Comparative Analysis of Alterations in Host Phenotype and Transcript Accumulation following Hypovirus and Mycoreovirus Infections of the Chestnut Blight Fungus Cryphonectria parasitica

ABSTRACT Infection of the chestnut blight fungus, Cryphonectria parasitica, by hypovirus CHV1-EP713 or by reovirus MyRV1-Cp9B21 or MyRV2-CpC18 results in reduced fungal virulence (hypovirulence). However, additional phenotypic changes caused by the two groups of mycoviruses are quite different. We now report that the loss of female fertility and the resulting absence of virus transmission through sexual spores observed after hypovirus infection was not observed for reovirus-infected C. parasitica. Consistent with this result, expression of two genes involved in sexual reproduction, the pheromone precursor gene, Mf2/1, and the yeast STE12-like transcriptional factor gene, cpst12, was less reduced in reovirus-infected strains than in the hypovirus CHV1-EP713-infected strain. Analysis with a custom microarray cDNA chip containing expressed sequence tag clones representing approximately 2,200 unique C. parasitica genes identified 140 and 128 host genes that were responsive to MyRV1-Cp9B21 or MyRV2-CpC18 infection, respectively. Comparison of these virus-responsive genes revealed an overlap of 85 genes, even though the nucleotide sequence identity for the two reoviruses is less than 50%. Significantly, 84 of the 85 genes were altered in the same direction. Further comparison revealed that 51% and 48% of the MyRV1-Cp9B21- and MyRV2-CpC18-responsive genes were also responsive to CHV1-EP713 infection. Finally, similar to results reported for CHV1-EP713 infection, a high percentage (59% and 66%) of the reovirus-responsive genes were also differentially expressed following disruption of the cellular G-protein signal transduction pathway. These data support the hypothesis that hypovirus and reovirus infections perturb common and specific C. parasitica regulatory pathways to cause hypovirulence and distinct sets of phenotypic changes.

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Infectious cDNA Clone of Hypovirus CHV1-Euro7: a Comparative Virology Approach To Investigate Virus-Mediated Hypovirulence of the Chestnut Blight Fungus Cryphonectria parasitica

Journal of Virology ◽

10.1128/jvi.73.2.985-992.1999 ◽

1999 ◽

Vol 73 (2) ◽

pp. 985-992 ◽

Cited By ~ 95

Author(s):

Baoshan Chen ◽

Donald L. Nuss

Keyword(s):

Cdna Clone ◽

Cryphonectria Parasitica ◽

Infectious Cdna Clone ◽

Chestnut Blight ◽

Phenotypic Traits ◽

Fungal Virulence ◽

Reading Frame ◽

Phenotypic Changes ◽

Chestnut Blight Fungus ◽

High Level

ABSTRACT We report the construction of a full-length infectious cDNA clone for hypovirus CHV1-Euro7, which is associated with reduced virulence (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica. Field strains infected with CHV1-Euro7 are more virulent and exhibit less severe phenotypic changes (hypovirulence-associated traits) than strains infected with the prototypic hypovirus CHV1-EP713, for which the first infectious cDNA clone was developed. These differences exist even though the two hypoviruses show extensive sequence identities: 87 to 93% and 90 to 98% at the nucleotide and amino acid levels, respectively. The relative contributions of viral and host genomes to phenotypic traits associated with hypovirus infection were examined by transfecting synthetic transcripts of the two hypovirus cDNAs independently into two different virus-free C. parasitica strains, EP155 and Euro7(−v). Although the contribution of the viral genome was clearly predominant, the final magnitude and constellation of phenotypic changes were a function of contributions by both genomes. The high level of sequence identity between the two hypoviruses also allowed construction of viable chimeras and mapping of the difference in symptom expression observed for the two viruses to the open reading frame B coding domain. Implications of these results for engineering enhanced biological control and elucidating the basis for hypovirus-mediated attenuation of fungal virulence are discussed.

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Specific and Common Alterations in Host Gene Transcript Accumulation following Infection of the Chestnut Blight Fungus by Mild and Severe Hypoviruses

Journal of Virology ◽

10.1128/jvi.78.8.4145-4155.2004 ◽

2004 ◽

Vol 78 (8) ◽

pp. 4145-4155 ◽

Cited By ~ 53

Author(s):

Todd D. Allen ◽

Donald L. Nuss

Keyword(s):

Transcriptional Response ◽

Cryphonectria Parasitica ◽

Chestnut Blight ◽

Gene Transcript ◽

Transcript Accumulation ◽

Transcriptional Responses ◽

Chestnut Blight Fungus ◽

Reverse Transcription Pcr ◽

Initial Assignment ◽

Severe Isolate

ABSTRACT We report the use of a cDNA microarray to monitor global transcriptional responses of the chestnut blight fungus, Cryphonectria parasitica, to infection by mild and severe isolates of virulence-attenuating hypoviruses that share 87 to 93% and 90 to 98% identity at the nucleotide and amino acid levels, respectively. Infection by the mild hypovirus isolate CHV1-Euro7 resulted in differential expression of 166 of the ca. 2,200 genes represented on the microarray (90 upregulated and 76 downregulated). This is roughly half the number of genes scored as differentially expressed after infection by the severe isolate, CHV1-EP713 (295 genes; 132 upregulated and 163 downregulated). Comparison of the lists of genes responsive to infection by the two hypovirus isolates revealed 80 virus-common responsive genes. Infection by CHV1-EP713 also caused changes in gene transcript accumulation that were, in general, of greater magnitude than those observed with CHV1-Euro7 infections. Thus, the host transcriptional response to infection by severe hypovirus CHV1-EP713 appears to be considerably more dynamic than the response to infection by the mild isolate CHV1-Euro7. Real-time reverse transcription-PCR was performed on 39 different clones, with false-positive rates of 3 and 8% observed for the microarray-predicted list of genes responsive to CHV1-EP713 and CHV1-Euro7 infections, respectively. This analysis has allowed an initial assignment for ca. 2,200 unique C. parasitica-expressed genes as being unresponsive to hypovirus infection, selectively responsive to a specific hypovirus, or generally responsive to hypovirus infection.

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Evidence for a Role of the Regulator of G-Protein Signaling Protein CPRGS-1 in Gα Subunit CPG-1-Mediated Regulation of Fungal Virulence, Conidiation, and Hydrophobin Synthesis in the Chestnut Blight Fungus Cryphonectria parasitica

Eukaryotic Cell ◽

10.1128/ec.3.6.1454-1463.2004 ◽

2004 ◽

Vol 3 (6) ◽

pp. 1454-1463 ◽

Cited By ~ 36

Author(s):

Gerrit C. Segers ◽

Jerome C. Regier ◽

Donald. L. Nuss

Keyword(s):

G Protein ◽

Pathogenic Fungus ◽

Aerial Mycelium ◽

Cryphonectria Parasitica ◽

Chestnut Blight ◽

G Protein Signaling ◽

Protein Signaling ◽

Fungal Virulence ◽

Chestnut Blight Fungus ◽

Rgs Protein

ABSTRACT We previously reported that the chestnut blight fungus Cryphonectria parasitica expresses at least three G-protein α subunits and that Gα subunit CPG-1 is essential for regulated growth, pigmentation, sporulation, and virulence. We now report the cloning and characterization of a C. parasitica regulator of G-protein signaling (RGS) protein, CPRGS-1. The phylogenetic relationship of CPRGS-1 to orthologs from other fungi was inferred and found to be generally concordant with species relationships based on 18S ribosomal sequences and on morphology. However, Hemiascomycotine RGS branch lengths in particular were longer than for their 18S sequence counterparts, which correlates with functional diversification in the signaling pathway. Deletion of cprgs-1 resulted in reduced growth, sparse aerial mycelium, and loss of pigmentation, sporulation, and virulence. Disruption of cprgs-1 was also accompanied by a severe posttranscriptional reduction in accumulation of CPG-1 and Gβ subunit CPGB-1 and severely reduced expression of the hydrophobin-encoding gene cryparin. The changes in phenotype, cryparin expression, and CPGB-1 accumulation resulting from cprgs-1 gene deletion were also observed in a strain containing a mutationally activated copy of CPG-1 but not in strains containing constitutively activated mutant alleles of the other two identified Gα subunits, CPG-2 and CPG-3. Furthermore, cprgs-1 transcript levels were increased in the activated CPG-1 strain but were unaltered in activated CPG-2 and CPG-3 strains. The results strongly suggest that CPRGS-1 is involved in regulation of Gα subunit CPG-1-mediated signaling and establish a role for a RGS protein in the modulation of virulence, conidiation, and hydrophobin synthesis in a plant pathogenic fungus.

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Ste12 Transcription Factor Homologue CpST12 Is Down-Regulated by Hypovirus Infection and Required for Virulence and Female Fertility of the Chestnut Blight Fungus Cryphonectria parasitica

Eukaryotic Cell ◽

10.1128/ec.00302-06 ◽

2006 ◽

Vol 6 (2) ◽

pp. 235-244 ◽

Cited By ~ 46

Author(s):

Fuyou Deng ◽

Todd D. Allen ◽

Donald L. Nuss

Keyword(s):

Transcription Factor ◽

Expressed Sequence Tag ◽

Substantial Reduction ◽

Cryphonectria Parasitica ◽

Female Fertility ◽

Chestnut Blight ◽

Cellular Transcription Factor ◽

Chestnut Blight Fungus ◽

High Level

ABSTRACT A putative homologue of the Saccharomyces cerevisiae Ste12 transcription factor was identified in a series of expressed sequence tag-based microarray analyses as being down-regulated in strains of the chestnut blight fungus, Cryphonectria parasitica, infected by virulence-attenuating hypoviruses. Cloning of the corresponding gene, cpst12, confirmed a high level of similarity to Ste12 homologues of other filamentous fungi. Disruption of cpst12 resulted in no alterations in in vitro growth characteristics or colony morphology and an increase in the production of asexual spores, indicating that CpST12 is dispensable for vegetative growth and conidiation on artificial medium. However, the disruption mutants showed a very substantial reduction in virulence on chestnut tissue and a complete loss of female fertility, two symptoms normally conferred by hypovirus infection. Both virulence and female fertility were restored by complementation with the wild-type cpst12 gene. Analysis of transcriptional changes caused by cpst12 gene disruption with a custom C. parastica cDNA microaray chip identified 152 responsive genes. A significant number of these putative CpST12-regulated genes were also responsive to hypovirus infection. Thus, cpst12 encodes a cellular transcription factor, CpST12, that is down-regulated by hypovirus infection and required for female fertility, virulence and regulated expression of a subset of hypovirus responsive host genes.

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Large-scale expressed sequence tag analysis for the chestnut blight fungus Cryphonectria parasitica

Fungal Genetics and Biology ◽

10.1016/j.fgb.2007.11.002 ◽

2008 ◽

Vol 45 (3) ◽

pp. 319-327 ◽

Cited By ~ 14

Author(s):

Jinjie Shang ◽

Xiaosong Wu ◽

Xiuwan Lan ◽

Yunyan Fan ◽

Haitao Dong ◽

...

Keyword(s):

Large Scale ◽

Expressed Sequence Tag ◽

Cryphonectria Parasitica ◽

Chestnut Blight ◽

Chestnut Blight Fungus ◽

Expressed Sequence Tag Analysis ◽

Expressed Sequence

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A Tannic Acid–Inducible and Hypoviral-Regulated Laccase3 Contributes to the Virulence of the Chestnut Blight Fungus Cryphonectria parasitica

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-12-1582 ◽

2008 ◽

Vol 21 (12) ◽

pp. 1582-1590 ◽

Cited By ~ 20

Author(s):

Hea-Jong Chung ◽

Bo-Ra Kwon ◽

Jung-Mi Kim ◽

Seung-Moon Park ◽

Jong-Kun Park ◽

...

Keyword(s):

Tannic Acid ◽

Constitutive Expression ◽

Morphological Characteristics ◽

Fungal Growth ◽

Cryphonectria Parasitica ◽

Chestnut Blight ◽

Loss Of Function ◽

Fungal Virulence ◽

Chestnut Blight Fungus ◽

Hypovirulent Strain

A new laccase gene (lac3) from the chestnut blight fungus Cryphonectria parasitica was induced by the presence of tannic acid, which is abundant in the bark of chestnut trees and is assumed to be one of the major barriers against pathogen infection. However, other commonly known laccase inducers, including ferulic acid, 2,5-xylidine, catechol, and pH, did not induce lac3 transcription. Moreover, the hypovirus modulated the induction of lac3 transcription, abolishing the transcriptional induction of the lac3 gene by tannic acid. A functional analysis of lac3 using a lac3-null mutant indicated that fungal growth and other morphological characteristics, including pigmentation and sporulation, were not affected. However, a virulence assay indicated that the loss of function of a tannic acid–inducible and hypoviral-regulated laccase resulted in reduced virulence without detectable changes in the morphological features. The constitutive expression of lac3 resulted in no significant differences in the necrotic lesions from those caused by the wild type, but its expression in the presence of the hypovirus led to larger lesions than those caused by the hypovirulent strain. These results suggest that the lac3 gene product may not be the only determinant of fungal virulence in chestnut trees but is an important factor.

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