Staphyloccocaceae, the species Staphylococcusaureus in particular was reported to cause severe infections. Although most of the other Staphylococcaceae members were not shown to cause severe illnesses, the transmission of AMR genes to harmful species might take place. Therefore, the monitoring of AMR potential in different environments is of high relevance. Mammaliicocci on dairy farms might represent such an AMR gene reservoir. Thus, in this study, the AMR potential of mammaliicocci isolates from German dairy farms was investigated. Whole-genome sequencing (WGS) of the isolates was conducted to evaluate the phylogenetic relationship of the isolates and analyze AMR genes. In addition, antimicrobial susceptibility testing was performed to compare the AMR genotype with the phenotype. It turned out that mammaliicocci may harbor large numbers of different AMR genes and exhibit phenotypic resistance to various antibiotics. Since some AMR genes are likely located on mobile genetic elements, such as plasmids, AMR gene transmission between members of the Staphylococcaceae family might occur.Worldwide, antimicrobial resistance (AMR) is of major concern for human and animal health since infections with multidrug-resistant bacteria are often more challenging and costly. In the family Staphylococcaceae, such as the potentially pathogenic species Staphylococcus aureus. The interest of this study was to analyze AMR profiles of mammaliicocci from German dairy farms to evaluate the AMR transmission potential. In total, 65 mammaliicocci isolates from 17 dairy farms with a history of MRSA detection were analyzed for AMR genotypes and phenotypes using whole genome sequencing and antimicrobial susceptibility testing against 19 antibiotics. The various genotypic and phenotypic AMR profiles of mammaliicocci from German dairy farms indicated the simultaneous occurrence of several different strains on the farms. The isolates exhibited a non-wildtype phenotype to penicillin (58/64), cefoxitin (25/64), chloramphenicol (26/64), ciprofloxacin (25/64), clindamycin (49/64), erythromycin (17/64), fusidic acid (61/64), gentamicin (8/64), kanamycin (9/64), linezolid (1/64), mupirocin (4/64), rifampicin (1/64), sulfamethoxazol (1/64), streptomycin (20/64), quinupristin/dalfopristin (26/64), tetracycline (37/64), tiamulin (59/64), and trimethoprim (30/64). Corresponding AMR genes against several antimicrobial classes were detected. Linezolid resistance was associated with the cfr gene in the respective isolate. However, discrepancies between genotypic prediction and phenotypic resistance profiles, such as for fusidic acid and tiamulin, were also observed. In conclusion, mammaliicocci from dairy farms may carry a broad variety of antimicrobial resistance genes and exhibit non-wildtype phenotypes to several antimicrobial classes; therefore, they may represent an important source for horizontal gene transfer of AMR genes to pathogenic Staphylococcaceae.Mammaliicocci might play a major role in antimicrobial resistance (AMR) gene transmission between organisms of the family  Staphylococcus (S.) aureus, which is the most prominent species in the family Staphylococcaceae. Along with its virulent character, the occurrence of methicillin-resistant S. aureus (MRSA) complicates the treatment of udder infections. MRSA carries a staphylococcal cassette chromosome (SCC) mec element, which harbors the mecA or mecC gene, transmitting resistance to virtually all beta-lactam antibiotics. Moreover, MRSA may exhibit resistance to various other antibiotics. MRSA and other Staphylococcaceae may also be transmitted from animals to humans and vice versa, illustrating their relevance with respect to a One Health approach.Bovine mastitis is frequently related to the presence of Staphylococcaceae. In particular, organisms of the so-called S. sciuri group were shown to inhabit various environments [Mammaliicoccus [Mammaliicoccus (M.) sciuri, M. fleurettii, M. lentus, M. vitulinus, and M. stepanovicii. These organisms were previously reported to harbor AMR genes against several antibiotics [mec elements, such as a SCCmec–mecC hybrid element [mecA genes harboring M. sciuri were recently found to be the most frequently detected species in quarter milk samples on German dairy farms with a history of MRSA detection [M. sciuri and M. lentus were the most frequently found species in the nasal swabs of calves [M. sciuri served as a mecA gene reservoir to S. aureus isolated from seabirds [aureus Staphylococcaceae, and native SCCmec elements were transmitted to S. aureus, which further developed into MRSA [Staphylococcaceae members, such as species of the genus Mammaliicoccus, from different environments gives further insights into this topic and reveals alternative AMR mechanisms that might be of public health relevance.Since antimicrobial resistance (AMR) genes are mostly located on mobile genetic elements such as plasmids or prophages, resistance may be transmitted between different species of the family ronments . Accordiiicoccus . The genibiotics  and show element ,4. Mamma element ,5,6. Howf calves . Next toseabirds . In genento MRSA . TherefoMammaliicoccus isolates from German dairy farms in order to evaluate the resistance potential and risk of AMR gene transmission to more harmful species, such as S. aureus.The aim of this study was to characterize the AMR genotype and AMR phenotype of Mammaliicoccus strains from 17 dairy farms (coded as A-R) located in nine German federal states were chosen for phylogenetic analyses and investigation of their AMR potential. Farms were pre-selected on the basis of a history of MRSA detection in the dairy cow herds [For this study, 64 ow herds . The strhttps://tygs.dsmz.de/, accessed on 8 August 2021) [M. sciuri and M. lentus isolates was conducted using CSI Phylogeny 1.4 from the Centre for Genomic Epidemiology  [M. sciuri NCTC12103 strain (NZ_LS483305.1) and M. lentus NCTC12102 strain (NZ_UHDR01000002.1) were used as reference genomes. Visualization of phylogenetic trees was performed in MEGA X version 10.1.7. Bacterial characterization was conducted with the in-house developed Bakcharak pipeline  using the NCBI AMRfinder database [dfrE gene (NZ_KZ846041.1) was performed using NCBI BLASTN. Detection of SCCmec elements was conducted using SCCmecFinder 1.2 .Mammaliicocci isolates were inoculated in 5 mL brain–heart-infusion broth and incubated at 37 °C for 24 h. The DNA of 1 mL culture was extracted using the Qiagen DNeasy Blood and Tissue Kit  according to the manufacturer’s protocol modified by adding 10 µL lysostaphin to the lysis buffer. The DNA library was prepared using an Illumina DNA Prep kit , and the 150 bp paired-end sequencing run was performed on an Illumina NextSeq 500 instrument. Raw Illumina reads were trimmed and assembled de novo with the in-house developed AQUAMIS pipeline [st 2021) . Phylogest 2021) . Completdatabase  for screS. aureus were used as follows: PEN, ≤0.125 mg/L; FOX, ≤4 mg/L; CHL, ≤16 mg/L; CIP, ≤1 mg/L; CLI, ≤0.25 mg/L; ERY, ≤1 mg/L; FUS, ≤0.5 mg/L; GEN, ≤2 mg/L; KAN, ≤8 mg/L; LZD, ≤4 mg/L; MUP, ≤1 mg/L; RIF, ≤0.016 mg/L; SMX, ≤128 mg/L; STR, ≤16 mg/L; SYN, ≤1 mg/L; TET, ≤1 mg/L; TIA, ≤2 mg/L; TMP, ≤2 mg/L; VAN, ≤2 mg/L. For quality control of resistance testing, the S. aureus isolates ATCC 29213 and ATCC 25923 were used.Antimicrobial susceptibility testing was performed by broth microdilution according to the CLSI guidelines (ISO 20776-1:2006 or CLSI M31-A3). It was carried out using a standardized antibiotic panel (EUST scheme) that is recommended by the European Food Safety Authority (EFSA) for resistance monitoring in MRSA from livestock and food . For intM. sciuri (26/64), M. lentus (22/64), M. fleurettii (15/64), and M. vitulinus (1/64). However, according to a variable G+C content, the taxonomic assignment of M. fleurettii isolates seemed to be unreliable; thus, these isolates were named M. sp. in the following.Presumptive mammaliicoccal isolates were verified by taxonomic assignment in TYGS. The analyzed isolates were further classified as M. sciuri and M. lentus isolates, which represent the major fractions of the analyzed isolates, show both genomic differences between the dairy farms and closely related strains within distinct farms. M. sciuri isolates from dairy farm D  clustered closely in the phylogenetic tree  were located on two different branches of the phylogenetic tree, indicating genomic distinction. Moreover, isolates from the farms I/N , E/O , N/Q , and A/C  clustered on the same branch.Separate phylogenetic trees of tic tree A. On theM. lentus isolates, several genotypically similar strains occurred on farms A , C , J , M , and R   were located on different branches in the phylogenetic tree. In addition, isolates from different dairy farms  clustered on the same branches, respectively.With regard to 64, M65) B. Other M. sciuri and M. lentus isolates. Fewer genes were found in M. spp and M. vitulinus genomes Ie, aadD1, aph(2”)Ia, spd, str), glycopeptide (bleO), macrolide (msr(A), mph(C), ermB, erm(43), erm(48)), trimethoprim , tetracycline , pleuromutilin–lincosamide–streptogramin (lnu(A), sal(A)), oxazolidinone (cfr), and phenicol .A broad range of AMR genes was detected in the genome sequences of  genomes . All isomec element was determined as type III with sequence identities of 67–90% to known S. aureus SCCmec element type III. In addition, one M. sciuri and one M. lentus isolate harbored a SCCmec–mecC hybrid element. In the remaining 27 isolates, no SCCmec element was detected.In 35/64 isolates, the SCCM. sciuri was resistant to five to twelve antimicrobial substances, M. lentus to between eight and eleven. By contrast, M. sp. and M. vitulinus isolates were never resistant to more than four antimicrobials.The phenotypic resistance observed was not always in line with the predicted AMR genes . The isoStaphylococcaceae other than S. aureus on human and animal health, such as bovine mastitis, is rarely investigated. Apart from the species S. haemolyticus and S. epidermidis, which were reported to exhibit various virulence factors [Staphylococcaceae were only occasionally associated with diseases such as bovine mastitis [Staphylococcaceae. Therefore, the monitoring of AMR in this group is important with respect to treatment options for harmful species, such as S. aureus.The impact of members of the family  factors ,16, othemastitis . HoweverIn this study, the phylogenetic relationship and AMR potential of mammaliicocci from German dairy farms were investigated. Since the visited dairy farms had a history of MRSA detection, it was assumed that mammaliicoccal species might also express distinct AMR profiles.S. sciuri group was recently reclassified to the new genus Mammaliicoccus [Mammaliicoccus consists of five species. In this study, M. sciuri, M. lentus, and M. vitulinus were detected in samples from dairy farms. Moreover, several isolates were typed as M. fleurettii with, however, unreliable species identification results according to a variable G+C content. The phylogenetic analyses of M. sciuri and M. lentus isolates showed broad genomic variability in these two species. On the one hand, similar strains were spread in different niches in one dairy farm, but otherwise, different strains were also present in the same farm. Moreover, the close phylogenetic relationship of some strains from different dairy farms indicates transmission of mammaliicocci between dairy farms. A spread of MRSA within and between German dairy farms was assumed in previous studies [Staphylococcaceae strains into dairy farms and spread within the farms may have different causes. Animal trading may contribute to bacterial introduction to the dairy herd and its environment [The iicoccus . The gen studies ,19; thusironment . InsuffiM. sciuri was also reported as a reservoir of the mecA gene for S. aureus [mec elements harboring mecA and mecC genes were reported in M. sciuri [M. sciuri and one M. lentus isolate in this study. In general, the detection of SCCmec elements using SCCmecFinder was difficult for the analyzed isolates. For most isolates, the prediction showed a SCCmec element type III; however, the sequence identity to known SCCmec elements type III from S. aureus was only 67–90%. Low homologies of M. sciuri SCCmec elements in comparison to SCCmec elements from S. aureus were also reported in a review on the evolution of beta-lactam resistance in staphylococci [mec elements originated from M. sciuri and were transmitted to S. aureus. Because of genomic adaptation processes in S. aureus with regard to stress responses, such as to antibiotics, SCCmec elements in S. aureus evolved, leading to differences in their genomic structure compared to the native SCCmec element in M. sciuri.The isolates of this study expressed phenotypic resistance to various antimicrobial substances. Impressively, 18/64 isolates showed non-wildtype phenotypes to ≥10 antibiotics. Mammaliicocci harbored AMR genes to several classes of antibiotics [. aureus . Moreove. sciuri ,4. This M. vitulinus and eight M. sp. isolates, the SCCmecFinder did not identify any SCCmec elements, indicating acquisition of the mecA gene independent from any known SCCmec element. The mecA gene is believed to originate from species of the previous S. sciuri group [mecA gene was not located in a SCCmec element [M. vitulinus and M. sp. isolates might still represent this original genomic status. Interestingly, all isolates that lacked an SCCmec element exhibited only low cefoxitin MICs of ≤4 mg/L. This indicates susceptibility to beta-lactam antibiotics despite the mecA gene. Most likely, the missing accompanying genes in the non-SCCmec-mecA isolates that are usually present in the SCCmec element lead to reduced beta-lactam resistance. Moreover, in general, the M. sp. and M. vitulinus isolates showed only low numbers of AMR genes and, accordingly, exhibited resistance to only a few antimicrobial substances. This might also indicate a native status of these isolates and might illustrate a lower competence of gene acquisition in these species.In addition, for one ri group , and the element . Therefoaac(6′)Ie, aadD1, aph(2”)Ia, and str genes. Resistance to kanamycin and gentamicin mostly co-occurred in the isolates.Only a few isolates expressed resistance to the aminoglycosides kanamycin (9/64) and gentamicin (8/64), whereas streptomycin resistance was detected in nearly a third of the isolates (20/64). Resistance to kanamycin, gentamicin, and streptomycin was encoded by the M. sciuri isolate in this study. Accordingly, the respective isolate harbored a cfr gene. Several studies describe the detection of cfr genes in members of the family Staphylococcaceae [cfr gene is usually located on plasmids, which can be transmitted between bacterial species. Thus, it must be considered that the occurrence of the cfr gene harboring mammaliicocci on dairy farms with a concurrent MRSA appearance, as presented in this study, may increase the risk of a linezolid resistance acquisition of MRSA.Resistance to linezolid is of high public relevance since it belongs to the last-resort antibiotics. Linezolid resistance was detected in one occaceae ,22. The erm genes and the msr(A) gene. In particular, erm genes play a major role in erythromycin resistance [erm genes were detected in 15/17 of erythromycin-resistant isolates in this study. The two other isolates harbored the msr(A) gene in their genomes. The erm genes may also confer inducible or constitutive resistance to lincosamides, such as clindamycin [erm genes. In addition, 13 isolates carried the lnu(A) gene, which may also provide resistance to lincosamides [Macrolide resistance was likely determined by different sistance . The ermndamycin . Clindamosamides .dfrG or dfrK genes (23/30 isolates). Prediction of AMR genes with regard to trimethoprim in the other seven isolates failed. It is likely that these isolates carry variable AMR genes, which are not yet covered by the NCBI AMRfinder database. Recently, a dfrE gene, which originates from Enterococcus faecalis, was discovered in a multidrug-resistant M. sciuri strain [dfrE gene search using NCBI BLASTN did not score a hit for this gene in the isolates of this study.Resistance to trimethoprim was mostly associated with i strain . HowevertetM, tetL, or tetK genes. Tetracycline is often used in livestock farming, increasing the pressure for bacteria to acquire resistance [By contrast, the prediction of tetracycline resistance was in good agreement with the respective phenotype. The resistant isolates harbored sistance . Althougsistance .sal, vga, and lsa genes [sal(A) gene was found in a part of the tiamulin-resistant isolates. Thus, it is very likely that tiamulin resistance is additionally affected by a completely different mechanism or encoded by so-far unknown resistance genes, which are not covered by the AMR gene database.Interestingly, the tiamulin resistance rate in the mammaliicocci in this study was also very high (92%), exceeding the rate of MRSA from the same German dairy farms . Tiamulisa genes . HoweverfexA gene was detected in all respective isolates. Moreover, one M. lentus isolate harbored the catA gene, which is also associated with phenicol resistance. However, only a lowered susceptibility of this isolate to chloramphenicol with a MIC of 16 mg/L was detected. The same reduced susceptibility was also found in three other isolates carrying the fexA gene.Chloramphenicol resistance was found in 26/64 isolates. Correspondingly, a fus genes. For other members of the family Staphylococcaceae, variations in the fus gene had already been detected [fus gene was not found in the mammaliicoccal isolates of this study. In accordance with the tiamulin resistance, for fusidic acid, it is also reasonable to assume that alternative AMR genes or mechanisms transmitted the phenotypic resistance.Although almost all isolates expressed resistance to fusidic acid, a respective AMR gene was not predicted. Fusidic acid resistance may be transmitted by detected . HoweverStaphylococcaceae, such as S. aureus, which might increase the risk of difficult-to-treat infections in humans and animals. With regard to the One Health approach, resistance to last-resort antibiotics, such as linezolid, is highly concerning. Mismatches of AMR gene prediction and the respective phenotype were occasionally found in the analyzed isolates. As already reported in other studies for different Staphylococcaceae, the AMR gene variability in this family is high, and recent databases do not fully cover the variations so far. In order to improve the monitoring of rarely investigated Staphylococcaceae genera with regard to the AMR potential, well-curated and complemented AMR gene databases are needed.This study illustrates that mammaliicocci from dairy farms may carry a broad variety of AMR genes and exhibit non-wildtype phenotypes to several antimicrobial classes. It cannot be ruled out that resistance genes are transmitted from mammaliicocci to more pathogenic species of the family


Enterococcus includes two Gram-positive pathogens of particular clinical relevance: E. faecalis and E. faecium. Infections with each of these pathogens are becoming more frequent, particularly in the case of hospital-acquired infections. Like most other bacterial species of clinical importance, antimicrobial resistance  is an increasing threat, with both species considered to be of particular importance by the World Health Organization and the US Centers for Disease Control. The threat of antimicrobial resistance is exacerbated by the staggering difference in the speeds of development for the discovery and development of the antimicrobials versus resistance mechanisms. In the search for alternative strategies, modulation of host-pathogen interactions in general, and virulence inhibition in particular, have drawn substantial attention. Unfortunately, these approaches require a fairly comprehensive understanding of virulence determinants. This requirement is complicated by the fact that enterococcal infection models generally require vertebrates, making them slow, expensive, and ethically problematic, particularly when considering the thousands of animals that would be needed for the early stages of experimentation. To address this problem, we developed the first high-throughput C. elegans–E. faecium infection model involving host death. Importantly, this model recapitulates many key aspects of murine peritonitis models, including utilizing similar virulence determinants. Additionally, host death is independent of peroxide production, unlike other E. faecium–C. elegans virulence models, which allows the assessment of other virulence factors. Using this system, we analyzed a panel of lab strains with deletions of targeted virulence factors. Although removal of certain virulence factors  was sufficient to affect virulence, multiple deletions were generally required to affect pathogenesis, suggesting that host-pathogen interactions are multifactorial. These data were corroborated by genomic analysis of selected isolates with high and low levels of virulence. We anticipate that this platform will be useful for identifying new treatments for E. faecium infection.The genus  Enterococcus faecalis and E. faecium, are categorized as serious threats by the Centers for Disease Control and Prevention and since the 1970s have risen to be some of the most commonly isolated Gram-positive organisms responsible for nosocomial infections. In 2017 alone, there were approximately 55,000 infections with enterococcal species in the United states, resulting in 5,400 deaths (E. faecium) often develop resistance to other antibiotics, leading to the requirement for intensive and invasive treatments. Resistance prolongs hospitalization and increases the likelihood for patients to contract secondary infections.Enterococci, particularly 0 deaths . Infecti0 deaths . Healthc0 deaths . Frequen0 deaths . Enteroc0 deaths . In fact0 deaths  and vancE. faecalis and E. faecium, both of which are commonly found as intestinal commensals    , and surms, esp) . Most ofE. faecium virulence are predominantly mammals. Although effective, these models are strongly constrained in terms of throughput, are expensive, and are fraught with ethical concerns. This makes them inappropriate for large scale screens to identify novel virulence factor or therapeutic compounds ], N2 wild type, and pmk-1(km25) were maintained on standard nematode growth medium (NGM) -based assays (pmk-1(km25), sterility was induced by feeding worms with cdc-25.1(RNAi)-expressing E. coli. Absence of progeny facilitates analysis of assay outcomes.um (NGM)  seeded wse noted . C. elegd assays . For expE. faecium strains used in this study were collected over the past 30 years. The goal of our study was to compare the virulence of E. faecium belonging to different clades in C. elegans model. Therefore the strains representing clinical strains (mostly clade A1), commensal strains from healthy volunteers (mostly clade B), and isolates from animal sources or published clade A2 strains were selected. These isolates included 75 health care-derived isolates (mostly clade A1) from USA and Colombia  using previously published methods  (fms11 (predicted pilus-associated) (scm (second collagen adhesin of E. faecium) , TX6114 (E1162 Δfms15), TX6109 (E1162 Δscm), TX6110 , TX6094 , and TX 6097 (TX0082 Δfms11 Δscm) and have been listed in strain list in Gene deletion mutants of previously described cell wall anchored (CWA) surface proteins with Ig-like folds  were giv methods . DeletioMSCRAMM) ; fms11 (ociated)  and scm faecium) . Two subAMM gene  using puAMM gene . These sE. faecium strains were grown in deep-well plates at 700 rpm for 16 h. S Basal medium was mixed with brain-heart infusion (BHI) medium (10%) and Enterococcus faecalis or E. faecium  were then added into each well. Plates were incubated at 25°C until assay completion. At various time points, bacterial OD was measured with a Cytation5 (BioTek Instruments) plate reader and then plates were washed three times. Sytox Orange nucleic acid stain was added into each well and incubated for 12 - 16 h to stain dead worms. Plates were then washed three times, worms were transferred into a new 384-well plate, washed three more times, and imaged with Cytation5 automated microscope. Dead worms were quantified with CellProfiler software, similar to previously established pipelines  was supplemented in S Basal, BHI, Enterococcus-BHI plates and incubated at 25°C. Worms were scored every day for survival; dead worms were removed from assay plates.For killing on agar, 50 young adult worms were transferred onto E. faecium strains, TX0016 (clade A) and a commensal isolate, TX1330 (clade B) were tested following our previously published method , a bioinformatic tools for rapid bacterial variant calling. Meanwhile, FASTA files were processed with prokka  for genome annotation, roary  for genomic alignment, and RAxML  to construct a maximum-likelihood phylogenetic tree. Genomic analysis was performed in the usegalaxy.org platform. FASTQ files generated from NGS were submitted for SNPs analysis to E. faecium E007 in the liquid-based assay, worms were washed three times to remove residual bacteria, incubated for one hour with or without 20 µg/mL gentamicin, washed three times, and stained with 40 µM acridine orange for 4 hours. Worms were then washed three more times to remove unbound dye. Worms were imaged using a Zeiss ApoTome.2 Imager.M2 fluorescent microscope  with a 20x objective magnification.Upon 48 h of infection with E. coli HT115 were cultured and seeded onto NGM plates supplemented with 25 μg/mL carbenicillin and 1 mM IPTG. 8,000 synchronized L1 larvae were transferred onto RNAi plates and grown at 25°C for 64 hours prior to exposure to pathogens.RNAi-expressing RNAi experiments in this study were conducted by using RNAi-competent HT115 obtained from the Ahringer RNAi library  and wereWith the exception of the mouse infection model, all experiments were performed in at least three independent biological replicates. The number of worms used per biological replicates is listed in the corresponding legends.t-test analysis was performed to calculate the p-values when comparing two groups in an experimental setting. p-values were indicated in graphs as follows: NS not significant, *p < 0.05, **p < 0.01, and ***p < 0.001.Student’s p-value between C. elegans survival and E. faecium OD600 were calculated and indicated on the figure.p-values between Clade A1 and Clade B were indicated on graphs.E. faecium virulence determinants, we developed the first high-throughput infection model for E. faecium that uses C. elegans as a host. In brief  , mdt-15(RNAi) significantly increased host mortality in both liquid- and agar-based assays , and B   . Althougstrains) , a more strains) . PathogeAn initial analysis of the pathogenicity of ~30 isolates in our liquid-based model was performed. K-means clustering was then used to group the strains based on virulence, resulting in strong preferential grouping of A1 and A2 clades in high virulence and B strains in low virulence  and the lowest  virulence, and infected C. elegans with them in the liquid assay . To ensure that only bacteria from the worm alimentary track, but not those that adhere to the cuticle, contributed to CFU counts, we performed the same assay except that worms were treated with gentamicin for 1 h after the first three washes  survival . The WxLsurvival . Three sE. faecium surface proteins (Fms) of the MSCRAMM  family were more likely to be involved in pathogenesis in this E. faecium – C. elegans killing assay. fms genes are tightly associated with E. faecium clinical isolates , two , or three genes mutated . Among the strains tested, the single deletion of fms15, an adhesin gene, significantly reduced E. faecium virulence  reduced pathogenicity as well  and TX2051 (higher virulence) (To further understand sorB (a PTS system sorbose-specific EIIB component) and another in mazE (the gene encoding the MazE Type II antitoxin). TX2051 acquired a frameshift in araQ (L-arabinose transport system permease protein) and a stop codon in wecA .Genomic analysis showed that TX2046 contained two frameshift mutations: a one nucleotide insertion in mazE would result in greatly diminished bacterial survival. We performed growth analysis of the OD 600 for the two strains TX2046 and TX2051. We observed differences in growth rates, with TX2051 growing faster than TX2046 , host responses , and metabolic adjustments seen in mammals , showed a correlation between faster growth and higher virulence, there was no overall trend. This is consistent with our observation that secreted virulence factors do not seem responsible and that colonization is required, as shown by diminished pathogenesis by the fms15 mutant.Interestingly, we found no correlation between the initial bacterial titer of E. faecium in this assay, was unexpected, given our previous results with a C. elegans – P. aeruginosa assay that has been utilized by our lab for a number of years. In the P. aeruginosa liquid pathogenesis assay, the living bacteria present in the intestine appear to be transient and have escaped the digestive process, but are not establishing a significant colonization. Consequently, isolates or mutants that would be attenuated on agar may have full virulence in liquid, and vice versa. In the case of P. aeruginosa, growth on agar (or in the host) leads to high bacterial density and key involvement of quorum-sensing pathways  to dispensable (E. faecium infection in liquid) to detrimental (P. aeruginosa intoxication in liquid). Involvement of the Mediator complex subunit MDT-15/Med15 was recently shown to be required for increased survival of C. elegans exposed to both Gram-negative and Gram-positive bacteria  in liquid when treated with immune-stimulatory compounds  lacked a clear phenotype, this is likely due to genetic redundancy. Combining multiple mutations in a common pathway reduced pathogenesis. For example, combining mutations in fmebpABC (or empABC or fms1-5-9) and acm-fms11-scm of E. faecium considerably limited lethality. These surface proteins are enriched in clinical isolates and aid in attachment to extracellular matrix proteins (ebpABCfm significantly attenuated biofilm formation and reduced virulence in a murine UTI model (E. faecium when developing future therapies, so that this redundancy does not compromise treatment effect.We validated our screen by confirming the above-mentioned adhesion-related virulence factors that play a role in proteins . The losTI model . This foThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Animal Welfare committee, University of Texas Health Science Center at Houston.E. faecium isolates and mouse data. KS participated in manuscript writing. BH performed sequencing experiments. NK performed overall design of the study, contributed to data analysis and manuscript writing and editing. All authors contributed to the article and approved the submitted version.AR performed a majority of the experiments. ET performed some of the experiments. AR and ET wrote the first draft of the manuscript. BM and KS contributed to conception of the study and provided This work was supported by a John S. Dunn Foundation Award to NK and BM and NIH NIGMS Award R35GM129294 to NK. Funders had no role in study design, data collection or analysis, decision to publish or preparation of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ectropis obliqua is a destructive leaf-eating pest that is widely distributed in China’s tea gardens. This pest shows remarkable resistance against multiple insecticides. As an environmentally friendly entomopathogen, Beauveria bassiana has been widely used to prevent agricultural pest infestations. However, the molecular mechanism of B. bassiana against E. obliqua remains unclear. We firstly isolated and identified a highly virulent B. bassiana strain. Using a transcriptome, we analyzed the differences of immune gene expression levels in fat bodies and hemocytes of E. obliqua that were infected by the B. bassiana, which provide molecular insights into the insect–pathogen interaction.Ectropis obliqua is a destructive masticatory pest in China’s tea gardens. Beauveria bassiana as microbial insecticides can effectively control E. obliqua larvae; however, the immune response of this insect infected by B. bassiana are largely unknown. Here, after isolating a highly virulent strain of B. bassiana from E. obliqua, the changes in gene expression among different tissues, including hemocytes and fat bodies, of E. obliqua larvae infected by the entomopathogen were investigated using transcriptome sequencing. A total of 5877 co-expressed differentially expressed genes (DEGs) were identified in hemocytes and fat bodies, of which 5826 were up-regulated in hemocytes and 5784 were up-regulated in fat bodies. We identified 249 immunity-related genes, including pattern recognition receptors, immune effectors, signal modulators, and members of immune pathways. A quantitative real-time PCR analysis confirmed that several pattern recognition receptors were upregulated in hemocytes and fat bodies; however, others were downregulated. The investigated immune effectors (ATT and PPO-1) were suppressed. The results showed that there were tissue differences in the expression of immune genes. This study provides a large number of immunity-related gene sequences from E. obliqua after being infected by B. bassiana, furthering the understanding of the molecular mechanisms of E. obliqua defenses against B. bassiana. Ectropis obliqua (Lepidoptera: Geometridae) is the most harmful leaf-feeding pest of tea gardens in China, owing to its wide distribution and great destructiveness [tiveness ,2. The ltiveness ,4. In retiveness ,6,7. Thetiveness .Beauveria bassiana, an entomopathogenic fungus, has a wide host range, is environmentally friendly, and markedly improves pest control efficacies, resulting in its use as a biological control agent worldwide [Ectropis obliqua [Rhynchophorous ferrugineus [Spodoptera frugiperda [Tetranychus urticae [Tuta absoluta [B. bassiana is as follows: the fungal conidia adhere to the host surface, proteases and chitinases are then secreted to hydrolyze the host cuticle, and then, the fungi invade and colonize the host hemocoel. They grow by absorbing host nutrients, and eventually the host is killed by the secreted toxins and physiological starvation [Helicoverpa armigera [Clanis bilineata [Galleria mellonella [Riptortus pedestris [Ostrinia furnacalis [Hypothenemus hampei [B. bassiana. These data provide clues and gene candidates for further exploration on the molecular mechanisms of entomopathogenicity by this fungus. However, the immune response mechanisms of E. obliqua after infection by the pathogenic fungi B. bassiana remain unclear.orldwide ,10. It h obliqua , Rhynchorugineus , Spodoptugiperda , Tetrany urticae , and Tutabsoluta ,16. Genearvation ,18,19,20armigera , Clanis ilineata , Gallerillonella , Riptortedestris , Ostrinirnacalis , and Hyps hampei  infectedE. obliqua larvae to the adherence of B. bassiana conidia, and then, the transcriptomic profiles of hemocytes and fat body tissues from E. obliqua infected by B. bassiana were analyzed using a high-throughput RNA sequencing method. We identified potential immunity-related genes through comparisons with homologous sequences of other insects. Finally, the expression levels of several key host immune genes were verified using quantitative real-time PCR (qRT-PCR). This study provides a theoretical basis for understanding the immune mechanisms of E. obliqua against B. bassiana, providing molecular insight into the complicated interaction between host and fungal pathogen.In this study, we determined the sensitivity of fifth-instar Ectropis obliqua larvae was collected from tea plant leaves in the wild at Dayangzhen Tea Garden in Hefei, China . Entomopathogenic fungi were isolated from the infected Ectropis obliqua larvae. Mycelium on the surfaces of E. obliqua were inoculated into potato dextrose agar medium and cultured in an incubator at 28 °C for 5 days. The isolated strains were purified three times. The benzyl chloride method was used to extract fungal DNA [Fungal-infected ngal DNA . Using tE. obliqua moths were acquired from the State Key Laboratory of Tea Plant Biology, Anhui Agricultural University, Hefei, China . The larvae were reared on tea leaves at 23 ± 2 °C and 70–80% relative humidity with a 16 h light/8 h dark photoperiod in the insect-rearing laboratory. Tea leaves used in the experiment were inserted into flower mud for storage. A total of 120 fifth-instar larvae were randomly selected and were injected with B. bassiana conidial suspension  and Tween 80 solution (2 μL) using microliter syringes  (n = 60). The numbers of dead larvae were recorded for 10 days. During this period, the dead larvae were collected and kept moisturized to observe whether the white hyphae could grow from the larvae.g for 5 min at 4 °C to collect hemocytes [At 0 h and 48 h of infection, fat body tissues and hemolymph samples of larvae were collected. Fat body tissues from ten larvae were collected under the microscope with dissecting forceps as one sample, respectively. Hemolymph collected from ten larvae was pooled into a 1.5 mL RNA-free microcentrifuge tube containing 0.1% 1-phenyl-2-thiourea and centrifuged at 500× emocytes . Each sa−5) [Before assembly, to obtain satisfactory determination results, low-quality sequences , sequences with nitrogen contents over 10%, and adapter sequences were removed. Trinity software was used to de novo assemble all the clean reads to produce contigs and singletons . By comp−5) . The gen−5) ,20. The −5) .p-value < 0.05) were used to identify the DEGs [E. obliqua unigenes were obtained using the tblastn method. Subsequently, the E. obliqua immunity-related genes were manually confirmed.Differentially expressed genes (DEGs) were calculated using the fragments per kilobases per million mapped reads (FPKM) method, and the screening conditions  and Tween 80 solution (n = 30). Ten larvae in each treatment were randomly chosen, and total RNA was extracted from fat body tissues and hemolymph. Each sample was performed in triplicate. In total, 1 μg of RNA was reverse transcribed using the StarScript II First-strand cDNA Synthesis Kit . Quantitative real-time PCR (qRT-PCR) was performed on an ABI 7300 Real-Time PCR System  using GoTaq qPCR Master Mix  in a 20-µL volume. Each well plate was loaded with 1μL cDNA. The reaction steps were as follows: 95 °C for 2 min and 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The data were analyzed using the 2−ΔΔCt method [β-actin gene of E. obliqua was used as the reference gene. The primers were described previously   A. After nfection B.Ectropis obliqua, the hemocytes and fat body tissues were collected from B. bassiana-infected larvae after 48 h. A total of 56,412 unigenes were annotated using BLASTX searches. A Venn diagram analysis indicated that 5877 genes were co-expressed between hemocyte and fat body samples (5826 upregulated and 51 downregulated in hemocytes and 5784 upregulated and 93 downregulated in fat bodies) (To obtain a transcriptome of  bodies) . The DEG bodies) .B. bassiana-infected larvae, a hierarchical clustering analysis of the DEGs was performed using fragments per kilobases per million mapped reads. Four major gene clusters were identified that exhibited distinct expression patterns among the different groups (B. bassiana-infected larvae. The 5275 genes in cluster 1 showed significantly greater RNA levels in hemocytes from entomopathogenic fungi-infected larvae compared with those of non-infected larvae. Cluster 4 (96 genes) showed identical expression patterns in the hemocytes and fat bodies of B. bassiana- and Tween-infected larvae. Four gene clusters were categorized into thirteen functional pathways using KEGG classifications. Most of the upregulated genes in clusters 1 and 2 were involved in immune responses, energy, and carbohydrate, lipid, and amino acid metabolic processes  were similar to those of L. dispar (150). The number of immunity-related genes was highest in D. melanogaster (180) and lowest in A. mellifera (63) , Coleopastaneum  and Dends valens ), Hymenoellifera ), and Di gambiae  and Drosnogaster ). The imera (63) B. bassiana-infected larvae’s genes involved in the recognition of C-type lectin CTL-10 were markedly upregulated, whereas the peptidoglycan recognition protein PGRP-5 and PGRP-6 genes were dramatically downregulated in hemocytes and fat bodies. The GNBP-2 gene was downregulated in hemocytes but upregulated in fat bodies. While SPH-5 was upregulated, serpin-7 was not differentially expressed in fat bodies. The immune response effector genes ATT and PPO-1 were significantly downregulated in hemocytes and fat bodies. Among the Toll pathway genes, SPZ 1–5 genes were all mostly up-regulated in hemocytes and down-regulated in fat bodies, according to our transcriptome data. SPZ-2 gene was further verified by qRT-PCR, which was consistent with the transcriptome data  were downregulated in all the samples infected by B. bassiana. We speculate that the 48 h sample time point allowed a sufficient amount of time for the release of entomopathogenic toxins.Insects perceive the presence of pathogens through intracorporal pattern recognition receptors in combination with pathogen-associated molecular patterns, which activate immune responses. Insect pattern recognition receptors mainly include CTLs, β-1,3-glucan recognition proteins, and PGRPs. CTLs recognize pathogens, enhance host phenol oxidase activity levels, and activate encapsulation and melanization reactions ,44. The vates PO . In this pathway . The GNB pathway . As showSPH-5, SPZ-2, and serpin-7 all showed upregulated trends in the immune responses of E. obliqua. Signal transduction molecules are the most common type of immunity-related genes, and those in the Toll and IMD signaling pathways mostly showed upregulated trends. In addition, in this transcriptome data, the JNK pathway is activated after pathogen recognition, leading to the activation of the transcription factor to turn on expression of antimicrobial peptides (AMP), such as cecropin and gloverin. Both gloverin and cecropin of antimicrobial peptides were down-regulated in the hemocytes after B. bassiana infection.After insects recognize pathogens, the protease activity signals of serine proteases (SPs) and their homologs  are amplified step by step and finally transmitted to the Toll pathway’s extracellular ligands SPZ or PPO. The enzyme-linked reactions of SPs are usually regulated by SP inhibitors (serpins). These proteases play crucial roles in the reduction or amplification of immune signals . In thisE. obliqua against B. bassiana, which is insufficient to fully understand when the insect immune is initially responding and how immune genes are temporally expressed to counter the invasion of B. bassiana. Furthermore, we aimed at identifying a highly virulent B. bassiana strain, profiling the immune system of E. obliqua, and partly revealing the immune reaction of the pest against the entomopathogen. The difference of the immune reaction in E. obliqua against high and low virulence strains is an interesting question and deserves investigation. In summary, our study provided new insights into the expression profiles of host defense genes in non-model insects exposed to pathogenic fungi, allowing us to further investigate the functions of antifungal immunity-related genes and to improve our understanding of host–pathogen interactions.In this study, we chose only one time point (48 h post-infection) to profile and characterize the immune reaction of B. bassiana from E. obliqua, we identified 249 immunity-related genes from E. obliqua infected by the B. bassiana using a transcriptome and compared their expression with those of homologous genes in other insects. Then, we confirmed the expression patterns of certain genes using qRT-PCR. Our study provides a molecular basis for the function studies of antifungal immunity-related genes and advances the understanding of host–pathogen interactions of E. obliqua.After the isolation of a highly virulent strain of


Phylum Cnidaria is an ancient venomous group defined by the presence of cnidae, specialised organelles that serve as venom delivery systems. The distribution of cnidae across the body plan is linked to regionalisation of venom production, with tissue-specific venom composition observed in multiple actiniarian species. In this study, we assess whether morphological variants of tentacles are associated with distinct toxin expression profiles and investigate the functional significance of specialised tentacular structures. Using five sea anemone species, we analysed differential expression of toxin-like transcripts and found that expression levels differ significantly across tentacular structures when substantial morphological variation is present. Therefore, the differential expression of toxin genes is associated with morphological variation of tentacular structures in a tissue-specific manner. Furthermore, the unique toxin profile of spherical tentacular structures in families Aliciidae and Thalassianthidae indicate that vesicles and nematospheres may function to protect branched structures that host a large number of photosynthetic symbionts. Thus, hosting zooxanthellae may account for the tentacle-specific toxin expression profiles observed in the current study. Overall, specialised tentacular structures serve unique ecological roles and, in order to fulfil their functions, they possess distinct venom cocktails. Anemonia sulcata and Heteractis crispa, but in the mesenterial filaments of Megalactis griffithsi and the acrorhagi of Actinia tenebrosa [Venom composition tends to be dynamic, varying across geographic locations and ontogenic stages, and between individuals in venomous taxa ,4,5,6,7.enebrosa ,15. HoweGalaxea fasicularis revealed that toxin genes are differentially expressed across the two tentacle subsets [While tentacles are used primarily for prey capture and defence against predators (feeding tentacles), contact with competitors can induce the development of a secondary tentacle type—catch tentacles—in actiniarians ,17,18,19 subsets  indicatiDofleinia and Macrodactyla (Dofleinia armata has a discrete battery of cells (papillae) covering their tentacles which are laden with nematocysts [Cryptodendrum adhaesivum and Heterodactyla hemprichii multiple tentacles are associated with each endocoel  completeness scores between 88.9 and 97.2%. The assembly N50 scores ranged from 609 to 2823 base pairs (bp). Although the N50 for C. adhaesivum  than between the body column and tentacles in C. adhaesivum (3672). However, in both species, comparable numbers of transcripts were upregulated in each of the structures examined  enrichment analysis supported functionally discrete roles for the structures analysed. Patterns of transcript expression differed significantly across the structures examined in all taxa. When comparing gene expression between inner and outer tentacles of . semoni . In H. hectively . Compariexamined .H. hemprichii and P. semoni than between inner and outer tentacles of D. cf. armata and M. doreensis. Extracellular region (GO:0005576) was found to be enriched in outer tentacles of D. cf. armata and M. doreensis. While no GO terms were enriched for inner tentacles of D. cf. armata, photosynthesis (GO:0015979) and related terms were enriched in inner tentacles of M. doreensis. Similarly, chlorophyll binding (GO:0016168) was enriched in nematospheres of C. adhaesivum and vesicles of P. semoni. The enrichment of photosynthesis-related terms likely reflects the presence of algal symbionts in C. adhaesivum, M. doreensis and P. semoni. Heterodactyla hemprichii also hosts zooxanthellae species, although the specimen included in this study had undergone bleaching prior to tissue dissection and therefore no photosynthetic-related GO terms were found to be enriched in this individual. Venom-related GO terms, including nematocyst (GO:0042151) and toxin activity (GO:0090729), were significantly enriched in multiple structures across C. adhaesivum, H. hemprichii and P. semoni.GO enrichment analysis of differentially expressed genes also supported greater differences among tentacular structures of H. hemprichii to 119 in C. adhaesivum and P. semoni (Transcripts with homology to known toxins (toxin-like) were identified and extracted from the transcriptome assembly of each taxon. The number of toxin-like transcripts compiled for each species ranged from 60 in . semoni , with apC. adhaesivum, D. cf. armata and H. hemprichii. In particular, sea anemone type 3 (BDS) potassium channel toxin-like transcripts were numerous in all three species. Conversely, membrane-active toxins were the most abundant category of toxins identified in the transcriptome of M. doreensis. The Membrane Attack Complex/Perforin (MACPF) toxin family is expanded in this species, with MACPF toxin-like transcripts also highly abundant in the transcriptome of P. semoni. Auxiliary toxins comprised a much higher proportion of the venom arsenal in P. semoni compared to the other four species; this can be explained by the large number of toxin-like transcripts with homology to peptidase M12A.In order to summarise the distinct venom profile of each actiniarian species, toxin-like transcripts were categorised into toxin families and then assigned one of six biological functions: auxiliary, membrane-active, mixed function enzymes, neurotoxin, protease inhibitor and unknown . Toxin fD. cf. armata and M. doreensis compared to all other species. Only a single toxin-like transcript was found to be significantly differentially expressed  between inner and outer tentacles. The toxin-like transcript that was upregulated in the inner tentacles of D. cf. armata has high sequence similarity to nematocyst expressed protein 6 (K7Z9Q9), a member of the peptidase M12A toxin family , a boundless beta-hairpin type sea anemone neurotoxin . More toxin-like transcripts were differentially expressed between endocoelic tentacles and nematospheres (15) than between endocoelic tentacles and exocoelic tentacles (13). Overall, the highest number of differentially expressed toxin-like transcripts was observed when comparing the body column and nematospheres (24), and the lowest when comparing exocoelic tentacles and nematospheres (1).Cutting the dendrogram at 50% of its height led to partitioning of the toxin-like transcripts into five subclusters with the following expression patterns: upregulated in exocoelic tentacles and nematospheres ; upregulated in the body column and endocoelic tentacles ; upregulated in exocoelic tentacles and body ; upregulated in all tentacle types ; and upregulated in the body column .2 toxins were found in three subclusters, the most of any toxin family. Thus, most toxin families are associated with distinct expression patterns across the envenomating structures examined in H. hemprichii.When examining the subclusters in conjunction with the biological function of toxins, a pattern emerged in that a single functional category was generally not present in more than two subclusters. The exceptions to this were neurotoxins and mixed function enzymes, which were present in four and three subclusters, respectively. Interestingly, auxiliary function toxin-like transcripts were only found in cluster 5, while the two membrane-active toxin families were consistently upregulated across exocoelic tentacles and nematospheres. Further interrogating the differential expression data, we established that 5, 3, 2, 2, and 3 toxin protein families were found in subclusters 1, 2, 3, 4 and 5, respectively. Additionally, many toxin families were found to be isolated within a single subcluster. Sea anemone type 3 (BDS) potassium channel and phospholipase AD. cf. armata and M. doreensis are simple unbranched tentacles, differentiated only by size, indicating that morphologically similar tentacles have comparable toxin profiles. As different cnidae type are associated with different venom profiles in cnidarians [D. cf. armata and M. doreensis. However, more pronounced differences in gene expression are observed when substantial morphological variation exists within tentacles.Differential expression of toxins between discrete anatomical regions has been established in the actiniarian families Actiniidae, Actinodendridae and Stichodactylidae ,15. Likeidarians ,38, the H. hemprichii individual indicates that endocoelic tentacles, exocoelic tentacles and nematospheres are each associated with a unique toxin profile. Differences in toxin expression between endocoelic and the other tentacle types is more pronounced than between exocoelic tentacles and nematospheres. Tissue-specific toxin expression with similar profiles have been observed between functionally similar structures, such as acrorhagi and tentacles in A. tenebrosa [H. hemprichii.Members of Thalassianthidae possess three morphologically distinct types of tentacles: endocoelic tentacles, exocoelic tentacles and nematospheres . Endocoeenebrosa ,15. GiveBerghia stephania resulted in fully retracted tentacles being concealed by the sphincter [Anemonia sulcata due to anatomical differences [C. adhaesivum and H. hemprichii. However, it has been observed that the oral disc of C. adhaesivum expands and contracts in response to light conditions. When exposed to low light conditions, the oral disc folds in on itself, with the region bearing nematospheres curling towards the mouth and concealing the majority of endocoelic tentacles [The location of nematospheres and exocoelic tentacles at the oral disc margin may provide insight into some of the functional demands of these two tentacle types. Actiniarians are soft-bodied creatures that rely on venom for multiple ecological functions, including defence against predators ,25. Nevephincter ,39. Howeentacles . Therefoentacles .P. semoni and offer no protection against predators [P. semoni participates in predatory feeding behaviour at night when the tentacles are extended [P. semoni tentacle and tentacle-like structures can be explained by the discrete key roles of these structures in predatory and defensive behaviour, respectively. Thus, nematospheres and vesicles share a common functional role in defence, but these structures are taxonomically restricted within two different actiniarian superfamilies  [Similarly, vesicles and pseudotentacles in species from Aliciidae are predicted to have a major role in predator deterrence. During the day, tentacles are retracted in redators ,36. Convredators , these “extended ,36, so vctively) ,42 and lC. adhaesivum we find an enzymatic toxin family upregulated. Conversely, in H. hemprichii, membrane-active toxins and neurotoxins are the predominant functional classes of toxins significantly upregulated in the nematospheres compared to other structures. Variation in the cnidome of tentacular structures between species may account for these differences, with different cnidae types associated with different venom profiles in cnidarians [C. adhaesivum and H. hemprichii but vary in size, with the largest basitrichs observed in the nematospheres [C. adhaesivum but rare in H. hemprichii [C. adhaesivum relative to H. hemprichii [Phylogenetic analysis conducted by Crowther  demonstridarians ,38. Basiospheres ,43,44. Wmprichii ,44,45. Amprichii ,44. ThesPristhesancus plagipennis and the cone snail Conus geographus produce distinct venoms specialised for predation and defence [C. adhaesivum, H. hemprichii and P. semoni to possess morphologically diverse tentacular structures will provide insights into the functional basis of the tentacle-specific toxin expression profiles observed in the current study.Regionalisation of venom production has been observed in other venomous taxa, but in all instances there is an underlying functional basis for producing multiple venoms with distinct compositions. For example, the assassin bug  defence ,47. Diff defence . While e defence ,25,48,49H. hemprichii specimen used to generate the transcriptomic dataset had bleached, but the enrichment of photosynthetic-related gene GO terms in the nematospheres of C. adhaevisum supports the presence of zooxanthellae in this structure. The endoderm of tentacles in C. adhaevisum and H. hemprichii has previously been noted to contain a high proportion of zooxanthellae compared to other regions [P. semoni. Therefore, morphological diversity of tentacles supports hosting of endosymbionts in the families Thalassianthidae and Aliciidae.Crowther demonstrated that convergent morphological adaptations can be observed in distantly related sea anemone families due to similar ecological demands . Actinia regions . Zooxant regions  and photP. semoni and Actinodendron plumosum resulting in severe skin ulceration [P. semoni has been reported [C. adhaesivum, H. hemprichii and P. semoni, and thereby protects a dependable energy source. Thus, hosting zooxanthellae appears to underpin both the morphological variation in tentacular structures and the distinct toxin profiles associated with each tentacular subtype in the subtidal species examined.Defensive spheres have convergently evolved at least three times in Actiniaria , resulticeration ,54,55,56ceration ,58, whilreported . The potActinodendron species and the haloclavid genera Anemonactis and Haloclava [Telmatactis is defined by club-like swellings at the distal portion of tentacles [C. adhaesivum, H. hemprichii and P. semoni proved challenging and thus we were unable to verify findings across individuals from the same species. Future research should employ biological replicates to determine whether the tissue-specific toxin expression profiles associated with tentacular structures is subject to intraspecific variability. Furthermore, investigating the phylogenetic relationship of toxin families differentially expressed across tentacular structures may also provide greater insights into venom diversity within order Actiniaria.Acrospheres are swellings of the tentacles, characterised by a unique cnidome, which are present in orders Actiniaria and Corallimorpharia ,51,60,61aloclava ,51. Addientacles  and, whiHeterodactyla hemprichii, Cryptodendrum adhaesivum and Phyllodiscus semoni were collected by Cairns Marine Pty Ltd. from the Great Barrier Reef, QLD, Australia and housed in holding tanks at the Queensland University of Technology under standard aquarium conditions [H. hemprichii, tissue from the body column, endocoelic tentacles, exocoelic tentacles and nematospheres was isolated  and sequenced using 75 bp single-end chemistry on an Illumina NextSeq 500. Sequencing libraries for C. adhaesivum and P. semoni were prepared using TruSeq Stranded mRNA Library Prep  and sequenced using 150 bp paired-end chemistry on an Illumina HiSeq X10.Total RNA was extracted from homogenised tissue using the TRIzol/chloroform protocol  followedDofleinia cf. armata (identified by sea anemone taxonomist M.L.M.) was collected from Southport, QLD, Australia under Queensland Museum permit 160782. After a 72-h starvation period, inner and outer tentacles . Preserved tissue was lysed using a TissueLyser II  and total RNA extracted using a QIAGEN RNeasy Mini Kit. Extracted RNA with an integrity number (RIN) above 8.5 was used for library preparation. Individual cDNA libraries were prepared using PolyA-enriched RNA-seq stranded sample prep . Libraries were sequenced using 100 bp, paired-end sequencing on an Illumina HiSeq 2500. Preserved animals (QM G335858 & QM G335860) are lodged in the Queensland Museum. Full details of D. cf. armata extraction and sequencing methods have been published previously [entacles a from tweviously .Macrodactyla doreensis were collected from North Stradbroke Island, QLD, Australia under the permit QS2014/MAN259 and kept in aquaria at The University of Queensland. Tissue from inner and outer tentacles from two individual specimens was collected with tweezers and flash frozen before total RNA was extracted using TRIzol  and enriched for mRNA using a DynaBeads Direct mRNA kit  as described [Two specimens of escribed . cDNA lihttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 23 March 2018) and tissue-specific reads were pooled prior to transcriptome assembly for each species. Sequence reads were filtered , with non-biological sequences and low-quality reads removed using Trimmomatic [Raw reads were quality checked using FastQC  were removed using CD-Hit EST v4.6.4 . BUSCO vM. doreensis and species from the families Thalassianthidae (C. adhaesivum and H. hemprichii) and Aliciidae (P. semoni) are known to contain symbiotic dinoflagellate algae [https://github.com/sylvainforet/psytrans, accessed on 5 April 2017) was employed to remove potential symbiont contamination from the assembled transcriptomes of M. doreensis and H. hemprichii. Host reference sequences were derived from the Nematostella vectensis genome proteins models [Symbiodinium microadriaticum, S. minutum and S. kawagutii [te algae ,35,71. Ts models . The comawagutii ,74,75 se−5. Protein family (Pfam) domains were identified using HMMER 3.1b2 software [Functional annotation was conducted using the Trinotate protocol. Open reading frames (ORFs) encoding more than 65 amino acid residues were extracted from assemblies using TransDecoder 3.0.1 . To detesoftware . Using Tsoftware , GO termC. adhaesivum and P. semoni [Bowtie2 was used to align raw reads to assembled contigs , followe. semoni . Differe. semoni . Using t. semoni . These t. semoni .GO enrichment analysis was performed by running GOSeq on significantly differentially expressed transcripts . SignifiTranscripts with homology to known toxins were identified from BLASTp searches against the UniProt database by filtering for transcripts annotated with the ‘toxin activity’ GO term (GO:0090729), excluding bacterial toxins. To limit results to lineage-specific toxin-like transcripts, the translated ORFs generated by TransDecoder were then used as BLASTp queries against database of cnidarian toxins from ToxProt (accessed on 21 April 2021) using BLAST+ 2.3.0. Output from edgeR was then filtered to include only toxin-like transcripts and using Trinity PtR, heatmaps were generated and used to visualise differentially expressed toxin-like transcripts. Protein family designations for toxin-like transcripts were assigned using data available in the UniProt database and toxin families were further categorised by biological function as in previous work ,84.


Elizabethkingia meningosepticum (E.meningosepticum) is a ubiquitous microorganism previously known as Chryseobacterium meningosepticum. It is emerging as a pathogen responsible for bacteremia in the immunocompromised population, particularly in cancer patients and those with a history of prolonged hospital stay and frequent instrumentations. A retrospective chart review of all cases over 10 years at the Moffitt Cancer Center showed a total of three patients with E.meningosepticum infection. The first patient (history of multiple myeloma) underwent endoscopy complicated by aspiration pneumonia and positive blood culture for E.meningosepticum infection. The second patient (current acute myelogenous leukemia) had neutropenic fever in the setting of a recent chest port infection. Blood culture from the chest port showed E. meningosepticum. The third patient  had a history of recent pneumonia and cellulitis who presented with recurrent neutropenic fever. Blood culture was positive for E. meningosepticum. E.meningosepticum bacteremia has a high 28-day mortality rate (41%). As these three cases illustrate, early identification of the pathogen along with empiric treatment with a fluoroquinolone and/or minocycline is indicated to reduce morbidity and mortality. Elizabethkingia is ubiquitously found in nature and is not a part of the normal human flora ), and temperature of 35.9° C. Labs were notable for leukocytosis , pre-renal acute kidney injury (blood urea nitrogen (BUN) 21 mg/dL, creatinine 1.4 mg/dL), and respiratory acidosis . CT of the thorax, abdomen, and pelvis showed extensive consolidation predominantly in the left lung and the right lower lobe consistent with multifocal pneumonia . She was transitioned to ciprofloxacin, meropenem, and minocycline. She later developed neutropenic fever for which voriconazole and acyclovir were added. Repeat blood culture was negative and she was discharged to hospice after relapsed leukemia was noted per bone marrow biopsy after the initiation of cladribine-based salvage chemotherapy.A 50-year-old female with relapsed acute myelogenous leukemia (AML), previously treated with 7+3 induction therapy and high-dose cytarabine cycle for consolidation was admitted for cladribine-based salvage chemotherapy. She was recently hospitalized at another institute for a chest port infection. She was treated with intravenous vancomycin, but the port was not removed. At the time of admission, she was febrile (38.5° C), normotensive (118/73 mmHg), had tachycardic (119 bpm), and had a normal respiratory rate. Labs were notable for bicytopenia  and a leukocyte count of 4,050/mL. She was started on empiric antibiotics with meropenem, minocycline, and tobramycin with the removal of the chest port. Blood cultures were positive for Case 3Pseudomonas aeruginosa followed by mild pneumonia and right lower extremity cellulitis. He recently completed a treatment course of daptomycin. On examination, he was normotensive (BP 140/70 mmHg), had tachycardia (110 bpm), and had a normal respiratory rate with an oxygen saturation of 97% on room air. Physical examination was notable for decreased breath sounds and a right upper chest port with no evidence of infection. His labs were significant for pancytopenia : 1010/mL, hemoglobin: 8.7 g/dL, platelets: 13,000/mL), and acute kidney injury (AKI) (creatinine: 1.4 mg/dL). CT of the thorax showed clustered ground glass nodularity in the left lower lobe indicating focal infection, pulmonary consolidation and adjacent atelectasis of the left lower lobe, and a small left pneumothorax  and AML was treated with 5-azacytidine (Vidaza®). He was admitted with a new onset of productive cough with blood-tinged sputum for two days and pleuritic chest pain. His past infectious history was notable for pneumonia caused by Infection is a well-known complication in cancer patients that can lead to prolonged hospital stays. Severe sepsis, defined as a systemic inflammatory response leading to acute organ dysfunction, accounts for 16.4 cases per 1000 people with cancer per year .Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. This is followed by Gram-positive bacteria, most commonly from coagulase-negative Staphylococcus. One of the newer and rarer causes of infection is the Gram-negative rod Elizabethkingia meningosepticum [E. meningosepticum infections in hospitalized individuals with severe chronic diseases, especially in the past five years. This suggests that E. meningosepticum is emerging as an important pathogen in the immunocompromised population. A recent study noted 118 cases of various presentations of E. meningosepticum in Taiwan and suggested a strong connection of the infection with underlying comorbidities like malignancy (36%) and diabetes mellitus (25%) [Common causes of bloodstream infections in neutropenic patients include Gram-negative bacteria such as septicum . There ius (25%) .E. meningosepticum bacteremia was associated with nosocomial infections such as pneumonia, endocarditis, soft tissue infections, osteomyelitis, and catheter-related bacteremia [E. meningosepticum infection; however, this may also be related to their hematological malignancies and chemotherapy and not the ongoing infection  and class B metallo-β-lactamases (MBLs) rendering them resistant to all β-lactam including carbapenems, aztreonam, tigecycline, and polymyxin [Pseudomonas or Klebsiella infection, which was being treated with colistin prior to acquiring an E. meningosepticum infection. Prior treatment with colistin had a strong association with the emergence of E. meningosepticum bacteremia in these cases owing to their unusual patterns of resistance [The etiology of sepsis in neutropenic patients has shifted from Gram-positive to Gram-negative bacteremia over the past few decades. Multidrug-resistant, Gram-negative bacilli are becoming increasingly challenging to treat and therefore current guidelines recommend the use of an anti-pseudomonal beta-lactamase with or without an additional antimicrobial as the empiric therapy in Gram-negative bacteremia . E. menicteremia . Many Elolymyxin . Some pasistance . High lesistance .E.meningosepticum shows varied degrees of susceptibility to antibiotics used to treat Gram-positive infections including piperacillin-tazobactam, fluoroquinolones, trimethoprim-sulfamethoxazole (TMP-SMX), vancomycin, and rifampicin [E. meningosepticum making these the empirical therapy of choice [E.meningosepticum bacteremia had 100% susceptibility to minocycline and 74.4% susceptibility to TMP-SMX and ciprofloxacin [fampicin . This unf choice . The supf choice . Anotherfloxacin . All theE. meningosepticum bacteremia. It has been suggested that prolonged therapy with combinations of rifampin with vancomycin, TMP-SMX, minocycline, or fluoroquinolones may have better clinical outcomes [E. meningosepticum bacteremia in healthcare-associated bacteremia in adults with underlying conditions [Therefore, a combination therapy is recommended over monotherapy in multidrug-resistant outcomes . There inditions . Given tE. meningosepticum bacteremia in cancer patients with multiple comorbidities due to prolonged hospitalization and frequent instrumentation. In this report, we present three patients with a hematologic malignancy and neutropenia who developed E. meningosepticum infection. Given the challenging clinical course of these patients, it is important to recognize this pathogen as an important cause of bacteremia in immunocompromised patients. Early identification of the pathogen along with empiric treatment with a fluoroquinolone and/or minocycline is imperative to reduce morbidity and mortality.There is a rising trend of


Haynaldia villosa, a wild grass species with a proven potential for wheat improvement, confers resistance to multiple diseases. So, genome-wide identification of the NLR gene family in Haynaldia villosa by SMRT-RenSeq can facilitate disease resistance genes exploration.Nucleotide-binding and leucine-rich repeat (NLR) genes have attracted wide attention due to their crucial role in protecting plants from pathogens. SMRT-RenSeq, combining PacBio sequencing after resistance gene enrichment sequencing (RenSeq), is a powerful method for selectively capturing and sequencing full-length NLRs. NLR complement of H. villosa. In total, 1320 NLRs were annotated in 1169 contigs, including 772 complete NLRs. All the complete NLRs were phylogenetically analyzed and 11 main clades with special characteristics were derived. NLRs could be captured with high efficiency when aligned with cloned R genes, and cluster expansion in some specific gene loci was observed. The physical location of NLRs to individual chromosomes in H. villosa showed a perfect homoeologous relationship with group 1, 2, 3, 5 and 6 of other Triticeae species, however, NLRs physically located on 4VL were largely in silico predicted to be located on the homoeologous group 7. Fifteen types of integrated domains (IDs) were integrated in 52 NLRs, and Kelch and B3 NLR-IDs were found to have expanded in H. villosa, while DUF948, NAM-associated and PRT_C were detected as unique integrated domains implying the new emergence of NLR-IDs after H. villosa diverged from other species.In this study, SMRT-RenSeq was performed to identify the genome-wide NLR genes from wild species using the baits of the evolutionary related species with reference sequences. The availability of the NLRs from H. villosa provide a valuable library for R gene mining and transfer of disease resistance into wheat.SMRT-RenSeq is a powerful tool to identify The online version contains supplementary material available at 10.1186/s12864-022-08334-w. Plants have evolved comprehensive mechanisms to protect themselves from attack by pests and pathogens . The firR genes) defined by genetics have been cloned from a wide range of plant species [NLR repertoires in plants. Typically, several hundred NLRs are found in a plant genome. For example, 149 NLRs were identified in Arabidopsis [Vitis vinifera, 330 in Populus trichocarpa [Glycine max [Manihot esculenta [NLR complements have also been studied in five species of Brassicaceae [Gossypium [Leguminosae [Solanaceae [Sorghum bicolor, Zea mays, Brachypodium distachion, cultivated and wild Oryza species, and several Triticeae species [NLRs were identified, the largest number reported thus far in a plant species [Carica papaya  and 54b genome ;). Howevnd apple , 73.Triticeae genomes, including more efficient and complete characterization of their NLR complements. The NLR contents have been identified in T. urartu, Ae. tauschii and T. aestivum by different researchers [R gene enrichment and sequencing (RenSeq) method provides a powerful and attractive tool for the identification of NLRs from plants without finished reference genome assemblies, in particular those plants with large genomes and higher ploidy levels, for example hexaploid wheat and octaploid strawberry [Solanum tuberosum, and it indicated that ~ 80% sequence identity between NLR genes and the corresponding oligonucleotide baits was sufficient for enrichment. This pioneering use of RenSeq increased the number of annotated NLRs in potato from 438 to 772, and facilitated the genetic mapping of NLRs associated with disease resistance to poorly or previously unannotated regions of the genome [Sr22 and Sr45 from hexaploid wheat [Aegilops tauschii), AgRenSeq, facilitated the rapid identification four stem rust resistance genes [Sr22 and Sr33 resulted in two and three contigs, respectively, with missing gaps corresponding largely to the introns. RenSeq combined with long read sequencing, such as PacBio single-molecule real-time (SMRT) or Oxford Nanopore Technology, mitigates these limitations by generating more complete assemblies including NLRs with novel integrated domains [Phytophothora infestans resistance gene Rpi-amr3i from the wild potato relative Solanum americanum [Potato Virus Y resistance genes RySto from the wild potato relative Solanum stoloniferum [Pm21 from the wild wheat relative Haynaldia villosa [Arabidopsis thaliana [With the recent rapid advances in bioinformatics and genomics, huge progress has been made in understanding earchers , 69. Howearchers , or a spearchers . The hige genome . MutRenSid wheat . More re domains , 76. SMRoniferum , the pow villosa , and a sthaliana .Haynaldia villosa  is a wild diploid species of wheat, which has been introduced into hexaploid bread wheat by the development of individual chromosome arm translocation lines between H. villosa and wheat. We previously reported the introgression from H. villosa into wheat of the powdery mildew resistance genes Pm21, Pm55 and Pm62, the yellow virus resistance gene Wss1 and the cereal cyst nematode resistance gene CreV [H. villosa chromatin in wheat have also been reported [Pm21 has been widely used in wheat breeding and more than 40 new varieties have been released and cultivated in regions of China where powdery mildew is prevalent. Pm55 and Wss1 have also been used in breeding programs and new wheat lines are currently under evaluation. Cloning of these resistance genes will accelerate their use by genetic engineering to reduce linkage drag. However, the lack of a reference genome and a high level of outcrossing of H. villosa makes resistance gene cloning in this species challenging.Two sequential polyploidization events followed by domestication and intensive breeding have narrowed the genetic diversity in cultivated wheat , 90. Thiene CreV , 83–85. reported , 52, 59.H. villosa accession 91C43. Then, NLR-Parser [https://github.com/steuernb/NLR-Annotator). The improved software differs from NLR-Parser in that it can distinguish the border between different NLRs located in long contigs [H. villosa, followed by determination of the NLR classes, assignment of NLRs to chromosomes, exploration of NLRs orthologous to cloned R genes, identification of NLR-IDs and comparison of NLRs among different Triticeae species. Our study provides a valuable resource to support R gene cloning in H. villosa and in H. villosa-wheat introgression lines.Previously, we used SMRT-RenSeq to de novo assemble the NLRs of the inbred R-Parser  was usedR-Parser . In 2018 contigs . Since m contigs , we hypoPreviously, 406 Mb of SMRT-RenSeq data consisting of 107,153 reads with an average length of 4.5 kb were generated and de novo assembled with Geneious v9.1.4 Fig. S. The datAe. tauschii using BLASTx. Then, the predicted H. villosa NLR protein sequences were obtained using FGENESH+ based on the homologous proteins. The 776 complete NLR protein candidates were analyzed by Plant_rgene to search for coiled-coil (CC), NB-ARC and integrated domains, then re-analyzed by InterProScan to search for the conserved LRR domains deposited in the SUPERFAMILY database. The results indicated that 772 members carried an NB-ARC, so these 772 candidates were considered to be bona fide NLRs. Then the 772 NLR proteins were further divided into five subclasses based on sub-domain analysis; 618 NLRs were typed as CC-NB-LRR (CNL), 98 as NB-LRR (NL), three as CC-NB (CN), one as NB-ARC (N) and 52 as NLRs with integrated domains (NLR-ID) , followed by the V genome with 772 NLRs  and B. distachyon (350 NLRs), suggesting that Triticeae species have experienced NLR expansion more rapidly after diverging from O. sativa and B. distachyon. An NLR expansion in the A and D genome was also observed after polyploidization of wheat . The conserved motifs from each complete NLR were displayed in the phylogenetic tree to track the evolutionary characteristics of each clade; 11 main clades were thus derived and displayed in different colors. The structure and characteristics of each clade was analyzed according to the motif classification by Jupe [All 772 NLRs of  by Jupe . Clade A by Jupe . In this by Jupe . AnotherH. villosa with PacBio SMRT chemistry. To detect expressed NLRs and to verify the predicted exon/intron boundaries, the 772 NLRs were searched against NLRs annotated in the transcriptome database. This revealed 91 NLRs with a transcript with more than > 95% identity and a query coverage > 90%. Sequence alignment between the transcript and the genomic DNA of these 91 NLRs indicated that the splicing pattern of 85 of the NLRs perfectly matched our predictions. The remaining 6 expressed NLRs, whose splicing patterns didn’t match with the predictions, were used to test whether specific splicing patterns happened in H. villosa by comparing their splicing patterns with those of the corresponding orthologous NLRs. The comparisons indicated that there were no specific splicing patterns happened in 3 NLRs, including Hv_Contig_1470_nlr_1, Hv_Contig_696_nlr_1 and Hv_Contig_1466_nlr_1, while the coding lengths were different between the predicted sequences and the expressed sequences. For Hv_Contig_249_nlr_2, its orthologous NLRs in wheat barley and Ae. tauschii contained A and B types of splicing patterns, and the previously predicted CDS was corresponding to the A type, while the expressed CDS was corresponding to the B type. So, there was no specific splicing pattern in Hv_Contig_249_nlr_2. However, the specific splicing patterns happened in Hv_Contig_670_nlr_1 and Hv_Contig_61_nlr_1. Knowledge of NLR expression can provide additional powerful support for R gene cloning.We sequenced a full-length transcriptome of H. villosa displays good chromosome collinearity with barley, Ae. tauchii, Triticum urartu and wheat. Therefore the nucleotide sequences of the 772 complete NLRs were used as a query for BLASTn analysis against the genome databases of these species for in silico prediction of chromosomal location. This procedure assigned all 772 complete NLRs to homoelogous chromosomes; 139 NLRs to group 1, 85 to group 2, 164 to group 3, 15 to group 4, 60 to group 5, 101 to group 6, and 208 to group 7 -markers based on single copy genes [ villosa , to physpy genes . The higH. villosa and other Triticum species  NLRs could be located to specific chromosome arms Fig. ; Table 2of wheat . Therefoof wheat , suggestMla genes were added manually. To test the efficiency of enrichment, the orthologous H. villosa Mla genes were obtained from our H. villosa NLR assembly. In total, 18 NLRs in H. villosa were predicted to be orthologous to Mla genes because they showed the highest homology to NLRs at the barley Mla locus at 30.2 Mb on chromosome 1H , and those proteins with IDs were then classified to be NLR-IDs . Among tAs shown by phylogenetic analysis based on all the NLRs in Fig. H. villosa was also conducted as shown in Fig. https://itol.embl.de/shared/2018201031). Most of the NLR-IDs with the same IDs could be phylogenetically clustered, especially those NLR-IDs containing DDE, B3 and Kelch clustered into large groups, respectively. The tightly clustered NLR-IDs with similar domain compositions in the tree were usually in silico located on the same chromosomes, indicating that these members likely expanded by tandem duplication after the NLR fused with the ID. There are exceptions, for example, NLR-IDs with Pkinase were clustered into different groups indicating independent cases of fusion. The direct evidence is that the tightly grouped NLR-IDs, Hv-contig-705, Hv-contig-869 and Hv-contig-1233, contained a Pkinase integrated into the CC-terminal, while Hv-contig-316 and Hv-contig-317 contained a Pkinase integrated into the LRR-terminal .The NLR-IDs were extracted from another six es Table . The numva Table . Among tNLR-IDs with physically tightly linked NLRs often form paired protein complexes. In this study, we found that nine NLR-IDs were located tandemly with another NLRs in the same contig, and in two contigs, namely Hv_Contig_48 and Hv_Contig_1157, NLR-IDs and its linked NLRs were arranged in a head-to-head pattern.We found that the homeologues of paired NLRs, such as RPS4/RRS1, Rpg5/RGA1, and RGA4/RGA5, usually displayed a pattern of head-to-head arrangement. Solanum tuberosum, a wild potato, indicated that ~ 80% sequence identity is enough for capturing NLR genes using oligonucleotide baits [H. villosa and barley and found a high degree of identity between these two closely related species [H. villosa. The enrichment efficiency analysis using the sequences of cloned R genes indicated that the homologous genes in H. villosa were successfully enriched. Therefore, the bait library designed based on a species with ample genomic resources could be used to isolate NLRs from an evolutionary closely related species.NLRs display high domain-conservation even across species. Previously, random isolation of NLRs from whole genomes was achieved using homology-based PCR in wide variety of species . In recede baits . Previou species . We therBefore annotation of the NLRs from the assembled contigs, the redundant contigs were removed using the cutoff value of 90 and 95%. Actually, the same 53 contigs were removed by both parameters. To avoid the situation that a recent duplication or residual heterozygosity perhaps could be ruled out as a source for some of the removed contigs, these 53 removed contigs were reanalyzed Table S. It was H. villosa using different reference assemblies and different pipelines, such as 463, 570, 563 or 558 NLRs in T. urartu, 842 or 738 NLRs in A. tauschii, 420, 336 or 462 NLRs in H. vulgare, 422 or 341 NLRs in S. bicolor, and 470 or 438 in S. italica [B. distachyon, 347 from O. sativa, 397 from H. vulgare, 530 from T. urartu, 572 from A. tauschii and 772 from H. villosa by the same pipeline. H. villosa contained relatively more NLRs than what has been reported in most diploid genomes. The study in Solanum tuberosum indicated that more NLRs were revealed by RenSeq than those predicted by the then available annotation software and genome reference suggesting that RenSeq facilitates the recovery of NLRs from the poorly or previously unannotated regions of the genome [H. villosa, might be accounted for by the NLR expansion that occurred at some loci.NLR complements have been identified in several diploid species related to  italica , 63. In Some of the in silico located NLRs were selected for physical location to specific chromosome arms, and good congruency was found for chromosomes 1 V, 2 V, 3 V, 5 V and 6 V suggesting these NLRs could be used as candidates for mining for R genes genetically assigned to these chromosomes. However, some of the NLRs, which were in silico located on 7 VS were physically located on 4VL. Therefore, NLRs in silico located on 7 VS could be used as candidates for mining for R genes which map to 4VL. Chromosomal rearrangements are commonly found in wheat and its wild relatives , and ourAvrPto injected into the host cell by the bacterial pathogen Pseudomonas syringae. The partner of Pto, Prf, an NLR receptor, senses the modification of Pto by AvrPto to trigger immune signaling [A. thaliana, carries a C-terminal WRKY domain which mimics the WRKY protein targeted by the PopP2 effector. Other examples include the NLRs RGA5 and Pik-1 in rice, which carry a heavy metal associated (HMA) domain targeted by AVR-Pia and AVR-Pik, respectively [In host-pathogen interactions, a decoy is used to describe those molecules which mimic a component manipulated during infection . The decignaling . Sometimectively , 53, 82.ectively .H. villosa. While the NLR-ID ratio was smaller in H. villosa compared to T. urartu and Ae. tauschii, the total number of NLR-IDs in H. villosa was the largest among all the seven species studied , among which Kelch and B3-type NLR-IDs experienced expansion, and three type of IDs were unique in H. villosa. This study gave an example to successfully capture the genome-wide NLRs in wild species using the baits from another species in Triticeae. The availability of the NLRs from H. villosa provides a valuable library for mining and transferring of disease resistance into wheat.In this study, the genome-wide Haynaldia villosa , with long hairs on the keel of the glume and apex of the lemma, is an annual diploid species which belonging to Triticinae of Triticeae in Gramineae. It is a vigorous ruderal wild plant growing on the harsh, moisture-stressed soils in the northeastern part of the Mediterranean region and Caucasus area [H. villosa accession 91C43 was obtained from Cambridge University, UK and propagated by artificial bagging and self-pollination for 6–7 successive generations to obtain an inbred line. Tissue from the 91C43 inbred line was used for DNA extraction. This accession was also used as the donor to develop 14 wheat-H.villosa translocation lines through wide-crosses and chromosome engineering, each involving one of the 14 chromosome arms of H. villosa, respectively [sus area . This spsus area . The H. ectively . This seH. villosa accession 91C43. The design of this NLR bait library has been previously described [Mla, Mlo, Rpg1, etc., were manually added. Repetitive and redundent sequences were removed to yield the final set of 99,421,100-mer RNA baits.The barley NLR bait library TSLMMHV1  was usedescribed , a tool escribed , was useescribed  . SMRT library preparation and NLR capture followed the procedure described by Witek et al. [Genomic DNA for SMRT library preparation was extracted from seedlings of k et al. . The enrwww.geneious.com) with defaults of 1% mismatch, 1% gap (no more than 3 bp) and minimum read length overlap greater than 100 nt with at least 98% identity. Only contigs assembled from at least five sequences and having a minimum coverage of two were considered for further analysis. A total of 1509 contigs fulfilling these criteria were obtained.The raw reads were screened by the SMRT Portal software with parameter settings > 3 full passes and > 90% accuracy to generate the inserted sequence ROI. The long ROI were analyzed and assembled using genetic R8 software (https://github.com/steuernb/NLR-Annotator).To obtain non-redundant NLR-containing contigs, the raw assembled contigs were analysed by the global alignment program Cd-hit (version 4.8.1)  to constftp://ftp.ensemblgenomes.org/pub/plants/release-46/fasta/hordeum_vulgare), Aegilops tauchii (ftp://ftp.ensemblgenomes.org/pub/plants/release-46/fasta/aegilops_tauschii), and common wheat (ftp://ftp.ensemblgenomes.org/pub/plants/release-46/fasta/triticum_aestivum) to find the orthologous genes from the three species. In each species, the proteins with the highest identity and longest coverage were selected as the putative orthologous proteins. The protein sequence from the species with the highest identity and longest coverage compared with H. villosa was used as the reference to predict the coding sequence of the corresponding H. villosa NLR using FGENESH+ (http://www.softberry.com/).The predicted NLR loci were extended backwards by 3000 bp from the left border and forwards by 3000 bp from the right border, respectively. The extended sequences were cut out for BLASTx against the database of barley  and the longest proteins corresponding to the longest transcripts of genes with alternative splicing were used for annotation analysis (Table Shttps://github.com/krasileva/plant_rgenes) and InterProScan (interproscan-version 5.30–69.0) were used to analyze the NLRs annotated as being complete. Plant_rgene program is a pipeline for analyzing the domain composition of putative NLRs, however, sometimes it cannot accurately output LRR domains. The InterProScan software [H. villosa NLRs and the proteomes from the six species listed above, were analyzed by the Plant_rgene software  to search for conserved domains . Then the same set of proteins were analyzed again by InterProScan to search for the conserved LRR domains deposited in the SUPERFAMILY database. The conserved domains output from both programs were integrated. Only those proteins containing an NB-ARC domain were designated as NLRs. If any atypical domain was additionally predicted in the NLRs, these domains were considered to be the integrated-decoys (IDs), and those NLRs containing ID(s) were then designated as NLR-IDs. The diagrams of all the 52 NLR-IDs were displayed by IBS (Version 1.0.3) [The protein sequences of six species, including s Table S. Plant_rn 1.0.3) .H. villosa and all the 315 NLR-IDs from seven Triticeae species were also constructed by the same method using the same parameters.For construction of the phylogenetic tree of all the 772 complete NLRs, the conserved NB-ARC domain was extracted from each NLR and a phylogenetic tree was constructed using MEGA7 by Neighbor-Joining method with 1000 bootstrap replicates . The treR genes, including Mla1 (AY009939.1), Sr50 (KT725812.1), Sr35 (KC573058.1), Pm3b (AY325736.1), RCR1 (KU161103.1), Lr1 (EF439840.1) and Yr7 (MN273771.1) were downloaded from the NCBI database, then the nucleotide sequences were used to perform a sequence alignment with the assembled SMRT RenSeq contigs. The sequences showing > 80% identity and > 60% coverage with the cloned R genes were screened from the annotated NLRs. Then the screened NLRs in silico located to the same homoeologous chromosome loci as the corresponding queries were considered as their putative orthologues respectively.The cloned ftp://ftp.ensemblgenomes.org/pub/plants/release-46/fasta/hordeum_vulgare), Aegilops tauchii (ftp://ftp.ensemblgenomes.org/pub/plants/release-46/fasta/aegilops_tauschii), Triticum urartu (ftp://ftp.ensemblgenomes.org/pub/plants/release-46/fasta/triticum_urartu) and common wheat (ftp://ftp.ensemblgenomes.org/pub/plants/release-46/fasta/triticum_aestivum) to locate NLRs to chromosomes. Then, a subset of the NLRs were selected to determine the physical location by PCR analysis using 14 wheat-H. villosa translocation lines each involving one of the 14 chromosome arms of H. villosa. The PCR primers were designed according to specific deletions or insertions in the H. villosa NLRs when compared to the presumed orthologous genes in Chinese Spring . The mixed tissue sample included seedling leaves, seedling stems, seedling roots, immature spikes, seedling leaves inoculated with Bgt for 6 and 24 h, seedling leaves and roots treated with 200 mM NaCl for 3 days, and seedling leaves and roots treated with 16% PEG4000 for 3 days. The PacBio trasncriptome sequences were screened by NLR-Annotator. Then the annotated NLRs of SMRT RenSeq were used to search against the annotated transcribed NLRs. When an NLR from SMRT RenSeq could find a corresponding transcribed NLR with an identity higher than 95% and coverage longer than 90%, then the enriched NLR was considered to be expressed. The sequences of all the expressed NLRs were compared with their genomic counterparts one by one to analyze the accuracy of the protein prediction based on the genomic sequence.PacBio sequencing of the full-length transcriptome extracted from ten mixed tissues of Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.Additional file 6.Additional file 7.Additional file 8.