Using chimeric hypoviruses to fine-tune the interaction between a pathogenic fungus and its plant host

Abstract

Infectious cDNA clones of mild (CHV1-Euro7) and severe (CHV1-EP713) hypovirus strains responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica were used to construct viable chimeric viruses. Differences in virus-mediated alterations of fungal colony morphology, growth rate, and canker morphology were mapped to a region of open reading frame B extending from nucleotides 2,363 to 9, 904. By swapping domains within this region, it was possible to generate chimeric hypovirus-infected C. parasitica isolates that exhibited a spectrum of defined colony and canker morphologies. Several severe strain traits were observed to be dominant. It was also possible to uncouple the severe strain traits of small canker size and suppression of asexual sporulation. For example, fungal isolates infected with a chimera containing nucleotides 2363 through 5310 from CHV1-Euro7 in a CHV1-713 background formed small cankers that were similar in size to that caused by CHV1-EP713-infected isolates but with the capacity for producing asexual spores at levels approaching that observed for fungal isolates infected with the mild strain. These results demonstrate that hypoviruses can be engineered to fine-tune the interaction between a pathogenic fungus and its plant host. The identification of specific hypovirus domains that differentially contribute to canker morphology and sporulation levels also provides considerable utility for continuing efforts to enhance biological control potential by balancing hypovirulence and ecological fitness.

FIG. 1

Schematic diagram of parental and chimeric hypoviruses used in this study. The position of restriction sites used to generate chimeric viruses from CHV1-Euro7 (coding regions are indicated as white boxes, and noncoding regions are indicated as gray lines) and CHV1-EP713 (coding regions are presented as gray boxes, and noncoding regions are presented as black lines) are indicated at the top. The unique NotI site fused to a minimal T7 polymerase promoter was introduced immediately upstream of the viral sequence in both parental cDNAs to facilitate swapping of the 5′ portions and in vitro transcription. Similarly, the SpeI site was introduced immediately after the viral poly(A) tail to allow linearization of the plasmid in preparation for in vitro transcription and to aid in swapping of the 3′ portions of the viruses. The CHV1-Euro7 map positions for the three restriction sites common to the two viruses are nt 3575 for XhoI, nt 5310 for NarI, and nt 9898 for NsiI. Since CHV1-Euro7 is 11 nt shorter than CHV1-EP713 (12,701 nt versus 12,712 nt), the map position numbering for the two viruses differs slightly (8). The approximate positions of the p48, putative RNA-dependent RNA polymerase (Pol), and putative RNA helicase (Hel) coding domains are indicated at the bottom of the figure (20, 25).

FIG. 2

Colony morphologies on PDA conferred by parental and chimeric hypoviruses. A colony of virus-free C. parasitica isolate EP155 is shown at the top left (A1). Colonies transfected with parental viruses CHV1-Euro7 and CHV1-EP713 are shown at coordinates A2 and A3, respectively. Colonies infected with chimeric viruses are shown at the following coordinates: R13, A4; R14, A5; R12, B2; R6, B3; R10, B4; R5, B5; R7, C2; R3, C3; R8, C4; and R9, C5. The photograph was taken on day 7 of culture on PDA.

FIG. 3

Agarose gel electrophoretic analysis of dsRNAs recovered from transfected C. parasitica isolates. The full-length viral dsRNAs are the slowest-migrating band in each lane. The faster-migrating species observed in lanes 2, 4, and 13 correspond to internally deleted defective viral RNAs previously identified in hypovirus-infected fungal isolates (5, 26). The presence of these defective RNAs has not been associated with any changes in fungal phenotype. Lane M, 200 ng of 1-kb DNA ladder (Gibco BRL) as relative size markers, with an asterisk indicating the position of the 4-kb band. Samples (1 μg) of partially purified viral dsRNA recovered from liquid cultures of individual transfected isolates were treated with S1 nuclease (5 U) and analyzed on 0.8% agarose gels. Lane 1, virus-free isolate EP155; lane 2, CHV1-EP713 transfectant; lane 3, CHV1-Euro7 transfectant; lane 4, R13 transfectant; lane 5, R14 transfectant; lane 6, R12 transfectant; lane 7, R6 transfectant; lane 8, R10 transfectant; lane 9, R5 transfectant; lane 10, R7 transfectant; lane 11, R3 transfectant; lane 12, R8 transfectant; lane 13, R9 transfectant.

FIG. 4

Representative cankers formed by virus-free and transfected C. parasitica isolates. The same coordinate system for strains transfected with chimeric viruses used in Fig. 2 is repeated here: EP155, A1; CHV1-Euro7, A2; CHV1-EP713, A3; R13, A4; R14, A5; R12, B2; R6, B3; R10, B4; R5, B5; R7, C2; R3, C3; R8, C4; and R9, C5. Cankers were photographed 30 days postinoculation. Cankers caused by R12 and R6 transfected isolates are enlarged at the bottom of the figure to illustrate contrast and to allow a closer inspection of the stromal pustules that contain spore-forming bodies, termed pycnidia, and the ridged margins of the canker formed by the R6 transfectant.