Isolation and characterization of a virus-resistant mutant of Cryphonectria parasitica

Hypovirulent strain NB58 of Cryphonectria parasitica contains a dsRNA virus with a genome size of approximately 12.5 kb. Although NB58 is very stable in culture, a phenotypically-distinct sector arose which was found to be dsRNA-free. Attempts to infect the mutant strain, termed NB58F, by pairing with the parent strain (NB58) or other conversion-compatible, virus-containing strains have been unsuccessful. DNA fingerprint analysis showed that NB58, NB58F, and a representative dsRNA-free single-conidial isolate of NB58 termed NB58-19, were isogenic. The mutant culture was phenotypically stable, and all single-conidial progeny had the NB58F morphology. NB58F was intermediate between NB58 and NB58-19 in laccase production and virulence. Pigmentation and sporulation of NB58F, however, were reduced to near the level of NB58. In mating studies, NB58F functioned only as the male in sexual crosses. The mutant phenotype (F) predominated by a ratio of 5:2 among the ascospore progeny of F-type x wild-type crosses. These data suggest the lesion is nuclear and may be associated with a chromosomal abnormality. Attempts to infect the NB58F-type ascospore progeny failed, whereas the wild-type progeny were successfully infected with strains compatible with one or the other parent at a frequency of about 34%. Hyphal anastomosis and movement of cytoplasmic material occurred when NB58F was paired with a compatible strain, suggesting that the lesion is involved in viral maintenance as opposed to initial virus infection. NB58F represents the first virus-resistant isolate of C. parasitica to be described.     

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

Summary An efficient DNA-mediated transformation system for the pathogen of chestnut, Cryphonectria parasitica, is reported. Ten vectors, each containing a promoter from Cochliobolus heterostrophus, Aspergillus nidulans, Ustilago maydis, Cephalosporium acremonium, Neurospora crassa or cauliflower mosaic virus, were creened for their ability to confer resistance to hygromycin B, benomyl or G418 sulfate. Transformants were obtained with all vectors screened and, in each case, transformation occurred by integration of the foreign DNA into the host genome. The initial transformation efficiency ranged from approximately 1–60 transformants/g circular DNA. Under optimized transformation conditions, the transformation rate of the vector pDH25, which contains the trpC promoter and terminator of A. nidulans, exceeded 10⁵ transformants/g DNA. The ease with which C. parasitica is transformed should greatly facilitate the genetic manipulation of this fungal plant pathogen.     

Deletion of the <Emphasis Type="Italic">cpku80</Emphasis> gene in the chestnut blight fungus, <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis>, enhances gene disruption efficiency

The chestnut blight fungus, Cryphonectria parasitica, and associated virulence-attenuating hypoviruses have emerged as an important model system for studying molecular mechanisms underlying fungal–plant pathogenic interactions. As more gene sequence information becomes available as a result of C. parasitica express sequence tags (ESTs) and ongoing whole genome sequencing projects, the development of an efficient gene disruption system has become an urgent need for functional genomics studies of this important forestry pathogen. Here, we report the cloning of the C. parasitica gene cpku80 that encodes a key component of the nonhomologous end joining DNA repair pathway and the construction of a corresponding deletion mutant strain. The cpku80 mutant was indistinguishable from the parental wild-type strain EP155 in colony morphology, ability to support hypovirus replication, conidiation and virulence. As predicted, the Δcpku80 strain did exhibit an increased sensitivity to the mutagen methyl methanesulfonate. A test with three selected genes resulted in a gene disruption efficiency of about 80% for the Δcpku80 strain, a significant increase over the 2–5% levels of homologous recombination generally observed for the wild-type strain EP155. This efficient homologous recombination system provides a powerful tool for large-scale analysis of gene functions in C. parasitica. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. The authors Xiuwan Lan and Ziting Yao contributed equally to this work.     

Transfer of genetic information from the mycoparasite Parasitella parasitica to its host Absidia glauca

The infection of the model organism Absidia glauca by P. parasitica is accompanied by the fusion of both mycelia. By two lines of evidence we were able to show that this process is associated with the transfer of genes. First, auxotrophically labelled A. glauca mutants are efficiently complemented as a consequence of transfer of the parasite's genetic material. Second, for a plasmidcoded dominant marker (neomycin resistance), which is expressed in either organism, we proved the presence of plasmid DNA in recombinant recipients by molecular analysis at the DNA level. We propose the term para-recombinants for describing recombinant inter-generic chimærae, which are generated as a consequence of mycoparasitism.     

Cloning and sequence analysis of the gene encoding invertase from the yeast Schwanniomyces occidentalis

Summary The gene encoding invertase (INV) has been cloned from Schwanniomyces occidentalis. The enzyme consists of 533 amino acids, 8 potential glycosylation sites and has a 45 % identity with the invertase from Saccharomyces cerevisiae. The proenzyme has a 22 amino acid signal sequence that has a high -helical transmembrane potential which differs significantly from that predicted for the Saccharomyces cerevisiae enzyme.     

Genome analysis of Cryphonectria hypovirus 4, the most common hypovirus species in North America

Many different viruses that reduce virulence and alter the phenotype to varying extents have been identified in the chestnut blight fungus Cryphonectria parasitica. Most viruses identified in this fungus fall within the Hypoviridae family of positive-sense RNA viruses, which contains one genus and four species. Different species predominate in different geographic locations in chestnut-growing areas around the world. In this paper, we describe the genome organization and some variants of Cryphonectria hypovirus 4 (CHV-4), the species most commonly found in eastern North America. CHV-4 is distinguished from other hypoviruses by having little effect on fungal virulence and colony morphology. The 9.1-kb genome of strain CHV-4/SR2 is the smallest of any member of the family characterized to date. Like the recently characterized species CHV-3, a single ORF was predicted from deduced translations of CHV-4/SR2. Sequence analysis revealed the presence of a putative glucosyltransferase domain in both CHV-4 and in CHV-3, but no such homolog was detected in the more thoroughly examined CHV-1 or in CHV-2. Alignments with 8 other CHV-4 isolates from different regions of eastern North America revealed sequence diversity within the species and the likelihood that RNA recombination has led to this diversity.     

Physical and genetic map of the mitochondrial genome of Cryphonectria parasitica Ep155

 In the chestnut-blight fungus, Cryphonectria parasitica, a cytoplasmically transmissible (infectious) form of hypovirulence is associated with mitochondrial DNA (mtDNA) mutations that cause respiratory deficiencies. To facilitate the characterization of such mutations, a restriction map including the probable location of 13 genes was constructed for a relatively well-characterized virulent strain of the fungus, Ep155. The physical map is based on the order of all fragments generated by cleavage of the mtDNA by the PstI restriction endonuclease and includes some of the cleavage sites for HindIII, EcoRI, and XbaI. It was constructed from hybridization patterns of cloned mtDNA fragments with Southern blots of mtDNA digested with the four restriction enzymes. On this map, the probable locations of genes commonly found in the mitochondrial genomes of ascomycetes were determined by low-stringency hybridization of cloned Neurospora crassa mitochondrial gene probes to Southern blots of C. parasitica mtDNA. The data indicate that the mtDNA of strain Ep155 is a circular molecule of approximately 157 kbp and ranks among the largest mitochondrial chromosomes observed so far in fungi. The mtDNAs of 11 different C. parasitica isolates range in size from 135 to 157 kbp and in relatedness from 68 to 100 percent, as estimated from restriction-fragment polymorphisms. In addition to the typical mtDNA, the mitochondria of some isolates of the fungus contain double-stranded DNA plasmids consisting of nucleotide sequences not represented in the mtDNA of Ep155. Received: 19 September 1995/4 January 1996     

Variability in genome organization of the zygomycete Parasitella parasitica

In addition to conventional methods for the identification of fungi, molecular techniques at the DNA level are increasingly being employed. In order to check the validity of such experimental approaches, we have analyzed the well-defined species Parasitella parasitica, which belongs to the family Mucoraceae (Mucorales, Zygometes). The seven strains of this species, which are available from international strain collections, were analyzed by several molecular methods: restriction fragment length polymorphism analysis (RFLP), the random primer-dependent polymerase chain reaction (RAPD-PCR), and electrophoretic karyotyping. Unexpectedly, these strains are highly diverse at the molecular level. By these techniques they can be divided consistently into two different groups. Nevertheless, all seven strains belong to a single species. They show no morphological differences and sexual spores (zygospores) were found in all possible combinations either within or between the two groups. Southern-blot analysis of genomic DNA of all P. parasitica strains with RAPD-PCR-derived labelled probes shows the existence of repetitive elements characteristic for only one group of P. parasitica. In addition, chromosome sizes, which were separated by rotating-field electrophoresis, were highly divergent, and ranged from 3 to 6.5 Mb in one group and between 2 and 4.5 Mb in the other. The RAPD-PCR patterns also discriminate both groups of P. parasitica. However, they are very similar if strains of a single group are compared. Therefore, we propose that the determination of fungal species by molecular techniques should be vetted at least by morphological and physiological parameters and, whenever possible, by mating experiments.     

Cloning of a Candida utilis gene which complements leu2 mutation in Saccharomyces cerevisiae

Summary DNA fragments containing the LEU2 gene of Candida utilis have been isolated, utilizing the genome library (constructed in YRp12) of this organism. Two recombinant plasmids pZR84 and pZR32, containing the cloned LEU2 gene, were 4.24 kb and 10.4 kb, respectively, and were shown to complement leu2 mutation in Saccharomyces cerevisiae and leuB mutation in Escherichia coli. The cloned fragment in pZR84 contained one restriction site each for EcoRI and PvuII, and two for HindIII, but none for SalI, BamHI or Pstl. This cloned fragment hybridized with the total DNA from C. utilis and from Leu⁺ transformants of S. cerevisiae, but not with that from untransformed S. cerevisiae. Subcloning analyses showed that a 2.34 kb BamHI HindIII fragment of the cloned C. utilis sequence contains the region essential for the expression of the LEU2 gene.Journal Article No. 11669 from the Michigan Agricultural Experiment Station     

Cloning,sequencing and analysis of the yeastS. uvarum ERG10 gene encoding acetoacetyl CoA thiolase

Summary TheERG10 gene specific toS. uvarum, a brewing yeast, has been cloned by complementation of anS. cerevisiae erg10 mutant.S. uvarum contains two differentERG10 genes. One of these is similar to theS. cerevisiae ERG10 gene; they are structurally different, but functionally homologous. The clonedERG10 gene has been located on chromosome XVI, and we have shown that it is allelic to the previously isolatedtsm0115 mutants. Northern blot and sequence analysis indicate that theERG10 gene is highly expressed, and biochemical and genetic evidence show that it encodes the cytoplasmic acetoacetyl CoA thiolase.     

Cloning and expression of a chitinase gene from the hyperparasitic fungus Aphanocladium album

Summary Recombinant clones from a cDNA library of an Aphanocladium album chitinase-overproducing mutant strain were isolated by screening with antiserum against a 39 kDa chitinase purified from this hyperparasitic fungus. Analysis of the isolated positive clones indicated that most of them carried the same cDNA. A cDNA from this group was used as a hybridization probe to isolate an 8 kb DNA fragment from a genomic library of the wild-type strain. The chitinase 1 gene was mapped to this fragment by two independent approaches. Its partial DNA sequence was in perfect agreement with an amino-terminal peptide sequence obtained by sequencing 23 amino acids of the 39 kDa chitinase. Its transfer in Fusarium oxysporum resulted in a transformant producting both a protein of about 39 kDa that cross-reacted with the chitinase antiserum and a chitinase activity that was inhibited by the same antiserum. Northern blot analysis indicates that the cloned chitinase gene was subject to catabolite repression and appeared inducible by chitin.     

Conserved and variable structural elements in the 5' untranslated region of two hypoviruses from the filamentous fungus Cryphonectria parasitica

Virulence-attenuating viruses (hypoviruses) of the filamentous fungus Cryphonectria parasitica, the causative agent of chestnut blight, have become a premier model for understanding the molecular biology of mycoviruses. However, a major gap exists in current understanding of structure and function of the untranslated regions (UTRs) of the hypovirus RNA genome, despite considerable evidence that secondary and tertiary UTR structure plays a crucial role in the control of translation and genome replication in other systems. In this study we have used structure prediction software coupled with RNase digestion studies to develop validated structural models for the 5′ UTRs of the two best-characterized members of the Hypoviridae, CHV1-EP713 and CHV1-Euro7. These two hypovirus strains exhibit significant variation in virulence attenuation despite sharing >90% sequence identity. Our models reveal highly structured regions in the 5′ UTR of both strains, with numerous stem-loops suggestive of internal ribosome entry sites. However, considerable differences in the size and complexity of structural elements exist between the two strains. These data will guide future, mutagenesis-based studies of the structural requirements for hypovirus genome replication and translation.     

Cloning,characterization, and use in strain improvement of the Cephalosporium acremonium gene cefG encoding acetyl transferase

Summary A long open reading frame (ORF) closely linked to the Cephalosporium acremonium gene cefEF was identified by DNA sequencing. The cefEF gene encodes the enzyme involved in cephalosporin C (CPC) biosynthesis known as expandase/hydroxylase. Complementation of a C. acremonium cefG mutant, as well as expression of the gene in Aspergillus niger, showed this ORF to be the cefG gene, encoding cephalosporin C acetyltransferase, which catalyzes the last step in CPC biosynthesis. Analysis of transformants containing additional copies of this gene showed that a direct relationship exists between cefG copy number, cefG message levels, and CPC titers. This gene encodes an enzyme for what may be a rate-limiting step in CPC production.     

Cloning and mapping of the CYS4 gene of Saccharomyces cerevisiae

Summary A DNA fragment containing the CYS4 gene of Saccharomyces cerevisiae was isolated from a genomic library. The cloned fragment hybridized to the transverse-alternating-field-electrophoresis band corresponding to chromosomes VII and XV. According to the 2 m DNA chromosome-loss procedure, the cys2 and cys4 mutations, which are linked together and co-operatively confer cysteine dependence, were assigned to chromosome VII. By further mapping involving tetrad analysis, the cys2-cys4 pair was localized between SUP77 (SUP166) and ade3 on the right arm of chromosome VII.     

Cloning the Schizosaccharomyces pombe lys2 + gene and construction of new molecular genetic tools

Molecular genetic analyses in Schizosaccharomyces pombe rely on selectable markers that are used in cloning vectors or to mark targeted gene deletions and other integrated constructs. In this study, we used genetic mapping data and genomic sequence information to predict the identity of the S. pombe lys2 ⁺ gene, which is homologous to Saccharomyces cerevisiae LYS4 ⁺. We confirmed this prediction, showing that the cloned SPAC343.16 gene can complement a lys2-97 mutant allele, and constructed the lys2 ⁺-based cloning vector pRH3. In addition, we deleted the S. pombe his7 ⁺ gene with a lys2 ⁺-marked polymerase chain reaction (PCR) product and the S. pombe lys2 ⁺ gene with a his7 ⁺-marked PCR product. Strains carrying these deletions of lys2 ⁺ or his7 ⁺ serve as relatively efficient hosts for the deletion of the ade6 ⁺ gene by lys2 ⁺- or his7 ⁺-marked PCR products when compared with hosts carrying lys2 or his7 point mutations. Therefore, these studies provide plasmids and strains allowing the use of lys2 ⁺ as a selectable marker, along with improved strains for the use of his7 ⁺ to mark gene deletions.     

Cloning, expression, and characterization of two novel cuticle-degrading serine proteases from the entomopathogenic fungus Cordyceps sinensis

The entomopathogenic fungus Cordyceps sinensis has been important in traditional Chinese medicine, but is yet to be commercially cultivated. Difficulty in cultivation results in part from the low percentage of fungal infection on artificially inoculated host insects. To better understand the infection mechanism, we cloned two cuticle-degrading serine protease genes (csp1 and csp2) from C. sinensis. These enzymes are novel members of the S8A subfamily of proteases. Identities of cDNA or amino acid sequences between Csp1 and Csp2 were 72.9% and 68.9%, respectively. After successful expression in the yeast Pichia pastoris, recombinant enzymes were purified and characterized using the synthetic substrate N-suc-AAPF-p-NA. Both were chymotrypsin-like serine proteases with an optimum pH of 7.0 and an optimal temperature of 40 degrees C (Csp1) or 50 degrees C (Csp2). Bioassay revealed that Csp1 and Csp2 degraded the cuticle proteins of larval Hepialus sp. in vitro. This is the first report of serine proteases from C. sinensis.