Basis Of Pathogenicity Research Articles

Genome characterization of Shewanella algae in Hainan Province, China.

Shewanella algae is an emerging marine zoonotic pathogen. In this study, we first reported the Shewanella algae infections in patients and animals in Hainan Province, China. Currently, there is still relatively little known about the whole-genome characteristics of Shewanella algae in most tropical regions, including in southern China. Here, we sequenced the 62 Shewanella algae strains isolated from Hainan Province and combined with the whole genomes sequences of 144 Shewanella algae genomes from public databases to analyze genomic features. Phylogenetic analysis revealed that Shewanella algae is widely distributed in the marine environments of both temperate and tropical countries, exhibiting close phylogenetic relationships with genomes isolated from patients, animals, and plants. Thereby confirming that exposure to marine environments is a risk factor for Shewanella algae infections. Average nucleotide identity analysis indicated that the clonally identical genomes could be isolated from patients with different sample types at different times. Pan-genome analysis identified a total of 21,909 genes, including 1,563 core genes, 8,292 strain-specific genes, and 12,054 accessory genes. Multiple putative virulence-associated genes were identified, encompassing 14 categories and 16 subcategories, with 171 distinct virulence factors. Three different plasmid replicon types were detected in 33 genomes. Eleven classes of antibiotic resistance genes and 352 integrons were identified. Antimicrobial susceptibility testing revealed a high resistance rate to imipenem and colistin among the strains studied, with 5 strains exhibiting multidrug resistance. However, they were all sensitive to amikacin, minocycline, and tigecycline. Our findings clarify the genomic characteristics and population structure of Shewanella algae in Hainan Province. The results offer insights into the genetic basis of pathogenicity in Shewanella algae and enhance our understanding of its global phylogeography.

A nonsense variant in KRT31 is associated with autosomal-dominant monilethrix.

Monilethrix is a rare hereditary hair disorder that is characterised by a beaded hair shaft structure and increased hair fragility. Patients may also present with keratosis pilaris and nail changes. Research has identified three genes for autosomal-dominant monilethrix (KRT81, KRT83, and KRT86), and one gene for the autosomal-recessive form (DSG4). To investigate the genetic basis of autosomal-dominant monilethrix in families with no pathogenic variants in any of the known monilethrix genes, and to understand the mechanistic basis of variant pathogenicity using a cellular model. Nine affected individuals from four unrelated families were included in this study. A clinical diagnosis of monilethrix was assigned based on clinical examination and/or trichoscopy. Exome sequencing (ES) was performed in six individuals to identify pathogenic variants, and Sanger sequencing was used for co-segregation and haplotype analyses. Cell culture experiments (immunoblotting, immunofluorescence, and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analyses) were used to confirm variant pathogenicity, to determine expression and subcellular localisation of proteins, and to identify a possible nonsense-mediated mRNA decay. In six affected individuals with clinically suggested monilethrix, ES led to the identification of the nonsense variant c.1081G>T; p.(Glu361*) in KRT31, which was subsequently identified in other affected members of these families by Sanger sequencing. This variant led to the abolition of both the last three amino acids of the 2B subdomain and the complete C-terminal tail domain of keratin 31. Immunoblotting demonstrated that when co-expressed with its binding partner keratin 85, the truncated keratin 31 was still expressed, albeit less abundantly than the wild type protein. Immunofluorescence revealed that p.(Glu361*) keratin 31 had an altered cytoskeletal localisation and formed vesicular-like structures in the cell cytoplasm near the cell membrane. RT-qPCR analysis did not generate evidence for a nonsense mediated decay of the mutant transcript. This study is the first to identify pathogenic variants in KRT31 as a cause of autosomal-dominant monilethrix. This highlights the importance of hair keratin proteins in hair biology, and will increase the molecular diagnostic yield for rare ectodermal phenotypes of hair and nail tissues.

Disruption of a DNA G-quadruplex causes a gain-of-function SCL45A1 variant relevant to developmental disorders.

SLC45A1 encodes a glucose transporter protein highly expressed in the brain. Mutations in SLC45A1 may lead to neurological diseases and developmental disorders, but its exact role is poorly understood. DNA G-quadruplexes (DNA G4s) are stable structures formed by four guanine bases and play a role in gene regulation and genomic stability. Changes in DNA G4s may affect brain development and function. The mechanism linking alterations in DNA G-quadruplex structures to SLC45A1 pathogenicity remains unknown. In this study, we identify a functional DNA G-quadruplex and its key binding site on SLC45A1 (NM_001080397.3: exon 2: c.449 G>A: p.R150K). This variant results in the upregulation of mRNA and protein expression, which may lead to intellectual developmental disorder with neuropsychiatric features. Mechanistically, the mutation is found to disrupt DNA G-quadruplex structures on SLC45A1, leading to transcriptional enhancement and a gain-of-function mutation, which further causes increased expression and function of the SLC45A1 protein. The identification of the functional DNA G-quadruplex and its effects on DNA G4s may provide new insights into the genetic basis of SLC45A1 pathogenicity and highlight the importance of DNA G4s of SLC45A1 in regulating gene expression and brain development.

Revealing the genome of the microsporidian Vairimorpha bombi, a potential driver of bumble bee declines in North America.

Pollinators are vital for food security and the maintenance of terrestrial ecosystems. Bumblebees are important pollinators across northern temperate, arctic, and alpine ecosystems, yet are in decline across the globe. Vairimorpha bombi is a parasite belonging to the fungal class Microsporidia that has been implicated in the rapid decline of bumblebees in North America, where it may be an emerging infectious disease. To investigate the evolutionary basis of pathogenicity of V. bombi, we sequenced and assembled its genome using Oxford Nanopore and Illumina technologies and performed phylogenetic and genomic evolutionary analyses. The genome assembly for V. bombi is 4.73 Mb, from which we predicted 1,870 protein-coding genes and 179 tRNA genes. The genome assembly has low repetitive content and low GC content. V. bombi's genome assembly is the smallest of the Vairimorpha and closely related Nosema genera, but larger than those found in the Encephalitozoon and Ordospora sister clades. Orthology and phylogenetic analysis revealed 18 core conserved single-copy microsporidian genes including the histone acetyltransferase (HAT) GCN5. Surprisingly, V. bombi was unique to the microsporidia in not encoding the second predicted HAT ESA1. The V. bombi genome assembly annotation included 265 unique genes (i.e. not predicted in other microsporidia genome assemblies), 20% of which encode a secretion signal, which is a significant enrichment. Intriguingly, of the 36 microsporidian genomes we analyzed, 26 also had a significant enrichment of secreted signals encoded by unique genes, ranging from 6 to 71% of those predicted genes. These results suggest that microsporidia are under selection to generate and purge diverse and unique genes encoding secreted proteins, potentially contributing to or facilitating infection of their diverse hosts. Furthermore, V. bombi has 5/7 conserved spore wall proteins (SWPs) with its closest relative V. ceranae (that primarily infects honeybees), while also uniquely encoding four additional SWPs. This gene class is thought to be essential for infection, providing both environmental protection and recognition and uptake into the host cell. Together, our results show that SWPs and unique genes encoding a secretion signal are rapidly evolving in the microsporidia, suggesting that they underpin key pathobiological traits including host specificity and pathogenicity.

Structural implications of amyloidogenic rare variants Ser282Leu and Gln356Arg identified in h-BRCA1.

Preliminary studies have shown BRCA1 (170-1600) residues to be intrinsically disordered with unknown structural details. However, thousands of clinically reported variants have been identified in this central region of BRCA1. Therefore, we aimed to characterize h-BRCA1(260-553) to assess the structural basis for pathogenicity of two rare missense variants Ser282Leu, Gln356Arg identified from the Indian and Russian populations respectively. Small-angle X-ray scattering analysis revealed WT scores Rg -32 Å, Dmax -93 Å, and Rflex-51% which are partially disordered, whereas Ser282Leu variant displayed a higher degree of disorderedness and Gln356Arg was observed to be aggregated. WT protein also possesses an inherent propensity to undergo a disorder-to-order transition in the presence of cruciform DNA and 2,2,2-Trifluoroethanol (TFE). An increased alpha-helical pattern was observed with increasing concentration of TFE for the Gln356Arg mutant whereas Ser282Leu mutant showed significant differences only at the highest TFE concentration. Furthermore, higher thermal shift was observed for WT-DNA complex compared to the Gln356Arg and Ser282Leu protein-DNA complex. Moreover, mature amyloid-like fibrils were observed with 30 μM thioflavin T (ThT) at 37°C for Ser282Leu and Gln356Arg proteins while the WT protein exists in a protofibril state as observed by TEM. Gln356Arg formed higher-order aggregates with amyloidogenesis over time as monitored by ThT fluorescence. In addition, computational analyses confirmed larger conformational fluctuations for Ser282Leu and Gln356Arg mutants than for the WT. The global structural alterations caused by these variants provide a mechanistic approach for further classification of the variants of uncertain clinical significance in BRCA1 into amyloidogenic variants which may have a significant role in disease pathogenesis.

Isolation, identification, molecular and pathogenicity characteristics of an infectious laryngotracheitis virus from Hubei province, China

Multiple outbreaks of avian infectious laryngotracheitis (ILT) in chickens, both domestically and internationally, have been directly correlate to widespread vaccine use in affected countries and regions. Phylogenetic and recombination event analyses have demonstrated that avian infectious laryngotracheitis virus (ILTV) field strains are progressively evolving toward the chicken embryo-origin (CEO) vaccine strain. Even with standardized biosecurity measures and effective prevention and control strategies implemented on large-scale farms, continuous ILT outbreaks result in significant economic losses to the poultry industry worldwide. These outbreaks undoubtedly hinder efforts to control and eradicate ILTV in the future. In this study, an ILTV isolate was successfully obtained by laboratory PCR detection and virus isolation from chickens that exhibited dyspnea and depression on a broiler farm in Hubei Province, China. The isolated strain exhibited robust propagation on chorioallantoic membranes of embryonated eggs, but failed to establish effective infection in chicken hepatocellular carcinoma (LMH) cells. Phylogenetic analysis revealed a unique T441P point mutation in the gJ protein of the isolate. Animal experiments confirmed the virulence of this strain, as it induced mortality in 6-wk-old chickens. This study expands current understanding of the epidemiology, genetic variations, and pathogenicity of ILTV isolates circulating domestically, contributing to the elucidate of ILTV molecular basis of pathogenicity and development of vaccine.

Genomic and Transcriptomic Survey Provides Insights into Molecular Basis of Pathogenicity of the Sunflower Pathogen Phoma macdonaldii

Phoma macdonaldii (teleomorph Leptosphaeria lindquistii) is the causal agent of sunflower (Helianthus annuus L.) black stem. In order to investigate the molecular basis for the pathogenicity of P. ormacdonaldii, genomic and transcriptomic analyses were performed. The genome size was 38.24 Mb and assembled into 27 contigs with 11,094 putative predicted genes. These include 1133 genes for CAZymes specific for plant polysaccharide degradation, 2356 for the interaction between the pathogen and host, 2167 for virulence factors, and 37 secondary metabolites gene clusters. RNA-seq analysis was conducted at the early and late stages of the fungal spot formation in infected sunflower tissues. A total of 2506, 3035, and 2660 differentially expressed genes (DEGs) between CT and each treatment group (LEAF-2d, LEAF-6d, and STEM) were retrieved, respectively. The most significant pathways of DEGs from these diseased sunflower tissues were the metabolic pathways and biosynthesis of secondary metabolites. Overall, 371 up-regulated DEGs were shared among LEAF-2d, LEAF-6d, and STEM, including 82 mapped to DFVF, 63 mapped to PHI-base, 69 annotated as CAZymes, 33 annotated as transporters, 91 annotated as secretory proteins, and a carbon skeleton biosynthetic gene. The most important DEGs were further confirmed by RT-qPCR. This is the first report on the genome-scale assembly and annotation for P. macdonaldii. Our data provide a framework for further revealing the underlying mechanism of the pathogenesis of P. macdonaldii, and also suggest the potential targets for the diseases caused by this fungal pathogen.

In situ architecture and membrane fusion of SARS-CoV-2 Delta variant

Among the current five Variants of Concern, infections caused by SARS-CoV-2 B.1.617.2 (Delta) variant are often associated with the greatest severity. Despite recent advances on the molecular basis of elevated pathogenicity using recombinant proteins, the architecture of intact Delta virions remains veiled. Moreover, pieces of molecular evidence for the detailed mechanism of S-mediated membrane fusion are missing. Here, we showed the pleomorphic nature of Delta virions from electron beam inactivated samples and reported the insitu structure and distribution of S on the authentic Delta variant. We also captured the virus-virus fusion events, which provided pieces of structural evidence for Delta's attenuated dependency on cellular factors for fusion activation, and proposed a model of S-mediated membrane fusion. Besides, site-specific glycan analysis revealed increased oligomannose-type glycosylation of native Delta S than that of the WT S. Together, these results disclose distinctive factors of Delta being the most virulent SARS-CoV-2 variant.

PPNet: Identifying Functional Association Networks by Phylogenetic Profiling of Prokaryotic Genomes.

Identification of microbial functional association networks allows interpretation of biological phenomena and a greater understanding of the molecular basis of pathogenicity and also underpins the formulation of control measures. Here, we describe PPNet, a tool that uses genome information and analysis of phylogenetic profiles with binary similarity and distance measures to derive large-scale bacterial gene association networks of a single species. As an exemplar, we have derived a functional association network in the pig pathogen Streptococcus suis using 81 binary similarity and dissimilarity measures which demonstrates excellent performance based on the area under the receiver operating characteristic (AUROC), the area under the precision-recall (AUPR), and a derived overall scoring method. Selected network associations were validated experimentally by using bacterial two-hybrid experiments. We conclude that PPNet, a publicly available (https://github.com/liyangjie/PPNet), can be used to construct microbial association networks from easily acquired genome-scale data. IMPORTANCE This study developed PPNet, the first tool that can be used to infer large-scale bacterial functional association networks of a single species. PPNet includes a method for assigning the uniqueness of a bacterial strain using the average nucleotide identity and the average nucleotide coverage. PPNet collected 81 binary similarity and distance measures for phylogenetic profiling and then evaluated and divided them into four groups. PPNet can effectively capture gene networks that are functionally related to phenotype from publicly prokaryotic genomes, as well as provide valuable results for downstream analysis and experiment testing.

Drosophila melanogaster larvae are tolerant to oral infection with the bacterial pathogen Photorhabdus luminescens.

The fruit fly Drosophila melanogaster is an excellent model for dissecting the molecular and functional bases of bacterial pathogenicity and host antibacterial immune response. The Gram-negative bacterium Photorhabdus luminescens is an insect-specific pathogen that forms a mutualistic relationship with the entomopathogenic nematode Heterorhabditis bacteriophora . Here we find that oral infection of D. melanogaster larvae with P. luminescens moderately reduces their survival ability while the bacteria replicate efficiently in the infected insects. This information will contribute towards understanding host gut immunity against potent bacterial pathogens.

The nematode parasite Steinernema hermaphroditum is pathogenic to Drosophila melanogaster larvae without activating their immune response.

Entomopathogenic nematodes are commonly used to control insect pest populations in the field. They also contribute substantially to understanding the molecular basis of nematode pathogenicity and insect anti-nematode immunity. Here, we tested the effect of the entomopathogenic nematode Steinernema hermaphroditum on the survival and immune signaling regulation of Drosophila melanogaster wild type larvae. Our results indicate that S. hermaphroditum infective juveniles are pathogenic toward D. melanogaster larvae, but they fail to activate certain immune pathway readout genes. These findings imply that S. hermaphroditum employs mechanisms that allow these parasitic nematodes to interfere with the D. melanogaster immune system.

Study of the effect of the combined fungicide Abacus Ultra on the population structure of the wheat leaf rust pathogen

Aim. To study the variability of the intrapopulation structure of the causative agent of wheat leaf rust (Puccinia triticina Erikss.) on the basis of pathogenicity under the influence of the combined fungicide Abacus Ultra, SE.Material and Methods. The studies were carried out in the greenhouse complex of the Federal Research Center of Biological Plant Protection on the winter wheat variety Krasnodarskaya 99 which is susceptible to leaf rust. The material studied was the combined fungicide Abacus Ultra, SE with application rates of 0.75; 1.0; 1.25; 1.5; 1.75 l/ha. The work was carried out according to generally accepted methods.Results. The influence of the impact of different rates of application of the fungicide Abacus Ultra, SE on the average virulence of the P. triticina population was established, this indicator varying from 40.0% (control [without treatment]) to 19.5% (fungicide application rate 1.75 l/ha). As a result of the analysis of the phenotypic composition of the population, 5 phenotypes were identified. A decrease in the viability of spores of the fungus P. triticina with an increase in the rate of application of the fungicide was established, this indicator varying from 69.1% (0.75 l/ha) to 29.2% (1.75 l/ha). A decrease in the aggressiveness of the population of the causative agent of leaf rust with an increase in the rate of application of the drug was established. The biological effectiveness of the fungicide Abacus Ultra, SE against the pathogen was 95.3–97.8% at the recommended application rate of 1.0–1.5 l/ha.Conclusion. The study showed that the population of the leaf rust pathogen treated with the chemical fungicide Abacus Ultra, SE is characterised by a change in structure in terms of aggressiveness and virulence, while the sensitivity remains high due to the combination of the active substances pyraclostrobin and epoxiconazole with a different mechanism of action in the preparation.

A forward genetics approach identifies pathogenicity genes in Fusarium oxysporum f. sp. carthami, a fungus causing vascular wilt disease of safflower

Vascular wilt disease caused by Fusarium oxysporum f. sp. carthami (Foc) is one of the biggest constraints for safflower production in India. Understanding the basis of pathogenicity and molecular dissection of its complex processes is of immense economic importance for the effective management of the wilt disease in safflower. In this study, a forward genetic approach was employed as an unbiased tool to identify the candidate pathogenicity-related genes. Agrobacterium mediated random T-DNA mutagenesis in Foc resulted in the generation of 178 Foc transformants. A hydroponics-based pathogenicity screening of generated mutants led to the identification of 12 avirulent mutants. Genome walking with two of the single insertion mutants revealed T-DNA insertion in the intergenic region of one mutant, while in the other mutant T-DNA was inserted in the coding region of a transcription factor. The genes identified in the present study can be targeted by host-delivered RNAi to generate transgenic safflower lines resistant to Foc.

Transcriptome Analysis of Wheat-Tilletia indica Interaction Provides Defense and Pathogenesis-Related Genes.

Karnal bunt (Tilletia indica Mitra) is an internationally quarantined disease of wheat. Until now, very little information has been available on the molecular basis of resistance and pathogenicity of T. indica. To investigate the molecular basis of host-pathogen interaction, the transcriptome of T. indica inoculated resistant (HD29) and susceptible (WH542) genotypes of wheat were analyzed. Approximately 58 million reads were generated using RNA sequencing by the Illumina NextSeq500 platform. These sequence reads were aligned to a reference genome of wheat to compare the expression level of genes in resistant and susceptible genotypes. The high-quality reads were deposited in the NCBI SRA database (SRP159223). More than 80,000 genes were expressed in both the resistant and susceptible wheat genotypes. Of these, 76,088 were commonly expressed genes, including 3184 significantly upregulated and 1778 downregulated genes. Four thousand one hundred thirteen and 5604 genes were exclusively expressed in susceptible and resistant genotypes, respectively. Based on the significance, 503 genes were upregulated and 387 genes were downregulated. Using gene ontology, the majority of coding sequences were associated with response to stimuli, stress, carbohydrate metabolism, developmental process, and catalytic activity. Highly differentially expressed genes (integral component of membrane, exonuclease activity, nucleic acid binding, DNA binding, metal ion binding) were validated in resistant and susceptible genotypes using qPCR analysis and similar expression levels were found in RNA-Seq. Apart from the wheat, the mapping of T. indica was 7.07% and 7.63% of resistant and susceptible hosts, respectively, upon infection, which revealed significant pathogenesis-related genes. This first study provided in-depth information and new insights into wheat-T. indica interaction for managing Karnal bunt disease of wheat.

Clinical Implications of a New DDX58 Pathogenic Variant That Causes Lupus Nephritis due to RIG-I Hyperactivation.

Lupus nephritis (LN) is one of the most severe complications of systemic lupus erythematosus, with heterogeneous phenotypes and different responses to therapy. Identifying genetic causes of LN can facilitate more individual treatment strategies. We performed whole-exome sequencing in a cohort of Chinese patients with LN and identified variants of a disease-causing gene. Extensive biochemical, immunologic, and functional analyses assessed the effect of the variant on type I IFN signaling. We further investigated the effectiveness of targeted therapy using single-cell RNA sequencing. We identified a novel DDX58 pathogenic variant, R109C, in five unrelated families with LN. The DDX58 R109C variant is a gain-of-function mutation, elevating type I IFN signaling due to reduced autoinhibition, which leads to RIG-I hyperactivation, increased RIG-I K63 ubiquitination, and MAVS aggregation. Transcriptome analysis revealed an increased IFN signature in patient monocytes. Initiation of JAK inhibitor therapy (baricitinib 2 mg/d) effectively suppressed the IFN signal in one patient. A novel DDX58 R109C variant that can cause LN connects IFNopathy and LN, suggesting targeted therapy on the basis of pathogenicity. This article contains a podcast at.

S1.3a Genetics andgenomics of Malassezia species

S1.3 Malassezia: genetics, genomics, and biology, September 21, 2022, 11:00 AM - 12:30 PM Malassezia includes yeasts belonging to the subphylum Ustilaginomycotina within the Basidiomycota. Malassezia yeasts are attracting the interest of both basic and applied scientists for their unique biological features, and for their importance in clinical and cosmetic settings. Although Malassezia yeasts are commonly found as commensal on human and animal skin, they are also associated with several skin disorders, such as dandruff/seborrheic dermatitis, atopic eczema, pityriasis versicolor, and folliculitis. More recently, an association of Malassezia with Crohn's disease, pancreatic ductal adenocarcinoma, and psoriasis exacerbation has been reported. To understand the genetic basis of Malassezia commensalism and pathogenicity, the availability of genomic and molecular tools plays a crucial role. Genomics advances in Malassezia reveal karyotype variations and gene turnover events, including genes horizontally transferred from bacteria. Moreover, the increasing availability of transcriptomic data allows us to prioritize studies on novel key genes that potentially characterize the pathophysiology of Malassezia fungi. For gene function studies, protocols for Agrobacterium tumefaciens-mediated transformation were developed and utilized in strategies of random insertional mutagenesis or targeted gene replacement through CRISPR/Cas9. Developed tools can be combined with the use of host-pathogen interaction models, such as the easy-to-use wax moth larvae of Galleria mellonella or the more complex murine skin model, enabling the characterization of both the fungal components that trigger skin damage and inflammation, and the inflammatory and antifungal response of the host to prevent fungal infection through immunological and molecular analyses of experimentally infected tissue.

Excreted secreted products from the parasitic nematode Steinernema carpocapsae manipulate the Drosophila melanogaster immune response

Steinernema carpocapsae is an entomopathogenic nematode (EPN) that rapidly infects and kills a wide range of insect hosts and has been linked to host immunosuppression during the initial stages of infection. The lethal nature of S. carpocapsae infections has previously been credited to its symbiotic bacteria; however, it has become evident that the nematodes are able to effectively kill their hosts independently through their excretion/secretion products (ESPs). Here we examined how the adult Drosophila melanogaster immune system is modulated in response to S. carpocapsae ESPs in an attempt to ascertain individual pathogenic contributions of the isolated compound. We found that the S. carpocapsae ESPs decrease the survival of D. melanogaster adult flies, they induce the expression of certain antimicrobial peptide-encoding genes, and they cause significant reduction in phenoloxidase enzyme activity and delay in the melanization response in males flies. We also report that S. carpocapsae ESPs affect hemocyte numbers in both male and female individuals. Our results indicate the manipulative role of EPN ESPs and reveal sex-specific differences in the host response against nematode infection factors. These findings are beneficial as they promote our understanding of the molecular basis of nematode pathogenicity and the parasite components that influence nematode-host interactions.

Recovery and genetic characterization of black queen cell virus.

Black queen cell virus (BQCV) is a severe threat to the honeybee (Apis mellifera) worldwide. Although several BQCV strains have been reported in China, the molecular basis for BQCV pathogenicity has not been well understood. Thus, a reverse genetic system of BQCV is required for studying viral replication and its pathogenic mechanism. Here, the complete genome sequence of BQCV was obtained from honeybees using reverse transcription PCR (RT-PCR), namely a BQCV China-GS1 strain (KY741959). Then, a phylogenetic tree was built to analyse the genetic relationships among BQCV strains from different regions. Our results showed that the BQCV China-GS1 contained two ORFs, consistent with the known reference strains, except for the BQCV China-JL1 strain (KP119603). Furthermore, the infectious clone of BQCV was constructed based on BQCV China-GS1 using a low copy vector pACYC177 and gene recombination. Due to the lack of culture cells for bee viruses, we infected the healthy bees with infectious clone of BQCV, and the rescued BQCV resulted in the recovery of recombinant virus, which induced higher mortality than those of the control group. Immune response after inoculated with BQCV further confirmed that the infectious clone of BQCV caused the cellular and humoral immune response of honeybee (A. mellifera). In conclusion, the full nucleotide sequence of BQCV China-GS1 strain was determined, and the infectious clone of BQCV was constructed in this study. These data will improve the understanding of pathogenesis and the host immune responses to viral infection.

Genome Analyses of Two Blueberry Pathogens: Diaportheamygdali CAA958 and Diaporthe eres CBS 160.32.

The genus Diaporthe includes pathogenic species distributed worldwide and affecting a wide variety of hosts. Diaporthe amygdali and Diaporthe eres have been found to cause cankers, dieback, or twig blights on economically important crops such as soybean, almond, grapevine, and blueberry. Despite their importance as plant pathogens, the strategies of species of Diaporthe to infect host plants are poorly explored. To provide a genomic basis of pathogenicity, the genomes of D. amygdali CAA958 and D. eres CBS 160.32 were sequenced and analyzed. Cellular transporters involved in the transport of toxins, ions, sugars, effectors, and genes implicated in pathogenicity were detected in both genomes. Hydrolases and oxidoreductases were the most prevalent carbohydrate-active enzymes (CAZymes). However, analyses of the secreted proteins revealed that the secretome of D. eres CBS 160.32 is represented by 5.4% of CAZymes, whereas the secreted CAZymes repertoire of D. amygdali CAA958 represents 29.1% of all secretomes. Biosynthetic gene clusters (BGCs) encoding compounds related to phytotoxins and mycotoxins were detected in D. eres and D. amygdali genomes. The core gene clusters of the phytotoxin Fusicoccin A in D. amygdali are reported here through a genome-scale assembly. Comparative analyses of the genomes from 11 Diaporthe species revealed an average of 874 CAZymes, 101 secondary metabolite BGCs, 1640 secreted proteins per species, and genome sizes ranging from 51.5 to 63.6 Mbp. This study offers insights into the overall features and characteristics of Diaporthe genomes. Our findings enrich the knowledge about D. eres and D. amygdali, which will facilitate further research into the pathogenicity mechanisms of these species.

Two functionally distinct heme/iron transport systems are virulence determinants of the fish pathogen Flavobacterium psychrophilum

ABSTRACT Bacterial pathogens have a critical impact on aquaculture, a sector that accounts for half of the human fish consumption. Flavobacterium psychrophilum (phylum Bacteroidetes) is responsible for bacterial cold-water disease in salmonids worldwide. The molecular factors involved in host invasion, colonization and haemorrhagic septicaemia are mostly unknown. In this study, we identified two new TonB-dependent receptors, HfpR and BfpR, that are required for adaptation to iron conditions encountered during infection and for virulence in rainbow trout. Transcriptional analyses revealed that their expression is tightly controlled and upregulated under specific iron sources and concentrations. Characterization of deletion mutants showed that they act without redundancy: BfpR is required for optimal growth in the presence of high haemoglobin level, while HfpR confers the capacity to acquire nutrient iron from haem or haemoglobin under iron scarcity. The gene hfpY, co-transcribed with hfpR, encodes a protein related to the HmuY family. We demonstrated that HfpY binds haem and contributes significantly to host colonization and disease severity. Overall, these results are consistent with a model in which both BfpR and Hfp systems promote haem uptake and respond to distinct signals to adapt iron acquisition to the different stages of pathogenesis. Our findings give insight into the molecular basis of pathogenicity of a serious pathogen belonging to the understudied family Flavobacteriaceae and point to the newly identified haem receptors as promising targets for antibacterial development.