Infectious cDNA clone of hypovirus CHV1-Euro7: a comparative virology approach to investigate virus-mediated hypovirulence of the chestnut blight fungus Cryphonectria parasitica

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.

FIG. 1

Comparison of the CHV1-Euro7 cDNA sequence information determined in this study with that of two previously reported full-length hypovirus cDNA sequences: CHV1-EP713 (41) and CHV2-NB58 (25). (A) Similarities at the nucleotide levels. Previously identified protein coding regions are noted within the open boxes representing the viral genome (34). The lengths in nucleotides for the 5′ and 3′ noncoding (nc) regions and ORFs A and B for CHV1-EP713 are indicated at the top. The numbers of nucleotides for comparable regions of the other hypoviruses were 494, 1,869, 9,494, and 844, respectively, for CHV1-Euro7 and 487, 1,314, 9,873, and 831, respectively, for CHV2-NB58. The percent nucleotide identity for different coding and noncoding regions is indicated between the different viral genome diagrams being compared. (B) Similar information at the deduced amino acid levels. Note that CHV2-NB58 lacks a p29 homolog and contains p50 and p52 as the homologs of p40 and p48 found in CHV1-Euro7 and CHV1-EP713.

FIG. 2

Colony morphology for virus-free C. parasitica EP55 and Euro7(−v) and related hypovirus transfectants. (Top row) Colonies of virus-free strain EP155 (center) and strain EP155 transfected with CHV1-EP713, CHV1-Euro7, chimeric virus AE7B713, or chimeric virus A713BE7. (Bottom row) Colonies of virus-free strain Euro7(−v) (center) and strain Euro7(−v) transfected with CHV1-EP713, CHV1-Euro7 chimeric virus AE7B713, and chimeric virus A713BE7. Photographs were taken on day 7.

FIG. 3

Agarose gel electrophoretic analysis of dsRNAs recovered from transfected C. parasitica strains. The migration position of the full-length hypovirus dsRNA is indicated by the arrow on the right. Lane M contains 200 ng of a 1-kb DNA ladder (Gibco BRL) as relative size markers. dsRNA preparations recovered from equal volumes of cultured virus-free and transfected strains were loaded in the following order: lane 1, EP155; lane 2, EP155–CHV1-EP713; lane 3, EP155–CHV1-Euro7; lane 4, EP155–AE7B713; lane 5, EP155–A713BE7; lane 6, Euro7(−v); lane 7, Euro7(−v)–CHV1-EP713; lane 8, Euro7(−v)–CHV1-Euro7; lane 9, Euro7(−v)–AE7B713; and lane 10, Euro7(−v)–A713BE7. The faster-migrating species observed in lanes 4 and 7 correspond to internally deleted defective viral RNAs previously identified in hypovirus-infected strains (7, 40). The presence of these deletion dsRNAs has not been associated with any change in phenotypic traits.

FIG. 4

Gallery of representative cankers formed by virus-free and transfected C. parasitica strains. (Top row) Typical cankers formed by virus-free strain EP155 (center) and strain EP155 transfected with CHV1-EP713, CHV1-Euro7, chimeric virus AE7B713, and chimeric virus A713BE7. (Bottom row) Cankers formed by virus-free strain Euro7(−v) and the corresponding set of Euro7(−v) transfectants (as detailed for top row). Cankers were photographed 30 days postinoculation after wetting with ethanol to enhance color contrast of cankers and the surrounding area. Stromal protrusions (stromata that contain asexual spore-forming bodies termed pycnidia) are prominent features of the surface of cankers caused by virus-free strains EP155 and Euro7(−v) as well as the CHV1-Euro7 and A713BE7 transfectants. Spiral structures, termed ceri, composed of conidia are seen extruded from some stromata. These structures are rarely observed on the surface of cankers formed by CHV1-EP713 or AE7B713 transfectants.