with a core genome size of 1,536,569 bp [Bacterial Features—Infections that are due to 6,569 bp , that ca6,569 bp .S. pneumoniae does not express siderophores, which is unlike other microbes [S. pneumoniae , which code for proteins involved in iron uptake [Pit1 and pit2, which are S. Pneumoniae loci which code for an ABC transporter, enables the Gram-positive bacteria to acquire iron from hemoglobin [S. pneumoniae was inoculated intranasally and a systemic model in which S. pneumoniae was inoculated via the intraperitoneal cavity), double knockouts of pit and pit2 led to a marked reduction in S. pneumoniae virulence [S. pneumoniae, was performed in the presence of the iron chelator, 2,2′-bipyridine, to limit iron content within the environment of the pathogen [S. pneumoniae pathogenesis as well as those involved in cell division  were downregulated [Contribution of Iron—microbes ,145. To activity )) to eliactivity . S. pneurom heme . A mutann uptake . Pit1 anmoglobin . Using tirulence . A protepathogen . Under tegulated ; in contegulated .S. pneumoniae.Altogether, these studies suggest that targeting the iron uptake pathways and/or use of iron chelators could antagonize the growth and virulence of Mycobacterium tuberculosis is a pathogen with a genome size (H37Rv strain) of 44.1 Mbp [M. tuberculosis can also be disseminated extracellularly into the blood to secondary locations  [M. tuberculosis resistant strains [Bacterial Features—44.1 Mbp . This ba44.1 Mbp . Howeversystems) . Current strains .M. tuberculosis activate a host defense pathway that limits serum iron availability causing “anemia of chronic disease” [M. tuberculosis has evolved mechanisms to acquire intracellular iron within host macrophages and myeloid dendritic cells via a siderophore-mediated process involving mycobactin and carboxymycobactin [M. tuberculosis [M. tuberculosis strains harboring loss of iron acquisition and uptake systems (located within the ESX-3 type VII secretion system) to restrict iron accessibility, the bacterial burden was markedly reduced [Contribution of Iron—Infections mediated by disease” . To overcobactin ,153. An cobactin . Thus, iM. tuberculosis.Further work is needed to determine clinical efficacy of targeting the above described pathway in patients infected with Fungal Cell Structure—Since the fungal cell wall is the primary barrier that is encountered in response to anti-fungals, its characteristics have been investigated in an effort to understand its role in mediating anti-fungal resistance . It is iSaccharomyces cerevisiae, a model organism [Cryptococcus neoformans and Aspergillus fumigatus with the past decade [Fungal Iron Uptake Pathways—The virulence and growth potential of fungi depend on specific metal ions, such as copper, zinc, manganese, nickel, and of relevance to this review, iron . Althougorganism , greatert decade . The mect decade .FRE genes); this is then followed by its uptake into the cells by the permease FTR1 along with the oxidation of the iron via the activity of a ferroxidase (Fet3) [S. cerevisiae, it primarily employs both low-affinity and high-affinity non-reductive iron transport systems in conjunction with metalloreductases (which reduce iron) and siderophore mediated iron transport mechanisms [C. albicans and S. cerevisiae and components of the endosomal sorting (ESCRT-I) complex are involved in C. neoformans [The most commonly described mechanisms across fungal species include the (1) reductive iron assimilation (RIA) and (2) non-reductive (siderophore-mediated) iron uptake . The RIAe (Fet3) . With rechanisms ,41,157. chanisms ,159. Thechanisms ; the prechanisms . The majDue to the vast array of iron acquisition mechanisms employed by fungi, these pathways involved in iron accumulation contributing to fungal virulence could be potentially targeted to formulate novel treatment strategies.Cryptococcus neoformans, a basidiomycete fungus involved in meningitis, can lead to a poor outcome in patients who are immunocompromised [Fungal Features—promised . The trepromised ) and flupromised ). Howevepromised  and thusC. neoformans [Contribution of Iron—It is well established that the iron permease (Cft1) and ferroxidase (Cfo1) are involved in the iron uptake pathway in oformans . Cft1 muoformans .C. neoformans containing a mutation in cft1 markedly reduced fungal virulence [C. neoformans with a deficiency of Cfo1 (but not Cfo2) into mice led to a reduction in virulence but also increased the sensitivity to amphotericin B and fluconazole [C. neoformans was noted with genetic deficits in these fungal iron uptake proteins coinciding with reduced resistance to the anti-fungal agents [In a mouse model, intranasal inoculation of irulence . Intranaconazole . The mecconazole . Furtherconazole . In suppl agents ,163,164.C. neoformans in vitro [Chelation of intracellular iron with bathophenanthroline disulfonate (BPS) or DFP, in combination with fluconazole or miconazole, synergistically altered the growth capacity of in vitro . Based oin vitro .Aspergillus fumigatus, an airborne saprophytic fungus that is responsible for the development of invasive pulmonary aspergillosis, is common in patients characterized by iron overload and with blood cancers [Fungal Features— cancers . First-l cancers .A. fumigatus is unable to acquire host iron directly, specifically from transferrin, ferritin, or heme, it has developed efficient iron uptake mechanisms including (1) RIA and (2) siderophore-mediated processes [3+ to Fe2+ via ferrireductases that are present within the fungal cell membrane; the imported iron is then oxidized by a ferroxidase and a permease, FetC and FtrA, respectively [A. fumigatus is also capable of acquiring iron via extracellular siderophores, fusarinine C (FsC) and triacetylfusarinine C (TAFC), which are then imported into the fungus via transporters in the membrane  [A. fumigatus is regulated by the GATA transcriptional regulator, SreA [A. fumigatus deficient in SreA does not differ to the wild type strain in terms of virulence [A. fumigatus led to a reduction in the spread of the fungal infection [Contribution of Iron—Since rocesses . As descectively . A. fumisporter) . Althougsporter) . The bioor, SreA ; howeverirulence . Using tnfection . HapX isnfection .A. fumigatus.The combination of the iron chelator DFRA, with a liposomal preparation of Amphotericin B, was effective in reducing fungal infections in murine models of pulmonary Aspergillosis which supported murine survival . AlthougRhizopus oryzae is a filamentous fungus involved in the development of mucormycosis, a common infection in (a) patients with diabetic ketoacidosis [Fungal Features—acidosis , (b) thoacidosis , or (c) acidosis ,175. Curacidosis . Unfortuacidosis .R. oryzae has identified several genes involved in iron uptake including three ferric reductases, six copper oxidases, a high-affinity iron permease, siderophore permeases, SreA, and genes involved in the uptake of iron from heme [R. oryzae as well as fungal infections in a murine model of diabetic ketoacidosis infected with spores via the tail vein or intranasal instillation [Contribution of Iron—The genome sequencing project for rom heme ,179. Therom heme  in orderrom heme . Loss ofillation .R. oryzae spores into the tail vein, FOB1 and FOB2 deficient fungi were essential for mediating virulence in the DFO-model only [R. oryzae [R. oryzae in immunocompetent guinea pigs [In a diabetic ketoacidosis mouse model and a DFO-treated mouse model injected with del only . Further. oryzae . However. oryzae ,183. It . oryzae , increas. oryzae . Treatmenea pigs  and albinea pigs .R. oryzae. Collectively, targeting these fungal iron pathway holds promise to improving existing therapeutic modalities for overcoming the detrimental health consequences of R. oryzae fungal infections.The oral administration of DFRA, an iron chelator, resulted in clinical improvement in a 40-year old patient infected with Rhinocerebral mucormycosis  in combination with liposomal amphotericin B . This onAlthough limited applications for iron chelation therapeutic approaches are noted in clinical practice for the topics presented herein, evidence from in vitro and in vivo animal models, which cover a wide range of diseases, provides a strong positive foundation for future clinical applications. With respect to iron availability, it is essential that iron levels in the host be tightly controlled to hinder the development of microorganismal infections within the host by (1) supporting the capacity of iron-binding proteins in the host plasma to limit iron availability to hinder the growth and virulence of microorganisms and (2) diminishing the intracellular host iron by targeting TBI or NTBI uptake processes, increasing ferritin-bound iron/reducing ferritinophagic processes, and increasing FPN-mediated iron export. Although there is evidence for iron chelators in hindering microorganismal virulence, this is an area that would benefit from further research investigation. Furthermore, the identification of the iron chelating components in the natural compounds derived from plants  would also be beneficial in the identification of naturally occurring iron chelators.


Caenorhabditis elegans is a free-living, bacterivorous nematode that frequently encounters pathogens while foraging for food in decomposing vegetation. Like other invertebrates, C. elegans entirely relies on its innate immune system to combat invading pathogens. A basal flight or fight response of animals is also observed in worms against infection. The former is an aversion response of C. elegans against select pathogens, and the latter is an inducible innate response comprising of pathogen-specific effectors including lysozymes, lectins, antimicrobial peptides (AMPs), and cytoprotective molecules. Although pathogen recognition in worms is poorly understood, various signaling pathways and immune effectors facilitating defense response are well studied, making this nematode an excellent model to study host–microbe interactions. In higher vertebrates such as mice and humans, sensing of infection through pathogen-associated molecular patterns (PAMPs) or host damage-associated molecular patterns (DAMPs) is primarily mediated by the toll-like receptors (TLRs), nod-like receptors (NLRs), RIG-I like receptors (RLRs), C-type lectin domain (CTLD) proteins, and AIM-like receptors (ALRs) [C. elegans, does not seem to be essential during infections with most pathogens, except during Salmonella enterica [Serratia marcescens [C. elegans RIG-I like receptor DRH-1 detects products of viral replication and activates an intracellular pathogen response [C. elegans, are essential for immune responses against S. marcescens and are known to bind live bacteria [C. elegans during a majority of infections remain elusive. In this review, we examine the roles of G protein-coupled receptors (GPCRs) as noncanonical pattern recognition receptors (PRRs) and also discuss how GPCR signaling in C. elegans regulates various immune processes.s (ALRs) . Humans enterica  and Serrrcescens  infectioresponse . CLEC-39bacteria . DespiteC. elegans encodes approximately 1,300 genes encoding putative GPCRs [C. elegans have revealed the importance of GPCRs and their signaling in host defense. In this review, we examine the role of GPCRs in innate immunity via the modulation of “flight” or “fight” responses of C. elegans.GPCRs form the largest superfamily of cell surface receptors in eukaryotes; ve GPCRs . They arve GPCRs  and alsove GPCRs . In receC. elegans prompted many research groups on a quest to identify neuronal GPCRs or GPCR signaling components that are involved in immune regulation. A screen of 40 GPCR mutants by Styer and colleagues in 2008 led to the first demonstration of a neuronal GPCR as a regulator of innate immunity in C. elegans [Pseudomonas aeruginosa, Enterococcus faecalis, and S. enterica [C. elegans  in the ligand-binding domain. Powell and colleagues screened for LRR domain-containing receptors in response . fshr-1  F49F1.6  Fig 1C)C. elegan F49F1.6 . Furtherruginosa . These sDrechmeria coniospora is an ascomycetes fungus and a natural pathogen of C. elegans. The fungal conidia attach to the worm’s cuticle and germinate, causing the hyphae to penetrate the cuticle, eventually colonizing the entire body of the worm. In response to this invasion, worms up-regulate several AMPs such as neuropeptide-like protein NLP-29 in the epidermis. NLPs in this cluster are also up-regulated during epidermal wounding. The NLP-29 up-regulation during wounding and D. coniospora infection relies on Gα subunit GPA-12 and Gβ subunit RACK-1 [C. elegans hypodermis. HPLA levels increase during injury and D. coniospora infection, activating DCAR-1, and subsequently induces immune response in the hypodermis DrechmeriMicrobacterium nematophilum, a natural pathogen of C. elegans, results in deformed anal region (Dar). The Dar phenotype can be easily visualized due to the swelling in the rectal epithelium, adjacent to the tail of worms. This is caused by the attachment of bacterial cells to the rectal and post-anal cuticle, and the colonization of the rectal opening, causing swelling, a classic signature of inflammation. During M. nematophilum infection, Gαq subunit EGL-30 in the rectal epithelium initiates a cascade of signaling events leading to the Dar phenotype [o subunit, in the rectal epithelium, thereby suppressing the Dar phenotype and immunity [M. nematophilum from the rectal opening [M. nematophilum (lf) mutation completely abrogates the Dar phenotype, while PCDR-1 lf mutation only causes partial suppression of the Dar phenotype [Infections with coryneform bacterium henotype . This cahenotype . Serotonimmunity . The inv opening  Fig 1C)Microbact opening . PCDR-1 henotype . Given tC. elegans has a well-developed chemosensory system that enables it to detect food cues and pheromones. The nematode is repelled by various pathogens, a response termed as “aversion.” It is an example of the flight response, a behavioral strategy conserved in the animal kingdom. Aversion can be categorized into innate and learned aversion. The former is reliant on the worm’s innate ability to detect naturally repulsive components of the pathogen, thereby triggering an innate aversive response. On the other hand, learned aversion involves the process of associative learning upon exposure to pathogens, resulting in the ability to avoid the pathogen on subsequent encounters. Innate aversion is observed when worms are exposed to a lawn of pathogenic bacterium S. marcescens [S. marcescens) [tol-1 mutants are defective in the aversion response toward S. marcescens [P. aeruginosa [M. nematophilum [P. aeruginosa is of the “learned” nature. Worms are initially attracted to a lawn of P. aeruginosa, and only begin to avoid the pathogen after a prolonged exposure [P. aeruginosa. Intestinal infection and the subsequent bloating of the lumen trigger feedback systems that modulate the NPR-1 GPCR pathway that facilitates the regulation of aversive learning [rcescens ,25. Thisrcescens . An unidruginosa , while ttophilum  Fig 1C)C. eleganexposure . Recent learning ,28. The S. marcescens secretes 2-butanol and acetone that initially attracts worms, perhaps facilitating an opportunity for the bacteria to establish infection [Bacillus nematocida B16 attracts nematodes via the neuronal GPCR STR-2 [B. nematocida and other Bacillus species. The identification of microbial ligands inducing behavioral response in C. elegans is an area of active investigations in many labs with a promise to elucidate a GPCR-based model of pattern recognition in the animal kingdom.Interestingly, there are evidences that some pathogens exploit the nematode’s chemosensory system by secreting attractive metabolites to trap them. For example, nfection . SimilarCR STR-2 . RemarkaCR STR-2 . Since sCR STR-2 , it remaC. elegans demonstrating immune-modulatory roles of neuronal and nonneuronal GPCRs. Notably, some studies in drosophila, mice, and humans [C. elegans sensory neurons, serve as PAMPs in C. elegans and other animals. Worms provide us with a unique opportunity to identify additional GPCRs and their ligands with the view of devising pharmacological strategies for boosting innate immune responses and for suppressing inflammation.In this review, we examined a number of elegant studies in d humans  also sug


Bougainvillea (Nyctaginaceae) is a popular ornamental plant group primarily grown for its striking colorful bracts. However, despite its established horticultural value, limited genomic resources and molecular studies have been reported for this genus. Thus, to address this existing gap, complete chloroplast genomes of four species  and one Bougainvillea cultivar were sequenced and characterized. The Bougainvillea cp genomes range from 153,966 bp to 154,541 bp in length, comprising a large single-copy region  and a small single-copy region  separated by a pair of inverted repeats . All sequenced plastomes have 131 annotated genes, including 86 protein-coding, eight rRNA, and 37 tRNA genes. These five newly sequenced Bougainvillea cp genomes were compared to the Bougainvillea spectabilis cp genome deposited in GeBank. The results showed that all cp genomes have highly similar structures, contents, and organization. They all exhibit quadripartite structures and all have the same numbers of genes and introns. Codon usage, RNA editing sites, and repeat analyses also revealed highly similar results for the six cp genomes. The amino acid leucine has the highest proportion and almost all favored synonymous codons have either an A or U ending. Likewise, out of the 42 predicted RNA sites, most conversions were from serine (S) to leucine (L). The majority of the simple sequence repeats detected were A/T mononucleotides, making the cp genomes A/T-rich. The contractions and expansions of the IR boundaries were very minimal as well, hence contributing very little to the differences in genome size. In addition, sequence variation analyses showed that Bougainvillea cp genomes share nearly identical genomic profiles though several potential barcodes, such as ycf1, ndhF, and rpoA were identified. Higher variation was observed in both B. peruviana and B. pachyphylla cp sequences based on SNPs and indels analysis. Phylogenetic reconstructions further showed that these two species appear to be the basal taxa of Bougainvillea. The rarely cultivated and wild species of Bougainvillea  diverged earlier than the commonly cultivated species and cultivar . Overall, the results of this study provide additional genetic resources that can aid in further phylogenetic and evolutionary studies in Bougainvillea. Moreover, genetic information from this study is potentially useful in identifying Bougainvillea species and cultivars, which is essential for both taxonomic and plant breeding studies. Bougainvillea, a tropical and subtropical shrubby vine cultivated primarily for its colorful showy bracts. Their vibrant structures often mistaken as “flowers” are actually bracts or specialized leaves (ca. 0.5–2-inch long), in which the true flowers are attached at the mid-rib [Bougainvillea’s growth habit and attractive bracts, it became a widely known plant for landscaping [The family Nyctaginaceae, distributed primarily in the tropics and subtropics, contains around 400 species of trees, shrubs, and herbs classified in ca. 31 genera . NyctagiBougainvillea glabra and Bougainvillea spectabilis, are native to South America, but were brought and introduced to various countries all over the world. There are approximately 18 species of Bougainvillea, but only the above two species are well-known for cultivation [Bougainvillea is mostly ornamental, but recently a number of studies have explored the potential use of Bougainvillea as a medicinal plant and as a pollution-mitigating plant in industrial areas [Bougainvillea as an anti-inflammatory, anticancer, antioxidant, antimicrobial, and antihyperglycemic plant [Bougainvillea spectabilis has been well-known for its ability to lower blood sugar and improve liver function, while Bougainvillea cv. buttiana and Bougainvillea glabra exhibit significant anti-inflammatory activities [Bougainvillea, particularly regarding the wild species not utilized for cultivation.The horticulturally important species, such as tivation . The pural areas ,10,11. Ric plant ,10,11. Stivities ,9,10. ThBougainvillea focused on taxonomic descriptions were published decades ago [Bougainvillea. Phylogenetic studies on family Nyctaginaceae based on ndhF, rps16, rpl16, and nrITS revealed that Bougainvillea glabra and Bougainvillea infesta are actually closely related to Belemia and Phaeoptilum [Bougainvillea spectabilis as representative of Bougainvilleeae [Bougainvillea species.Most of the publications on ades ago ,13,14, aeoptilum . SimilarBougainvillea are rarely deposited in the database. The commercial value of Bougainvillea as an ornamental plant has overshadowed the need for genetic studies. Hence, the majority of the available sequences in the GenBank are from the commercially cultivated Bougainvillea. Short-genome announcement papers have presented the gene content and structure of B. glabra and B. spectabilis [Bougainvillea is clustered together with Acleisanthes, Mirabilis, and Nyctaginia; however, specific relationships cannot be inferred due to the lack of available sequences [Bougainvillea, particularly on their plastome features and specific phylogenetic placements.Even though next generation sequencing (NGS) has instigated a rapid increase in the available complete organelle genome sequences of plants that can be used for phylogenetic studies, sequences of ctabilis ,18. It wequences . CurrentBougainvillea species is an initial step that is needed to fill the gap in genomic resources before conducting further studies. Thus, this study aimed to characterize the cp genome sequences of several cultivated and wild Bougainvillea species . Specifically, the study aimed to discuss the structures and features of the five newly sequenced cp genomes, including the codon usage, RNA editing, simple sequence repeats, tandem repeats, IR contractions and expansion, divergent regions, SNPs, and indels. Phylogenetic analyses were also conducted to determine the relationship among cultivated and wild Bougainvillea in the family Nyctaginaceae. It has been established that plastomes, particularly chloroplast genomes, are useful in phylogenetic studies due to their ability to self-replicate, their conservative structure, and their slow evolutionary rate . InvestiBougainvillea glabra, Bougainvillea peruviana, Bougainvillea pachyphylla, Bougainvillea praecox, and Bougainvillea cv., together with the available sequence of Bougainvillea spectabilis, range from 153,966 bp to 154,541 bp. Similar to most sequenced Nyctaginaceae cp genomes, they all exhibit the typical quadripartite structure, consisting of a large single-copy (LSC) region , a small single-copy (SSC) region , and a pair of inverted repeats  , four rRNA , and seven tRNA  genes duplicated in the IR regions. Thus, there are 79 unique protein-coding genes that function primarily in photosynthesis and transcription–translation processes, while the remaining are transfer RNA genes (30), and ribosomal RNA genes (four) (accD) loss in some genera. Bougainvillea cp genomes have an intact accD gene, whereas Nycataginia capitata (MH286318.1) and Pisonia aculeata (MK397866.1) lack the accD gene. In general, the genome structure and features of the Bougainvillea cp genomes are highly similar to most Nyctaginaceae chloroplast genomes—no significant changes in the gene order or gene content have been observed.The assembled chloroplast genome sequences of ,427 bp) , Table 1s (four) . The numrps16, atpF, rpoC1, petB, petD, rpl16, rpl2, ndhB, ndhA, trnI-GAU, trnA-UGC, trnV-UAC, trnL-UAA, trnG-UCC, trnK-UUU) have one intron, while clpP and ycf3 have two introns each (trnK-UUU gene has the longest intron (2508–2524 bp), which encodes the matK ORF. This is normally perceived in published cp genomes, since plastid trn introns are relatively longer in comparison to nuclear tRNA introns [rps12 gene, with the 5′ exon positioned in the LSC region, while the 3′ exons are duplicated in the IR regions. The aforementioned genes with introns can be categorized into three types—genes for electron transfer, protein synthesis, and ATP synthesis. Most introns in general do not code for proteins, but recent studies have shown that they can enhance gene expression and regulation in specific locations [Bougainvillea.In addition, there are also 17 intron-containing genes identified in all six cp genomes, of which 15 genes  are higher compared to the LSC (34.2–34.3%) and SSC regions (29.5–29.6%). Higher GC contents in IR regions are typically linked to the presence of rRNA genes in the IRs or to the GC-biased conversion (gBGC) [Overall, the GC contents of the six n (gBGC) . The gBGn (gBGC) ,26,27.Bougainvillea cp genomes are comprised of protein-coding genes with 24,557–26,717 codons. Of these codons, leucine (10.50–10.64%) and isoleucine (8.67–8.76%) are the most abundant amino acids, whereas cysteine (1.10–1.15%) has the lowest frequency  and methionine (AUG) (RSCU = 1). There are 30 codons that are highly favored (RSCU > 1) and 32 codons that are less preferred (RSCU < 1). Moreover, it can be observed that out of the 30 preferred codons, 29 are A/U-ending codons, meaning that C/G-ending codons are less common in the chloroplast genomes. Several reported cp genomes from other families such as Zingiberaceae, Euphorbiaceae, and Asparagaceae have constantly reported the same occurrence [In addition, codon usage analysis also revealed that there are particular amino acid codons that are more frequently used or preferred . This cocurrence ,34,35. TBougainvillea cp genomes were predicted using the Predictive RNA Editor for Plants (PREP) software, which identified a total of 42 RNA editing sites. Parallel to other seed plants, the annotated RNA editing sites in the Bougainvillea cp genomes were C to U conversions located in the first or second position of the codons  of the cp genomes, particularly in ndhB (12 sites) [Nucleotide sequences in cpDNAs are commonly altered at the transcript level through RNA editing and RNA splicing. Thus, determining the RNA editing sites is necessary in order to understand coding information in the cp genomes. Putative RNA editing sites in the six e codons . C to U 2 sites) ,37. In a2 sites) ,38. RNA 2 sites) ,38.Bougainvillea glabra, Bougainvillea peruviana, Bougainvillea pachyphylla, Bougainvillea praecox, Bougainvillea cv., and Bougainvillea spectabilis, respectively  in ectively A–C. Boug genomes D. TrinucC region A,B. The ultivars .Bougainvillea glabra (26) has the highest number of identified tandem repeats, whereas Bougainvillea peruviana (17) and Bougainvillea pachyphylla (19) have the lowest. The remaining three  have the same number of tandem repeats. Similar to SSR, TRs in the six cp genomes are mostly distributed in the non-coding areas of the LSC region  were also determined in the six cp genomes using Tandem Repeats Finder v4.04. C region A,B. The C region C. All TR changes . TR muta changes .Bougainvillea cp genomes, the gene contents and arrangements are highly similar, however there are few contractions and expansions in the IR boundaries , hence producing long fragments of ycf1 in the IRb–SSC junction (JSB). It is also evident that the partial copies of ycf1 in the IRb regions of all cp genomes overlap with the ndhF gene. However, in five Bougainvillea cp genomes , the ycf1 fragments have two bp extension in the IRb–SSC junction (JSB), suggesting an infinitesimal expansion of the IR. This IR contraction and expansion pattern is usually observed in most angiosperms [Inverted repeat regions are generally conserved among land plants. They all contain four rRNA, five tRNA genes, and a few protein-coding genes as a result of some expansion and contraction in the IR junctions . Sequencundaries . For insiosperms ,44,45. ABougainvillea cp genomes was compared through multiple sequence alignment carried out in mVISTA. Generally, no significant rearrangements were observed among Bougainvillea cp genomes, but several regions displayed higher variation than others. Resulting alignment analysis using Bougainvillea glabra as a reference showed that the coding regions of the Bougainvillea cp genomes are less divergent compared to the non-coding regions, whereas the non-coding regions are more variable than the coding regions  values were calculated using DnaSP v5.10  positioned at the single copy regions exhibit higher Pi values (>0.005)  have high diversity values (>0.012) (To further examine the sequence divergence in the SP v5.10 . Similar(>0.005) A. On the(>0.012) B. Most oBougainvillea sequences was analyzed through mVISTA and DnaSP v5.10, however to further elucidate the differences among taxa, SNP/indel analysis was also conducted using MUMmer 4 and Geneious 2020.2. Using Bougainvillea glabra as a reference, the single-nucleotide polymorphisms (SNPs) and indels (insertions deletions) were identified in the other five Bougainvillea cp genomes. General results revealed that more SNPs and indels were detected in B. pachyphylla and B. peruviana in contrast to the other three plastomes. When aligned to B. glabra, B. pachyphylla has 571 SNPs in the non-coding regions and 317 SNPs in the coding regions, whilst B. peruviana has 545 SNPs in the non-coding regions and 320 SNPs coding regions , B. spectabilis , and B. cultivar , indicating that these three have higher sequence similarities to B. glabra , particularly rpoC1 and rpoC2, predominantly contain synonymous and non-synonymous SNPs , and B. pachyphylla (357), which have more indels in comparison to B. praecox (218), B. spectabilis (171), and B. cultivar (171) , B. cultivar (55 bp), B. pachyphylla (47 bp), and B. peruviana (29 bp) mainly differentiates these four from B. glabra  are discovered in B. peruviana and B. pachyphylla  exhibit a high degree of variation due to the presence of indels.A similar trend can be observed in ar (171) A. Both B. glabra . On the nd rps19 . Althoughyphylla C. The prhyphylla A,C. In ae indels B. Again,ycf1, ndhF, and rpoA are potential molecular markers. Consistent with all analyses, ycf1 has high sequence divergence, high diversity value, and has the most SNPs and indels  and six other species from the Nyctaginaceae family. Species from Petiveriaceae were used as outgroups. In addition, the highly variable regions from the same dataset were extracted and used to construct a phylogenetic tree. As a whole, the resulting ML and BI trees based on complete cp genomes have a consistent and well-supported topology  compared to other tribes within Nyctaginaceae. Within the Bougainvillea genus, Bougainvillea peruviana and Bougainvillea pachyphylla appear to be the basal taxa, while Bougainvillea praecox is a sister to two distinct subclades: the “glabra” and “spectabilis” subclades. Bougainvilleeae, as represented by the B. peruviana was assumed to be closely related to either B. glabra [B. pachyphylla [B. pachyphylla. The sequence variation analysis discussed earlier also showed that B. peruviana and B. pachyphylla are the two genomes that differ most from B. glabra. In terms of morphological structure, B. pachyphylla is not too distinct from B. peruviana—it differs only by having thick and leathery leaves and a densely puberulent perianth in comparison to B. peruviana [B. peruviana is considered one of the most stable species, as there is less variation in the shapes of the bracts and leaves [B. pachyphylla to B. peruviana must be taken into consideration in future taxonomic revisions for the genus Bougainvillea. Based on its morphology, . glabra ,47 or B.hyphylla , but moleruviana ,49. B. pBougainvillea praecox is actually distinct from other Bougainvillea by being sparsely spiny or unarmed [B. praecox is the sister to two distinct groups containing Bougainvillea glabra and Bougainvillea spectabilis.On the other hand,  unarmed . It is nBougainvillea glabra and Bougainvillea spectabilis is not surprising, as morphologically B. glabra is highly similar to B. spectabilis [B. glabra differs only by having puberulent to glabrate branches and leaves, while B. spectabilis has fulvous–villous branches and densely villous abaxial leaf surfaces [B. spectabilis has stouter spines and wavy bracts. The sequences from GenBank are mostly cultivated and are either classified as B. glabra or B. spectabilis. Similarly, the Bougainvillea cultivar clusters together with B. spectabilis and B. glabra, since most of the cultivars are actually crosses between B. spectabilis and B. glabra. Many crosses between the two species have produced new hybrids and horticultural cultivars. As presented earlier, less sequence variation was observed among these three species, so closer relationships are also evident in the resulting trees.The close relationship between ctabilis . B. glabsurfaces ,49. In aBougainvillea  diverged earlier than the commonly cultivated species of Bougainvillea. These results showed that information inferred from highly variable regions and complete cp genome sequences resulted in consistent phylogeny. The potential barcodes can also successfully differentiate Bougainvillea species and cultivars, meaning more samples can be sequenced to give a broader view of the evolutionary relationships within this genus.In general, it can be observed that rarely cultivated and wild species of Bougainvillea species and one cultivar used in this study were collected from Brazil, Peru, Ecuador, and China. Two samples were obtained from Brazil, namely Bougainvillea glabra (M.B.M. da Cruz 0001 NY) from Ilhéus, Bahia; and Bougainvillea praecox (Chen T. 2012063001 SZG) from Jardim Botanico Plantarum, Nova Odes. Bougainvillea pachyphylla  was from Chota, Cajamarca, Peru, while Bougainvillea peruviana  was collected on the way from Loja to Macara, Ecuador. Lastly, the Bougainvillea cultivar (Chen T. 2020031204 SZG), which is originally from India, was acquired from Fairy Lake Botanical Garden, Shenzhen, China. Fresh leaves of B. glabra, B. praecox, B. peruviana, and B. cultivar were dried in silica gel, while the B. pachyphylla leaf sample was obtained from herbarium material. Total genomic DNA was extracted from each sample through the modified CTAB (cetyl trimethylammonium bromide) method [The leaf samples of the four ) method , then thAfter the DNA quality assessment, DNA was sheared to fragments using a Covaris ultrasonic disruptor. Short-insert (350–400 bp) libraries were constructed using the Nextera XT DNA Library Preparation Kit. Sequencing was performed in Illumina Novseq 600 platform, and for each sample around 10.0 Gb of raw data were generated, with an average read length of 150 bp and sequencing depths of 263.8X–1627.3X. The Illumina raw sequence reads were filtered for adaptor sequences, undersized inserts, duplicated reads, and low-quality reads using the NGS-QC  toolkit . High-quBougainvillea cp genomes were annotated using Plann software [Bougainvillea cp genomes were generated using the online program OGDRAW v1.3.1 [The assembled software  and the software  and DOGMsoftware . Proteinsoftware  and tRNAsoftware  were useW v1.3.1 , then thBougainvillea cp genomes were identified using Predictive RNA Editor for Plants (PREP) suite (http://prep.unl.edu/), with a cutoff value of 0.8 [Relative synonymous codon usage (RSCU) was determined for all protein-coding genes using MEGAX software . Amino ae of 0.8 . The defBougainvillea cp genomes were identified using MISA (http://pgrc.ipk-gatersleben.de/misa/) [Simple sequence repeats (SSRs) in the e/misa/) , with moe/misa/)  using deBougainvillea cp genomes were compared to the available cpDNA sequence of Bougainvillea spectabilis. For comparison, sequence alignment was carried out in mVISTA (http://genome.lbl.gov/vista/mvista/about.shtml) using Shuffle-LAGAN mode [Bougainvillea glabra was used as the reference. In addition, the nucleotide diversity (Pi) values of the cp genomes were determined to detect various divergence hotspots. Using MAFFT v7.388 [Bougainvillea cp sequences were aligned, then DnaSP v5.10 [https://irscope.shinyapps.io/irapp/) [Bougainvillea cp genomes, SNPs and indels were also identified and located using MUMmmer 4 [Bougainvillea glabra cp genome was also used as the reference.The five newly sequenced GAN mode . BougainT v7.388 , BougainSP v5.10  was util/irapp/) . To furtUMmmer 4  and GeneUMmmer 4 . The BouBougainvillea cp genomes, three available Bougainvillea sequences and six Nyctaginaceae sequences deposited in NCBI GenBank were included in the dataset. Four complete cp genomes from the closely related family Petiveriaceae were used as outgroups. From these sequences, 79 protein-coding regions were extracted using Geneious Prime 2020.2. Multiple alignments of these extracted regions were conducted in MAFFT v7.388 [ndhF, rpoA, and ycf1 were extracted and concatenated using Geneious Prime 2020.2 [Together with the five newly sequenced T v7.388 . AmbiguoT v7.388 , which ge 2020.2 Maximum likelihood (ML) analyses were performed using RAxML 8.2.11  and IQ-TBougainvillea is deemed to be one of the most popular genera in the Nyctaginaceae family, as it is used mainly as an ornamental plant. However, despite its horticultural value, its molecular and phylogenetic aspects are not well-researched. Hence, in this paper, the complete chloroplast genomes of four species  and one cultivar were sequenced and analyzed. In general, all Bougainvillea cp genomes newly sequenced from GenBank, including Bougainvillea spectabilis, have similar genome structures and features. They all display the typical quadripartite structure and have the same numbers of genes (131) and introns (17). Highly similar patterns were also obtained from codon usage, RNA editing, and repeat analyses. Although the Bougainvillea cp genomes are highly conserved and no rearrangement was observed, several highly divergent regions were identified. Moreover, phylogenetic analyses revealed the early divergence of the rarely cultivated or wild Bougainvillea . B. pachyphylla and B. peruviana were shown to be the basal taxa of Bougainvillea. Close relationships among Bougainvillea glabra, Bougainvillea spectabilis, and Bougainvillea cultivar were also confirmed. These results show that chloroplast genomes can provide sufficient information that can be used in phylogenetic studies. However, more cp genome sequences are needed to further elucidate relationships among Bougainvillea species and cultivars.


Humulus yunnanensis is an endemic species in Yunnan, China, which is used for beer and pharmacology industry. The phylogenetic position of this species in Humulus remains controversial. The complete chloroplast (cp) genome sequence of H. yunnanensis was reported and characterized in this study. The cp genome is 153,612 bp in length and contains a pair of inverted repeats  separated by a large  and small  single-copy regions. A total of 112 unique genes were predicted, including 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. The phylogenetic analysis revealed that H. yunnanensis is more closely related to H. scandens than H. lupulus. Humulus is a small genus of Cannabaceae native to temperate regions of Northern Hemisphere. Three recognized species, H. lupulus, H. scandens, and H. yunnanensis are widely used in brewing, medicinal, and pharmaceutical industries. However, the phylogenetic relationships within Humulus have been questioned  indicated that H. yunnanensis and H. lupulus consisted of the sister group , Boutain  genome of H. yunnanensis using high-throughput sequencing technology. Boutain  validateH. yunnanensis was collected from Kunming . Specimens (14CS9673) were deposited in the herbarium of the Kunming Institute of Botany, CAS (KUN). Total genomic DNA was extracted with a modified CTAB method (Doyle and Doyle The fresh leaf of H. yunnanensis (GenBank accession number: MK423880) is 153,612 bp in length and has a typical quadripartite structure. It comprises a large single-copy region  and a small single-copy region  separated by a pair of inverted repeats . The overall GC content of the cp genome is 36.9%, while that of IRs (42.5%) is higher than that of LSC (34.6%) and SSC (30.8%) regions. The H. yunnanensis cp genome encodes 112 unique genes including 78 protein-coding genes, 30 tRNA, and 4 rRNA genes. There are 19 intron-containing genes, in which 10 protein-coding  and 6 tRNA  genes has a single intron and 3 genes  has two introns.The complete cp genome of H. yunnanensis and two previously released cp genomes of H. scandens and H. lupulus. Eleven species of other five genera in Cannabaceae were used as outgroups. The identical maximum likelihood (ML) and Bayesian phylogenetic trees indicated that three species from the genus Humulus formed a monophyletic clade with 100% bootstrap and 1.0 posterior probability support, respectively (H. yunnanensis was supported more closely related to H. scandens than H. lupulus (In this study, the phylogenetic analysis was performed with the newly sequenced cp genome of ectively . Moreove lupulus .


Cheirotonus gestroi is an endangered large insect in southwestern China and neighboring countries. Herein, we present the first complete mitochondrial genome of C. gestroi. The 16,899 bp long circular genome consists of 2 ribosomal RNA genes, 22 transfer RNA genes, 13 protein-coding genes, and a non-coding control region. Phylogenetic analysis showed that C. gestroi shared the closest evolutionary relationship with C. jansoni and that Scarabaeidae and Lucanidae were correctly identified within superfamily Scarabaeoidea. The complete mitogenome sequence will provide a basis for further phylogenetic studies and conservation genetics of the genus Cheirotonus.The long-armed scarab  Cheirotonus gestroi (Coleoptera: Euchiridae), is an endangered large insect, mainly living in the mountainous areas of southwestern China and neighboring countries , which make them the top list of insect fans’ collections. In last few decades, the population size of C. gestroi has substantially decreased due to illegal collections and habitats fragmentation. The species has been listed as the State Second-Class Protected Animal in China since 1989 (http://www.forestry.gov.cn/main/3951/20180104/1063898.html). Mitogenomics play important roles in conservation biology   as the reference genome , Yunnan, China in June 2018 and deposited in the herbarium of Southwest Forestry University (specimen code: BJG_HL2018_032). Genomic DNA was extracted from the muscles of a single adult’s chest using the QIAamp DNA Micro Kit  and then sequenced using Illumina HiSeq X10 . Genes were assembled by the Assembly by Reduced Complexity (ARC) pipeline . The base composition is 38.8% for A, 31.9% for T, 18.5% for C and 10.7% for G. The genome contains 37 genes, including 13 PCGs, 22 tRNAs, two rRNAs, and a non-coding control region (D-loop). J-strand (majority strand) encodes nine PCGs , while N-strand (minority strand) encodes four PCGs . The gene arrangement of C. gestroi mitogenome is identical to the most insect mitogenomes  were used to construct the maximum likelihood (ML) tree. The ML inference was performed using IQ-TREE (Nguyen et al. C. gestroi has the closest evolutionary relationship with the same genus species C. jansoni. Furthermore, two clades of Scarabaeidae and Lucanidae were correctly identified with high bootstrap values (C. gestroi present in this study will be beneficial to the further phylogenetic studies of Scarabaeidae and the conservation genetics of the genus Cheirotonus species.In order to validate the phylogenetic position of p values . The mit


Melilotus albus is an annual or biennial legume species that adapts to extreme environments via its high stress tolerance. NAC and MYB transcription factors (TFs) are involved in the regulation of lignin biosynthesis, which has not been studied in M. albus. A total of 101 MaNAC and 299 MaMYB members were identified based on M. albus genome. Chromosome distribution and synteny analysis indicated that some genes underwent tandem duplication. Ka/Ks analysis suggested that MaNACs and MaMYBs underwent strong purifying selection. Stress-, hormone- and development-related cis-elements and MYB-binding sites were identified in the promoter regions of MaNACs and MaMYBs. Five MaNACs, two MaMYBs and ten lignin biosynthesis genes were identified as presenting coexpression relationships according to weighted gene coexpression network analysis (WGCNA). Eleven and thirteen candidate MaNAC and MaMYB genes related to lignin biosynthesis were identified, respectively, and a network comprising these genes was constructed which further confirmed the MaNAC and MaMYB relationship. These candidate genes had conserved gene structures and motifs and were highly expressed in the stems and roots, and qRT-PCR further verified the expression patterns. Overall, our results provide a reference for determining the precise role of NAC and MYB genes in M. albus and may facilitate efforts to breed low-lignin-content forage cultivars in the future. Arabidopsis transcription activation factor (ATAF1/2), and cup-shaped cotyledon (CUC2)) family members are plant-specific TFs. NAC TFs have a highly conserved N-terminal region and a variable C-terminal region; the N-terminal regions share a conserved DNA-binding region, whereas the C-terminal regions have various transcriptional regulatory domains [Transcription factors (TFs) play an important role in plant growth and development. NAC , ctor ATAF/2, and c domains . In addi domains ,5. These domains . ATAF1,  domains .Arabidopsis. AtMYB5 and AtMYB23 control trichome extension and branching [Arabidopsis enhanced drought resistance, with reduced lateral roots [The MYB gene family is one of the largest TF families. MYB proteins are characterized by a highly conserved DNA-binding domain that generally consists of up to four imperfect amino acid sequence repeats (R) of 50–53 amino acids, each encoding three α-helices . The secranching , and AtMranching . AtMYB33ranching . MYB96 mal roots .NAC and MYB gene regulatory network [Lignin is composed of phenylpropanoid-derived polymers and exists in the secondary cell walls of all vascular plants . Lignin  network . In the  network ,18,19,20 network . Further network . These p network .Melilotus spp., which comprises 19 species, are important annual or biennial legume crop species [Melilotus genus are highly stress tolerant and are adapted to extreme environments. Compared with traditional legume crop species, Melilotus species can grow in moderately saline soils while maintaining normal seed yields. Melilotus species are usually used as green manure crops, as a crop fertilizer (with a relatively high nitrogen fixation rate) and as a nectar source [Melilotus are significantly larger than those of Medicago sativa, and Melilotus produces yields of up to 640 kg of dry matter per mu, which is much greater than that produced by two-year-old M. sativa [Melilotus is greater than that of M. sativa. Lignin is the key element that limits cell wall digestibility and limits the digestible energy available to ruminants [Melilotus is widely distributed in tropical regions throughout both Asia and Europe. At present, the genetic diversity and population structure of Melilotus have been studied [Melilotus genus have been identified [Melilotus species have been constructed [Melilotus albus has been evaluated [M. albus have been analyzed [ species . The memr source . In addi. sativa . Howeveruminants . Melilot studied , interspentified ,29, the structed , the genvaluated , and theanalyzed ,33. HoweM. albus. A total of 101 MaNAC TFs and 299 MaMYB TFs were identified, and the gene structure, phylogenetics, promoter region, evolution and coexpression relationships were analyzed. Eleven and thirteen candidate MaNAC and MaMYB genes, respectively, that may regulate lignin biosynthesis were identified, and their networks were constructed. This genome-wide analysis of the MaNAC and MaMYB TF families might contribute to the future functional characterization of M. albus and lay a foundation for future studies on lignin biosynthesis. This research may provide a reference for molecular breeding to develop low-lignin-content forage cultivars of M. albus.In this study, we conducted a genome-wide analysis of the NAC and MYB TF families in M. albus CDSs and protein sequences predicted approximately 400 sequences that contained Myb or Myb-like repeats. The domains of candidate MaMYB proteins were further examined via Pfam analysis. A total of 299 nonredundant MaMYB genes were identified and used for further analysis. The MaMYB genes were classified into three distinct subfamilies: R1-MYB genes (175), R2R3-MYB genes (123), and R1R2R3-MYB genes based on the number of Myb repeats  and protein sequences against the  repeats . Using s domains .MaNACs and 287 MaMYBs were mapped onto eight chromosomes  and 15 (14.85%), respectively, were the result of tandem duplications  phylogenetic tree was constructed , M. truncatula (185) and Arabidopsis (197) MYB family, the M. albus (299) MYB family is substantially larger. M. albus, rice, M. truncatula and Arabidopsis had independent clades, and clades that included only M. albus MYB members were more abundant than those that included the other three species  classification of MaNAC and MaMYB members was performed . The NACVND genes, one NST gene and four SND genes in M. albus  CDSs and protein sequences as queries, we identified six M. albus a. We subMEGA 7.0 a. The 24subgroup a. The sisubgroup . With thsubgroup b. Proteisubgroup c. All prsubgroup c. The NAsubgroup d. All amArabidopsis  regulatentified . On the entified a. The geMaMYB118 b. ExceptMaMYB118 c. The amM. albus, we analyzed RNA-seq data from different tissues. A total of 63 MaNAC genes and 185 MaMYBs were expressed in the roots, stems, flowers and seeds  [MaNACs, 34 MaMYBs, and 53 lignin biosynthesis-related genes were identified  ,37,38,39entified , which eentified a. The coentified a. MaNAC entified a. In parentified a.AtMYB58, AtMYB63, and AtMYB103 are direct targets of AtMYB46/83 [AtMYB20, AtMYB42, AtMYB43, and AtMYB85 activate AtMYB4, AtMYB7, and AtMYB32 and directly activate lignin biosynthesis genes during secondary wall formation in Arabidopsis [AtMYB58, AtMYB63, and AtMYB103 regulate lignin biosynthesis genes [AtMYB4, AtMYB32, and AtMYB7 negatively regulate lignin biosynthesis genes [MaNAC and MaMYB coregulation network (MaMYB234 was opposite that of MaNAC43 and MaNAC8 (VND) and MaMYB29. The expression pattern of MaMYB234 was similar to that of MaNAC78 and MaNAC32, MaMYB118, MaMYB235 and MaMYB249 and MaMYB266  were highly expressed in the roots. To further validate the reliability of gene expression data obtained by RNA-Seq analysis, four lignin biosynthesis-related NAC and MYB genes each carried out qRT–PCR analysis in five tissues  and the second layer of master switch genes (MYB46 and MYB83) regulate downstream genes to control lignin biosynthesis during secondary wall formation . MYB46 aMYB46/83 . AtMYB20bidopsis . AtMYB58is genes ,21,44, ais genes . On the  network b. The exMaMYB266 b. In the tissues . NAC andGlycine max, 123 and 185 in M. truncatula, 116 and 126 in Lotus japonicus, and 96 and 166 in Cicer arietinum . In the present study, based on whole-genome data of the M. albus, 101 MaNAC and 299 MaMYB genes were identified. Because G. max underwent independent whole-genome duplication, there are substantially more of these family members in this species than other legume species. The number of NACs is similar between M. albus and both L. japonicus and C. arietinum, but there are substantially more MYBs in M. albus than in M. truncatula, L. japonicus and C. arietinum. M. albus can adapt to drought, cold and high-salinity environments [M. albus. The phylogenetic tree M. albus, M. truncatula, rice and Arabidopsis showed that most clades contained NAC or MYB members of all three species, but some clades contained members of only one species. In addition, the intron/exon and motif structures of NAC and MYB genes were similar in one clade. Genes in the same clade may share the same ancestor before species divergence, and genes in independent clades with genes from only one species may have formed after species divergence.NACs and MYBs compose two large TF families. As an increasing number of genomes of legume species are sequenced, additional NAC and MYB TF family members are being identified. For instance, there are, respectively, 269 and 438 NACs and MYBs in ronments , and theMaNACs and MaMYBs contain stress-, development- and hormone-responsive cis-elements. Some of the gene promoter regions contain ARE cis-elements (>80%) and LTR cis-elements (>37%), which are involved in stress responses [MaNACs and MaMYBs are strongly associated with gene function. Furthermore, GO annotation of the MaNACs and MaMYBs indicated that they are associated with stimulus response and development, and tissue-specific expression of the MaNAC and MaMYB genes indicated that these genes may regulate organ development. Furthermore, 23 MaMYB genes were expressed only in the flowers, suggesting that they may regulate M. albus floral development. Therefore, MaNAC and MaMYB members may be involved in stress, development and hormone responses.Previous studies have shown that NAC TFs regulate cell division  and flowesponses . CAT-boxesponses . The varMaNACs and MaMYBs contained MBS and MBSI cis-elements. Moreover, WGCNA indicated that MaNAC and MaMYB members presented coexpression relationships. Our results are consistent with those of previous studies, which showed that NAC members regulate MYB members. On the basis of the relationship between MYBs and NACs, and MYBs in Arabidopsis, a network comprising MaNACs and MaMYBs with respect to lignin biosynthesis in M. albus was constructed which further confirmed the MaNAC and MaMYB relationship. Stems and roots are highly lignified, and our sequencing results are consistent with this fact. Comparing gene expression levels in different tissues, we identified genes that are highly expressed in the stems and roots, as well as flower-specific genes. Furthermore, the results of qRT-PCR also showed higher relative expression level in stems and roots. VND and NST subfamily members may function redundantly [Arabidopsis, only six VNDs and one NST were identified in M. albus. Two SND2s, two SND3s, two MYB83s, two MYB20s, and two MYB103s were identified that had expanded in M. albus compared with Arabidopsis. Except for MaMYB284, all these genes were expressed in different tissues. These expanded genes may increase M. albus lignin biosynthesis, as mature M. albus plants have a greater degree of lignification than mature Arabidopsis plants.Some NAC members target MYB members that are involved in lignin biosynthesis . Analysiundantly , compareArabidopsis, Oryza sativa and Medicago truncatula NAC and MYB TFs were downloaded from the Plant Transcription Factor Database . Similarly, gene sequences and coding sequences (CDSs) were downloaded from the Phytozome 12  database  with default parameters. NAC candidate sequences without an NAM domain and MYB candidate sequences without a Myb domain were discarded manually. Gene, coding, and protein sequences of NAC and MYB family members are deposited in NCBI.The protein sequences of database . Local BArabidopsis lignin biosynthesis-related NAC and MYB genes were retrieved from previous studies and used as queries to identify the corresponding M. albus genes. Local BLAST searches were performed: an e-value cut-off of 1e−100 and 1e−70 was applied to NAC and MYB homologous sequence recognition, respectively  (with the default parameters) to identify conserved motifs. To examine the conserved domains of M. albus in detail, multiple amino acid sequence alignment was performed via DNAMAN 6.0 software. The multiple alignment sequences were submitted to WebLogo  to create sequence logos of NAM and R2 and R3 Myb repeats. Furthermore, the CDSs of NAC and MYB were compared with their corresponding genomic sequences to determine all gene structures, which were visualized using the Gene Structure Display Server  with default parameters.Each rameters . The proArabidopsis, O. sativa and M. albus were aligned via the Muscle method, which is the MEGA 7.0 built-in method. Phylogenetic trees of NAC and MYB family members were then constructed by MEGA 7.0 [The NAC and MYB protein sequences of MEGA 7.0  with theMaNAC and MaMYB genes was extracted from the M. albus genome to identify the cis-elements within the promoter regions. The plant Cis-Acting Regulatory Element  database was used to identify cis-elements within the promoter regions of each gene (with the default parameters), and the function of each cis-element was annotated.The 2000-bp upstream of the transcriptional start site of −10), and the best matched gene pairs were defined as homologous pairs. The synonymous substitution rates (Ks) and nonsynonymous substitution rates (Ka) of the gene pairs were calculated by TBtools [Genes within families were aligned with one another by the use of BLAST  and provided a 16 h light/8 h dark photoperiod. During the inflorescence stage, tissue samples of fresh stems, flowers and roots were collected, immediately frozen in liquid nitrogen and then stored at −80 °C. Seed samples were mixed together, imbibed and then allowed to germinate for 2 days. Each sample was a composite of independent biological replicates.® Ultra™ Directional RNA Library Prep Kit for Illumina®  according to the manufacturer’s recommendations. The qualified cDNA libraries were constructed by PCR enrichment and sequenced on a HiSeq 2500 platform, with a read length of 125 bp. Three independent biological replicates were sequenced per sample. The RNA sequencing (RNA-seq) data of leaf samples from 5 different genotypes from a previous study were also included [Total RNA from each sample was isolated by the use of TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Messenger RNAs (mRNAs) were separated from the total RNA by oligo (dT) primers and were cleaved into short fragments at random. Sequencing libraries were generated via rRNA-depleted RNA with a NEBNextincluded .M. albus genome by HISAT2. StringTie 1.3.1 [Clean reads were obtained by removing reads containing adapters, reads containing poly-N sequences and reads of low quality from the raw reads. The clean reads were mapped to the ie 1.3.1  was usedAll gene expression levels were used to calculate the weight of gene pairs via WGCNA. The relationships of the MYB and NAC gene pairs were visualized by Cytoscape 3.5.1, and gene functions were annotated via the Gene Ontology (GO) database.® RT reagent Kit  was used to reverse transcribe RNA into first-stand cDNA. The product was used as a template for qRT-PCR with specific primers (PrimeScript primers  which weM. albus. Phylogenetic and evolutionary relationships, gene structure analysis and motif analysis indicated the conservation of MaNAC and MaMYB families and that some members diverged from their ancestors. MaNAC and MaMYB members experienced purifying selection and some of the members were functionally redundant. Promoter region analysis and GO functional annotation of MaNAC and MaMYB TFs suggested that they may be involved in stress, development and hormone responses. WGCNA revealed that MaNAC, MaMYB and lignin biosynthesis-related genes presented coexpression relationships, and a regulatory network comprising some of these genes in M. albus further confirmed their relationship. Gene expression levels in different tissues were compared, some of the MaNAC and MaMYB members presented tissue-specific expression, lignin biosynthesis-related MaNACs and MaMYBs were highly expressed in stem and root, and the relative expression of several members was also high in stem and root, which further confirmed that these candidate genes may regulated lignin biosynthesis. These results provide a useful framework for future research to understand the evolution and function of the MaNAC and MaMYB gene families and to understand the potential physiological role of the MaNAC and MaMYB genes during lignin biosynthesis. Since no MaNAC and MaMYB genes have been functionally characterized to date, our results may facilitate future efforts to breed forage cultivars with low amounts of lignin.In summary, a total of 101 MaNAC and 299 MaMYB family members were identified in


Aspergillus display a variety of lifestyles, ranging from saprobic to pathogenic on plants and/or animals. Increased genome sequencing of economically important members of the genus permits effective use of “-omics” comparisons between closely related species and strains to identify candidate genes that may contribute to phenotypes of interest, especially relating to pathogenicity. Protein-coding genes were predicted from 216 genomes of 12 Aspergillus species, and the frequencies of various structural aspects  and functional annotations  were compared.Members of the genus Aspergillus flavus. In contrast, Aspergillus fumigatus strains, which are pathogenic to animals but not plants, were assigned relatively more terms related to phosphate transferases, and carbohydrate and amino-sugar metabolism. Analyses of publicly available RNA-Seq data indicated that one A. fumigatus protein among 17 amino-sugar processing candidates, a hexokinase, was up-regulated during co-culturing with human immune system cells.Using principal component analyses, the three sets of functional annotations for each strain were clustered by species. The species clusters appeared to separate by pathogenicity on plants along the first dimensions, which accounted for over 20% of the variance. More annotations for genes encoding pectinases and secondary metabolite biosynthetic enzymes were assigned to phytopathogenic strains from species such as Aspergillus pathogenicity factors.Genes encoding hexokinases and other proteins of interest may be subject to future manipulations to further refine understanding of The online version contains supplementary material available at 10.1186/s12866-020-02031-y. Aspergillus provides an intriguing model for studying differentiation among opportunistic pathogens  and saprobic decomposers. All members of this genus live largely as saprobes. However, several species are able to cause rots on living plant tissues and/or invasive aspergillosis in immunocompromised mammals [A. fumigatus; less common Aspergillus causative agents are A. fischeri, A. flavus, A. nidulans, A. niger and A. terreus [Aspergillus species is complex and requires the normally saprotrophic Aspergillus spp. to adapt to the environment of the human lung [Aspergillus infections cost the United States an estimated 1.2 billion dollars annually [Aspergillus species, A. sydowii, is salt-tolerant pathogen of coral and humans that may become more destructive with continued global warming, along with other pathogenic species [Only a minority of fungi are pathogenic, mostly on plants, while the majority of fungal species are saprobes or mutualists . Numerou mammals –5. Syste mammals –8. Invas terreus . Human pman lung . Hospitaannually . One Asp species .Aspergillus species are also common after drought, insect damage or other environmental stresses. In particular, infection by A. flavus and A. parasiticus strains cause large economic losses in agriculture due to associated contamination with mycotoxins, notably aflatoxins. Aflatoxin contamination alone costs the United States 50 million to 1.7 billion dollars each year [A. flavus is not only the primary causative agent of Aspergillus infections and aflatoxin contamination in crops, it is simultaneously the second most common cause of aspergillosis in human patients. In contrast, while A. fumigatus is the most common cause of human and veterinary aspergillosis, it is not known to cause disease in any plant host [Aspergillus pathogens can be grouped by those that may infect both plants and animals (phytopathogenic), and those that are not known to cause diseases in plants (non-phytopathogenic). This distinction is not sufficiently explored in published research to date.Opportunistic plant infections by ach year . A. flavant host –16. EsseDifferent infection strategies are required to cause disease in different hosts; thus, pathogenic species and strains must have genes that enable disease-causing infections and suppress resistance responses. The expression of relevant genes may be influenced by environmental conditions such as nutrient composition and response to host defenses . MammaliA. flavus and A. fumigatus, were used to address the discrepancy between the host ranges of phytopathogenic and non-phytopathogenic Aspergillus species. The recent substantial increase in genomic sequencing of filamentous fungi permits more focused comparisons among strains between and within species [In this study, multiple comparisons of genome annotations, largely represented by  species . Both stAspergillus genus. The identified gene candidates are corroborated by reports on the effects of Aspergillus deletion mutants on pathogenicity or virulence, and by published transcriptomic data. To the authors’ knowledge, this the first report of a method to search for genes contributing to a phenotype of interest by gene annotation frequencies across a genus. The long-term objective of our research is to develop a pipeline for extracting new, mycologically-relevant information from the wealth of genomics data stored in public databases. These bioinformatics findings will guide hypotheses that can be tested later via methods such as gene deletion. Our immediate goals were to 1) conduct a comparative analysis of annotations for 216 Aspergillus genomes, 2) identify previously-unidentified pathogenicity factors and 3) use previously-reported information to determine if the candidate pathogenicity factors are known to affect virulence or change expression level during infection processes.The present study demonstrates the utility of this approach by looking for new pathogenicity genes within the Aspergillus genomes were initially included in the study . By t-test, there were no statistically significant differences between the species classified as phytopathogenic or not. Only A. terreus strains had significantly different exonic and gene GC content compared to all other species. Some codon bias in favor of AAG for lysine and GAG for glutamate instead of equivalent codons was observed  predicted secondary metabolite clusters  results explained 73.3% of the variance among structural annotations and 22.3 to 38.7% of the variances of relative frequencies of functional annotations. Both structural and functional annotations yielded grouping of strains that largely clustered with other members of the same species Fig. . There w00% Fig. . The incAspergillus genes in the PHI-base database. Limiting the search to only functionally annotated PHI-base genes that non-lethally affect pathogenicity and/or virulence of Aspergillus when mutated yielded 32 IPR, 20 GO terms and 52 KEGG DAAs  were defined as annotations with significantly different relative frequencies between phytopathogenic and non-phytopathogenic strains within a set of InterPro (IPR), Gene Ontology (GO) or Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation terms. Most 80.8 to 91.5%) of the AUGUSTUS-predicted genes in each strain were assigned at least one functional annotation term  and histidine metabolism (KO00340) were enriched in the phytopathogenic strains. Further, A. flavus, A. oryzae, A. parasiticus, A. sojae and A. tamarii had enrichment of a glycosaminoglycan degradation term (KO00531).DAA GO and KEGG terms related to peptidoglycan metabolic processes (GO:0009254), glycosaminoglycan metabolic processes (GO:0030203), carbohydrate metabolic processes (GO:0005975), peptidoglycan turnover (GO:0009254), amino sugar and nucleotide sugar metabolism (KO00520), and galactose metabolism (KO00052) were commonly enriched in the non-phytopathogenic strains A. flavus NRRL 3357 and A. fumigatus Af293. The strains had 226 and 131 proteins, respectively, back-matched from the DAAs. Sub-proteomic enrichment analyses identified many enriched GO and KEGG terms , transferase activity , 121 polyketide and secondary metabolite synthesis terms , carbohydrate metabolism , phosphorylation , nucleotide binding , oxidation-reduction , steroid biosynthesis  and chromatin structure (K14440).The major difference between the phytopathogens and non-phytopathogens was the number of translated amino acid sequences annotated to a broad category of amino and/or sugar (amino-sugar) metabolism and modification terms. This trend was also observed when comparing the enrichments between the full proteomes and sub-proteomes of rms Fig. . A. flavA. flavus NRRL 3357 and 17 A. fumigatus Af293 AUGUSTUS-predicted protein-coding genes. These predicted A. flavus and A. fumigatus genes were then matched to 9 and 17 NCBI-curated proteins of A. flavus NRRL 3357  and A. fumigatus Af293 , respectively.Annotation terms related to amino-sugar metabolism IPR037950, GO:0000270, GO:0009254, GO:0030203, K00844, K12407, K13748, K19223 and K21471 were assigned to 12 A. fumigatus genome were low, probably reflecting lower fungus to human ratios in the co-cultures compared to the experiments performed to yield the reads for the PRJEB1583 dataset  up-regulated when A. fumigatus was co-cultured with human dendritic cells for 4 h compared to incubation alone . Genes encoding these proteins were not in the PHI-base.The percentages of sequencing reads from NCBI SRA accession PRJNA560197 that mapped to the Aspergillus genus that may contribute to pathogenicity. A comparative protein annotation approach was employed in which differential frequencies of annotation assignments were used to find predicted annotations that may be over or underrepresented (DAAs) within a set of genomes. The structural annotations did not yield a meaningful distinction between the phytopathogenic and non-phytopathogenic Aspergillus strains, though the larger number of secondary metabolite gene clusters in the phytopathogenic species tracked with the fact that fungal plant pathogens have larger secretomes than non-phytopathogens [A. fischeri, A. fumigatus and A. sydowii. Six of the A. fumigatus predicted genes retrieved from PHI-base were predicted to be involved in sugar metabolism, being annotated as glycosyl transferases, glycoside hydrolases, glucan synthases or mannosidases. Five of these genes cause loss of virulence or death when mutated: FKS1 (PHI-base accession PHI2533), AGS1 (PHI3902), AGS2 (PHI3903), AGS3 (PHI3904) and GEL2 (PHI434) [tslA , increases virulence after mutation [A. flavus PHI-base gene  was a pectinase [The aim of this study was to identify candidate protein-coding genes within the athogens . In cont(PHI434) –29. One mutation . The oneectinase .A. fumigatus, which might be indicated by differential expression during infection and disease progression in human hosts compared to growth in single-species cultures. Briefly, invading A. fumigatus is subject to phagocytosis by macrophages mediated by antigen-presenting dendritic cells. Analyses of publicly available RNA-Seq datasets indicated that one A. fumigatus Af293 hexokinase gene encoding the protein XP_749720.1 out of 17 observed candidates has time-dependent increased expression when the fungus is incubated with human immune cells. This gene is not known to have been previously studied as a pathogenicity factor. Its nucleotide sequence did not match  any human genes in the NCBI database, indicating that human RNA sequencing reads extracted from co-cultures should not substantially map to this hexokinase gene in the A. fumigatus genome. If the hexokinase is a pathogenicity factor and has a sufficiently different structure from human proteins, it may be useful as a target for inhibitory drugs to treat aspergillosis.The agreement among predicted overrepresented annotations led to the hypothesis that the identified genes play important roles in the pathogenicity of A. fumigatus beyond energy production and storage, it can be hypothesized that the genes are involved in fungal pathogen signaling on the cell wall. Pathogenic fungi interact with host immune systems via chemical signatures called pattern-associated molecular patterns (PAMPs) and host pattern recognition receptors. Fungal PAMPs include complex carbohydrates in the cell walls which bind Toll-like receptors and C-type lectin receptors found on animal mononuclear phagocytes [While it is not immediately clear what roles sugar-metabolizing genes may play in aspergillosis caused by agocytes –36. Thisagocytes .N-acetylglucosamine synthesis and/or breakdown. This amino-sugar has structural and functional roles in fungal cell walls, and in cell signaling for expression of virulence genes of Candida albicans and pseudohyphal morphogenesis of Candida and Yarrowia species [N-acetylglucosamine metabolism in A. fumigatus and Candida albicans [A. fumigatus [A. fumigatus conidial surfaces have 3 to 20 times more sialic acids than the less virulent or non-pathogenic Aspergillus species A. auricomus, A. ornatus and A. wentii [A. fumigatus candidate pathogenicity factors identified here could be involved in the synthesis of those macromolecules. Fungal carbohydrates and bacterial peptidoglycans additionally induce plant innate immunity [, the overrepresentation of amino-sugar processing genes in A. fumigatus compared to A. flavus may suggest that A. fumigatus PAMPs are modified in unique, species-specific ways to be misrecognized or induce improper responses by immunocompromised animal hosts and/or are easily recognized by plant hosts. Further experimental study is required to properly test the above hypotheses and to understand the differences among the strains used here.The higher number of genes functioning in amino-sugar processing could also be related to  species . Hexokinalbicans –42. Therumigatus , 43–49. . wentii . Perhapsimmunity –53. AltoA. fumigatus, A. nidulans and A. sydowii can be endophytes [A. fumigatus has genes encoding cellulases, hemicellulases and pectinases, but no gene sets uniquely shared with non-Aspergillus human fungal pathogens [A. terreus was found to have a lower number of proteins annotated as pectinase-like proteins compared to other phytopathogenic species, which may reflect that A. terreus infects leaves where pectin levels are lower compared to pectin levels in fruits and seeds [A. nidulans had fewer amino-sugar metabolism genes than the other non-phytopathogenic species. A. nidulans, compared to A. fumigatus, induces a weaker oxidative burst by human immune cells and is phagocytosed at a slower rate by rodent macrophages [While the authors are unaware of strain idiosyncrasies that may result in one strain being better classified as plant pathogenic or not, the strains had noticeable differences in genome characterizations. The genus itself is not amenable to neat categorization between phytopathogenic and non-phytopathogenic species. Differences in strain virulence and/or host preference may be implied by isolation source . Aspergillus species were classified according to reported ability to cause disease and persistent rot in live plants in environmental settings. Therefore, A. flavus, A. niger, A. parasiticus, A. tamarii, A. terreus and A. tubingensis were labeled as phytopathogenic and were compared to the non-phytopathogenic species A. fischeri, A. fumigatus, A. nidulans and A. sydowii. A. oryzae and A. sojae comprise domesticated strains of A. flavus and A. parasiticus, respectively, and may not reflect natural loss of phytopathogenicity [Genomic sequences were retrieved from the NCBI Genome database . Only spgenicity –65. Thesgenicity .A. nidulans for all Aspergillus strains [− 50) IPR, GO and KEGG annotation term were counted. Frequencies of annotations for each strain were normalized as percentages of total predicted gene count for the strain. The 211 Aspergillus genes present in PHI-base version 4.9 were uploaded after being experimentally studied as pathogenicity or virulence factors by other researchers [A. flavus, 189 from A. fumigatus and 4 from A. nidulans. These genes were retrieved and subjected to annotation by InterProScan and KofamScan.Gene predictions were performed on both DNA strands using AUGUSTUS version 3.0 trained on  strains . The res strains , 69, andearchers . The PHIt-tests. Statistical tests were performed with counts of assigned annotations normalized as percentages of total annotations for a strain. Figures were generated using R packages ape, DECIPHER, dplyr, factoextra, FactoMineR, ggdendro and ggplot2 [t-tests, relative differences of at least 0.01% with Benjamini-Hochberg corrected p-values < 1− 4  were considered significant. Excluding the non-hierarchical IPR terms, GOATOOLS and Fisher’s exact test  were used to identify enriched GO and KEGG terms compared to the full list of proteomic annotations [Two-way analysis of variance (ANOVA) was used to determine if species identity was a factor in multiple genomic structural aspects. DAAs were identified using PCA and independent  ggplot2 –78. For otations . DAAs beotations .A. fumigatus incubated alone or with human dendritic cells , or with macrophage-like cells . The sequencing reads were processed as previously described with slight modifications [A. fumigatus Af293 genome (NCBI GenBank assembly GCA_000002655.1) guided by the respective GFF3 file [A. fumigatus Af293 proteins with enriched DAAs of interest were matched to accessions in the NCBI Protein and Nucleotide databases with a maximum E-value of 1− 100 [A. fumigatus spores incubated alone or co-cultured with human dendritic cells (5:1 spores:human cells) in complete RPMI 1640 medium at 37 °C for 4 h , and A. fumigatus cultured with a human leukemia cell line differentiated into macrophage-like cells (2:1 spores:human cells) for 0, 1 or 2 h at 37 °C in a modified RPMI 1640 medium . For the PRJEB1583 experiment, t-tests (α-level = 1− 3) were performed to compare gene expression quantified as transcripts per million reads.RNA-Seq reads were retrieved from the NCBI SRA database project accessions PRJEB1583 and PRJNA560197 , 81. TheFF3 file . UtiliziAdditional file 1: Table S1. Strains included in the present study.Additional file 2: Figure S1. Structural aspects of predicted Aspergillus genes. a. Number of genes; b. average exon count per gene; c. average intron count per gene; d. average gene length; e. average exon length per gene; f. average intron length per gene; g. average gene GC content; h. average exon GC content; i. average intron GC content.Additional file 3: Figure S2. Amino acid usage among predicted Aspergillus genes.Additional file 4: Figure S3. Codon usage among predicted Aspergillus genes.Additional file 5: Table S2. Overview of functional annotation of Aspergillus strains.Additional file 6: Figure S4. Structural and functional annotation cluster by Aspergillus species. PCA and scree plots for a., b. structural aspects; c. IPR terms; d., e. GO terms; f., g. KEGG terms.Additional file 7: Table S3. Functional annotation of pathogenicity/virulence Aspergillus genes.Additional file 8: Figure S5. Relative frequencies of a. GO and b. KEGG terms associated with virulence in Aspergillus. Annotation terms and definitions are listed in the same order in Additional file Additional file 9: Table S4. Definitions of annotations shown in Fig. Additional file 10: Table S5. Differentially-assigned annotations not assigned to PHI-base genes. Overrepresented IPR amino-sugar terms are highlighted in blue. Enriched GO and KEGG polyketide and secondary metabolite synthesis terms are highlighted in orange.Additional file 11: Figure S6. Original diagram of A. flavus KEGG metabolic pathways. Updates to the image are available via KEGG Mapper [G Mapper .Additional file 12: Table S6. RNA-Seq metadata and read mapping results.