101 Dothideomycetes genomes: A test case for predicting lifestyles and emergence of pathogens.

Dothideomycetes is the largest class of kingdom Fungi and comprises an incredible diversity of lifestyles, many of which have evolved multiple times. Plant pathogens represent a major ecological niche of the class Dothideomycetes and they are known to infect most major food crops and feedstocks for biomass and biofuel production. Studying the ecology and evolution of Dothideomycetes has significant implications for our fundamental understanding of fungal evolution, their adaptation to stress and host specificity, and practical implications with regard to the effects of climate change and on the food, feed, and livestock elements of the agro-economy. In this study, we present the first large-scale, whole-genome comparison of 101 Dothideomycetes introducing 55 newly sequenced species. The availability of whole-genome data produced a high-confidence phylogeny leading to reclassification of 25 organisms, provided a clearer picture of the relationships among the various families, and indicated that pathogenicity evolved multiple times within this class. We also identified gene family expansions and contractions across the Dothideomycetes phylogeny linked to ecological niches providing insights into genome evolution and adaptation across this group. Using machine-learning methods we classified fungi into lifestyle classes with >95 % accuracy and identified a small number of gene families that positively correlated with these distinctions. This can become a valuable tool for genome-based prediction of species lifestyle, especially for rarely seen and poorly studied species.

Deletion and complementation of the mating type (MAT) locus of the wheat head blight pathogen Gibberella zeae.

Gibberella zeae, a self-fertile, haploid filamentous ascomycete, causes serious epidemics of wheat (Triticum aestivum) head blight worldwide and contaminates grain with trichothecene mycotoxins. Anecdotal evidence dating back to the late 19th century indicates that G. zeae ascospores (sexual spores) are a more important inoculum source than are macroconidia (asexual spores), although the fungus can produce both during wheat head blight epidemics. To develop fungal strains to test this hypothesis, the entire mating type (MAT1) locus was deleted from a self-fertile (MAT1-1/MAT1-2), virulent, trichothecene-producing wild-type strain of G. zeae. The resulting MAT deletion (mat1-1/mat1-2) strains were unable to produce perithecia or ascospores and appeared to be unable to mate with the fertile strain from which they were derived. Complementation of a MAT deletion strain by transformation with a copy of the entire MAT locus resulted in recovery of production of perithecia and ascospores. MAT deletion strains and MAT-complemented strains retained the ability to produce macroconidia that could cause head blight, as assessed by direct injection into wheat heads in greenhouse tests. Availability of MAT-null and MAT-complemented strains provides a means to determine the importance of ascospores in the biology of G. zeae and perhaps to identify novel approaches to control wheat head blight.

Identity and conservation of mating type genes in geographically diverse isolates of Phaeosphaeria nodorum.

Mating type idiomorphs (MAT1-1 and MAT1-2) were identified from the heterothallic loculoascomycete Phaeosphaeria nodorum (wheat biotype) using DNA from a pair of isolates from Poland and Georgia, USA that are known to mate. MAT predicted proteins of P. nodorum are similar in sequence and in phylogenetic relationship to those described for other loculoascomycetes such as Cochliobolus spp., Alternaria alternata, and Didymella zeae-maydis. The organization of the MAT locus of the P. nodorum differs from these species in that its idiomorph begins within an adjacent upstream conserved ORF of unknown function. MAT-specific primers were used to identify isolates of both mating types in field populations, demonstrating that an absence of either mating type is not the reason that the teleomorph has not been found in New York. Portions of MAT1-1 and MAT1-2 were sequenced from geographically diverse isolates, including those from regions where the teleomorph has been reported. MAT was highly conserved and no significant differences in sequence were found.

Expression of recombinant aequorin as an intracellular calcium reporter in the phytopathogenic fungus Phyllosticta ampelicida.

Conidia of Phyllosticta ampelicida germinate only after they have made contact with a substratum. Previous work has shown that external free calcium must be available to the spore for germination to be initiated. Transgenic strains of P. ampelicida expressing apo-aequorin, a calcium-sensitive luminescent protein, were developed to monitor cytoplasmic free Ca(2+) ([Ca(2+)]c). Transformants were verified by PCR and Southern hybridization. Apo-aequorin production was quantified for each of 21 transformants. The transformant that emitted the most light per unit of protein was found to contain 0.59 mg apo-aequorin/g total protein. To ascertain the feasibility of aequorin-based [Ca(2+)]c quantification, [Ca(2+)]c changes were measured in mycelia during various physiologically perturbing treatments: exposure to high concentrations of external Ca(2+), hypoosmotic shock, and mechanical perturbation. This is the first report of a plant pathogenic fungus for which aequorin-based Ca(2+) measurement protocols have been developed.

Analysis of mating-type genes in the chestnut blight fungus, Cryphonectria parasitica.

In nature, the chestnut blight fungus, Cryphonectria parasitica, has a mixed mating system; i.e., individuals in the same population have the ability to self and outcross. In the laboratory, C. parasitica appears to have a bipolar self-incompatibility system, typical of heterothallic ascomycetes; selfing is rare, although demonstrable. In this report we describe the cloning and sequencing of both mating-type idiomorphs and their flanking regions at the MAT locus in C. parasitica. The two idiomorphs, MAT1-1 and MAT1-2, are structurally similar to those of other pyrenomycetes described to date. MAT1-1 encodes three genes (MAT1-1-1, MAT1-1-2, and MAT1-1-3) and MAT1-2 encodes a single gene (MAT1-2-1). Unlike MAT idiomorphs in some ascomycetes, the sequences at both ends of the idiomorphs in C. parasitica show a relatively gradual, rather than abrupt, transition from identity in the flanking regions to almost complete dissimilarity in the coding regions. The flanking regions have repetitive polypyrimidine (T/C) and polypurine (A/G) tracts; the significance of these repetitive tracts is unknown. Although we found repetitive tracts in the flanks and gradual transition zones at the ends of the idiomorphs, we found no special features that would explain how selfing occurs in an otherwise self-incompatible fungus.

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.

Molecular organization of mating type loci in heterothallic, homothallic, and asexual Gibberella/Fusarium species.

Mating type (MAT) genes were cloned from three members of the Gibberella/Fusarium complex that differ in reproductive mode: heterothallic G. fujikuroi, homothallic G. zeae, and asexual F. oxysporum. The G. fujikuroi MAT locus organization is typical of other heterothallic pyrenomycetes characterized to date; i.e., there are three genes at MAT1-1 and one at MAT1-2. G. zeae has homologues of all four genes encoded by the two G. fujikuroi MAT idiomorphs, tightly linked on the same chromosome, interspersed with sequences unique to G. zeae. Field isolates of F. oxysporum, although asexual, have either the MAT1-1 or the MAT1-2 genes found in sexual species and these genes are highly similar to those of heterothallic G. fujikuroi. RT-PCR analysis proved that the F. oxysporum MAT genes are expressed and that all putative introns found in each of the four MAT genes in G. fujikuroi and F. oxysporum are removed. Apparent failure of F. oxysporum to reproduce sexually could not be attributed to mutations in the MAT genes themselves.

Characterization of 6-epi-3-anhydroophiobolin B from Cochliobolus heterostrophus.

The new sesterterpenoid 6-epi-3-anhydroophiobolin B (1) and six known ophiobolins were isolated from the extracts of the fungus Cochliobolus heterostrophus race O. The structure of 6-epi-3-anhydroophiobolin B was deduced from analysis of spectral data and the structural characterization of dehydration and dimerization products. Ophiobolin A (2) showed potent activity in cytotoxicity assays and marginal activity in antimalarial assays.

Evolution of the fungal self-fertile reproductive life style from self-sterile ancestors.

In most fungal ascomycetes, mating is controlled by a single locus (MAT). Fungi requiring a partner to mate are heterothallic (self-sterile); those not requiring a partner are homothallic (self-fertile). Structural analyses of MAT sequences from homothallic and heterothallic Cochliobolus species support the hypothesis that heterothallism is ancestral. Homothallic species carry both MAT genes in a single nucleus, usually closely linked or fused, in contrast to heterothallic species, which have alternate MAT genes in different nuclei. The structural organization of MAT from all heterothallic species examined is highly conserved; in contrast, the organization of MAT in each homothallic species is unique. The mechanism of conversion from heterothallism to homothallism is a recombination event between islands of identity in otherwise dissimilar MAT sequences. Expression of a fused MAT gene from a homothallic species confers self-fertility on a MAT-null strain of a heterothallic species, suggesting that MAT alone is sufficient to change reproductive life style.

A G protein alpha subunit from Cochliobolus heterostrophus involved in mating and appressorium formation.

A Galpha subunit-encoding gene (CGA1) was cloned from Cochliobolus heterostrophus, a heterothallic foliar pathogen of corn. The deduced amino acid sequence showed similarity to Galpha proteins from other filamentous fungi and suggested that CGA1 is a member of the Galphai class. cga1 mutants had reduced ability to form appressoria on glass surfaces and on corn leaves; mutants nevertheless caused lesions on corn plants like those of wild type. cga1 mutants were female sterile; sexual development was completely abolished when the mutant allele was homozygous in a cross. Ascospores produced in crosses heterozygous at Cga1 were all wild type. The signal transduction pathway represented by CGA1 appears to be involved in developmental pathways leading to either appressorium formation or mating; in sexual development CGA1 is required for both fertility and ascospore viability.

The translocation-associated tox1 locus of Cochliobolus heterostrophus is two genetic elements on two different chromosomes.

Previously, Tox1 was defined as a single genetic element controlling the difference between races of Cochliobolus heterostrophus: race T is highly virulent on T-cytoplasm corn and produces the polyketide T-toxin; race O is weakly virulent and does not produce T-toxin. Here we report that Tox1 is two loci, Tox1A and Tox1B, on two different chromosomes. Evidence for two loci derives from: (1) the appearance of 25% Tox+ progeny in crosses between induced Tox1(-) mutants, one defective at Tox1A, the other at Tox1B; (2) the ability of Tox1A- + Tox1B- heterokaryons to complement for T-toxin production; and (3) electrophoretic karyotypes proving that Tox1(-) mutations are physically located on two different chromosomes. Data showing Tox1 as a single genetic element are reconciled with those proving it is two loci by the fact that Tox1 is inseparably linked to the breakpoints of a reciprocal translocation; the translocation results in a four-armed linkage group. In crosses where the translocation is heterozygous (i.e., race T by race O), all markers linked to the four-armed intersection appear linked to each other; in crosses between induced Tox1(-) mutants, complications due to the translocation are eliminated and the two loci segregate independently.

Single mating type-specific genes and their 3' UTRs control mating and fertility in Cochliobolus heterostrophus.

To determine the number of proteins required for mating type (MAT) locus-regulated control of mating in Cochliobolus heterostrophus, MAT fragments of various sizes were expressed in MAT deletion strains. As little as 1.5 kb of MAT sequence, encoding a single unique protein in each mating type (MAT-1 and MAT-2), conferred mating ability, although an additional 160 bp of 3' UTR was needed for production of ascospores. No other mating type-specific genes involved in mating identity or fertility were found. Thus, although homologs of the C. heterostrophus MAT-1 and MAT-2 genes exist in the filamentous ascomycetes Neurospora crassa and Podospora anserina, C. heterostrophus does not appear to have mating type-specific homologs of two additional genes required by both N. crassa and P. anserina for successful sexual reproduction. Three genes were identified in the common DNA flanking the MAT locus: a gene encoding a GTPase-activating protein and an ORF of unknown function lie 5' while a beta-glucosidase encoding gene lies found 3'. None of these genes appears to be involving in the mating process.

A cytoplasmic dynein required for mitotic aster formation in vivo.

An astral pulling force helps to elongate the mitotic spindle in the filamentous ascomycete, Nectria haematococca. Evidence is mounting that dynein is required for the formation of mitotic spindles and asters. Obviously, this would be an important mitotic function of dynein, since it would be a prerequisite for astral force to be applied to a spindle pole. Missing from the evidence for such a role of dynein in aster formation, however, has been a dynein mutant lacking mitotic asters. To determine whether or not cytoplasmic dynein is involved in mitotic aster formation in N. haematococca, a dynein-deficient mutant was made. Immunocytochemistry visualized few or no mitotic astral microtubules in the mutant cells, and studies of living cells confirmed the veracity of this result by revealing the absence of mitotic aster functions in vivo: intra-astral motility of membranous organelles was not apparent; the rate and extent of spindle elongation during anaphase B were reduced; and spindle pole body separation almost stopped when the anaphase B spindle in the mutant was cut by a laser microbeam, demonstrating unequivocally that no astral pulling force was present. These unique results not only provide a demonstration that cytoplasmic dynein is required for the formation of mitotic asters in N. haematococca; they also represent the first report of mitotic phenotypes in a dynein mutant of any filamentous fungus and the first cytoplasmic dynein mutant of any organism whose mitotic phenotypes demonstrate the requirement of cytoplasmic dynein for aster formation in vivo.

Role of fungal dynein in hyphal growth, microtubule organization, spindle pole body motility and nuclear migration.

Cytoplasmic dynein is a microtubule-associated motor protein with several putative subcellular functions. Sequencing of the gene (DHC1) for cytoplasmic dynein heavy chain of the filamentous ascomycete, Nectria haematococca, revealed a 4,349-codon open reading frame (interrupted by two introns) with four highly conserved P-loop motifs, typical of cytoplasmic dynein heavy chains. The predicted amino acid sequence is 78.0% identical to the cytoplasmic dynein heavy chain of Neurospora crassa, 70.2% identical to that of Aspergillus nidulans and 24.8% identical to that of Saccharomyces cerevisiae. The genomic copy of DHC1 in N. haematococca wild-type strain T213 was disrupted by inserting a selectable marker into the central motor domain. Mutants grew at 33% of the wild-type rate, forming dense compact colonies composed of spiral and highly branched hyphae. Major cytological phenotypes included (1) absence of aster-like arrays of cytoplasmic microtubules focused at the spindle pole bodies of post-mitotic and interphase nuclei, (2) limited post-mitotic nuclear migration, (3) lack of spindle pole body motility at interphase, (4) failure of spindle pole bodies to anchor interphase nuclei, (5) nonuniform distribution of interphase nuclei and (6) small or ephemeral Spitzenkörper at the apices of hyphal tip cells. Microtubule distribution in the apical region of tip cells of the mutant was essentially normal. The nonuniform distribution of nuclei in hyphae resulted primarily from a lack of both post-mitotic nuclear migration and anchoring of interphase nuclei by the spindle pole bodies. The results support the hypothesis that DHC1 is required for the motility and functions of spindle pole bodies, normal secretory vesicle transport to the hyphal apex and normal hyphal tip cell morphogenesis.

A fungal kinesin required for organelle motility, hyphal growth, and morphogenesis.

A gene (NhKIN1) encoding a kinesin was cloned from Nectria haematococca genomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed that NhKIN1 belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1 by transformation-mediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkörper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkörper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubule-based motor protein is essential for normal positioning of the Spitzenkörper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology.

Application of mating type gene technology to problems in fungal biology.

In ascomycetes, the single mating type locus (MAT) controls sexual development. This locus is structurally unusual because the two alternate forms ("alleles") are completely dissimilar sequences, encoding different transcription factors, yet they occupy the same chromosomal position. Recently developed procedures allow efficient cloning of MAT genes from a wide array of filamentous ascomycetes, thereby providing MAT-based technology for application to several ongoing issues in fungal biology. This article first outlines the basic nature of MAT genes, then addresses the following topics: efficient cloning of MAT genes; the unusual molecular characteristics of these genes; phylogenetics using MAT; the issues of why some fungi are self-sterile, others self-fertile, and yet others asexual; the long-standing mystery of possible mating type switching in filamentous fungi; and finally the evolutionary origins of pathogenic capability.

Efficient cloning of ascomycete mating type genes by PCR amplification of the conserved MAT HMG Box.

Cloning of mating type (MAT) genes from ascomycetes has been hampered by low conservation among them. One of the pair of MAT genes, represented by MAT-2 of Cochliobolus heterostrophus (a loculoascomycete) and mt a of Neurospora crassa (a pyrenomycete), encodes a protein with a conserved DNA binding motif called the high mobility group (HMG) box. PCR with primer pairs corresponding to the borders of the C. heterostrophus and the N. crassa HMG boxes generated an approximately 0.3-kb product from genomic DNAs of MAT-2 and mt a strains, respectively, but not from MAT-1 and mt A strains. The C. heterostrophus primers amplified approximately 0.3-kb products from DNA of most loculoascomycete genera tested but not from DNA of pyrenomycete genera; this specificity was reversed with the N. crassa primers. The validity of the PCR procedure was documented by near sequence identity between the C. heterostrophus MAT-2 HMG box and PCR products from several Cochliobolus spp. and by cosegregation of the PCR product with mating type in progeny of Setosphaeria turcica and of Cryphonectria parasitica. Regions of the MAT locus flanking the HMG box were readily cloned using the TAIL-PCR procedure with a combination of random and specific primers.

Transcripts at the mating type locus of Cochliobolus heterostrophus.

The single mating type locus (MAT) of the heterothallic ascomycete Cochliobolus heterostrophus is composed of a pair of unlike sequences called idiomorphs, each of which encodes one MAT-specific gene (MAT-1 and MAT-2). MAT transcripts were observed in blots of poly(A)+ RNA isolated from cultures grown in minimal medium, but were not detectable after growth of the fungus in complete medium, suggesting that transcription of MAT is tightly regulated. The idiomorphs (MAT-1 = 1297-bp, MAT-2 = 1171-bp) encode transcripts of 2.2 kb (MAT-1) and 2.1 kb (MAT-2), which start 5' and end 3' of the idiomorph within sequences common to both mating types. Analyses of MAT-1 and MAT-2 cDNAs revealed obligatory splicing of one intron (55-bp in MAT-1, 52-bp in MAT-2) within each MAT-specific ORF and optional splicing of two introns (63 and 79-bp) in the long (approximately 0.55 kb) 5' untranslated leader sequences; the 3' untranslated region is 0.46 kb long. Transcription start sites were found 5' of, and within, the 79-bp intron. Optional splicing of the upstream introns and at least two transcription start sites result in three types of transcript: Type I with both 5' introns spliced, Type II with only the 63-bp intron spliced, and Type III with neither 5' intron spliced. The three transcript types are distinguished by various combinations of four short ORFs encoded by the corresponding genomic DNA, in the leader sequences of the MAT mRNAs. The transcript structure suggests several mechanisms by which expression of the MAT genes might be regulated at the level of translation during sexual development.

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.