Transformation of the fungal pathogen Cryphonectria parasitica with a variety of heterologous plasmids

Churchill, Alice C. L.; Ciuffetti, Lynda M.; Hansen, Dane R.; Etten, H. D. Van; Alfen, Neal K. Van

doi:10.1007/bf00313245

Cited by 159 publications

(127 citation statements)

References 37 publications

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“…Preparation and transformation of C. parasitica spheroplasts were carried out essentially according to Churchill et al (37). Hygromycin (40 g/mL), benomyl (0.4 g/mL), or G418 (20 g/mL), depending on the selectable marker used, was included in the growth medium to provide selection of transformants.…”

Section: Methodsmentioning

confidence: 99%

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

Sun

Choi

Nuss

2009

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

144

191

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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...

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Section: Methodsmentioning

confidence: 99%

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

Sun

Choi

Nuss

2009

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

144

191

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“…Genomic DNA from C. parasitica was extracted using the method of Churchill et al (1990). DNA (10 mg) was digested with restriction enzymes, blotted onto nylon membrane and hybridized with a radioactively labelled cpmk2 fragment.…”

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confidence: 99%

“…Protoplast preparation and transformation were performed as described previously (Churchill et al, 1990;Kim et al, 1995). Transformants were selected from agar plates that were supplemented with 150 mg hygromycin B ml 21 (Calbiochem) or 1?5 mg benomyl ml 21 (DuPont) as appropriate, passaged three or four times on selective media and single-spore-isolated, as described previously (Kim et al, 1995(Kim et al, , 2002.…”

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confidence: 99%

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

et al. 2005

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The Cryphonectria parasitica gene cpmk2, which encodes a mitogen-activated protein kinase belonging to the yeast extracellular signalling-regulated kinase (YERK1) subfamily, was isolated and its biological function was examined. Disruption of cpmk2 resulted in impaired pigmentation and abolished conidiation. Growth defects were observed in the cpmk2 mutant grown on solid plates, but growth of the mutant appeared normal in liquid media, including EP complete and PD broth, suggesting that the cpmk2 gene is involved in sensing and responding to growth conditions. The mutant's production of laccase, as measured by the size of the coloured area produced on tannic-acid-supplemented plates, was significantly reduced compared with the wild-type, but the intensity of the coloured area was unchanged, suggesting that the reduced laccase activity was owing to reduced growth on solid media rather than transcriptional downregulation. A dramatic reduction observed in the canker area produced by the cpmk2 mutant compared with the wild-type, even more severe than that of a hypovirulent strain, can also be ascribed to defective growth on solid surfaces rather than to impairments in a virulence factor(s). Downregulation of the pheromone gene Mf2/1 was also observed in the mutant, indicating a possible explanation for the regulation of the pheromone precursor gene in filamentous fungi and suggesting the presence of the yeast-like pheromone-responsive pathway in C. parasitica. Immunoblot analyses revealed that the phosphorylation level of CpMK2 increased in both virus-free and virus-containing strains in liquid cultures of up to 5 days old and decreased in older cultures. Moreover, the CpMK2 phosphorylation level increased in both strains after transfer from liquid to solid medium. However, levels of phosphorylated CpMK2 were similar in the two strains, suggesting that CpMK2, unlike CpMK1, is not under the direct control of a hypovirus.

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“…Disruption of both vic candidate genes vic3a-2 and vic3b-2 was also accomplished in strain EU60. DNA-mediated transformation was performed according to the method of Churchill et al (1990), followed by selection of putative transformants grown in the presence of 40 mg/ml hygromycin B or 25 mg/ml G418 for neomycinresistance selection. Gene disruption was confirmed by PCR analysis of the putative transformants, followed by selection of uninuclear single conidial isolates on antibioticcontaining PDA to eliminate heterokaryons and by a final PCR confirmation.…”

Section: Disruption Of Candidate Vic Genesmentioning

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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Transformation of the fungal pathogen Cryphonectria parasitica with a variety of heterologous plasmids

Cited by 159 publications

References 37 publications

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

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

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

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

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