An ordered collection of expressed sequences from Cryphonectria parasitica and evidence of genomic microsynteny with Neurospora crassa and Magnaporthe grisea

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2373-2384 ◽  
Author(s):  
Angus L. Dawe ◽  
Vanessa C. McMains ◽  
Maria Panglao ◽  
Shin Kasahara ◽  
Baoshan Chen ◽  
...  
Keyword(s):  
Magnaporthe Grisea ◽  
Experimental System ◽  
Cryphonectria Parasitica ◽  
Genomic Region ◽  
Wild Type Virus ◽  
Global Changes ◽  
Cdna Clones ◽  
Sequence Information ◽  
Protein Database ◽  
Fungal Virulence

Cryphonectria parasitica, the causative agent of chestnut blight, has proven to be a tractable experimental system for studying fungal pathogenesis. Moreover, the development of infectious cDNA clones of C. parasitica hypoviruses, capable of attenuating fungal virulence, has provided the opportunity to examine molecular aspects of fungal plant pathogenesis in the context of biological control. In order to establish a genomic base for future studies of C. parasitica, the authors have analysed a collection of expressed sequences. A mixed cDNA library was prepared from RNA isolated from wild-type (virus-free) and hypovirus-infected C. parasitica strains. Plasmid DNA was recovered from individual transformants and sequenced from the 5′ end of the insert. Contig analysis of the collected sequences revealed that they represented approximately 2200 individual ORFs. An assessment of functional diversity present in this collection was achieved by using the blast software utilities and the NCBI protein database. Candidate genes were identified with significant potential relevance to C. parasitica growth, development, pathogenesis and vegetative incompatibility. Additional investigations of a 12·9 kbp genomic region revealed microsynteny between C. parasitica and both Neurospora crassa and Magnaporthe grisea, two closely related fungi. These data represent the largest collection of sequence information currently available for C. parasitica and are now forming the basis of further studies using microarray analyses to determine global changes in transcription that occur in response to hypovirus infection.

scholarly journals Using Chimeric Hypoviruses To Fine-Tune the Interaction between a Pathogenic Fungus and Its Plant Host

2000 ◽  
Vol 74 (16) ◽  
pp. 7562-7567 ◽  
Author(s):  
Baoshan Chen ◽  
Lynn M. Geletka ◽  
Donald L. Nuss
Keyword(s):  
Pathogenic Fungus ◽  
Cryphonectria Parasitica ◽  
Cdna Clones ◽  
Plant Host ◽  
Reading Frame ◽  
Ecological Fitness ◽  
Severe Strain ◽  
Fungal Isolates ◽  
Virulence Attenuation ◽  
Fine Tune

ABSTRACT Infectious cDNA clones of mild (CHV1-Euro7) and severe (CHV1-EP713) hypovirus strains responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica were used to construct viable chimeric viruses. Differences in virus-mediated alterations of fungal colony morphology, growth rate, and canker morphology were mapped to a region of open reading frame B extending from nucleotides 2,363 to 9,904. By swapping domains within this region, it was possible to generate chimeric hypovirus-infectedC. parasitica isolates that exhibited a spectrum of defined colony and canker morphologies. Several severe strain traits were observed to be dominant. It was also possible to uncouple the severe strain traits of small canker size and suppression of asexual sporulation. For example, fungal isolates infected with a chimera containing nucleotides 2363 through 5310 from CHV1-Euro7 in a CHV1-713 background formed small cankers that were similar in size to that caused by CHV1-EP713-infected isolates but with the capacity for producing asexual spores at levels approaching that observed for fungal isolates infected with the mild strain. These results demonstrate that hypoviruses can be engineered to fine-tune the interaction between a pathogenic fungus and its plant host. The identification of specific hypovirus domains that differentially contribute to canker morphology and sporulation levels also provides considerable utility for continuing efforts to enhance biological control potential by balancing hypovirulence and ecological fitness.

scholarly journals Recovery of Virulent and RNase-Negative Attenuated Type 2 Bovine Viral Diarrhea Viruses from Infectious cDNA Clones

2002 ◽  
Vol 76 (16) ◽  
pp. 8494-8503 ◽  
Author(s):  
Christiane Meyer ◽  
Martina von Freyburg ◽  
Knut Elbers ◽  
Gregor Meyers
Keyword(s):  
Cdna Clone ◽  
Infectious Cdna Clone ◽  
Wild Type Virus ◽  
Bovine Viral Diarrhea ◽  
Cdna Clones ◽  
Wild Type ◽  
Viral Glycoprotein ◽  
Diarrhea Virus ◽  
Viral Diarrhea

ABSTRACT Cloned cDNA derived from the genome of the virulent type 2 bovine viral diarrhea virus (BVDV) strain NY'93/C was sequenced and served for establishment of the infectious cDNA clone pKANE40A. Virus recovered from pKANE40A exhibited growth characteristics similar to those of wild-type BVDV NY'93/C and proved to be clinically indistinguishable from the wild-type virus in animal experiments. A virus mutant in which the RNase residing in the viral glycoprotein Erns was inactivated, revealed an attenuated phenotype. The plasmid pKANE40A represents the first infectious cDNA clone established for a type 2 BVDV and offers a variety of new approaches to analyze the mechanisms of BVDV-induced disease in cattle.

scholarly journals Targeted Disruption of a Fungal G-Protein β Subunit Gene Results in Increased Vegetative Growth but Reduced Virulence

1997 ◽  
Vol 10 (8) ◽  
pp. 984-993 ◽  
Author(s):  
Shin Kasahara ◽  
Donald L. Nuss
Keyword(s):  
Amino Acid ◽  
G Protein ◽  
Vegetative Growth ◽  
Synthetic Medium ◽  
Cryphonectria Parasitica ◽  
Β Subunit ◽  
Subunit Gene ◽  
Targeted Disruption ◽  
Fungal Virulence ◽  
Α Subunit

Targeted disruption of two G-protein α subunit genes in the chestnut blight fungus Cryphonectria parasitica revealed roles for the Giα subunit CPG-1 in fungal reproduction, virulence, and vegetative growth. A second Gα subunit, CPG-2, was found to be dispensable for these functions. We now report the cloning and targeted disruption of a C. parasitica G-protein β subunit gene. The deduced amino acid sequence encoded by this gene, designated cpgb-1, was found to share 66.2, 65.9, and 66.7% amino acid identity with Gβ homologues from human, Drosophila, and Dictyostelium origins, respectively, but only 39.7% identity with the Saccharomyces cerevisiae Gβ homologue STE4 product. Low stringency Southern hybridization failed to detect any related Gβ subunit genes in C. parasitica. Targeted disruption of cpgb-1 resulted in several of the changes previously reported to accompany disruption of the C. parasitica Giα subunit gene cpg-1. These included very significant reductions in pigmentation, asexual sporulation, and virulence. In contrast to results obtained for Giα gene disruption, the reduction in virulence resulting from the disruption of a Gβ gene was accompanied by increased, rather than decreased, vegetative growth on synthetic medium. The relevance of these results to mechanisms of fungal virulence is considered.

scholarly journals G Protein α Subunit Genes Control Growth, Development, and Pathogenicity of Magnaporthe grisea

1997 ◽  
Vol 10 (9) ◽  
pp. 1075-1086 ◽  
Author(s):  
Shaohua Liu ◽  
Ralph A. Dean
Keyword(s):  
Neurospora Crassa ◽  
G Protein ◽  
Vegetative Growth ◽  
Magnaporthe Grisea ◽  
Cryphonectria Parasitica ◽  
Appressorium Formation ◽  
Α Subunit ◽  
Targeted Deletion ◽  
Control Growth ◽  
G Protein Α Subunit

Three G protein α subunit genes have been cloned and characterized from Magnaporthe grisea: magA is very similar to CPG-2 of Cryphonectria parasitica; magB is virtually identical to CPG-1 of Cryphonectria parasitica, to gna1 of Neurospora crassa, and to fadA of Emericella nidulans; and magC is most similar to gna2 of Neurospora crassa. Homologous recombination resulting in targeted deletion of magA had no effect on vegetative growth, conidiation, or appressorium formation. Deletion of magC reduced conidiation, but did not affect vegetative growth or appressorium formation. However, disruption of magB significantly reduced vegetative growth, conidiation, and appressorium formation. magB¯ transformants, unlike magA¯ and magC¯ transformants, exhibited a reduced ability to infect and colonize susceptible rice leaves. G protein α subunit genes are required for M. grisea mating. magB¯ transformants failed to form perithecia, whereas magA¯ and magC¯ transformants did not produce mature asci. These results suggest that G protein α subunit genes are involved in signal transduction pathways in M. grisea that control vegetative growth, conidiation, conidium attachment, appressorium formation, mating, and pathogenicity.

Gene expression in the rat supraoptic nucleus induced by chronic hyperosmolality versus hyposmolality

2000 ◽  
Vol 279 (4) ◽  
pp. R1239-R1250 ◽  
Author(s):  
Eric Glasgow ◽  
Takashi Murase ◽  
Bingjun Zhang ◽  
Joseph G. Verbalis ◽  
Harold Gainer
Keyword(s):  
Gene Expression ◽  
Supraoptic Nucleus ◽  
Cdna Libraries ◽  
Global Changes ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Magnocellular Neurons ◽  
In Situ Hybridization Histochemistry ◽  
Functional Classes

Magnocellular neurons of the hypothalamo-neurohypophysial system play a fundamental role in the maintenance of body homeostasis by secreting vasopressin and oxytocin in response to systemic osmotic perturbations. During chronic hyperosmolality, vasopressin and oxytocin mRNA levels increase twofold, whereas, during chronic hyposmolality, these mRNA levels decrease to 10–20% of that of normoosmolar control animals. To determine what other genes respond to these osmotic perturbations, we have analyzed gene expression during chronic hyper- versus hyponatremia. Thirty-seven cDNA clones were isolated by differentially screening cDNA libraries that were generated from supraoptic nucleus tissue punches from hyper- or hyponatremic rats. Further analysis of 12 of these cDNAs by in situ hybridization histochemistry confirmed that they are osmotically regulated. These cDNAs represent a variety of functional classes and include cytochrome oxidase, tubulin, Na+-K+-ATPase, spectrin, PEP-19, calmodulin, GTPase, DnaJ-like, clathrin-associated, synaptic glycoprotein, regulator of GTPase stimulation, and gene for oligodendrocyte lineage-myelin basic proteins. This analysis therefore suggests that adaptation to chronic osmotic stress results in global changes in gene expression in the magnocellular neurons of the supraoptic nucleus.

scholarly journals Vaccinia Virus G1 Protein, a Predicted Metalloprotease, Is Essential for Morphogenesis of Infectious Virions but Not for Cleavage of Major Core Proteins

2004 ◽  
Vol 78 (13) ◽  
pp. 6855-6863 ◽  
Author(s):  
Camilo Ansarah-Sobrinho ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Virus Replication ◽  
Protein A ◽  
Proteolytic Processing ◽  
Wild Type Virus ◽  
Sequence Information ◽  
Wild Type ◽  
Active Site Residues ◽  
Virus Particles ◽  
Core Proteins

ABSTRACT Genes encoding orthologs of the vaccinia virus G1 protein are present in all poxviruses for which sequence information is available, yet neither the role of the protein nor its requirement for virus replication is known. G1 was predicted to be involved in the cleavage of core proteins, based on a transfection study and the presence of an HXXEH motif found in a subset of metallopeptidases. In the present study, we engineered a recombinant vaccinia virus containing a single copy of the G1L gene with a C-terminal epitope tag that is stringently regulated by the Escherichia coli lac repressor. In the absence of inducer, expression of G1 was repressed and virus replication was inhibited. Rescue of infectious virus was achieved by expression of wild-type G1 in trans, but not when the putative protease active site residues histidine-41, glutamate-44, or histidine-45 were mutated. Nevertheless, the synthesis and proteolytic processing of major core and membrane proteins appeared unaffected under nonpermissive conditions, distinguishing the phenotype of the G1L mutant from one in which the gene encoding the I7 protease was repressed. Noninfectious virus particles, assembled in the absence of inducer, did not attain the oval shape or characteristic core structure of mature virions. The polypeptide composition of these particles, however, closely resembled that of wild-type virus. Full-length and shorter forms of the G1 protein were found in the core fraction of virus particles assembled in the presence of inducer, suggesting that G1 is processed by self-cleavage or by another protease.

scholarly journals A Viral Double-Stranded RNA Up Regulates the Fungal Virulence of Nectria radicicola

2001 ◽  
Vol 14 (4) ◽  
pp. 496-507 ◽  
Author(s):  
II-Pyung Ahn ◽  
Yong-Hwan Lee
Keyword(s):  
Protein Kinase ◽  
Pathogenic Fungi ◽  
Ginseng Root ◽  
Cdna Clones ◽  
Fungal Virulence ◽  
Double Stranded Rna ◽  
Kinase C ◽  
Causal Fungus ◽  
Dependent Protein ◽  
Biochemical Analyses

Double-stranded RNAs (dsRNAs) are widespread in plant pathogenic fungi, but their functions in fungal hosts remain mostly unclear, with a few exceptions. We analyzed dsRNAs from Nectria radicicola, the causal fungus of ginseng root rot. Four distinct sizes of dsRNAs, 6.0, 5.0, 2.5, and 1.5 kbp, were detected in 24 out of the 81 strains tested. Curing tests of individual dsRNAs suggested that the presence of 6.0-kbp dsRNA was associated with high levels of virulence, sporulation, laccase activity, and pigmentation in this fungus. The 6.0-kbp dsRNA-cured strains completely lost virulence-related phenotypes. This 6.0-kbp dsRNA was reintroduced by hyphal anastomosis to a dsRNA-cured strain marked with hygromycin resistance, which resulted in the restoration of virulence-related phenotypes. These results strongly suggest that 6.0-kbp dsRNA up regulates fungal virulence in N. radicicola. Sequencing of several cDNA clones derived from 6.0-kbp dsRNA revealed the presence of a RNA-dependent RNA polymerase (RDRP) gene. Phylogenetic analysis showed that this gene is closely related to those of plant cryptic viruses. Biochemical analyses suggested that the 6.0-kbp dsRNA may regulate fungal virulence through signal-transduction pathways involving cyclic AMP-dependent protein kinase and protein kinase C.

scholarly journals Characterization of the transcriptome and EST-SSR development in Boea clarkeana, a desiccation-tolerant plant endemic to China

2016 ◽  
Author(s):  
Ying Wang ◽  
Kun Liu ◽  
De Bi ◽  
Biao Shou Zhou ◽  
Wen Jian Shao
Keyword(s):  
De Novo ◽  
Gene Annotation ◽  
Sequence Similarity ◽  
Molecular Study ◽  
Sequence Information ◽  
Sequencing Data ◽  
Protein Database ◽  
Illumina Hiseq ◽  
Significant Similarity ◽  
Assembly Technology

Background. Resurrection plants constitute a unique cadre within angiosperms. Boea clarkeana Hemsl. (Boea, Gesneriaceae) is a desiccation-tolerant dicotyledonous herb that is endemic to China. Although research on angiosperms with DT could be instructive for crops, genomic resources for B. clarkeana remain scarce. In addition, transcriptome sequencing could be an effective way to study desiccation-tolerant plants. Methods. In the present study, we used the platform Illumina HiSeqTM 2000 and de novo assembly technology to obtain leaf transcriptomes of B. clarkeana and conducted a BLASTX alignment of the sequencing data and protein databases for sequence classification and annotation. Then, based on the sequence information obtained, we developed EST-SSR markers by means of EST-SSR mining, primer design and polymorphism identification. Results. A total of 91,449 unigenes were generated from the leaf cDNA library of B. clarkeana in this study. Based on a sequence similarity search with a known protein database, 72,087 unigenes were annotated. Among the annotated unigenes, a total of 71,170 unigenes showed significant similarity to known proteins of 463 popular model species in the Nr database, and 59,962 unigenes and 32,336 unigenes were assigned to GO classifications and COG, respectively. In addition, 44,924 unigenes were mapped in 128 KEGG pathways. Furthermore, a total of 7,610 unigenes with 8,563 microsatellites were found. Seventy-four primer pairs were selected from 436 primer pairs designed for polymorphism validation. SSRs with higher polymorphism rates were concentrated on dinucleotides, pentanucleotides and hexanucleotides. Finally, 17 pairs with highly polymorphic and stable loci were selected for polymorphism screening. There were a total of 65 alleles, with 2–6 alleles at each locus. Mainly due to the unique biological characteristics of plants, the HE, HO and PIC per locus were very low, ranging from 0 to 0.196, 0.082 to 0.14 and 0 to 0.155, respectively. Discussion. A substantial fraction transcriptome sequences of B. clarkeana were generated in this study, which is the first molecular-level analysis of this plant. These sequences are valuable resources for gene annotation and discovery and molecular marker development. These sequences could also provide a valuable basis for the future molecular study of B. clarkeana.

scholarly journals Distinct Roles of Two DNA Methyltransferases from Cryphonectria parasitica in Fungal Virulence, Responses to Hypovirus Infection, and Viral Clearance

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yo-Han Ko ◽  
Kum-Kang So ◽  
Jeesun Chun ◽  
Dae-Hyuk Kim
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Cryphonectria Parasitica ◽  
Dna Methyltransferases ◽  
Viral Clearance ◽  
Biological Functions ◽  
Fungal Virulence ◽  
Content Type ◽  
Genome Defense ◽  
Fungal Dna

ABSTRACT Two DNA methyltransferase (DNMTase) genes from Cryphonectria parasitica have been previously identified as CpDmt1 and CpDmt2, which are orthologous to rid and dim-2 of Neurospora crassa, respectively. While global changes in DNA methylation have been associated with fungal sectorization and CpDmt1 but not CpDmt2 has been implicated in the sporadic sectorization, the present study continues to investigate the biological functions of both DNMTase genes. Transcription of both DNMTases is regulated in response to infection with the Cryphonectria hypovirus 1 (CHV1-EP713). CpDmt1 is upregulated and CpDmt2 is downregulated by CHV1 infection. Conidium production and response to heat stress are affected only by mutation of CpDmt1, not by CpDmt2 mutation. Significant changes in virulence are observed in opposite directions; i.e., the CpDmt1-null mutant is hypervirulent, while the CpDmt2-null mutant is hypovirulent. Compared to the CHV1-infected wild type, CHV1-transferred single and double mutants show severe growth retardation: the colony size is less than 10% that of the parental virus-free null mutants, and their titers of transferred CHV1 are higher than that of the wild type, implying that no defect in viral replication occurs. However, as cultivation proceeds, spontaneous viral clearance is observed in hypovirus-infected colonies of the null mutants, which has never been reported in this fungus-virus interaction. This study demonstrates that both DNMTases are significant factors in fungal development and virulence. Each fungal DNMTase affects fungal biology in both common and separate ways. In addition, both genes are essential to the antiviral responses, including viral clearance which depends on their mutations. IMPORTANCE Although relatively few in number, studies of DNA methylation have shown that fungal DNA methylation is implicated in development, genome integrity, and genome defense. While fungal DNMTase has been suggested as playing a role in genome defense, studies of the biological function of fungal DNMTase have been very limited. In this study, we have shown distinct biological functions of two DNA methyltransferases from the chestnut blight fungus C. parasitica. We have demonstrated that DNMTases are important to fungal development and virulence. In addition, these genes are shown to play an important role in the fungal response to hypoviral CHV1 infection, including severely retarded colonial growth, and in viral clearance, which has never been previously observed in mycovirus infection. These findings provide a better understanding of the biological functions of fungal DNA methyltransferase and a basis for clarifying the epigenetic regulation of fungal virulence, responses to hypovirus infection, and viral clearance.

scholarly journals CSTDB: A Crop Stress-tolerance Gene and Protein Database Integrated by Convolutional Neural Networks

2018 ◽  
Author(s):  
Di Zhang ◽  
Yi Yue ◽  
Yang Zhao ◽  
Chao Wang ◽  
Xi Cheng ◽  
...  
Keyword(s):  
Stress Tolerance ◽  
Abiotic Stress Tolerance ◽  
Sequence Information ◽  
Protein Database ◽  
Crop Species ◽  
Biotic Stresses ◽  
Tolerance Gene ◽  
As Stress ◽  
User Friendly ◽  
Id Protein

AbstractNumerous studies have shown that many genes and proteins in plants are involved in the regulation of plant resistance to abiotic and biotic stresses. The researches on the stress tolerance of crops are also the focus of many researchers. To provides a reliable platform for collecting and retrieving genetic and protein information related to stress tolerance found in crops, we constructed CSTDB(Crops Stress-tolerance Database), an integrated database that includes stress-tolerance genes and proteins for many crop species. The database was developed based on convolutional neural network technology. It is a web-accessible database that contains detailed information on the stress-tolerance genes and proteins of major crop species. Currently, the database records four major crops containing 1,371 abiotic stress-tolerance genes or proteins, and 207 genes or proteins associated with biotic stress. Each gene and protein has detailed functional information and sequence information, such as stress types, Genbank ID, Pubmed ID, Protein ID, 3D model picture and FASTA files. As a user-friendly browsing tool, this database provides search functions, BALST functions and file download functions. CSTDB can be a valuable resource, which is designed to meet the broad needs of researchers working on crops stress-tolerance experiments. Database URL: http://pcsb.ahau.edu.cn:8080/CSTDB

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