Concordance of gene genealogies reveals reproductive isolation in the pathogenic fungus Coccidioides immitis

Simple cladogenetic theory suggests that gene genealogies can be used to detect mixis in a population and delineate reproductively isolated groups within sexual taxa. We have taken this approach in a study of Coccidioides immitis, an ascomycete fungus responsible for a recent epidemic of coccidioidomycosis (Valley fever) in California. To test whether this fungus represents a single sexual species throughout its entire geographic range, we have compared genealogies from fragments of five nuclear genes. The five genealogies show multiple incompatibilities indicative of sex, but also share a branch that partitions the isolates into two reproductively isolated taxa, one centered in California and the other outside California. We conclude that coccidioidomycosis can be caused by two distinct noninterbreeding taxa. This result should aid the future study of the disease and illustrates the utility of the genealogical approach in population genetics.

The mating-type and pathogenicity locus of the fungus Ustilago hordei spans a 500-kb region

The fungal pathogen Ustilago hordei causes the covered smut disease of barley and oats. Mating and pathogenicity in this fungus are controlled by the MAT locus, which contains two distinct gene complexes, a and b. In this study, we tagged the a and b regions with the recognition sequence for the restriction enzyme I-SceI and determined that the distance between the complexes is 500 kb in a MAT-1 strain and 430 kb in a MAT-2 strain. Characterization of the organization of the known genes within the a and b gene complexes provided evidence for nonhomology and sequence inversion between MAT-1 and MAT-2. Antibiotic-resistance markers also were used to tag the a gene complex in MAT-1 strains (phleomycin) and the b gene complex in MAT-2 strains (hygromycin). Crosses were performed with these strains and progeny resistant to both antibiotics were recovered at a very low frequency, suggesting that recombination is suppressed within the MAT region. Overall, the chromosome homologues carrying the MAT locus of U. hordei share features with primitive sex chromosomes, with the added twist that the MAT locus also controls pathogenicity.

Multiple Sex Pheromones and Receptors of a Mushroom-producing Fungus Elicit Mating in Yeast

The mushroom-producing fungus Schizophyllum commune has thousands of mating types defined, in part, by numerous lipopeptide pheromones and their G protein-linked receptors. Compatible combinations of pheromones and receptors encoded by different mating types regulate a pathway of sexual development leading to mushroom formation and meiosis. A complex set of pheromone–receptor interactions maximizes the likelihood of outbreeding; for example, a single pheromone can activate more than one receptor and a single receptor can be activated by more than one pheromone. The current study demonstrates that the sex pheromones and receptors of Schizophyllum, when expressed in Saccharomyces cerevisiae, can substitute for endogenous pheromone and receptor and induce the yeast pheromone response pathway through the yeast G protein. Secretion of active Schizophyllum pheromone requires some, but not all, of the biosynthetic machinery used by the yeast lipopeptide pheromone a-factor. The specificity of interaction among pheromone–receptor pairs in Schizophyllum was reproduced in yeast, thus providing a powerful system for exploring molecular aspects of pheromone–receptor interactions for a class of seven-transmembrane-domain receptors common to a wide range of organisms.

Expression of alfalfa mosaic virus coat protein in tobacco mosaic virus (TMV) deficient in the production of its native coat protein supports long-distance movement of a chimeric TMV

Alfalfa mosaic virus (AlMV) coat protein is involved in systemic infection of host plants, and a specific mutation in this gene prevents the virus from moving into the upper uninoculated leaves. The coat protein also is required for different viral functions during early and late infection. To study the role of the coat protein in long-distance movement of AlMV independent of other vital functions during virus infection, we cloned the gene encoding the coat protein of AlMV into a tobacco mosaic virus (TMV)-based vector Av. This vector is deficient in long-distance movement and is limited to locally inoculated leaves because of the lack of native TMV coat protein. Expression of AlMV coat protein, directed by the subgenomic promoter of TMV coat protein in Av, supported systemic infection with the chimeric virus in Nicotiana benthamiana, Nicotiana tabacum MD609, and Spinacia oleracea. The host range of TMV was extended to include spinach as a permissive host. Here we report the alteration of a host range by incorporating genetic determinants from another virus.

Changes in left ventricular structure and function in patients with white coat hypertension: cross sectional survey

Objectives: To assess the relation between white coat hypertension and alterations of left ventricular structure and function.

The surface coat of procyclic Trypanosoma brucei: Programmed expression and proteolytic cleavage of procyclin in the tsetse fly

Trypanosoma brucei, the protozoan parasite causing sleeping sickness, is transmitted by a tsetse fly vector. When the tsetse takes a blood meal from an infected human, it ingests bloodstream form trypanosomes that quickly differentiate into procyclic forms within the fly's midgut. During this process, the parasite loses the 107 molecules of variant surface glycoprotein that formed its surface coat, and it develops a new coat composed of several million procyclin molecules. Procyclins, the products of a small multigene family, are glycosyl phosphatidylinositol-anchored proteins containing characteristic amino acid repeats at the C terminus [either EP (EP procyclin, a form of procyclin rich in Glu-Pro repeats) or GPEET (GPEET procyclin, a form of procyclin rich in Glu-Pro-Glu-Glu-Thr repeats)]. We have used a sensitive and accurate mass spectrometry method to analyze the appearance of different procyclins during the establishment of midgut infections in tsetse flies. We found that different procyclin gene products are expressed in an orderly manner. Early in the infection (day 3), GPEET2 is the only procyclin detected. By day 7, however, GPEET2 disappears and is replaced by several isoforms of glycosylated EP, but not the unglycosylated isoform EP2. Unexpectedly, we discovered that the N-terminal domains of all procyclins are quantitatively removed by proteolysis in the fly, but not in culture. These findings suggest that one function of the protease-resistant C-terminal domain, containing the amino acid repeats, is to protect the parasite surface from digestive enzymes in the tsetse fly gut.

Hypersensitive Cell Death and Papilla Formation in Barley Attacked by the Powdery Mildew Fungus Are Associated with Hydrogen Peroxide but Not with Salicylic Acid Accumulation1

We analyzed the pathogenesis-related generation of H2O2 using the microscopic detection of 3,3-diaminobenzidine polymerization in near-isogenic barley (Hordeum vulgare L.) lines carrying different powdery mildew (Blumeria graminis f.sp. hordei) resistance genes, and in a line expressing chemically activated resistance after treatment with 2,6-dichloroisonicotinic acid (DCINA). Hypersensitive cell death in Mla12 and Mlg genotypes or after chemical activation by DCINA was associated with H2O2 accumulation throughout attacked cells. Formation of cell wall appositions (papillae) mediated in Mlg and mlo5 genotypes and in DCINA-activated plants was paralleled by H2O2 accumulation in effective papillae and in cytosolic vesicles of up to 2 μm in diameter near the papillae. H2O2 was not detected in ineffective papillae of cells that had been successfully penetrated by the fungus. These findings support the hypothesis that H2O2 may play a substantial role in plant defense against the powdery mildew fungus. We did not detect any accumulation of salicylic acid in primary leaves after inoculation of the different barley genotypes, indicating that these defense responses neither relied on nor provoked salicylic acid accumulation in barley.

Arabidopsis Sec21p and Sec23p Homologs. Probable Coat Proteins of Plant COP-Coated Vesicles1

Intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus and within the Golgi apparatus is facilitated by COP (coat protein)-coated vesicles. Their existence in plant cells has not yet been demonstrated, although the GTP-binding proteins required for coat formation have been identified. We have generated antisera against glutathione-S-transferase-fusion proteins prepared with cDNAs encoding the Arabidopsis Sec21p and Sec23p homologs (AtSec21p and AtSec23p, respectively). The former is a constituent of the COPI vesicle coatomer, and the latter is part of the Sec23/24p dimeric complex of the COPII vesicle coat. Cauliflower (Brassica oleracea) inflorescence homogenates were probed with these antibodies and demonstrated the presence of AtSec21p and AtSec23p antigens in both the cytosol and membrane fractions of the cell. The membrane-associated forms of both antigens can be solubilized by treatments typical for extrinsic proteins. The amounts of the cytosolic antigens relative to the membrane-bound forms increase after cold treatment, and the two antigens belong to different protein complexes with molecular sizes comparable to the corresponding nonplant coat proteins. Sucrose-density-gradient centrifugation of microsomal cell membranes from cauliflower suggests that, although AtSec23p seems to be preferentially associated with ER membranes, AtSec21p appears to be bound to both the ER and the Golgi membranes. This could be in agreement with the notion that COPII vesicles are formed at the ER, whereas COPI vesicles can be made by both Golgi and ER membranes. Both AtSec21p and AtSec23p antigens were detected on membranes equilibrating at sucrose densities equivalent to those typical for in vitro-induced COP vesicles from animal and yeast systems. Therefore, a further purification of the putative plant COP vesicles was undertaken.

The role of haustoria in sugar supply during infection of broad bean by the rust fungus Uromyces fabae

Biotrophic plant pathogenic fungi differentiate specialized infection structures within the living cells of their host plants. These haustoria have been linked to nutrient uptake ever since their discovery. We have for the first time to our knowledge shown that the flow of sugars from the host Vicia faba to the rust fungus Uromyces fabae seems to occur largely through the haustorial complex. One of the most abundantly expressed genes in rust haustoria, the expression of which is negligible in other fungal structures, codes for a hexose transporter. Functional expression of the gene termed HXT1 in Saccharomyces cerevisiae and Xenopus laevis oocytes assigned a substrate specificity for d-glucose and d-fructose and indicated a proton symport mechanism. Abs against HXT1p exclusively labeled haustoria in immunofluorescence microscopy and the haustorial plasma membrane in electron microscopy. These results suggest that the fungus concentrates this transporter in haustoria to take advantage of a specialized compartment of the haustorial complex. The extrahaustorial matrix, delimited by the plasma membranes of both host and parasite, constitutes a newly formed apoplastic compartment with qualities distinct from those of the bulk apoplast. This organization might facilitate the competition of the parasite with natural sink organs of the host.

Yeast Gga Coat Proteins Function with Clathrin in Golgi to Endosome Transport

Gga proteins represent a newly recognized, evolutionarily conserved protein family with homology to the “ear” domain of the clathrin adaptor AP-1 γ subunit. Yeast cells contain two Gga proteins, Gga1p and Gga2p, that have been proposed to act in transport between the trans-Golgi network and endosomes. Here we provide genetic and physical evidence that yeast Gga proteins function in trans-Golgi network clathrin coats. Deletion of Gga2p (gga2Δ), the major Gga protein, accentuates growth and α-factor maturation defects in cells carrying a temperature-sensitive allele of the clathrin heavy chain gene. Cells carrying either gga2Δ or a deletion of the AP-1 β subunit gene (apl2Δ) alone are phenotypically normal, but cells carrying both gga2Δ and apl2Δ are defective in growth, α-factor maturation, and transport of carboxypeptidase S to the vacuole. Disruption of both GGA genes and APL2 results in cells so severely compromised in growth that they form only microcolonies. Gga proteins can bind clathrin in vitro and cofractionate with clathrin-coated vesicles. Our results indicate that yeast Gga proteins play an important role in cargo-selective clathrin-mediated protein traffic from the trans-Golgi network to endosomes.

Molecular evidence that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina.

The relationship of the important cellulase producing asexual fungus Trichoderma reesei to its putative teleomorphic (sexual) ancestor Hypocrea jecorina and other species of the Trichoderma sect. Longibrachiatum was studied by PCR-fingerprinting and sequence analyses of the nuclear ribosomal DNA region containing the internal transcribed spacers (ITS-1 and ITS-2) and the 5.8S rRNA gene. The differences in the corresponding ITS sequences allowed a grouping of anamorphic (asexual) species of Trichoderma sect. Longibrachiatum into Trichoderma longibrachiatum, Trichoderma pseudokoningii, and Trichoderma reesei. The sexual species Hypocrea schweinitzii and H. jecorina were also clearly separated from each other. H. jecorina and T. reesei exhibited identical sequences, suggesting close relatedness or even species identity. Intraspecific and interspecific variation in the PCR-fingerprinting patterns supported the differentiation of species based on ITS sequences, the grouping of the strains, and the assignment of these strains to individual species. The variations between T. reesei and H. jecorina were at the same order of magnitude as found between all strains of H. jecorina, but much lower than the observed interspecific variations. Identical ITS sequences and the high similarity of PCR-fingerprinting patterns indicate a very close relationship between T. reesei and H. jecorina, whereas differences of the ITS sequences and the PCR-fingerprinting patterns show a clear phylogenetic distance between T. reesei/H. jecorina and T. longibrachiatum. T. reesei is considered to be an asexual, clonal line derived from a population of the tropical ascomycete H. jecorina.

Nonclathrin coat protein gamma, a subunit of coatomer, binds to the cytoplasmic dilysine motif of membrane proteins of the early secretory pathway.

Coatomer, a cytosolic heterooligomeric protein complex that consists of seven subunits [alpha-, beta-, beta'-, gamma-, delta-, epsilon-, and zeta-COP (nonclathrin coat protein)], has been shown to interact with dilysine motifs typically found in the cytoplasmic domains of various endoplasmic-reticulum-resident membrane proteins [Cosson, P. & Letourneur, F. (1994) Science 263, 1629-1631]. We have used a photo-cross-linking approach to identify the site of coatomer that is involved in binding to the dilysine motifs. An octapeptide corresponding to the C-terminal tail of Wbp1p, a component of the yeast N-oligosaccharyltransferase complex, has been synthesized with a photoreactive phenylalanine at position -5 and was radioactively labeled with [125I]iodine at a tyrosine residue introduced at the N terminus of the peptide. Photolysis of isolated coatomer in the presence of this peptide and immunoprecipitation of coatomer from photo-cross-linked cell lysates reveal that gamma-COP is the predominantly labeled protein. From these results, we conclude that coatomer is able to bind to the cytoplasmic dilysine motifs of membrane proteins of the early secretory pathway via its gamma-COP subunit, whose complete cDNA-derived amino acid sequence is also presented.

An abundantly expressed mucin-like protein from Toxocara canis infective larvae: the precursor of the larval surface coat glycoproteins.

Evasion of host immunity by Toxocara canis infective larvae is mediated by the nematode surface coat, which is shed in response to binding by host antibody molecules or effector cells. The major constituent of the coat is the TES-120 glycoprotein series. We have isolated a 730-bp cDNA from the gene encoding the apoprotein precursor of TES-120. The mRNA is absent from T. canis adults but hyperabundant in larvae, making up approximately 10% of total mRNA, and is trans-spliced with the nematode 5' leader sequence SL1. It encodes a 15.8-kDa protein (after signal peptide removal) containing a typical mucin domain: 86 amino acid residues, 72.1% of which are Ser or Thr, organized into an array of heptameric repeats, interspersed with proline residues. At the C-terminal end of the putative protein are two 36-amino acid repeats containing six Cys residues, in a motif that can also be identified in several genes in Caenorhabditis elegans. Although TES-120 displays size and charge heterogeneity, there is a single copy gene and a homogeneous size of mRNA. The association of overexpression of some membrane-associated mucins with immunosuppression and tumor metastasis suggests a possible model for the role of the surface coat in immune evasion by parasitic nematodes.

Null mutation of endothelin receptor type B gene in spotting lethal rats causes aganglionic megacolon and white coat color.

Mutations in the gene encoding the endothelin receptor type B (EDNRB) produce congenital aganglionic megacolon and pigment abnormalities in mice and humans. Here we report a naturally occurring null mutation of the EDNRB gene in spotting lethal (sl) rats, which exhibit aganglionic megacolon associated with white coat color. We found a 301-bp deletion spanning the exon 1-intron 1 junction of the EDNRB gene in sl rats. A restriction fragment length polymorphism caused by this deletion perfectly cosegregates with the sl phenotype. The deletion leads to production of an aberrantly spliced EDNRB mRNA that lacks the coding sequence for the first and second putative transmembrane domains of the G-protein-coupled receptor. Radioligand binding assays revealed undetectable levels of functional EDNRB in tissues from homozygous sl/sl rats. We conclude that EDNRB plays an essential role in the normal development of two neural crest-derived cell lineages, epidermal melanocytes and enteric neurons, in three mammalian species--humans, mice, and rats. The EDNRB-deficient rat may also prove valuable in defining the postnatal physiologic role of this receptor.