Hypovirulence-Associated Suppression of Host Functions in<i>Cryphonectria parasitica</i>Can be Partially Relieved by High Light Intensity

Hillman, Bradley I.; Shapira, Roni; Nuss, Donald L.

doi:10.1094/phyto-80-950

Cited by 149 publications

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“…C. parasitica strain EP155 and dicer gene mutant strains ⌬dcl-1 and ⌬dcl-2 (14) were maintained on potato-dextrose agar (PDA; Difco) at 22°C to 24°C as described previously (32). Strains were cultured on cellophane membrane overlaid on the PDA medium for 7 days for RNA and protein isolation.…”

Section: Methodsmentioning

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

Nuss

2008

Proc. Natl. Acad. Sci. U.S.A.

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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 ...

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Section: Methodsmentioning

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

Nuss

2008

Proc. Natl. Acad. Sci. U.S.A.

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“…Colony morphology and virulence on dormant chestnut stems were assessed according to standard procedures (41). Virus transmission assays were performed as described in Choi et al (22).…”

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

Zhang

Nuss

2016

Proc. Natl. Acad. Sci. U.S.A.

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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) ...

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“…Colony morphology, asexual conidia production, and fungal virulence were assessed according to standard procedures (Hillman et al 1990). Mycelial incompatibility and virus transmission assays were performed as described in Choi et al (2012).…”

Section: Analysis Of Fungal Phenotype Mycelial Incompatibility and mentioning

confidence: 99%

Vegetative Incompatibility Loci with Dedicated Roles in Allorecognition Restrict Mycovirus Transmission in Chestnut Blight Fungus

et al. 2014

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Vegetative incompatibility (vic), a form of nonself allorecognition, operates widely in filamentous fungi and restricts transmission of virulence-attenuating hypoviruses in the chestnut blight fungus Cryphonectria parasitica. We report here the use of a polymorphism-based comparative genomics approach to complete the molecular identification of the genetically defined C. parasitica vic loci with the identification of vic1 and vic3. The vic1 locus in the C. parasitica reference strain EP155 consists of a polymorphic HET-domain-containing 771-aa ORF designated vic1a-2, which shares 91% identity with the corresponding vic1a-1 allele, and a small (172 aa) idiomorphic DUF1909-domain-containing ORF designated vic1b-2 that is absent at the vic1-1 locus. Gene disruption of either vic1a-2 or vic1b-2 in strain EP155 eliminated restrictions on virus transmission when paired with a vic1 heteroallelic strain; however, only disruption of vic1a-2 abolished the incompatible programmed cell death (PCD) reaction. The vic3 locus of strain EP155 contains two polymorphic ORFs of 599 aa (vic3a-1) and 102 aa (vic3b-1) that shared 46 and 85% aa identity with the corresponding vic3a-2 and vic3b-2 alleles, respectively. Disruption of either vic3a-1 or vic3b-1 resulted in increased virus transmission. However, elimination of PCD required disruption of both vic3a and vic3b. Additional allelic heterogeneity included a sequence inversion and a 8.5-kb insertion containing a LTR retrotransposon sequence and an adjacent HET-domain gene at the vic1 locus and a 7.7-kb sequence deletion associated with a nonfunctional, pseudo vic locus. Combined gene disruption studies formally confirmed restriction of mycovirus transmission by five C. parasitica vic loci and suggested dedicated roles in allorecognition. The relevance of these results to the acquisition and maintenance of vic genes and the potential for manipulation of vic alleles for enhanced mycovirus transmission are discussed.A LLORECOGNITION genetic systems, which provide the ability to distinguish self from nonself, play important functional roles in microbial and multicellular organisms. These systems range from restriction endonucleases in bacteria (Meselson and Yuan 1968) to somatic histocompatibility in protocordates (De Tomaso et al. 2005), self-infertility in plants (Nasrallah 2005), and innate immunity in vertebrates (Medzhitov and Janeway 2002) (reviewed by Aanen et al. 2008;Nydam and De Tomaso 2011;Rosengarten and Nicotra 2011). Allorecognition operates widely in filamentous fungi in both the sexual and the vegetative growth phases (Saupe 2000). The role of the mating-type locus in controlling sexual recognition and promoting outbreeding in yeast and filamentous fungi is well understood (reviewed by Coppin et al. 1997). It is also known that somatic or vegetative fusion of fungal cells (termed "anastomosis") occurs at a high frequency within and between individuals promoting network formation (Rayner 1996) and facilitating foraging, the pooling of resources (Rayner 1996), ...

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Hypovirulence-Associated Suppression of Host Functions inCryphonectria parasiticaCan be Partially Relieved by High Light Intensity

Cited by 149 publications

References 0 publications

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

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

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

Vegetative Incompatibility Loci with Dedicated Roles in Allorecognition Restrict Mycovirus Transmission in Chestnut Blight Fungus

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