ABSTRACT
Tuberculosis (TB) is one of the leading infectious diseases of global concern, and one quarter of the world's population are TB carriers. Biotin metabolism appears to be an attractive anti-TB drug target. However, the first-stage of mycobacterial biotin synthesis is fragmentarily understood. Here we report that three evolutionarily-distinct BioH isoenzymes (BioH1 to BioH3) are programmed in biotin synthesis of Mycobacterium smegmatis. Expression of an individual bioH isoform is sufficient to allow the growth of an Escherichia coli ΔbioH mutant on the non-permissive condition lacking biotin. The enzymatic activity in vitro combined with biotin bioassay in vivo reveals that BioH2 and BioH3 are capable of removing methyl moiety from pimeloyl-ACP methyl ester to give pimeloyl-ACP, a cognate precursor for biotin synthesis. In particular, we determine the crystal structure of dimeric BioH3 at 2.27Å, featuring a unique lid domain. Apart from its catalytic triad, we also dissect the substrate recognition of BioH3 by pimeloyl-ACP methyl ester. The removal of triple bioH isoforms (ΔbioH1/2/3) renders M. smegmatis biotin auxotrophic. Along with the newly-identified Tam/BioC, the discovery of three unusual BioH isoforms defines an atypical 'BioC-BioH(3)' paradigm for the first-stage of mycobacterial biotin synthesis. This study solves a long-standing puzzle in mycobacterial nutritional immunity, providing an alternative anti-TB drug target.
Subject(s)
Antitubercular Agents , Biotin , Biotin/chemistry , Biotin/metabolism , Escherichia coli/metabolism , Esters/metabolism , Isoenzymes/metabolismABSTRACT
The transferability of bacterial resistance to tigecycline, the 'last-resort' antibiotic, is an emerging challenge of global health concern. The plasmid-borne tet(X) that encodes a flavin-dependent monooxygenase represents a new mechanism for tigecycline resistance. Natural source for an ongoing family of Tet(X) resistance determinants is poorly understood. Here, we report the discovery of 26 new variants [tet(X18) to tet(X44)] from the poultry pathogen Riemerella anatipestifer, which expands extensively the current Tet(X) family. R. anatipestifer appears as a natural reservoir for tet(X), of which the chromosome harbours varied copies of tet(X) progenitors. Despite that an inactive ancestor rarely occurs, the action and mechanism of Tet(X2/4)-P, a putative Tet(X) progenitor, was comprehensively characterized, giving an intermediate level of tigecycline resistance. The potential pattern of Tet(X) dissemination from ducks to other animals and humans was raised, in the viewpoint of ecological niches. Therefore, this finding defines a large pool of natural sources for Tet(X) tigecycline resistance, heightening the need of efficient approaches to manage the inter-species transmission of tet(X) resistance determinants.
Subject(s)
Poultry Diseases , Riemerella , Animals , Anti-Bacterial Agents/pharmacology , Ducks , Microbial Sensitivity Tests , Poultry , Riemerella/genetics , Tigecycline/pharmacologyABSTRACT
BACKGROUND: Grey mould is an important plant disease worldwide, caused by Botrytis cinerea, resulting in serious economic loss. Wuyiencin, a low toxicity, high efficiency, and broad-spectrum agricultural antibiotic, has been demonstrated effectiveness against B. cinerea. RESULTS: Wuyiencin treatment inhibited growth and sporulation of B. cinerea, specifically altering hypha morphology and intracellular structures. These changes were accompanied by differential expression (fold change > 2.0) of 316 proteins identified by iTRAQ-labelling LC-MS/MS analysis (P < 0.05). Up-regulation of 14 proteins, including carbohydrate metabolism proteins and cell wall stabilization proteins, was validated by parallel reaction monitoring (PRM). Down-regulation of 13 proteins was validated by PRM, including regulators of energy metabolism, nucleotide/protein synthesis, and the biosynthesis of mediators of plant stress and decay. CONCLUSION: Our results confirm the inhibitory biological effects of wuyiencin on B. cinereal and elaborate on the differentially expressed proteins and associated pathways implicated in the capacity of wuyiencin to debilitate the growth and pathogenicity of grey mould. This study provides validated candidates for further targeted exploration with the goal of optimizing wuyiencin as a safe, low-toxicity agent for biological control.
Subject(s)
Botrytis/drug effects , Fungicides, Industrial/pharmacology , Proteomics , Agriculture , Botrytis/growth & development , Cell Wall/drug effects , Chromatography, Liquid , Hyphae/drug effects , Plant Diseases/microbiology , Tandem Mass SpectrometryABSTRACT
The vacuolar H+-ATPases (V-ATPases) are conserved ATP-dependent proton pumps that acidify intracellular compartments in eukaryotic cells. The role of Cpvma1, a V-ATPase catalytic subunit A of Cryphonectria parasitica, was investigated by generating cpvma1-overexpressing and cpvma1-silenced strains. The mutant strains were evaluated for phenotypic characteristics, V-ATPase activity, response to elevated pH and Ca2+ in the medium, virulence on chestnut, and accumulation of hypovirus RNA in the cells. Compared with the wild-type strain, cpvma1-overexpressing strains showed no significant difference in phenotype; however, cpvma1-silenced strains exhibited a phenotype of reduced growth rate, lower level of sporulation, and a marked decrease in V-ATPase activity and virulence. In addition, silencing of cpvma1 increased sensitivity to elevated pH and Ca2+, implicating an important role for Cpvma1 in pH adaptation and Ca2+ homeostasis. Furthermore, silencing of cpvma1 resulted in significantly decreased accumulation of hypoviral RNA. Taken together, our results indicate that Cpvma1 plays an important role in the regulation of phenotypic traits and virulence and the accumulation of hypovirus RNA in C. parasitica.
Subject(s)
RNA Viruses , Vacuolar Proton-Translocating ATPases , Catalytic Domain , Plant Diseases/microbiology , RNA , RNA Viruses/enzymology , VirulenceABSTRACT
The investigation of the interacting proteins with testis-specific calcium-binding protein CBP86-IV (CABYR) was carried out in human spermatozoa. The total RNA from human spermatozoa was extracted, and the ORF sequence of TSCBP86-IV gene was amplified and cloned into expression vector pET-28a. The positive recombinant clones were transformed into Escherichia coli strain BL21 (DE3) to express fusion protein. Then, co-immunoprecipitation (Co-IP) of TSCBP86-IV was performed in BL21 cell lysate expressing CBP86-IV recombinant protein. The immune complex was captured and identified by mass spectrometry. Reverse Co-IP of potential interacting proteins was performed in human sperm cell lysate. The potential protein interactions were confirmed by yeast two-hybrid system. Thirteen proteins were successfully identified in immune complex from E. coli cell lysate. Phosphoglycerate kinase 2 (PGK2) further showed positive results both in reverse Co-IP and yeast two-hybrid experiments and was confirmed to be interacted with TSCBP86-IV in human sperm cells.
Subject(s)
Calcium-Binding Proteins/metabolism , Isoenzymes/metabolism , Phosphoglycerate Kinase/metabolism , Spermatozoa/metabolism , Adult , Calcium-Binding Proteins/isolation & purification , Humans , Male , Protein Binding , Recombinant Proteins , Semen , Two-Hybrid System Techniques , Young AdultABSTRACT
OBJECTIVES: To investigate the expression level of testis-specific calcium-binding protein CBP86-IV in normal and asthenozoospermic human sperm. METHODS: The total RNA was extracted from human sperm, and target cDNA was obtained by reverse transcription-polymerase chain reaction. Then the cDNA was used for quantitative PCR analysis and cloned into the prokaryotic expression vector pET-28a, respectively. The fusion protein was induced and expressed as inclusion body which was used to produce the polyclonal antibody against TSCBP86-IV. The protein expression level of TSCBP86-IV from normal human sperm and idiopathic asthenozoospermic samples was detected by the purified antibody. RESULTS: The experimental results showed that the protein expression of TSCBP86-IV was reduced in idiopathic asthenozoospermia and consistent with the transcriptional changing tendency which was detected by quantitative PCR analysis. CONCLUSIONS: The stable and reliable change of TSCBP86-IV may be taken as a new molecular marker for clinical diagnosis of idiopathic asthenozoospermia.
Subject(s)
Asthenozoospermia/diagnosis , Asthenozoospermia/metabolism , Calcium-Binding Proteins/metabolism , Phosphoproteins/metabolism , Testis/metabolism , Adult , Biomarkers/metabolism , Calcium-Binding Proteins/genetics , Case-Control Studies , Gene Expression Regulation , Humans , Male , Middle Aged , Phosphoproteins/genetics , RNA, Messenger/metabolism , Sperm MotilityABSTRACT
OBJECTIVE: To evaluate the accuracy of parents' perception of whether their child is a picky eater and the specific food category the children avoideating according to the food frequency questionnaire. METHODS: This research selected 1 663 infants aged 4-36 months receiving non-diary complimentary food from maternal, infants, nutrition and growth study (MING Study) in 8 Chinese cities in which a combination of systematic cluster random sampling and purposive sampling was used. The general information, dietary status and picky eating status were collected through a self-designed questionnaire from the caregiver of the children. According to the parents' perception, the children were classified into picky/non-picky groups or avoid/non-avoid to a specific food category groups. Mann-Whitney U test was used to compare between the groups. RESULTS: The reported percentage of picky eaters increased from 7.37% in 4-6 months old infants to 36.20% in 25-36 months old infants. Most picky infants in 4-6 months and 7-12 months old infants avoided eating dairy food (25% and 24%); while most picky toddlers aged 13-24 months and 25-36 months avoided eating vegetables (26.92% and 47.46%). The infants aged 4-6 months and 7-12 months who were perceived as picky by their parents took more kinds of food (8 and 19.5 kinds) than those who were not (6 and 18 kinds), while the picky toddlers aged 13-24 months and 25-36 months took fewer kinds of food (28.5 and 34 kinds for picky eaters, 31 and 37 kinds for non-picky eaters). The parents of infants aged 4-6 months judged correctly in every category of food without any statistical significance; the parents of 7-12 months old infants judged correctly only in dairy food and eggs with statistical significance; those of 13-24 months old infants judged correctly in every food category except for vegetables with statistically significant difference in the category of eggs; those of 25-36 months old toddlers misjudged in dairy, beans and grains with no statistically significant difference in every category. CONCLUSION: Parents tend to misjudge their children's picky eating behavior before the first 12 months of the child, and tend to make a more accurate perception after the 12th month.
Subject(s)
Child Behavior , Food Preferences , Parents , Child, Preschool , Humans , Infant , VegetablesABSTRACT
OBJECTIVE: Chestnut blight fungus Cryphonectria parasitica and hypovirus constitute a model system to study fungal pathogenesis and host-virus interaction. Proteomic analysis of chestnut blight fungus upon hypovirus infection was conducted to find the differentially expressed host proteins. METHODS: According to the characteristics of this filamentous fungus, an optimized extraction protocol for fungal total protein was developed. Two-dimensional electrophoresis (2-DE) was used for comparative proteomic analysis of wild strain EP155 and hypovirus-infected strain EP713. The quantitative RT-PCR was applied to analyze mRNA expression level of protein-coding genes. RESULTS: In total 71 protein spots were detected to be differentially expressed on the base of EP155 of which 19 up-regulated and 52 down-regulated. Fifty-eight unique proteins were identified by mass spectrometry. Further study on quantitative RT-PCR indicated that the regulation of related host genes by hypovirus occurred at different levels. CONCLUSION: The TCA cycle of C. parasitica was weakened after hypovirus infection and the process of methylation was regulated by hypovirus. Meanwhile, viral regulation of virulence factors also contributed to the phenomenon of hypovirulence.
Subject(s)
Ascomycota/genetics , Ascomycota/virology , Fungal Proteins/genetics , RNA Viruses/physiology , Ascomycota/chemistry , Ascomycota/metabolism , Citric Acid Cycle , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Molecular Sequence Data , Proteomics , RNA Viruses/pathogenicity , VirulenceABSTRACT
Porcine epidemic diarrhea virus (PEDV) and rotavirus has posed a significant threat to the pig industry annually across different nations, resulting in huge economic losses. The frequent co-infection of these two viruses in clinical settings complicates the process of differential diagnoses. Rapid and accurate detection of PEDV and rotavirus is in great demand for timely diarrhea disease prevention and control. In this study, tris stabilized AuNPs were prepared and a sensitive lateral flow immunoassay (LFIA) sensor was developed for the simultaneous and rapid detection of PEDV and rotavirus on site. After the system optimization, the established LFIA can simultaneously identify PEDV and rotavirus with limits of detection (LOD) of 1.25 × 103 TCID50 mL-1 and 3.13 × 102 pg mL-1, respectively. When applying for clinical samples, the LFIA show a concordance of 95 % and 100 % to reverse transcript polymerase chain reaction (RT-PCR) for PEDV and rotavirus respectively. Therefore, this LFIA can qualitatively detect PEDV and rotavirus in 18 min with high sensitivity and accuracy without any sophisticated equipment and operation, making it a promising candidate for the early diagnosis of PEDV or/and rotavirus diarrhea on site.
Subject(s)
Chromatography, Affinity , Gold , Metal Nanoparticles , Porcine epidemic diarrhea virus , Rotavirus , Gold/chemistry , Porcine epidemic diarrhea virus/isolation & purification , Rotavirus/isolation & purification , Animals , Metal Nanoparticles/chemistry , Swine , Chromatography, Affinity/methods , Limit of Detection , Rotavirus Infections/diagnosis , Rotavirus Infections/veterinary , Rotavirus Infections/virology , Swine Diseases/diagnosis , Swine Diseases/virology , Immunoassay/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Coronavirus Infections/veterinaryABSTRACT
PURPOSE: Idiopathic asthenozoospermia is considered as one of the causes of male infertility and characterized by reduced sperm motility. For a better determination of pathogenic mechanism of asthenozoospermia, the exploration of differentially expressed proteins in normal sperm motility and idiopathic asthenozoospermia was conducted in our study. METHODS: Sperm proteins were extracted and isolated by two-dimensional electrophoresis. All significantly changed protein spots were picked up from 2D gels and identified by tandem mass spectrometry. Sixteen of the thirty-three total differentially expressed protein spots were successfully identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. RESULTS: Sixteen proteins identified belonged to 15 unique protein groups. GRP78, lactoferrin, SPANXB, PGK2, flagellin, DJ-1, XPA binding protein 2, CAB2, GPX4, and GAPDH were the first to be identified as differentially expressed proteins in idiopathic asthenospermia patients. Meanwhile, the analysis of quantitative RT-PCR was carried out to compare the protein levels, and the results indicated that the expression levels of the gene and protein were not entirely consistent. CONCLUSIONS: These experimental results expand the scope of the protein database, generating targets for further investigation of the pathogenic mechanism of idiopathic asthenozoospermia.
Subject(s)
Asthenozoospermia/genetics , Asthenozoospermia/physiopathology , Gene Expression Regulation/genetics , Proteomics , Sperm Motility/genetics , Sperm Motility/physiology , Spermatozoa/metabolism , Antigens, CD/genetics , Antigens, CD/metabolism , Case-Control Studies , Endoplasmic Reticulum Chaperone BiP , Flagellin/genetics , Flagellin/metabolism , Gene Expression Regulation/physiology , Glutathione Peroxidase/genetics , Glutathione Peroxidase/metabolism , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Male , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Phosphoglycerate Kinase/genetics , Phosphoglycerate Kinase/metabolism , Phospholipid Hydroperoxide Glutathione Peroxidase , Protein Deglycase DJ-1 , RNA Splicing Factors , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Spermatozoa/pathology , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Introduction: The chestnut blight fungus, Cryphonectria parasitica, and hypovirus have been used as a model to probe the mechanism of virulence and regulation of traits important to the host fungus. Previous studies have indicated that mitochondria could be the primary target of the hypovirus. Methods: In this study, we report a comprehensive and comparative study comprising mitochondrion quantification, reactive oxygen species (ROS) and respiratory efficiency, and quantitative mitochondrial proteomics of the wild-type and virus-infected strains of the chestnut blight fungus. Results and discussion: Our data show that hypovirus infection increases the total number of mitochondria, lowers the general ROS level, and increases mitochondrial respiratory efficiency. Quantification of mitochondrial proteomes revealed that a set of proteins functioning in energy metabolism and mitochondrial morphogenesis, as well as virulence, were regulated by the virus. In addition, two viral proteins, p29 and p48, were found to co-fractionate with the mitochondrial membrane and matrix. These results suggest that hypovirus perturbs the host mitochondrial functions to result in hypovirulence.
ABSTRACT
Based on genetic and molecular analyses, the ABC model was proposed to explain the genetic control of floral development. The C-class MADS box gene AGAMOUS (AG) plays crucial roles in Arabidopsis thaliana development through regulation of the organ identity of stamens and gynoecium. The present research reports for the first time the cloning of an AG homologue (HpAG) from Hosta plantaginea Aschers. Phylogenetic analysis shows HpAG is a homologue of AG that is closely related to C-lineage AG homologues from monocot species. Semi-quantitative PCR and real-time PCR analyses show that HpAG is stamen- and gynoecium-specific in expression and has spatial and temporal expression patterns in the reproductive organs of H. plantaginea. The transcriptional activation property of HpAG is also verified by a yeast one-hybrid. Functional analysis is carried out in Arabidopsis by overexpression of HpAG. The homeotic transformations of petals into staminoid organs are observed in 35S::HpAG transgenic plants. All these results show that HpAG1 plays a crucial role in stamen specification and gynoecium development.
Subject(s)
Flowers/metabolism , Gene Expression Regulation, Plant/genetics , Hosta/genetics , MADS Domain Proteins/genetics , Phylogeny , Arabidopsis , Cloning, Molecular , Cluster Analysis , Computational Biology , DNA Primers/genetics , Flowers/genetics , Flowers/growth & development , Plants, Genetically Modified , Real-Time Polymerase Chain Reaction , Two-Hybrid System TechniquesABSTRACT
Duo1, a major component of the Dam1 complex which has been found in two species of yeast (the budding yeast Saccharomyces cerevisae and the fission yeast Schizosaccharomyces pombe), is involved in mitosis-related chromosome segregation, while its relevance to pathogenicity in filamentous fungi remains unclear. This report elucidated this very fact in the case of the rice blast fungus Magnaporthe oryzae. A gene designated MoDUO1 that encodes a Duo1-like homolog (MoDuo1) was discovered in the M. oryzae genome. Two types of MoDUO1 mutants were obtained using genetic approaches of Agrobacterium-mediated gene disruption and homologous recombination. Both disruption and deletion of MoDUO1 can exert profound effects on the formation pattern of conidiophores and conidial morphology, such as abnormal nucleic numbers in conidia and delayed extension of infectious hyphae. Intriguingly, plant infection assays demonstrated that inactivation of MoDUO1 significantly attenuates the virulence in its natural host rice leaves, and functional complementation can restore it. Subcellular localization assays showed that MoDuo1 is mainly distributed in the cytosol of fungal cells. Proteomics-based investigation revealed that the expression of four mitosis-related proteins is shut down in the MoDUO1 mutant, suggesting that MoDuo1 may have a function in mitosis. In light of the fact that Duo1 orthologs are widespread in plant and human fungal pathogens, our finding may represent a common mechanism underlying fungal virulence. To the best of our knowledge, this is the first example of linking a Duo1-like homolog to the pathogenesis of a pathogenic fungus, which might provide clues to additional studies on the role of Dam1 complex in M. oryzae and its interaction with rice.
Subject(s)
Fungal Proteins/genetics , Fungal Proteins/metabolism , Magnaporthe/genetics , Magnaporthe/pathogenicity , Oryza/microbiology , Transcription Factors/genetics , Transcription Factors/metabolism , Amino Acid Sequence , Gene Deletion , Molecular Sequence Data , Mutation , Phenotype , Proteomics , Sequence Alignment , Transcription Factors/chemistry , Virulence Factors/genetics , Virulence Factors/metabolismABSTRACT
Heat-stable antifungal factor (HSAF) is produced by the fermentation of Lysobacter enzymogenes, which is known for its broad-spectrum antifungal activity and novel mode of action. However, studies on the separation of HSAF have rarely been reported. Herein, alteramide B (the main byproduct) was removed firstly from the fermentation broth by photodegradation to improve the purity of HSAF. Then, the separation of HSAF via adsorption by macroporous adsorption resins (MARs) was evaluated and NKA resin showed highest static adsorption and desorption performances. After optimizing the static and dynamic adsorption characteristics, the content of HSAF in the purified product increased from 8.67 ± 0.32% (ethyl acetate extraction) to 31.07 ± 1.12% by 3.58-fold. These results suggest that the developed strategy via photodegradation and macroporous resin adsorption is an effective process for the separation of HSAF, and it is also a promising method for the large-scale preparation of HSAF for agricultural applications.
ABSTRACT
BACKGROUND: Chitinases are enzymes which degrade ß-1,4-glycosidid linkages in chitin. The enzymatic degradation of shellfish waste (containing chitin) to chitooligosaccharides is used in industrial applications to generate high-value-added products from such waste. However, chitinases are currently produced with low efficiency and poor tolerance, limiting the industrial utility. Therefore, identifying chitinases with higher enzymatic activity and tolerance is of great importance. METHODS: Primers were designed using the genomic database of Paenibacillus chitinolyticus NBRC 15660. An exochitinase (CHI) was cloned into the recombinant plasmid pET-22b (+) to form pET-22b (+)-CHI, which was transformed into Escherichia coli TOP10 to construct a genomic library. Transformation was confirmed by colony-polymerase chain reaction and electrophoresis. The target sequence was verified by sequencing. Recombinant pET-22b (+)-CHI was transformed into E. coli Rosetta-gami B (DE3) for expression of chitinase. Recombinant protein was purified by Ni-NTA affinity chromatography and enzymatic analysis was carried out. RESULTS: The exochitinase CHI from P. chitinolyticus strain UMBR 0002 was successfully cloned and heterologously expressed in E. coli Rosetta-gami B (DE3). Purification yielded a 13.36-fold enrichment and recovery yield of 72.20%. The purified enzyme had a specific activity of 750.64 mU mg-1. The optimum pH and temperature for degradation of colloidal chitin were 5.0 and 45 °C, respectively. The enzyme showed high stability, retaining >70% activity at pH 4.0-10.0 and 25-45 °C (maximum of 90 min). The activity of CHI strongly increased with the addition of Ca2+, Mn2+, Tween 80 and urea. Conversely, Cu2+, Fe3+, acetic acid, isoamyl alcohol, sodium dodecyl sulfate and ß-mercaptoethanol significantly inhibited enzyme activity. The oligosaccharides produced by CHI from colloidal chitin exhibited a degree of polymerization, forming N-acetylglucosamine (GlcNAc) and (GlcNAc)2 as products. CONCLUSIONS: This is the first report of the cloning, heterologous expression and purification of a chitinase from P. chitinolyticus strain UMBR 0002. The results highlight CHI as a good candidate enzyme for green degradation of chitinous waste.
ABSTRACT
Autophagy is a degradation system in the cell, involved in the turnover of cellular components, development, differentiation, immune responses, protection against pathogens, and cell death. Autophagy is induced by nutrient starvation, in which cytoplasmic components and organelles are digested via vacuoles/lysosomes. In this study, by using electron microscopy, we observed that hypovirus CHV1-EP713 infection of Cryphonectria parasitica, the causative agent of chestnut blight disease, caused proliferation of autophagic-like vesicles. This phenomenon could be mimicked by treating the wild-type strain of the fungus EP155 with the autophagy induction drug rapamycin. Some of the hypovirulence-associated traits, including reduced pigmentation and conidiation, were also observed in the rapamycin-treated EP155. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) revealed that genes involved in autophagy were up-regulated in expression. Deletion of cpatg8, a gene encoding a homolog of ATG8 in Saccharomyces cerevisiae, resulted in attenuation of virulence and reduction in sporulation, as well as accumulation of the double-stranded viral RNA. Furthermore, virus-encoded p29 protein was found to co-localize with CpATG8, implying that the viral protein may interfere with the function of CpATG8. Taken together, these findings show that cpatg8 can be regulated by the hypovirus and is required for virulence and development of the fungus and accumulation of viral dsRNA in chestnut blight fungus.
Subject(s)
Ascomycota/pathogenicity , Ascomycota/virology , Autophagy-Related Protein 8 Family/metabolism , Autophagy , RNA, Viral/metabolism , Thorax/microbiology , Ascomycota/genetics , Ascomycota/metabolism , Autophagy-Related Protein 8 Family/genetics , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , RNA, Viral/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Transcriptional Activation , Viral Proteins/metabolism , Virulence/geneticsABSTRACT
Ubiquitination plays key roles in eukaryotic growth, stress adaptation, and metabolic regulation. In our previous work, ubiquitin was found to be secreted in the hypovirus-infected strain of Cryphonectria parasitica, a phytopathogenic filamentous fungus responsible for the chestnut blight. Here we report the functional and molecular characterization of a polyubiquitin gene, cpubi4, in C. parasitica. The expression of cpubi4 was upregulated by the infection of a hypovirus. Deletion of cpubi4 resulted in abnormal morphology, reduced sporulation, attenuation of virulence, and significant reduction in ubiquitination. A total of 378 sites in 236 proteins were identified to be significantly decreased in ubiquitination in the absence of cpubi4. Quantitative proteome analysis revealed that 285 in 4,776 identified proteins changed in abundance (1.5-fold, P < 0.05) in the cpubi4 null mutant, as compared with the wild-type strain.
ABSTRACT
Low temperature at the seedling stage is a major damaging factor for rice production in southern China. To better understand the cold response of cultivated and wild rice, cold-sensitive cultivar 93-11 (Oryza sativa L. ssp. Indica) and cold-resistant hybrid wild rice DC907 with a 93-11 genetic background were used for a quantitative proteomic analysis with tandem mass tags (TMT) in parallel. Rice seedlings grown for four weeks at a normal temperature (25°C) were treated at 8-10°C for 24, 72 and 120 h. The number of differentially expressed proteins increased gradually over time in the cold-exposed rice in comparison with the untreated rice. A total of 366 unique proteins involved in ATP synthesis, photosystem, reactive oxygen species, stress response, cell growth and integrity were identified as responding to cold stress in DC907. While both DC907 and 93-11 underwent similar alterations in proteomic profiles in response to cold stress, DC907 responded in a prompter manner in terms of expressing cold-responding proteins, maintained a higher level of photosynthesis to power the cells, and possessed a stable and higher level of DIR proteins to prevent the plant from obtaining irreversible cell structure damage. The observations made in this study may lay a new foundation for further investigation of cold sensitivity or tolerance mechanisms in rice.
Subject(s)
Cold-Shock Response/physiology , Oryza/physiology , Proteome/metabolism , Seedlings/physiology , Photosynthesis/physiology , ProteomicsABSTRACT
Simian immunodeficiency virus (SIV) in the rhesus macaque is regarded as a classic animal model, playing a crucial role in HIV vaccine strategies and therapeutics by characterizing various cytotoxic T-lymphocyte (CTL) responses in macaque monkeys. However, the availability of well documented structural reports focusing on rhesus macaque major histocompatibility complex class I (MHC I) molecules remains extremely limited. Here, a complex of the rhesus macaque MHC I molecule (Mamu-A*02) with human beta2m and an immunodominant SIV-Gag nonapeptide, GESNLKSLY (GY9), has been crystallized. The crystal diffracts X-rays to 2.7 A resolution and belongs to space group C2, with unit-cell parameters a = 124.11, b = 110.45, c = 100.06 A, and contains two molecules in the asymmetric unit. The availability of the structure, which is being solved by molecular replacement, will provide new insights into rhesus macaque MHC I (Mamu-A*02) presenting pathogenic SIV peptides.
Subject(s)
Gene Products, gag/chemistry , Histocompatibility Antigens Class I/chemistry , Immunodominant Epitopes/chemistry , Peptide Fragments/chemistry , Simian Immunodeficiency Virus/chemistry , Simian Immunodeficiency Virus/immunology , Animals , Antigen Presentation , Crystallization , Crystallography, X-Ray , Gene Products, gag/immunology , Gene Products, gag/metabolism , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/isolation & purification , Immunodominant Epitopes/immunology , Immunodominant Epitopes/metabolism , Macaca mulatta , Peptide Fragments/immunology , Peptide Fragments/metabolismABSTRACT
To understand the impact of a hypovirus infection on the secretome of the chestnut blight fungus, Cryphonectria parasitica, a phytopathogenic filamentous fungus, two-dimensional electrophoresis (2-DE) and isobaric tag for relative and absolute quantitation (iTRAQ) technology were employed to identify and quantify the secreted proteins. A total of 403 unique proteins were identified from the secretome of the wild type virus-free strain EP155. Of these proteins, 329 were predicted to be involved in known secretory pathways and they are primarily composed of metabolic enzymes, biological regulators, responders to stimulus and components involved in plant-pathogen interactions. When infected with the hypovirus CHV1-EP713, 99 proteins were found to be differentially expressed as compared to the wild type strain EP155. These proteins were mainly related to plant cell wall degradation, response to host defense, fungal virulence and intracellular structure. The effects of CHV1 on secreted proteins may reveal a relationship between physiological pathways and hypovirulence.