O. C. Yoder

Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycetes

Fungal type I polyketides (PKs) are synthesized by PK synthases (PKSs) and include well known secondary metabolites such as the anticholesterol drug lovastatin and the potent natural carcinogen aflatoxin. Other type I PKs are known to be virulence factors for some plant pathogens and pigments such as melanin. In this study, a phylogenomic approach was used to investigate the origin and diversity of fungal genes encoding putative PKSs that are predicted to synthesize type I PKs. The resulting genealogy, constructed by using the highly conserved PKS ketosynthase (KS) domain, indicated that: (i) Species within subphylum Pezizomycotina (phylum Ascomycota) but not early diverging ascomycetes, like Saccharomyces cerevisiae (Saccharomycotina) or Schizosaccharomyces pombe (Taphrinomycotina), had large numbers (7–25) of PKS genes. (ii) Bacteria and fungi had separate groups of PKS genes; the few exceptions are the likely result of horizontal gene transfer from bacteria to various sublineages of fungi. (iii) The bulk of genes encoding fungal PKSs fell into eight groups. Four groups were predicted to synthesize variously reduced PKs, and four groups were predicted to make unreduced PKs. (iv) Species within different classes of Pezizomycotina shared the same groups of PKS genes. (v) Different fungal genomes shared few putative orthologous PKS genes, even between closely related genomes in the same class or genus. (vi) The discontinuous distributions of orthologous PKSs among fungal species can be explained by gene duplication, divergence, and gene loss; horizontal gene transfer among fungi does not need to be invoked.

Split-Marker Recombination for Efficient Targeted Deletion of Fungal Genes

A commonly used method for fungal gene deletion is introduction of linear DNA consisting of a selectable marker gene flanked on both sides by short stretches of DNA that target a gene of interest (W irsel et al 1996 Curr. Genet 29:241-249). Gene deletion in Cochliobolus heterostrophus and Gibberella zeae occurs efficiently with this approach. To facilitate deletion construct synthesis, we have applied the split-marker” deletion strategy previously developed for Saccharomyces cerevisiae (Fairhead et al. 1996 Y east 12:1439-57; Fairhead et al. 1998 Gene 223:33-46). Here, we describe both fusion PCR-based and plasmid-based deletion methods using this strategy with PEG-mediated protoplast transformation (Turgeon et al, 1985 M ol. Gen. Genet. 201:450-453). These methods are predicted to work well with any transformable fungus that undergoes homologous recombination between chromosomal and introduced DNA sequences.

Whole-Genome Analysis of Two-Component Signal Transduction Genes in Fungal Pathogens

ABSTRACT Two-component phosphorelay systems are minimally comprised of a histidine kinase (HK) component, which autophosphorylates in response to an environmental stimulus, and a response regulator (RR) component, which transmits the signal, resulting in an output such as activation of transcription, or of a mitogen-activated protein kinase cascade. The genomes of the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans encode one, three, and three HKs, respectively. In contrast, the genome sequences of the filamentous ascomycetes Neurospora crassa, Cochliobolus heterostrophus (Bipolaris maydis), Gibberella moniliformis (Fusarium verticillioides), and Botryotinia fuckeliana (Botrytis cinerea) encode an extensive family of two-component signaling proteins. The putative HKs fall into 11 classes. Most of these classes are represented in each filamentous ascomycete species examined. A few of these classes are significantly more prevalent in the fungal pathogens than in the saprobe N. crassa, suggesting that these groups contain paralogs required for virulence. Despite the larger numbers of HKs in filamentous ascomycetes than in yeasts, all of the ascomycetes contain virtually the same downstream histidine phosphotransfer proteins and RR proteins, suggesting extensive cross talk or redundancy among HKs.

Isolation of DNA from filamentous fungi and separation into nuclear, mitochondrial, ribosomal, and plasmid components

A general procedure for purifying and efficiently separating four types of DNA from filamentous fungi has been developed. The protocol involves (i) disruption of mycelial cells by blending in liquid nitrogen followed by suspension of cell contents in buffer containing high concentrations of protease and EDTA; (ii) deproteinization with phenol; (iii) cesium chloride/bisbenzimide density gradient centrifugation to separate nuclear DNA, mitochondrial DNA, and ribosomal DNA; and (iv) agarose gel electrophoresis to identify and purify plasmid DNA, if present. All DNAs are suitable for digestion with restriction endonucleases, ligation, and cloning in Escherichia coli, and DNAs from step three are recovered in high-molecular-weight form. The procedure has been used successfully with several dozen isolates of the plant pathogenic fungus Cochliobolus heterostrophus (including both laboratory strains and isolates collected directly from the field), and has been found to be equally suitable for C. carbonum, Neurospora crassa, N. tetrasperma, and Nectria haematococca.

A polyketide synthase is required for fungal virulence and production of the polyketide T-toxin.

Race T of the fungal pathogen Cochliobolus heterostrophus is highly virulent toward Texas male sterile (T) maize and differs from its relative, race O, at a locus (Tox1) that is responsible for the production of T-toxin, a family of linear long-chain (C35 to E41) polyketides. In a previous study, the restriction enzyme-mediated integration procedure was used to mutagenize and tag Tox1. Here, we report that the DNA recovered from the insertion site of one mutant encodes a 7.6-kb open reading frame (2530 amino acids) that identifies a multifunctional polyketide synthase (PKS)-encoding gene (PKS1) with six catalytic domains arranged in the following order, starting at the N terminus: beta-ketoacyl synthase, acyltransferase, dehydratase, enoyl reductase, beta-ketoacyl reductase, and acyl carrier protein. PKS1 is interrupted by four apparent introns (74, 57, 49, and 41 bp) and exists in the genome as a single copy surrounded by highly repetitive, A + T-rich DNA. When PKS1 in race T was inactivated by targeted gene disruption, T-toxin production and high virulence were eliminated, indicating that this PKS is required for fungal virulence. Race O strains, which do not produce T-toxin, lack a detectable homolog of PKS1, suggesting that race T may have acquired PKS1 by horizontal transfer of DNA rather than by vertical inheritance from an ancestral strain.

Cloning and analysis of the mating type genes from Cochliobolus heterostrophus

Cochliobolus heterostrophus, a heterothallic Ascomycete, has a single mating type locus with two alternate forms called MAT-1 and MAT-2. MAT-1 was cloned by complementing a MAT-2 strain using a cosmid library from a MAT-1 strain and screening for a homothallic transformant. The cosmid recovered from this transformant was able to re-transform a MAT-2 strain to homothallism and MAT identity was proven by restriction fragment length polymorphism and conventional genetic mapping. All homothallic transformants could mate with either MAT-1 or MAT-2 strains, although the number of ascospores produced by self matings or crosses to MAT-2 strains was low. Progeny of selfed homothallic transformants were themselves homothallic. MAT-2 was cloned by probing a cosmid library from a MAT-2 strain with a fragment of insert DNA from the MAT-1 cosmid. A 1.5 kb subclone of either MAT-containing cosmid was sufficient to confer mating function in transformants. Examination of the DNA sequence of these subclones revealed that MAT-1 and MAT-2 contain 1297 by and 1171 bp, respectively, of completely dissimilar DNA flanked by DNA common to both mating types. Putative introns were found (one in each MAT gene) which, when spliced out, would yield open reading frames (ORFs) that occupied approximately 90% of the dissimilar DNA sequences. Translation of the MAT-1 ORF revealed similarity to the Neurospora crassa MATA, Podospora anserina mat−, and Saccharomyces cerevisiae MATα1 proteins; translation of the MAT-2 ORF revealed similarity to the N. crassa MATa, P. anserina mat+, and Schizosaccharomyces pombe mat-Mc proteins. These gene products are all proven or proposed DNA binding proteins. Those with similarity to MAT-2 are members of the high mobility group.

Transformation of the fungal maize pathogen Cochliobolus heterostrophus using the Aspergillus nidulans amdS gene

SummaryCochliobolus heterostrophus protoplasts transformed with a plasmid carrying the Aspergillus nidulans amdS gene (Hynes et al. 1983) gave rise to colonies on a selective medium that did not support significant growth of wild type cells. The plasmid integrated at a single chromosomal locus in each transformant analyzed and the site of integration differed among transformants. Some transformants had one copy of the plasmid, others had multiple copies tandemly arranged and oriented head-to-tail. Both single and multiple copies segregated meiotically as single genes and were mitotically stable on either selective or nonselective medium. The andS gene is advantageous for transformation of genetically undeveloped fungi because it is selectable in wild type cells in organisms that lack a functional amdS gene, thus eliminating the need for induced mutations in recipient strains. Moreover, there is no background due to reversion of a counter-selected mutant allele.

Fungal genomics and pathogenicity

The filamentous fungal genetics community has enthusiastically embraced the utilization of genomics technologies to resolve long-standing issues in fungal biology. For example, such technologies have been proposed to study the mechanics of tip growth, photoreception, gene silencing, the molecular basis of conidiation, the pathway leading to sexual reproduction, and mechanisms of pathogenesis. These studies have provided a refreshing change of pace in research on filamentous fungi, which has lagged behind that on other eukaryotes in the exploitation of genome-wide methodologies. Despite the late start, several fungal genome sequencing projects are underway. The resulting databases will allow the comprehensive analysis of developmental processes that are characteristic of fungi, including the molecular nature of pathogenicity. DNA databases underpin analyses of the fungal transcriptome, proteome, and metabolome. This combined information will contribute to our basic understanding of not only the mechanics of infection but also the evolution of pathogenicity.

Functional Analysis of All Nonribosomal Peptide Synthetases in Cochliobolus heterostrophus Reveals a Factor, NPS6, Involved in Virulence and Resistance to Oxidative Stress

ABSTRACT Nonribosomal peptides, made by nonribosomal peptide synthetases, have diverse biological activities, including roles as fungal virulence effectors. Inspection of the genome of Cochliobolus heterostrophus, a fungal pathogen of maize and a member of a genus noted for secondary metabolite production, revealed eight multimodular nonribosomal peptide synthase (NPS) genes and three monomodular NPS-like genes, one of which encodes a nonribosomal peptide synthetase/polyketide synthase hybrid enzyme presumed to be involved in synthesis of a peptide/polyketide molecule. Deletion of each NPS gene and phenotypic analyses showed that the product of only one of these genes, NPS6, is required for normal virulence on maize. NPS6 is also required for resistance to hydrogen peroxide, suggesting it may protect the fungus from oxidative stress. This and all other nps mutants had normal growth, mating ability, and appressoria. Real-time PCR analysis showed that expression of all NPS genes is low (relative to that of actin), that all (except possibly NPS2) are expressed during vegetative growth, and that expression is induced by nitrogen starvation. Only NPS6 is unfailingly conserved among euascomycete fungi, including plant and human pathogens and saprobes, suggesting the possibility that NPS6 activity provides oxidative stress protection during both saprobic and parasitic growth.