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.

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 Inactivation of hantaviruses by N-ethylmaleimide preserves virion integrity

2011 ◽  
Vol 92 (5) ◽  
pp. 1189-1198 ◽  
Author(s):  
Tomas Strandin ◽  
Jussi Hepojoki ◽  
Hao Wang ◽  
Antti Vaheri ◽  
Hilkka Lankinen
Keyword(s):  
Cell Attachment ◽  
Buoyant Density ◽  
Structural Proteins ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Thiol Groups ◽  
Wild Type ◽  
Enveloped Viruses ◽  
Nitrobenzoic Acid ◽  
The Family

Thiol groups of cysteine residues are crucial for the infectivity of various enveloped viruses, but their role in the infectivity of viruses of the family Bunyaviridae has thus far not been studied. This report shows that thiol groups are essential to the infectivity of hantaviruses. Alkylation of the thiol functional groups using the membrane-permeable compound N-ethylmaleimide (NEM) and membrane-impermeable compound 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) showed NEM to be a highly effective inactivator of Puumala and Tula hantaviruses. The NEM-inactivated hantavirus maintained the buoyant density of the wild-type virus. Furthermore, the antigenicity of glycoproteins and the cell attachment capacity of virions were retained at NEM concentrations that totally abolished virus infectivity. These results signified preservation of virion integrity following inactivation with NEM, making chemically inactivated virions valuable research antigens. It was demonstrated with biotin-conjugated maleimide, a mechanistic analogue of NEM, that all the structural proteins of hantavirus were sensitive towards thiol alkylation. In contrast to hantaviruses, NEM did not abolish Uukuniemi phlebovirus infectivity to the same extent. This indicates differences in the use of free thiols in virus entry among members of the family Bunyaviridae.

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 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 Hypovirus Virulence and Vegetative Incompatibility in Populations of the Chestnut Blight Fungus

2012 ◽  
Vol 102 (12) ◽  
pp. 1161-1167 ◽  
Author(s):  
Sarah Franziska Bryner ◽  
Daniel Rigling
Keyword(s):  
Biocontrol Agent ◽  
Cryphonectria Parasitica ◽  
Vegetative Compatibility ◽  
Chestnut Blight ◽  
Vegetative Incompatibility ◽  
Tree Disease ◽  
Chestnut Blight Fungus ◽  
Fungal Populations ◽  
Cryphonectria Hypovirus ◽  
High Diversity

Cryphonectria hypovirus 1 hyperparasitizes the chestnut blight fungus Cryphonectria parasitica and acts as a biocontrol agent for this serious tree disease. The virus is transmitted cytoplasmatically between fungal individuals. However, highly virulent viruses strongly debilitate their host and, thus, reduce their own transmission probability. Furthermore, vegetative incompatibility between fungi is an important transmission barrier. Therefore, virulent viruses are expected to be strongly selected against in fungal populations with high levels of vegetative incompatibility, eventually leading to the erosion of biocontrol. To test this prediction, we assessed the virulence of the virus in four European C. parasitica populations with high diversity of vegetative compatibility types and in four populations with low diversity. We expected the degree of virus virulence to be lower in fungal populations with high levels of vegetative incompatibility. However, our results did not reveal such a trend. No significant differences in virus virulence between populations with low versus high diversity of vegetative compatibility types were observed. There was no evidence for an erosion of disease control due to the presence of these transmission barriers. Thus, the findings of this study are promising for the sustainability of Cryphonectria hypovirus 1 as a biocontrol agent for chestnut blight in Europe.

scholarly journals Role of Fresh Dead Wood in the Epidemiology and the Biological Control of the Chestnut Blight Fungus

Plant Disease ◽  
2019 ◽  
Vol 103 (3) ◽  
pp. 430-438 ◽  
Author(s):  
Joana Beatrice Meyer ◽  
Loïc Chalmandrier ◽  
Fabio Fässler ◽  
Christopher Schefer ◽  
Daniel Rigling ◽  
...  
Keyword(s):  
Biological Control ◽  
Dead Wood ◽  
Biocontrol Agent ◽  
Castanea Sativa ◽  
Cryphonectria Parasitica ◽  
Chestnut Blight ◽  
Chestnut Blight Fungus ◽  
Study Sites ◽  
Cryphonectria Hypovirus

The invasive fungus Cryphonectria parasitica, the causal agent of chestnut blight, is able to survive and sporulate on the bark of fresh dead Castanea sativa wood for at least 2 years. Here, we experimentally investigated the role of fresh dead wood in the epidemiology of chestnut blight, specifically in the spread of the hyperparasitic virus Cryphonectria hypovirus 1, which acts as biocontrol agent of C. parasitica. A total of 152 artificially initiated, virulent bark cankers in four chestnut stands were treated with virus-infected asexual spores originating either from sporulating dead wood or from a spore suspension. Molecular markers for both the virus and the fungal carrier were used to examine the spread of the applied biocontrol virus. Fourteen months after treatment, 42 to 76% of the conidial spray-treated cankers and 50 to 60% of the cankers exposed to a sporulating dead stem had been virus infected by the applied hypovirulent conidia in all four study sites. Virus infection reduced canker expansion and promoted canker healing (callusing). Thus, fresh chestnut dead wood may play an important role in supporting the successful spread of natural hypovirulence in chestnut forests. Further, combined with the application of virus-infected conidial suspensions, it may help promote the establishment of artificially released hypoviruses in chestnut stands to control chestnut blight.

Occurrence of Cryphonectria hypovirus 1 in the chestnut blight fungus in Macedonia

2006 ◽  
Vol 36 (2) ◽  
pp. 136-143 ◽  
Author(s):  
K. Sotirovski ◽  
M. G. Milgroom ◽  
D. Rigling ◽  
U. Heiniger
Keyword(s):  
Chestnut Blight ◽  
Chestnut Blight Fungus ◽  
Cryphonectria Hypovirus

scholarly journals Identification of an RNA Silencing Suppressor Encoded by a Symptomless Fungal Hypovirus, Cryphonectria Hypovirus 4

Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 100
Author(s):  
Annisa Aulia ◽  
Kiwamu Hyodo ◽  
Sakae Hisano ◽  
Hideki Kondo ◽  
Bradley I. Hillman ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Expression System ◽  
Cryphonectria Parasitica ◽  
Inhibitory Effect ◽  
Chestnut Blight ◽  
Silencing Suppressor ◽  
Terminal Portion ◽  
Transgenic Expression ◽  
Chestnut Blight Fungus ◽  
Cryphonectria Hypovirus

Previously, we have reported the ability of a symptomless hypovirus Cryphonectria hypovirus 4 (CHV4) of the chestnut blight fungus to facilitate stable infection by a co-infecting mycoreovirus 2 (MyRV2)—likely through the inhibitory effect of CHV4 on RNA silencing (Aulia et al., Virology, 2019). In this study, the N-terminal portion of the CHV4 polyprotein, termed p24, is identified as an autocatalytic protease capable of suppressing host antiviral RNA silencing. Using a bacterial expression system, CHV4 p24 is shown to cleave autocatalytically at the di-glycine peptide (Gly214-Gly215) of the polyprotein through its protease activity. Transgenic expression of CHV4 p24 in Cryphonectria parasitica suppresses the induction of one of the key genes of the antiviral RNA silencing, dicer-like 2, and stabilizes the infection of RNA silencing-susceptible virus MyRV2. This study shows functional similarity between CHV4 p24 and its homolog p29, encoded by the symptomatic prototype hypovirus CHV1.

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.

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