Attenuation of Fungal Virulence by Synthetic Infectious Hypovirus Transcripts

2008

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Defective interfering (DI) RNAs, helper virus-dependent deletion mutant RNAs derived from the parental viral genomic RNA during replication, have been described for most RNA virus taxonomic groups. We now report that DI RNA production in the chestnut blight fungus, Cryphonectria parasitica, persistently infected by virulence-attenuating positive sense RNA hypoviruses, depends on one of two host dicer genes, dcl-2. We further report that nonviral sequences that are rapidly deleted from recombinant hypovirus RNA virus vectors in wild-type and dicer gene dcl-1 deletion mutant strains are stably maintained and expressed in the ⌬dcl-2 mutant strain. These results establish a requirement for dcl-2, the C. parasitica dicer gene responsible for antiviral defense and generation of virus-derived small interfering RNAs, in DI RNA production and recombinant virus vector RNA instability.defective interfering RNA ͉ hypovirus ͉ RNA silencing ͉ RNA recombination V irus RNA recombination is an important component of virus evolution that contributes to the emergence of new viruses (reviewed in 1) and the generation of internally deleted mutant RNAs, termed defective interfering (DI) RNAs, that are derived from, and are dependent on, the parental viral genomic RNA (2). The presence of DI RNAs can suppress parental virus RNA accumulation, leading to attenuation of symptoms (3) and persistent virus infections (4, 5). Rapid recombination also appears to be an underlying cause of the instability and deletion of foreign, nonviral sequences from recombinant viral RNA vectors (6; reviewed in 7). Genome instability presents one of the most important obstacles to the use of recombinant RNA viruses as gene expression vectors for various practical applications, including gene therapy (8-10).Single-strand, positive sense mycoviruses in the family Hypoviridae, hypoviruses that persistently infect and attenuate virulence of the chestnut blight fungus, Cryphonectria parastica, generate internally deleted DI RNAs at a very high frequency (11,12). Efforts to use recombinant hypoviruses to express foreign genes also have encountered the limitation of instable nonviral nucleotide sequences (13).We recently reported that disruption of one of two C. parasitica dicer genes, dcl-2, results in increased susceptibility to mycovirus infection (14). We subsequently showed that dcl-2 functions to process mycovirus RNAs into virus-derived small interfering RNAs (vsRNAs) as part of an inducible RNA silencing antiviral response (15). Cloning and characterization of vsRNAs generated from hypovirus CHV1-EP713 revealed a nonrandom distribution of vsRNAs along the 12.7-kb genome RNA. Conspicuous was the very low representation of vsRNA mapping to an internal portion of the 12.7-kb genome RNA that encodes the viral polymerase and helicase domains, from map position 7500 to 11000 (15). It has been postulated that the propensity of hypoviruses to generate internally deleted DI RNAs results in a lower level of substrate for vsRNA biogenesis from this region (15). Here ...

Section: Discussionmentioning

confidence: 99%

A host dicer is required for defective viral RNA production and recombinant virus vector RNA instability for a positive sense RNA virus

Zhang

2008

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“…Infection of fungal strains with hypovirus CHV1-EP713 and hypovirus vector Ctp40[2AEGFP] (33) was initiated by transfection of fungal spheroplasts with hypovirus transcripts generated in vitro from a viral cDNA clone using the protocol developed by Chen et al (30). Transfected fungal strains were transferred to new PDA plates every 7 days.…”

Section: Methodsmentioning

confidence: 99%

“…The four agl gene disruption mutant strains were independently infected with hypovirus CHV1-EP713, either by transfection of spheroplasts with infectious transcripts (30) or by anastomosis with CHV1-EP713 infected strain EP155 (31). Mutant strains ⌬agl1, ⌬agl3, and ⌬agl4 all responded to CHV1-EP713 infection with phenotypic changes that were indistinguishable from that exhibited by CHV1-EP713-infected wildtype strain EP155.…”

Section: Cryphonectria Parasitica Encodes Four Argonaute-like Proteinmentioning

confidence: 99%

A single Argonaute gene is required for induction of RNA silencing antiviral defense and promotes viral RNA recombination

Sun

Choi

2009

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143

191

Dicer gene dcl2, required for the RNA silencing antiviral defense response in the chestnut blight fungus Cryphonectria parasitica, is inducible upon mycovirus infection and promotes viral RNA recombination. We now report that the antiviral defense response requires only one of the four C. parasitica Argonaute-like protein genes, agl2. The agl2 gene is required for the virus-induced increase in dcl2 transcript accumulation. Agl2 and dcl2 transcripts accumulated to much higher levels in response to hairpin RNA production or infection by a mutant CHV1-EP713 hypovirus lacking the suppressor of RNA silencing p29 than to wild-type CHV1-EP713. Similar results were obtained for an agl2-promoter/EGFP-reporter construct, indicating that p29-mediated repression of agl2 transcript accumulation is promoter-dependent. Significantly, the agl2 deletion mutant exhibited stable maintenance of non-viral sequences in recombinant hypovirus RNA virus vectors and the absence of hypovirus-defective interfering (DI) RNA production. These results establish a key role for an Argonaute gene in the induction of an RNA silencing antiviral defense response and the promotion of viral RNA recombination. They also provide evidence for a mechanism by which a virus-encoded RNA silencing suppressor represses the transcriptional induction of an RNA silencing component.Cryphonectria parasitica ͉ mycovirus ͉ defective interfering RNA ͉ silencing suppressor ͉ RNA virus vector A n RNA-based antiviral defense response, related to RNA interference (1), serves as a key component of the innate immunity repertoire in plants, invertebrates, and fungi (2, 3). Common elements of this response across Kingdoms include the action of conserved ribonucleases: members of the Dicer-like and Argonaute-like protein families (4). Dicer nucleases recognize viral double-stranded (ds) and structured RNAs and use the associated RNase III-type activity to process these RNAs into small RNAs of 21-24 nts in length, termed virus-derived small (vs) RNAs. The vsRNAs are incorporated into an effector complex with the aid of an Argonaute family protein. One strand of the vsRNA is removed and the remaining guide strand then targets the effector complex to the cognate viral RNA, which is cleaved, or sliced, by the Argonaute-associated RNase H-like activity.Four Dicer proteins drive the RNA silencing pathways in the model plant, Arabidopsis thaliana (5). All four Dicers have been implicated in antiviral RNA silencing (3, 6-8). Studies with Drosophila melanogaster (9, 10) and with mosquitoes (11) have identified a role for Dicer-2, the Dicer involved in siRNA production, in insect antiviral defense. The contribution to antiviral defense of the second Dicer, Dicer-1, remains unclear due to its essential role in microRNA processing and development (12).Antiviral RNA silencing has been demonstrated in the filamentous Ascomycete fungi Cryphonectria parasitica, the chestnut blight fungus (13), and Aspergillus nidulans (14). Only one of the two C. parasitica Dicer genes, dcl2, was shown to be...

“…Virus transmission assays were performed as described in Choi et al (22). Infections by hypoviruses CHV-1/EP713 were initiated in C. parasitica strain EP155 by transfecting fungal spheroplasts with viral transcripts generated in vitro from full-length viral cDNA as described by Chen et al (42). Hypoviruses were then introduced into the SD strains by anastomosis.…”

mentioning

confidence: 99%

Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic genes

Zhang

2016

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Transmission of mycoviruses that attenuate virulence (hypovirulence) of pathogenic fungi is restricted by allorecognition systems operating in their fungal hosts. We report the use of systematic molecular gene disruption and classical genetics for engineering fungal hosts with superior virus transmission capabilities. Four of five diallelic virus-restricting allorecognition [vegetative incompatibility (vic)] loci were disrupted in the chestnut blight fungus Cryphonectria parasitica using an adapted Cre-loxP recombination system that allowed excision and recycling of selectable marker genes (SMGs). SMG-free, quadruple vic mutant strains representing both allelic backgrounds of the remaining vic locus were then produced through mating. In combination, these super donor strains were able to transmit hypoviruses to strains that were heteroallelic at one or all of the virus-restricting vic loci. These results demonstrate the feasibility of modulating allorecognition to engineer pathogenic fungi for more efficient transmission of virulence-attenuating mycoviruses and enhanced biological control potential.M ycovirus infections have been reported to reduce virulence (hypovirulence) for a wide range of plant pathogenic fungi, providing potential for biological disease control (1-6). For hypovirulence to be effective, the virulence-attenuating viruses must be efficiently transmitted from infected hypovirulent strains to uninfected virulent strains (5, 7). Mycoviruses generally have evolved exclusive intracellular lifestyles (8). With very few exceptions (9), mycovirus infections cannot be initiated by exposure of uninfected hyphae to cell extracts or secretions from infected fungal isolates. Transmission is limited to intracellular mechanisms, vertical transmission through asexual spores, and horizontal transmission through anastomosis (fusion of hyphae).Horizontal mycovirus transmission to uninfected isolates of the same fungal species is complicated by nonself allorecognition genetic systems, termed "heterokaryon" or "vegetative incompatibility" (vic), which operate widely in filamentous fungi (10). Interactions between genetically distinct individuals of the same species result in an incompatible reaction that triggers localized programmed cell death (PCD), forming a line of demarcation, or barrage, along the zone of contact (10-12) and restricting cytoplasmic transmission of viruses and other cytoplasmic elements (1,(13)(14)(15).A negative correlation between vic diversity and virus transmission has been reported for several fungal hosts (1, 16), but has most extensively been demonstrated for the chestnut blight fungus Cryphonectria parasitica infected with virulence-attenuating hypoviruses (7,(17)(18)(19)(20). Genetic analyses have defined six diallelic vic genetic loci for C. parasitica (21). These loci and associated genes were recently identified at the molecular level through a comparative genomics approach (22, 23) ( Table 1). Independent gene disruption analysis of 12 genes associated with these loci (22, 23) ...