scholarly journals Investigation of Host Range of and Host Defense against a Mitochondrially Replicating Mitovirus

2019 ◽  
Vol 93 (6) ◽  
Author(s):  
Sabitree Shahi ◽  
Ana Eusebio-Cope ◽  
Hideki Kondo ◽  
Bradley I. Hillman ◽  
Nobuhiro Suzuki
Keyword(s):  
Rna Polymerase ◽  
Host Range ◽  
Protoplast Fusion ◽  
Rna Silencing ◽  
Cryphonectria Parasitica ◽  
Chestnut Blight ◽  
Rna Dependent Rna Polymerase ◽  
Content Type ◽  
Host Interactions ◽  
Chestnut Blight Fungus

ABSTRACT Mitoviruses (genus Mitovirus, family Narnaviridae) are mitochondrially replicating viruses that have the simplest positive-sense RNA genomes of 2.2 to 4.4 kb with a single open reading frame (ORF) encoding an RNA-dependent RNA polymerase. Cryphonectria parasitica mitovirus 1 (CpMV1) from U.S. strain NB631 of the chestnut blight fungus, Cryphonectria parasitica, was the first virus identified as a mitochondrially replicating virus. Despite subsequent discovery of many other mitoviruses from diverse fungi, no great advances in understanding mitovirus biology have emerged, partly because of the lack of inoculation methods. Here we developed a protoplast fusion-based protocol for horizontal transmission of CpMV1 that entailed fusion of recipient and donor protoplasts, hyphal anastomosis, and single-conidium isolation. This method allowed expansion of the host range to many other C. parasitica strains. Species within and outside the family Cryphonectriaceae, Cryphonectria radicalis and Valsa ceratosperma, also supported the replication of CpMV1 at a level comparable to that in the natural host. No stable maintenance of CpMV1 was observed in Helminthosporium victoriae. PCR-based haplotyping of virus-infected fungal strains confirmed the recipient mitochondrial genetic background. Phenotypic comparison between CpMV1-free and -infected isogenic strains revealed no overt effects of the virus. Taking advantage of the infectivity to the standard strain C. parasitica EP155, accumulation levels were compared among antiviral RNA silencing-proficient and -deficient strains in the EP155 background. Comparable accumulation levels were observed among these strains, suggesting the avoidance of antiviral RNA silencing by CpMV1, which is consistent with its mitochondrial replication. Collectively, the results of study provide a foundation to further explore the biology of mitoviruses. IMPORTANCE Capsidless mitoviruses, which are ubiquitously detected in filamentous fungi, have the simplest RNA genomes of 2.2 to 4.4 kb, encoding only RNA-dependent RNA polymerase. Despite their simple genomes, detailed biological characterization of mitoviruses has been hampered by their mitochondrial location within the cell, posing challenges to their experimental introduction and study. Here we developed a protoplast fusion-based protocol for horizontal transfer of the prototype mitovirus, Cryphonectria parasitica mitovirus 1 (CpMV1), which was isolated from strain NB631 of the chestnut blight fungus (Cryphonectria parasitica), a model filamentous fungus for studying virus-host interactions. The host range of CpMV1 has been expanded to many different strains of C. parasitica and different fungal species within and outside the Cryphonectriaceae. Comparison of CpMV1 accumulation among various RNA silencing-deficient and -competent strains showed clearly that the virus was unaffected by RNA silencing. This study provides a solid foundation for further exploration of mitovirus-host interactions.

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 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.

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 The IDR-containing protein PID-2 affects Z granules and is required for piRNA-induced silencing in the embryo

2020 ◽  
Author(s):  
Maria Placentino ◽  
António Miguel de Jesus Domingues ◽  
Jan Schreier ◽  
Sabrina Dietz ◽  
Svenja Hellmann ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Caenorhabditis Elegans ◽  
Rna Polymerase ◽  
Small Rna ◽  
Rna Silencing ◽  
Rna Dependent Rna Polymerase ◽  
Prolyl Aminopeptidase ◽  
C Elegans

AbstractIn Caenorhabditis elegans, the piRNA (21U RNA) pathway is required to establish proper gene regulation and an immortal germline. To achieve this, PRG-1-bound 21U RNAs trigger silencing mechanisms mediated by RNA-dependent RNA polymerase (RdRP)-synthetized 22G RNAs. This silencing can become PRG-1-independent, and heritable over many generations. This state is named RNAe. It is unknown how and when RNAe is established, and how it is maintained. We show that maternally provided 21U RNAs can be sufficient to trigger RNAe in embryos. Additionally, we identify the IDR-containing protein PID-2, as a factor required to establish and maintain RNAe. PID-2 interacts with two novel, partially redundant, eTudor domain proteins, PID-4 and PID-5. Additionally, PID-5 has a domain related to the X-prolyl aminopeptidase protein APP-1, and binds APP-1, implicating N-terminal proteolysis in RNAe. All three proteins are required for germline immortality, localize to perinuclear foci, affect Z granules, and are required for balancing of 22G RNA populations. Overall, our study identifies three new proteins with crucial functions in the C. elegans small RNA silencing network.

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.

scholarly journals Hypovirus Papain-Like Protease p29 Suppresses RNA Silencing in the Natural Fungal Host and in a Heterologous Plant System

2006 ◽  
Vol 5 (6) ◽  
pp. 896-904 ◽  
Author(s):  
Gerrit C. Segers ◽  
Rene van Wezel ◽  
Xuemei Zhang ◽  
Yiguo Hong ◽  
Donald L. Nuss
Keyword(s):  
Rna Silencing ◽  
Defense Mechanism ◽  
Fluorescent Protein ◽  
Cryphonectria Parasitica ◽  
Fungal Host ◽  
Chestnut Blight Fungus ◽  
Systemic Spread ◽  
Green Fluorescent ◽  
Fungal Viruses ◽  
Suppressor Of Rna Silencing

ABSTRACT Virulence-attenuating hypoviruses of the species Cryphonectria hypovirus 1 (CHV1) encode a papain-like protease, p29, that shares similarities with the potyvirus-encoded suppressor of RNA silencing HC-Pro. We now report that hypovirus CHV1-EP713-encoded p29 can suppress RNA silencing in the natural host, the chestnut blight fungus Cryphonectria parasitica. Hairpin RNA-triggered silencing was suppressed in C. parasitica strains expressing p29, and transformation of a transgenic green fluorescent protein (GFP)-silenced strain with p29 resulted in an increased number of transformants with elevated GFP expression levels. The CHV1-EP713 p29 protein was also shown to suppress both virus-induced and agroinfiltration-induced RNA silencing and systemic spread of silencing in GFP-expressing transgenic Nicotiana benthamiana line 16c plants. The demonstration that a mycovirus encodes a suppressor of RNA silencing provides circumstantial evidence that RNA silencing in fungi may serve as an antiviral defense mechanism. The observation that a phylogenetically conserved protein of related plant and fungal viruses functions as a suppressor of RNA silencing in both fungi and plants indicates a level of conservation of the mechanisms underlying RNA silencing in these two groups of organisms.

scholarly journals Crystal Structure of Classical Swine Fever Virus NS5B Reveals a Novel N-Terminal Domain

2018 ◽  
Vol 92 (14) ◽  
Author(s):  
Weiwei Li ◽  
Baixing Wu ◽  
Wibowo Adian Soca ◽  
Lei An
Keyword(s):  
Crystal Structure ◽  
Rna Polymerase ◽  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Structural Basis ◽  
Economic Losses ◽  
Rna Dependent Rna Polymerase ◽  
Content Type ◽  
Terminal Domain

ABSTRACTClassical swine fever virus (CSFV) is the cause of classical swine fever (CSF). Nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that is a key enzyme initiating viral RNA replication by ade novomechanism. It is also an attractive target for the development of anti-CSFV drugs. To gain a better understanding of the mechanism of CSFV RNA synthesis, here, we solved the first crystal structure of CSFV NS5B. Our studies show that the CSFV NS5B RdRp contains the characteristic finger, palm, and thumb domains, as well as a unique N-terminal domain (NTD) that has never been observed. Mutagenesis studies on NS5B validated the importance of the NTD in the catalytic activity of this novel RNA-dependent RNA polymerase. Moreover, our results shed light on CSFV infection.IMPORTANCEPigs are important domesticated animals. However, a highly contagious viral disease named classical swine fever (CSF) causes devastating economic losses. Classical swine fever virus (CSFV), the primary cause of CSF, is a positive-sense single-stranded RNA virus belonging to the genusPestivirus, familyFlaviviridae. Genome replication of CSFV depends on an RNA-dependent RNA polymerase (RdRp) known as NS5B. However, the structure of CSFV NS5B has never been reported, and the mechanism of CSFV replication is poorly understood. Here, we solve the first crystal structure of CSFV NS5B and analyze the functions of the characteristic finger, palm, and thumb domains. Additionally, our structure revealed the presence of a novel N-terminal domain (NTD). Biochemical studies demonstrated that the NTD of CSFV NS5B is very important for RdRp activity. Collectively, our studies provide a structural basis for future rational design of anti-CSFV drugs, which is critically important, as no effective anti-CSFV drugs have been developed.

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