scholarly journals Major impacts on the primary metabolism of the plant pathogen Cryphonectria parasitica by the virulence-attenuating virus CHV1-EP713

Microbiology ◽  
2009 ◽  
Vol 155 (12) ◽  
pp. 3913-3921 ◽  
Author(s):  
Angus L. Dawe ◽  
Wayne A. Van Voorhies ◽  
Tannia A. Lau ◽  
Alexander V. Ulanov ◽  
Zhong Li
Keyword(s):  
Metabolic Rate ◽  
Carbohydrate Metabolism ◽  
Cryphonectria Parasitica ◽  
Fungal Species ◽  
Primary Metabolism ◽  
Wild Type ◽  
Primary Metabolites ◽  
Antiviral Responses ◽  
Chestnut Blight Fungus ◽  
The Family

Cryphonectria parasitica, the chestnut blight fungus, can be infected by virulence-attenuating mycoviruses of the family Hypoviridae. Previous studies have led to the hypothesis that the hypovirus-infected phenotype is partly due to metabolic changes induced by the viral infection. To investigate this, we measured the metabolic rate and respiration of C. parasitica colonies grown on solid medium. These experiments supported historical observations of other fungal species done in liquid cultures that the metabolic rate steadily declines with age and differentiation of the mycelium. Hypovirus infection increased metabolic rate in the youngest mycelium, but a subsequent decline was also observed as the mycelium aged. By measuring both CO2 production and O2 consumption, we also observed that changes occur in carbohydrate metabolism as a result of ageing in both infected and uninfected mycelium. Mycelium on the periphery of the colony exploited fermentation pathways extensively, before transitioning to aerobic carbohydrate metabolism and finally lipid metabolism in the interior regions, despite abundant remaining glucose. However, the hypovirus affected the extent of these changes, with infected mycelium apparently unable to utilize lipid-related metabolic pathways, leading to an increased depletion of glucose. Finally, we used metabolic profifiling to determine the changes in accumulation of primary metabolites in wild-type and hypovirus-infected mycelium and found that approximately one-third of the 164 detected metabolites were affected. These results are consistent with those expected from the physiological measurements, with significant alterations noted for compounds related to lipid and carbohydrate metabolism. Additionally, we observed an increase in the accumulation of the polyamine spermidine in the presence of hypovirus. Polyamines have been implicated in antiviral responses of mammalian systems; therefore this may suggest a novel antiviral response mechanism in fungi.

scholarly journals Complete genome sequence of Mycoreovirus-1/Cp9B21, a member of a novel genus within the family Reoviridae, isolated from the chestnut blight fungus Cryphonectria parasitica

2004 ◽  
Vol 85 (11) ◽  
pp. 3437-3448 ◽  
Author(s):  
Nobuhiro Suzuki ◽  
S. Supyani ◽  
Kazuyuki Maruyama ◽  
Bradley I. Hillman
Keyword(s):  
Cryphonectria Parasitica ◽  
Chestnut Blight ◽  
Virus Family ◽  
Chestnut Blight Fungus ◽  
Choice Pattern ◽  
The Family ◽  
Isoelectric Points ◽  
Large Virus ◽  
Hypovirulent Strain ◽  
Cap Analysis

Mycoreovirus 1 (MYRV-1) is the type species of the newly described genus Mycoreovirus of the large virus family Reoviridae. The virus was isolated from a hypovirulent strain (9B21) of the chestnut blight fungus, Cryphonectria parasitica. A previous study showed that double-shelled particles introduced to fungal spheroplasts resulted in stably infected colonies. Of the 11 double-stranded RNA genomic segments (S1–S11), the three largest (S1–S3) were sequenced previously and shown to have moderate levels of similarity to the homologous segments of mammal-pathogenic coltiviruses (Eyach virus and Colorado tick fever virus) and another fungus-infecting reovirus, Mycoreovirus 3 of Rosellinia necatrix strain W370 (MYRV-3/RnW370). The sequences of the remaining segments (S4–S11) are reported here. All of the segments have single ORFs on their positive strands and the terminal sequences 5′-GAUCA----GCAGUCA-3′ are conserved among currently and previously sequenced segments. Oligo-cap analysis showed that the positive strands of the genomic segments are capped, whereas the negative strands are not. Similarities among the four evolutionarily related viruses include low or moderate levels of amino acid sequence identity (14·7–34·2 %) and isoelectric points among equivalent polypeptides, e.g. proteins encoded by segments S4 and S5 of the four viruses. Phylogenetic analysis indicated that MYRV-1/Cp9B21 is related more closely to MYRV-3/RnW370 than to the coltiviruses. An interesting dissimilarity is found in codon-choice pattern among the four viruses, i.e. MYRV-1/Cp9B21 segments have a lower frequency of [XYG+XYC] than corresponding segments of the other viruses, suggesting a possible adjustment of virus codon usage to their host environments.

scholarly journals Extending Chestnut Blight Hypovirus Host Range Within Diaporthales by Biolistic Delivery of Viral cDNA

2002 ◽  
Vol 15 (8) ◽  
pp. 780-789 ◽  
Author(s):  
Atsuko Sasaki ◽  
Mari Onoue ◽  
Satoko Kanematsu ◽  
Kouich Suzaki ◽  
Masaki Miyanishi ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Cryphonectria Parasitica ◽  
Fungal Species ◽  
Chestnut Blight ◽  
Practical Applications ◽  
Chestnut Blight Fungus ◽  
Delivery Technique ◽  
Virulence Attenuation ◽  
Viral Cdna ◽  
Valsa Ceratosperma

Biolistic bombardment was used to successfully transform three phytopathogenic fungal species with an infectious cDNA clone of the prototypic hypovirus, CHV1-EP713, a genetic element responsible for the virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica. The fungal species included two strains each of C. parasitica and Valsa ceratosperma, as well as one strain of Phomopsis G-type (teleomorph Diaporthe Nitschke); all are members of the order Diaporthales but classified into three different genera. A subset of transformants for each of the fungal species contained CHV1-EP713 dsRNA derived from chromosomally integrated viral cDNA. As has been reported for CHV1-EP713 infection of the natural host C. parasitica, biolistic introduction of CHV1-EP713 into the new fungal hosts V. ceratosperma and Phomopsis G-type resulted in altered colony morphology and, more importantly, reduced virulence. These results suggest a potential for hypoviruses as biological control agents in plant-infecting fungal pathogens other than the chestnut blight fungus and closely related species. In addition, the particle delivery technique offers a convenient means of transmitting hypoviruses to potential host fungi that provides new avenues for fundamental mycovirus research and may have practical applications for conferring hypovirulence directly on infected plants in the field.

scholarly journals A Host Factor Involved in Hypovirus Symptom Expression in the Chestnut Blight Fungus, Cryphonectria parasitica

2007 ◽  
Vol 82 (2) ◽  
pp. 740-754 ◽  
Author(s):  
M. Iqbal Faruk ◽  
Ana Eusebio-Cope ◽  
Nobuhiro Suzuki
Keyword(s):  
Insertion Site ◽  
Cryphonectria Parasitica ◽  
Inverse Pcr ◽  
Chestnut Blight ◽  
Wild Type ◽  
Targeted Disruption ◽  
Chestnut Blight Fungus ◽  
Virulence Attenuation ◽  
Symptom Modulation ◽  
Sequence Similarities

ABSTRACT The prototype hypovirus CHV1-EP713 causes virulence attenuation and severe suppression of asexual sporulation and pigmentation in its host, the chestnut blight fungus, Cryphonectria parasitica. We identified a factor associated with symptom induction in C. parasitica using a transformation of C. parasitica strain EP155 with a full-length cDNA clone from a mild mutant virus strain, Cys(72). This was accomplished by using mutagenesis of the transformant fungal strain TCys(72)-1 by random integration of plasmid pHygR, conferring hygromycin resistance. The mutant, namA (after nami-gata, meaning wave shaped), showed an irregular fungal morphology with reduced conidiation and pigmentation while retaining similar levels of virulence and virus accumulation relative to TCys(72)-1- or Cys(72)-infected strain EP155. However, the colony morphology of virus-cured namA (VC-namA) was indistinguishable from those of EP155 and virus-cured TCys(72)-1 [VC-TCys(72)-1]. The phenotypic difference between VC-namA and VC-TCys(72)-1 was found only when these strains infected with the wild type or certain mutant CHV1-EP713 strains but not when infected with Mycoreovirus 1. Sequence analysis of inverse-PCR-amplified genomic DNA fragments and cDNA identified the insertion site of the mutagenic plasmid in exon 8 of the nam-1 gene. NAM-1, comprising 1,257 amino acids, shows sequence similarities to counterparts from other filamentous fungi and possesses the CorA domain that is conserved in a class of Mg2+ transporters from prokaryotes and eukaryotes. Complementation assays using the wild-type and mutant alleles and targeted disruption of nam-1 showed that nam-1 with an extension of the pHygR-derived sequence contributed to the altered phenotype in the namA mutant. The molecular mechanism underlying virus-specific fungal symptom modulation in VC-namA is discussed.

scholarly journals A New Double-Stranded RNA Mycovirus in Cryphonectria naterciae Is Able to Cross the Species Barrier and Is Deleterious to a New Host

2021 ◽  
Vol 7 (10) ◽  
pp. 861
Author(s):  
Carolina Cornejo ◽  
Sakae Hisano ◽  
Helena Bragança ◽  
Nobuhiro Suzuki ◽  
Daniel Rigling
Keyword(s):  
De Novo ◽  
Rna Virus ◽  
Cryphonectria Parasitica ◽  
Fungal Species ◽  
Species Barrier ◽  
New Host ◽  
Double Stranded Rna ◽  
The Family ◽  
Rapid Amplification ◽  
Segmented Genome

Cryphonectria is a fungal genus associated with economically significant disease of trees. Herein we characterized a novel double-stranded RNA virus from the fungal species Cryphonectria naterciae, a species unexplored as a virus host. De novo assembly of RNA-seq data and Sanger sequencing of RACE (rapid amplification of cDNA ends) clones gave the complete, non-segmented genome (10,164 bp) of the virus termed Cryphonectria naterciae fusagravirus (CnFGV1) that was phylogenetically placed within the previously proposed viral family Fusagraviridae. Of 31 field-collected strains of C. naterciae, 40% tested CnFGV1-positive. Cocultivation resulted in within-species transmission of CnFGV1 to virus-free strains of C. naterciae. Comparison of the mycelium phenotype and the growth rate of CnFGV1-infected and virus-free isogenic strains revealed frequent sectoring and growth reduction in C. naterciae upon virus infection. Co-culturing also led to cross-species transmission of CnFGV1 to Cryphonectria carpinicola and Cryphonectria radicalis, but not to Cryphonectria parasitica. The virus-infected C. naterciae and the experimentally infected Cryphonectria spp. readily transmitted CnFGV1 through asexual spores to the next generation. CnFGV1 strongly reduced conidiation and in some cases vegetative growth of C. carpinicola, which is involved in the European hornbeam disease. This study is the first report of a fusagravirus in the family Cryphonectriaceae and lays the groundwork for assessing a hypovirulence effect of CnFGV1 against the hornbeam decline in Europe.

Characterization of the ERK homologue CpMK2 from the chestnut blight fungus Cryphonectria parasitica

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1349-1358 ◽  
Author(s):  
Eun-Sil Choi ◽  
Hea-Jong Chung ◽  
Myoung-Ju Kim ◽  
Seung-Moon Park ◽  
Byeong-Jin Cha ◽  
...  
Keyword(s):  
Growth Conditions ◽  
Cryphonectria Parasitica ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Growth Defects ◽  
Chestnut Blight Fungus ◽  
Phosphorylation Level ◽  
Solid Media ◽  
Mitogen Activated Protein ◽  
Hypovirulent Strain

The Cryphonectria parasitica gene cpmk2, which encodes a mitogen-activated protein kinase belonging to the yeast extracellular signalling-regulated kinase (YERK1) subfamily, was isolated and its biological function was examined. Disruption of cpmk2 resulted in impaired pigmentation and abolished conidiation. Growth defects were observed in the cpmk2 mutant grown on solid plates, but growth of the mutant appeared normal in liquid media, including EP complete and PD broth, suggesting that the cpmk2 gene is involved in sensing and responding to growth conditions. The mutant's production of laccase, as measured by the size of the coloured area produced on tannic-acid-supplemented plates, was significantly reduced compared with the wild-type, but the intensity of the coloured area was unchanged, suggesting that the reduced laccase activity was owing to reduced growth on solid media rather than transcriptional downregulation. A dramatic reduction observed in the canker area produced by the cpmk2 mutant compared with the wild-type, even more severe than that of a hypovirulent strain, can also be ascribed to defective growth on solid surfaces rather than to impairments in a virulence factor(s). Downregulation of the pheromone gene Mf2/1 was also observed in the mutant, indicating a possible explanation for the regulation of the pheromone precursor gene in filamentous fungi and suggesting the presence of the yeast-like pheromone-responsive pathway in C. parasitica. Immunoblot analyses revealed that the phosphorylation level of CpMK2 increased in both virus-free and virus-containing strains in liquid cultures of up to 5 days old and decreased in older cultures. Moreover, the CpMK2 phosphorylation level increased in both strains after transfer from liquid to solid medium. However, levels of phosphorylated CpMK2 were similar in the two strains, suggesting that CpMK2, unlike CpMK1, is not under the direct control of a hypovirus.

scholarly journals Functional Analysis of an Essential GSP1/Ran Ortholog Gene, CpRan1, from the Chestnut Blight Fungus Cryphonectria parasitica Using a Heterokaryon

2021 ◽  
Vol 7 (5) ◽  
pp. 332
Author(s):  
Dae-Hyuk Kim ◽  
Yo-Han Ko ◽  
Jeesun Chun
Keyword(s):  
Amino Acids ◽  
Functional Analysis ◽  
Mutant Allele ◽  
Microscopic Analysis ◽  
Essential Amino Acids ◽  
Cryphonectria Parasitica ◽  
Complementation Analysis ◽  
Null Mutant ◽  
Wild Type ◽  
Chestnut Blight Fungus

Functional analysis of a GSP1/Ran ortholog, CpRan1, from Cryphonectria parasitica was conducted. Genotype analysis revealed that the putative CpRan1-null mutant was a heterokaryotic transformant harboring two different types of nuclei, one with the wild-type CpRan1 allele and the other with the CpRan1-null mutant allele. The mycelial growth and colony morphology of the heterokaryotic transformant was normal. Microscopic analysis of the resulting conidia (aseptate and monokaryotic asexual spores) demonstrated that although normal germinating spores were observed from conidia harboring a nucleus with the wild-type CpRan1 allele, a number of residual conidia that did not germinate existed. Complementation analysis using protoplasts from the heterokaryon with the wild-type CpRan1 allele confirmed that the CpRan1 gene is essential to C. parasitica. Complementation analysis using the various CpRan1 chimera constructs allowed us to perform a functional analysis of essential amino acids of the CpRan1. Among the four suggested essential amino acids, Lys-97 for ubiquitination was determined to not be an essential residue. Moreover, the CpRan1-null mutant allele was successfully complemented with mouse Ran gene, which suggested that the biological function of Ran gene is evolutionary conserved and that our heterokaryon rescue can be applied for the functional analysis of heterologous genes.

scholarly journals A New Double-Stranded RNA Mycovirus in Cryphonectria naterciae is Able to Cross the Species Barrier and is Deleterious to a New Host

2021 ◽  
Author(s):  
Carolina Cornejo ◽  
Sakae Hisano ◽  
Helena Bragança ◽  
Nobuhiro Suzuki ◽  
Daniel Rigling
Keyword(s):  
De Novo ◽  
Rna Virus ◽  
Cryphonectria Parasitica ◽  
Fungal Species ◽  
Species Barrier ◽  
New Host ◽  
Double Stranded Rna ◽  
The Family ◽  
Rapid Amplification ◽  
Segmented Genome

Cryphonectria is a fungal genus associated with economically significant disease of trees. Herein we characterized a novel double-stranded RNA virus from the fungal species Cryphonectria naterciae, a species unexplored as a virus host. De novo assembly of RNA-seq data and Sanger sequencing of RACE (rapid amplification of cDNA ends) clones gave the complete, non-segmented genome (10,164 bp) of the virus termed Cryphonectria naterciae fusagravirus (CnFGV1) that was phylogenetically placed within the previously proposed viral family Fusagraviridae. Of 31 field-collected strains of C. naterciae, 40% tested CnFGV1-positive. Co-cultivation resulted in within-species transmission of CnFGV1 to virus-free strains of C. naterciae. Comparison of the mycelium phenotype and the growth rate of CnFGV1-infected and virus-free isogenic strains revealed frequent sectoring and growth reduction in C. naterciae. Co-culturing also led to cross-species transmission of CnFGV1 to Cryphonectria carpinicola and Cryphonectria radicalis, but not to Cryphonectria parasitica. The virus-infected C. naterciae and the experimentally infected Cryphonectria spp. readily transmitted CnFGV1 through asexual spores to the next generation. CnFGV1 strongly reduced conidiation and in some cases vegetative growth of C. carpinicola, which is involved in the European hornbeam disease. This study is the first report of a fusagravirus in the family Cryphonectriaceae and lays the groundwork for assessing a hypovirulence effect of CnFGV1 against the hornbeam decline in Europe.

scholarly journals Characterization of a Hypovirus-Regulated Septin Cdc11 Ortholog, CpSep1, from the Chestnut Blight Fungus Cryphonectria parasitica

2019 ◽  
Vol 32 (3) ◽  
pp. 286-295 ◽  
Author(s):  
Myeongjin Jo ◽  
Kum-Kang So ◽  
Yo-Han Ko ◽  
Jeesun Chun ◽  
Jung-Mi Kim ◽  
...  
Keyword(s):  
Cryphonectria Parasitica ◽  
Chestnut Blight ◽  
Null Mutant ◽  
Wild Type ◽  
Chestnut Blight Fungus ◽  
Germination Process ◽  
Cryphonectria Hypovirus ◽  
Null Mutants

We identified a protein spot showing downregulation in the presence of Cryphonectria hypovirus 1 and tannic acid supplementation as a septin subunit with the highest homology to the Aspergillus nidulans aspA gene, an ortholog of the Saccharomyces cerevisiae Cdc11 gene. To analyze the functional role of this septin component (CpSep1), we constructed its null mutant and obtained a total of eight CpSep1-null mutants from 137 transformants. All CpSep1-null mutants showed retarded growth, with fewer aerial mycelia and intense pigmentation on plates of potato dextrose agar supplemented with L-methionine and biotin. When the marginal hyphae were examined, hyperbranching was observed in contrast to the wild type. The inhibition of colonial growth was partially recovered when the CpSep1-null mutants were cultured in the presence of the osmostabilizing sorbitol. Conidia production of the CpSep1-null mutants was significantly increased by at least 10-fold more. Interestingly, the conidial morphology of the CpSep1-null mutants changed to circular in contrast to the typical rod-shaped spores of the wild type, indicating a role of septin in the spore morphology of Cryphonectria parasitica. However, no differences in the germination process were observed. Virulence assays using excised chestnut bark, stromal pustule formation on chestnut stems, and apple inoculation indicated that the CpSep1 gene is important in pathogenicity.

scholarly journals Comparisons of Ectomycorrhizal Colonization of Transgenic American Chestnut with Those of the Wild Type, a Conventionally Bred Hybrid, and Related Fagaceae Species

2014 ◽  
Vol 81 (1) ◽  
pp. 100-108 ◽  
Author(s):  
Katherine M. D'Amico ◽  
Thomas R. Horton ◽  
Charles A. Maynard ◽  
Stephen V. Stehman ◽  
Allison D. Oakes ◽  
...  
Keyword(s):  
Cryphonectria Parasitica ◽  
Oxalate Oxidase ◽  
Fungal Species ◽  
American Chestnut ◽  
Fungal Colonization ◽  
Wild Type ◽  
Root Tips ◽  
Oxidase Gene ◽  
Content Type ◽  
Ectomycorrhizal Colonization

ABSTRACTAmerican chestnut (Castanea dentata[Marsh.] Borkh.) dominated the eastern forests of North America, serving as a keystone species both ecologically and economically until the introduction of the chestnut blight,Cryphonectria parasitica, functionally eradicated the species. Restoration efforts include genetic transformation utilizing genes such as oxalate oxidase to produce potentially blight-resistant chestnut trees that could be released back into the native range. However, before such a release can be undertaken, it is necessary to assess nontarget impacts. Since oxalate oxidase is meant to combat a fungal pathogen, we are particularly interested in potential impacts of this transgene on beneficial fungi. This study compares ectomycorrhizal fungal colonization on a transgenic American chestnut clone expressing enhanced blight resistance to a wild-type American chestnut, a conventionally bred American-Chinese hybrid chestnut, and other Fagaceae species. A greenhouse bioassay used soil from two field sites with different soil types and land use histories. The number of colonized root tips was counted, and fungal species were identified using morphology, restriction fragment length polymorphism (RFLP), and DNA sequencing. Results showed that total ectomycorrhizal colonization varied more by soil type than by tree species. Individual fungal species varied in their colonization rates, but there were no significant differences between colonization on transgenic and wild-type chestnuts. This study shows that the oxalate oxidase gene can increase resistance againstCryphonectria parasiticawithout changing the colonization rate for ectomycorrhizal species. These findings will be crucial for a potential deregulation of blight-resistant American chestnuts containing the oxalate oxidase gene.

scholarly journals Rearrangements of mycoreovirus 1 S1, S2 and S3 induced by the multifunctional protein p29 encoded by the prototypic hypovirus Cryphonectria hypovirus 1 strain EP713

2011 ◽  
Vol 92 (8) ◽  
pp. 1949-1959 ◽  
Author(s):  
Toru Tanaka ◽  
Liying Sun ◽  
Kouhei Tsutani ◽  
Nobuhiro Suzuki
Keyword(s):  
Structural Proteins ◽  
Chestnut Blight ◽  
Wild Type ◽  
Internal Deletion ◽  
Multifunctional Protein ◽  
Chestnut Blight Fungus ◽  
A Genome ◽  
The Family ◽  
Aerial Hyphae ◽  
Cryphonectria Hypovirus

Mycoreovirus 1 (MyRV1), a member of the family Reoviridae possessing a genome consisting of 11 dsRNA segments (S1–S11), infects the chestnut blight fungus and reduces its virulence (hypovirulence). Studies have previously demonstrated reproducible induction of intragenic rearrangements of MyRV1 S6 (S6L: almost full-length duplication) and S10 (S10ss: internal deletion of three-quarters of the ORF), mediated by the multifunctional protein p29 encoded by the prototype hypovirus, Cryphonectria hypovirus 1 (CHV1) strain EP713, of the family Hypoviridae with ssRNA genomes. The current study showed that CHV1 p29 also induced rearrangements of the three largest MyRV1 segments, S1, S2 and S3, which encode structural proteins. These rearranged segments involved in-frame extensions of almost two-thirds of the ORFs (S1L, S2L and S3L, respectively), which is rare for a reovirus rearrangement. MyRV1 variants carrying S1L, S2L or S3L always contained S10ss (MyRV1/S1L+S10ss2, MyRV1/S2L+S10ss2 or MyRV1/S3L+S10ss2). Levels of mRNAs for the rearranged and co-existing unaltered genome segments in fungal colonies infected with each of the MyRV1 variants appeared to be comparable to those for the corresponding normal segments in wild-type MyRV1-infected colonies, suggesting that the rearranged segments were fully competent for packaging and transcription. Protein products of the rearranged segments were detectable in fungal colonies infected with S2L MyRV1/S2L+S10ss2 and S3L MyRV1/S3L+S10ss2, whilst S1L-encoded protein remained undetectable. S1L, S2L and S3L were associated with enhancement of the aerial hyphae growth rate. This study has provided additional examples of MyRV1 intragenic rearrangements induced by p29, and suggests that normal S1, S2 and S3 are required for the symptoms caused by MyRV1.

Sign in / Sign up

Export Citation Format

Share Document