Use of cDNA Microarrays To Monitor Transcriptional Responses of the Chestnut Blight FungusCryphonectria parasiticato Infection by Virulence-Attenuating Hypoviruses

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 downregulated. 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.Viruses are widely distributed throughout the kingdom Fungi (2). Members of the mycovirus family Hypoviridae are distinguished by the ability to persistently attenuate virulence (hypovirulence) and stably alter dependent developmental processes, e.g., asexual and sexual sporulation, of their host, the chestnut blight fungus Cryphonectria parasitica (13). They are also the first viruses of filamentous fungi for which a reverse genetics system was developed, providing the capability to manipulate the genomes of both a eukaryotic virus and its host. Consequently, hypoviruses are unique experimental tools for gaining deeper insights into virus-host interactions and revealing fundamental processes underlying fungal pathogenesis.The pleiotropic nature of hypovirus-mediated phenotypic changes suggested the perturbation of one or more key regulatory pathways (13). Subsequent studies implicated two principal signal transduction pathways in the manifestation of hypovirus-induced phenotypic changes: G-protein-linked, cyclic AMP (cAMP)-mediated (5, 7, 20, 37) and calcium/calmodulin/ inositol trisphosphate-dependent (31, 32) signaling cascades. However, evidence for hypovirus-mediated alteration of these pathways has relied predominantly on single-gene analyses to monitor pathway activity, e.g., the 13-1 gene for G-proteinlinked, cAMP-mediated signaling and the lac...

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

“…Microarray slide printing, fluorescent probe generation, microarray hybridization, scanning, and data analyses were performed as described by Allen et al (1). Integrated pixel intensity values for each spot were calculated by using TIGR Spotfinder software and saved in tab-delimited format for use by MIDAS software (http://www.tigr.org/software) from The Institute for Genomic Research (Rockville, Md.).…”

“…Cultures used for RNA isolation were grown on PDAcellophane for 6 days and harvested by freezing the mycelia in liquid nitrogen with immediate grinding of the mycelia into a fine powder by using a mortar and pestle. RNA isolation was performed as described by Allen et al (1).…”

“…Allen et al (1) recently used a spotted cDNA microarray containing ca. 2,200 C. parastica genes to monitor the change in host transcriptional profile after infection with severe hypovirus isolate CHV1-EP713.…”

Characterization of the ERK homologue CpMK2 from the chestnut blight fungus Cryphonectria parasitica

“…C. parasitica strains containing the double-stranded (ds) RNA virus Cryphonectria hypovirus (CHV) display characteristic symptoms of hypovirulence and hypovirulence-associated changes such as reduced sporulation, laccase production, oxalate accumulation and pigmentation (Havir & Anagnostakis, 1983;Elliston, 1985;Rigling et al, 1989). Interestingly, the symptoms caused by hypoviral infection appear to be the result of aberrant expression of specific fungal genes in the hypovirulent strain, including genes for cutinase, laccase, cryparin and mating pheromones (Rigling & Van Alfen, 1991;Choi et al, 1992;Varley et al, 1992;Zhang et al, 1993Zhang et al, , 1994Allen et al, 2003;Allen & Nuss, 2004). Thus, C. parasitica and its hypovirus are a useful model system to study the mechanisms of fungal gene regulation by a mycovirus.…”

Modified small-scale batch procedure for isolation of dsRNA from Cryphonectria parasitica

“…Although this method has been successfully utilized so far, several modifications to the procedure have been suggested (Allemann et al 1999;Allen et al 2003;Hillman et al 1990;Peever et al 1997;Rigling et al 1989). …”