Gil H. Choi

Virus-like genetic organization and expression strategy for a double-stranded RNA genetic element associated with biological control of chestnut blight.

The complete nucleotide sequence of the largest double‐stranded (ds) RNA present in hypovirulent strain EP713 of the chestnut blight pathogen, Cryphonectria parasitica, was determined and the predicted genetic organization was confirmed by translational mapping analysis. The deduced RNA sequence was 12 712 bp in length, excluding the terminal poly(A):poly(U) homopolymer domain. The strand terminating with 3′‐poly(A) contained two contiguous large open reading frames (ORF A and ORF B) beginning at nucleotide residues 496–498 and extending to nucleotide positions 11 859–11 861. The junction between ORF A and ORF B consisted of the sequence 5′‐UAAUG‐3′, where UAA served as the termination codon for ORF A and AUG was the 5′‐proximal initiation codon within ORF B. ORF A (622 codons in length, excluding the termination codon) was recently shown to encode two polypeptides, p29 and p40, which were generated from a nascent polyprotein by an autocatalytic event mediated by p29 (Choi et al., 1991). A similar autocatalytic event was observed during in vitro translation of ORF B (3165 codons in length) resulting in the release of a 48 kd polypeptide from the amino‐terminal portion of the ORF B‐encoded polyprotein. These results are discussed in terms of the opportunities they provide for elucidating the molecular basis of transmissible hypovirulence and possible origins of hypovirulence‐associated dsRNAs.

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

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.

The autocatalytic protease p29 encoded by a hypovirulence-associated virus of the chestnut blight fungus resembles the potyvirus-encoded protease HC-Pro

Gene expression by a viral-like double-stranded RNA genetic element associated with reduced virulence (hypovirulence) of the chestnut blight fungus was recently shown to involve an autoproteolytic event which resulted in the release of an encoded protease, designated p29, from a polyprotein during translation. Mutational analysis of p29, described in this report, revealed that residues Cys-162 and His-215 are essential for autocatalytic cleavage. The results were also consistent with previous predictions that cleavage occurs between Gly-248 and Gly-249. Interestingly, p29 bears a striking resemblance to the potyvirus-encoded protease HC-Pro. Both proteases autocatalytically cleave at glycine dipeptides. In addition, there is a significant degree of similarity in the amino acid sequences flanking the essential Cys and His residues of the two proteases and in the spacing of these residues from their respective cleavage sites.

Mitotic stability and nuclear inheritance of integrated viral cDNA in engineered hypovirulent strains of the chestnut blight fungus.

Transmissible hypovirulence is a novel form of biological control in which virulence of a fungal pathogen is attenuated by an endogenous RNA virus. The feasibility of engineering hypovirulence was recently demonstrated by transformation of the chestnut blight fungus, Cryphonectria parasitica, with a full‐length cDNA copy of a hypovirulence‐associated viral RNA. Engineered hypovirulent transformants were found to contain both a chromsomally integrated cDNA copy of the viral genome and a resurrected cytoplasmically replicating double‐stranded RNA form. We now report stable maintenance of integrated viral cDNA through repeated rounds of asexual sporulation and passages on host plant tissue. We also demonstrate stable nuclear inheritance of the integrated viral cDNA and resurrection of the cytoplasmic viral double‐stranded RNA form in progeny resulting from the mating of an engineered hypovirulent C. parasitica strain and a vegetatively incompatible virulent strain. Mitotic stability of the viral cDNA ensures highly efficient transmission of the hypovirulence phenotype through conidia. Meiotic transmission, a mode not observed for natural hypovirulent strains, introduces virus into ascospore progeny representing a spectrum of vegetative compatibility groups, thereby circumventing barriers to anastomosis‐mediated transmission imposed by the fungal vegetative incompatibility system. These transmission properties significantly enhance the potential of engineered hypovirulent C. parasitica strains as effective biocontrol agents.

Hypovirus-Responsive Transcription Factor Gene pro1 of the Chestnut Blight Fungus Cryphonectria parasitica Is Required for Female Fertility, Asexual Spore Development, and Stable Maintenance of Hypovirus Infection

ABSTRACT We report characterization of the gene encoding putative transcription factor PRO1, identified in transcriptional profiling studies as being downregulated in the chestnut blight fungus Cryphonectria parasitica in response to infection by virulence-attenuating hypoviruses. Sequence analysis confirmed that pro1 encodes a Zn(II)2Cys6 binuclear cluster DNA binding protein with significant sequence similarity to the pro1 gene product that controls fruiting body development in Sordaria macrospora. Targeted disruption of the C. parasitica pro1 gene resulted in two phenotypic changes that also accompany hypovirus infection, a significant reduction in asexual sporulation that could be reversed by exposure to high light intensity, and loss of female fertility. The pro1 disruption mutant, however, retained full virulence. Although hypovirus CHV1-EP713 infection was established in the pro1 disruption mutant, infected colonies continually produced virus-free sectors, suggesting that PRO1 is required for stable maintenance of hypovirus infection. These results complement the recent characterization of the hypovirus-responsive homologue of the Saccharomyces cerevisiae Ste12 C2H2 zinc finger transcription factor gene, cpst12, which was shown to be required for C. parasitica female fertility and virulence.

Molecular analysis and overexpression of the gene encoding endothiapepsin, an aspartic protease from Cryphonectria parasitica

The gene, epn-1, encoding endothiapepsin (Epn), an aspartic protease (AspP) synthesized and secreted by the ascomycete fungus responsible for chestnut blight, Cryphonectria (Endothia) parasitica, was identified and characterized. Inspection of the nucleotide and deduced amino acid (aa) sequences revealed perfect agreement with the experimentally derived 330-aa sequence of mature Epn [Barkholt, Eur. J. Biochem. 167 (1987) 327-338] and an additional 89 aa of putative preprosequence. Of the nine fungal AspP characterized to date, Epn was found to be most closely related to aspergillopepsin and penicillopepsin (52% and 55% identity, respectively), proteases produced by the ascomycetes Aspergillus awamori and Penicillium janthinellum, and least related to proteases produced by the yeasts Candida albicans and Saccharomyces cerevisiae (27% and 26% identity, respectively). Epn production was found to be the same in isogenic virus-free and virus-containing strains, indicating that this AspP is not down-regulated by the presence of a hypovirulence-associated viral double-stranded RNA, as has been reported for several other secreted C. parasitica gene products. Strains containing multiple copies of epn-1 were obtained by transformation with a plasmid vector containing the cloned epn-1. One of these strains was shown to produce seven to ten times more Epn than the parental wild-type strain.

RECENT ADVANCES IN EXPANDING AND UNDERSTANDING VIRUS­ MEDIATED ATTENUATION OF FUNGAL VIRULENCE

Strains of the chestnut blight fungus Cryphonectria parasitica that contain viral double-stranded (ds) RNA belonging to the genus Hypovirus exhibit reduced levels of virulence (hypovirulence) 1. These elements are transmissible to vegetatively compatible strains of C. parasitica by anastomosis, providing the basis for biological control 2. The potential for effective biological control of chestnut blight has been further advanced by the development of an infectious cDNA copy of a hypovirus dsRNA 3. Strains transformed with these infectious cDNA clones (engineered hypovirulent strains) are able to transmit hypovirus genetic information through mating, a mode of transmission not available to natural hypovirulent strains. This property circumvents barriers to cytoplasmic virus transmission imposed by the fungal vegetative incompatibility system 4,5. Two recent extensions of our studies on virus-mediated attenuation of fungal virulence are reviewed in this chapter. The first involves the development of an infectious hypovirus synthetic transcript and its use to extend fungal host range, thus expanding virus-mediated virulence attenuation to other fungal pathogens. The second concerns additional insights into the molecular mechanisms underlying virus-mediated virulence attenuation.