[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: Using an established spotted cDNA microarray platform, the nature of changes in the transcriptional profiles of 2200 unique genes from the chestnut blight fungus Cryphonectria parasitica in response to the absence of either the Galpha subunit CPG-1 or the Gbeta subunit CPGB-1 has been explored. It is reported that 216 transcripts were altered in accumulation in the Deltacpg-1 strain and 163 in the Deltacpgb-1 strain, with a considerable overlap (100 genes) that were changed in both cases. Of note, these commonly altered transcripts were changed in the same direction in every instance, thus suggesting a considerable redundancy in pathway control or extensive crosstalk. To further knowledge of the potential impact on G-protein-signalling of infection by hypovirus CHV1-EP713, the accumulation of CPG-1 and CPGB-1 was also investigated by Western analysis. It was demonstrated that both signalling components were reduced in abundance to approximately 25 % of wild-type levels, while their transcripts were slightly elevated. Comparison of a list of genes with altered expression in the presence of CHV1-EP713 to the data obtained in the absence of either G-protein subunit showed that more than one-half of all the transcripts changed by hypovirus infection were also changed in at least one G-protein mutant strain, with one-third being changed in both. Significantly, 95 % of the co-changed genes were altered in the same direction. These data provide the first evidence for modulation of Gbeta protein levels as well as the Gbetagamma-signalling pathways by hypovirus infection, and support the hypothesis that modification of G-protein-signalling via both Galpha and Gbetagamma provides for a significant contribution to hypovirus-mediated phenotype.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
Journal of Virology 05/2004; 78(8):4145-55. DOI:10.1128/JVI.78.8.4145-4155.2004 · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hypoviruses are a family of cytoplasmically replicating RNA viruses of the chestnut blight fungus Cryphonectria parasitica. Members of this mycovirus family persistently alter virulence (hypovirulence) and related fungal developmental processes, including asexual and sexual sporulation. In order to gain a better understanding of the molecular basis for these changes, we have developed a C. parasitica cDNA microarray to monitor global transcriptional responses to hypovirus infection. In this report, a spotted DNA microarray representing approximately 2,200 C. parasitica genes was used to monitor changes in the transcriptional profile after infection by the prototypic hypovirus CHV1-EP713. Altered transcript abundance was identified for 295 clones (13.4% of the 2,200 unique cDNAs) as a result of CHV1-EP713 infection-132 up-regulated and 163 down-regulated. In comparison, less than 20 specific C. parasitica genes were previously identified by Northern analysis and mRNA differential display as being responsive to hypovirus infection. A 93% validation rate was achieved between real-time reverse transcription-PCR results and microarray predictions. Differentially expressed genes represented a broad spectrum of biological functions, including stress responses, carbon metabolism, and transcriptional regulation. These findings are consistent with the view that infection by a 12.7-kbp hypovirus RNA results in a persistent reprogramming of a significant portion of the C. parasitica transcriptome. The potential impact of microarray studies on current and future efforts to establish links between hypovirus-mediated changes in cellular gene expression and phenotypes is discussed.