scholarly journals Hypovirus Papain-Like Protease p48 Is Required for Initiation but Not for Maintenance of Virus RNA Propagation in the Chestnut Blight Fungus Cryphonectria parasitica

2008 ◽  
Vol 82 (13) ◽  
pp. 6369-6378 ◽  
Author(s):  
Fuyou Deng ◽  
Donald L. Nuss
Keyword(s):  
Cryphonectria Parasitica ◽  
Mutant Virus ◽  
Chestnut Blight ◽  
Cdna Clones ◽  
Coding Region ◽  
Chestnut Blight Fungus ◽  
Virus Rna ◽  
Virulence Attenuation ◽  
Rna Accumulation ◽  
Suppressor Of Rna Silencing

ABSTRACT The prototypic hypovirus CHV1-EP713, responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica, encodes two papain-like proteases, p29 and p48. Protein p29 has been shown to be dispensable for hypovirus RNA replication and to act as a suppressor of RNA silencing. Here we describe a role for p48 in hypovirus RNA propagation. CHV1-EP713 infectious cDNA clones in which the p48 coding region was deleted, Δp48, were unable to establish infection in C. parasitica when introduced as a DNA form by transformation or as a coding strand transcript by electroporation. However, the Δp48 mutant virus RNA was rescued when p48 was provided in trans. Surprisingly, the Δp48 mutant viruses retained replication competence in the apparent absence of p48 following transmission to wild-type C. parasitica and successive subculturing. The replicating Δp48 mutant virus was reduced in RNA accumulation by 60% both in the absence and presence of p48 provided in trans and was transmitted through asexual spores (conidia) at a rate 3 to 8% of that for full-length CHV1-EP713. Complementary analysis of strains expressing p48 or containing the replicating Δp48 mutant virus showed that like p29, p48 contributes to virus-mediated suppression of host pigmentation and conidiation, although to a lesser extent, and is dispensable for hypovirus-mediated hypovirulence. The combined results suggest that papain-like protease p48 plays an essential role in the initiation but not the maintenance of virus RNA propagation and also contributes to the regulation of viral RNA accumulation and vertical transmission.

scholarly journals Synergism between a mycoreovirus and a hypovirus mediated by the papain-like protease p29 of the prototypic hypovirus CHV1-EP713

2006 ◽  
Vol 87 (12) ◽  
pp. 3703-3714 ◽  
Author(s):  
Liying Sun ◽  
Donald L. Nuss ◽  
Nobuhiro Suzuki
Keyword(s):  
Vertical Transmission ◽  
Cryphonectria Parasitica ◽  
Chestnut Blight Fungus ◽  
Virus Rna ◽  
The Family ◽  
Heterologous Virus ◽  
In Trans ◽  
Rna Accumulation ◽  
Suppressor Of Rna Silencing

Infection of the chestnut blight fungus, Cryphonectria parasitica, by the prototypic hypovirus Cryphonectria hypovirus 1-EP713 (CHV1-EP713) or by the type member, Mycoreovirus 1-Cp9B21 (MyRV1-Cp9B21), of a novel genus (Mycoreovirus) of the family Reoviridae results in hypovirulence, but with a different spectrum of phenotypic changes. The former virus depresses pigmentation and conidiation dramatically, whilst the latter virus has little effect on these processes. This study showed that double infection by the two viruses resulted in a phenotype similar to that of CHV1-EP713 singly infected colonies, but with further decreased levels of host conidiation and vegetative growth and increased levels of MyRV1-Cp9B21 genomic dsRNA accumulation (twofold) and vertical transmission (sixfold). In contrast, CHV1-EP713 RNA accumulation was not altered by MyRV1-Cp9B21 infection. It was also found that the papain-like cysteine protease p29, encoded by CHV1-EP713 ORF A, contributes to the phenotypic alterations and transactivation of MyRV1-Cp9B21 replication and transmission. Chromosomally expressed p29 was able to increase MyRV1-Cp9B21 vertical transmission by more than twofold and genomic RNA accumulation by 80 %. Transactivation was abolished by Cys→Gly mutations at p29 residues 70 and 72 located within the previously identified symptom-determinant domain required for suppression of host pigmentation and sporulation and p29-mediated in trans enhancement of homologous Δp29 mutant virus RNA replication. Transactivation was not altered by Ser substitutions at the p29 protease catalytic residue Cys162. These results indicated a link between p29-mediated enhancement of heterologous virus accumulation and transmission and p29-mediated host symptom expression. The role of p29 as a suppressor of RNA silencing is discussed.

scholarly journals Characterization of Hypovirus-Derived Small RNAs Generated in the Chestnut Blight Fungus by an Inducible DCL-2-Dependent Pathway

2008 ◽  
Vol 82 (6) ◽  
pp. 2613-2619 ◽  
Author(s):  
Xuemin Zhang ◽  
Gert C. Segers ◽  
Qihong Sun ◽  
Fuyou Deng ◽  
Donald L. Nuss
Keyword(s):  
Small Rnas ◽  
Rna Silencing ◽  
Cryphonectria Parasitica ◽  
Chestnut Blight ◽  
Fungal Host ◽  
Chestnut Blight Fungus ◽  
Mutant Strains ◽  
Suppressor Of Rna Silencing ◽  
Dependent Pathway

ABSTRACT The disruption of one of two dicer genes, dcl-2, of the chestnut blight fungus Cryphonectria parasitica was recently shown to increase susceptibility to mycovirus infection (G. C. Segers, X. Zhang, F. Deng, Q. Sun, and D. L. Nuss, Proc. Natl. Acad. Sci. USA 104:12902-12906, 2007). We now report the accumulation of virus-derived small RNAs (vsRNAs) in hypovirus CHV1-EP713-infected wild-type and dicer gene dcl-1 mutant C. parasitica strains but not in hypovirus-infected dcl-2 mutant and dcl-1 dcl-2 double-mutant strains. The CHV1-EP713 vsRNAs were produced from both the positive and negative viral RNA strands at a ratio of 3:2 in a nonrandom distribution along the viral genome. We also show that C. parasitica responds to hypovirus and mycoreovirus infections with a significant increase (12- to 20-fold) in dcl-2 expression while the expression of dcl-1 is increased only modestly (2-fold). The expression of dcl-2 is further increased (∼35-fold) following infection with a hypovirus CHV1-EP713 mutant that lacks the p29 suppressor of RNA silencing. The combined results demonstrate the biogenesis of mycovirus-derived small RNAs in a fungal host through the action of a specific dicer gene, dcl-2. They also reveal that dcl-2 expression is significantly induced in response to mycovirus infection by a mechanism that appears to be repressed by the hypovirus-encoded p29 suppressor of RNA silencing.

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 Linkage between Mitochondrial Hypovirulence and Viral Hypovirulence in the Chestnut Blight Fungus Revealed by cDNA Microarray Analysis

2004 ◽  
Vol 3 (5) ◽  
pp. 1227-1232 ◽  
Author(s):  
Todd D. Allen ◽  
Donald L. Nuss
Keyword(s):  
Mitochondrial Dysfunction ◽  
Cdna Microarray ◽  
Gene List ◽  
Chestnut Blight ◽  
Cdna Clones ◽  
Mtdna Mutation ◽  
Transcript Accumulation ◽  
Transcriptional Responses ◽  
Chestnut Blight Fungus ◽  
Virulence Attenuation

ABSTRACT The phenomenon of transmissible hypovirulence (virulence attenuation) associated with biological control of natural populations of the chestnut blight fungus Cryphonectria parasitica can be experimentally reproduced by infection with hypovirus cDNA clones (viral hypovirulence) or by mutation of mitochondrial DNA (mtDNA) in the absence of virus infection (mitochondrial hypovirulence). We now report the use of an established C. parasitica cDNA microarray to monitor nuclear transcriptional responses to an mtDNA mutation of C. parasitica strain EP155, designated EP155/mit2, which was previously shown to induce elevated alternative oxidase activity and hypovirulence (C. B. Monterio-Vitorello, J. A. Bell, D. W. Fulbright, and H. A. Bertrand, Proc. Natl. Acad. Sci. USA 92:5935-5939, 1995). Approximately 10% of the 2,200 genes represented on the microarray exhibited altered transcript accumulation as a result of the mit2 mtDNA mutation. While genes involved in mitochondrial function were clearly represented in the EP155/mit2-responsive gene list, direct parallels to the well-characterized Saccharomyces cerevisiae retrograde response to mitochondrial dysfunction were not observed. Remarkably, 47% of the genes that were differentially expressed following the infection of strain EP155 by the prototypic hypovirus CHV1-EP713 had similarly changed transcript accumulation in the virus-free EP155/mit2 mutant. These results establish a linkage between viral and mitochondrial hypovirulence and raise questions regarding the relationship between hypovirus infection and mitochondrial dysfunction. The combined set of transcriptional profile data provides a foundation for future studies on mitochondrion-to-nucleus communications in the context of hypovirus infection and senescence associated with mitochondrial dysfunction in filamentous fungi.

Crypt1, an active Ac-like transposon from the chestnut blight fungus, Cryphonectria parasitica

2001 ◽  
Vol 265 (4) ◽  
pp. 730-738 ◽  
Author(s):  
D. Linder-Basso ◽  
R. Foglia ◽  
P. Zhu ◽  
B.I. Hillman
Keyword(s):  
Cryphonectria Parasitica ◽  
Chestnut Blight ◽  
Chestnut Blight Fungus

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 Hard Mast Production Before and After the Chestnut Blight

2000 ◽  
Vol 24 (4) ◽  
pp. 196-201 ◽  
Author(s):  
Seth J. Diamond ◽  
Robert H. Giles ◽  
Roy L. Kirkpatrick ◽  
Gary J. Griffin
Keyword(s):  
Carrying Capacity ◽  
Basal Area ◽  
Cryphonectria Parasitica ◽  
Castanea Dentata ◽  
Chestnut Blight ◽  
Wildlife Species ◽  
Chestnut Blight Fungus ◽  
Southern Appalachian ◽  
Before And After ◽  
Mast Production

Abstract We estimated hard mast production of a Southern Appalachian forest for two 10 yr intervals: one before and one, 35 yr after, the chestnut blight fungus (Cryphonectria parasitica) (Murr.) Barr, had killed all mature chestnut trees. The basal area of hard mast-producing trees in the postblight forest was 28% less than in the preblight forest. The estimate of hard mast output was 34% less after the chestnut blight. Postblight production was less than preblight production for 8 of 10 yr. During 5 of these years, postblight production was only 5-27% of preblight production. Annual preblight mast production was relatively stable, whereas annual postblight production fluctuated substantially. Our findings suggest that the loss of mature chestnuts (Castanea dentata) markedly reduced the Southern Appalachian forest's carrying capacity for certain wildlife species. South. J. Appl. For 24(4):196-201.

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