PubMed Health⌕ Search

Biomedical subjects

J E Hamer

Publications and source records attributed to J E Hamer.

At least 19 recordsLinked to original sources
PubMed2001

Gene discovery and gene function assignment in filamentous fungi.

Filamentous fungi are a large group of diverse and economically important microorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (TI) during transformation. We developed transposon-arrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to investigate an amino acid oxidation pathway in two important fungal phytopathogens.

Alleles↗
PubMed2001

Recent advances in large-scale transposon mutagenesis.

Transposons were identified as mobile genetic elements over fifty years ago and subsequently became powerful tools for molecular-genetic studies. Recently, transposon-mutagenesis strategies have been developed to identify essential and pathogenicity-related genes in pathogenic microorganisms. Also, a number of in vitro transposition systems have been used to facilitate genome sequence analysis. Finally, transposon mutagenesis of yeast and complex eukaryotes has provided valuable functional genomic information to complement genome-sequencing projects.

Animals↗
PubMed2001

Characterization of a PR-10 pathogenesis-related gene family induced in rice during infection with Magnaporthe grisea.

A partial cDNA with homology to the PR-10 class of pathogenesis-related proteins was used to screen a rice genomic library. One 16-kb genomic clone contained three genes with PR-10 similarity. These genes, RPR10a, RPR10b, and RPR10c, were arranged in tandem and separated by approximately 2.5 kb. RPR10a cDNA was obtained by reverse transcription-polymerase chain reaction, and sequence analysis revealed that RPR10a and RPR10b encode predicted proteins of 158 and 160 amino acids, respectively, and share 71% amino acid identity. RPR10c appears to be a nonfunctional pseudogene. Gene-specific probes were used to study transcript accumulations of the three RPR10 genes in rice plants following inoculation with Magnaporthe grisea. RPR10a transcripts were induced from a low basal level within 12 h after inoculation and showed a second higher level induction at 48 h, which continued throughout the 144 h it was examined. In addition, RPR10a was induced strongly by salicylic and jasmonic acid applications to rice plants. Transcripts of RPR10b also were enhanced by M. grisea, but were not strongly visible until 48 h after inoculation. Tissue prints of M. grisea-infected rice leaves when the RPR10a-specific probe was used indicate that RPR10a is expressed most strongly in a localized fashion in response to the pathogen.

Base Sequence↗
PubMed2000

Hypomorphic bimA(APC3) alleles cause errors in chromosome metabolism that activate the DNA damage checkpoint blocking cytokinesis in Aspergillus nidulans.

The Aspergillus nidulans sepI(+) gene has been implicated in the coordination of septation with nuclear division and cell growth. We find that the temperature-sensitive (ts) sepI1 mutation represents a novel allele of bimA(APC3), which encodes a conserved component of the anaphase-promoting complex/cyclosome (APC/C). We have characterized the septation, nuclear division, cell-cycle checkpoint defects, and DNA sequence alterations of sepI1 (renamed bimA10) and two other ts lethal bimA(APC3) alleles, bimA1 and bimA9. Our observations that bimA9 and bimA10 strains had morphologically abnormal nuclei, chromosome segregation defects, synthetic phenotypes with mutations in the DNA damage checkpoint genes uvsB(MEC1/rad3) or uvsD(+), and enhanced sensitivity to hydroxyurea strongly suggest that these strains accumulate errors in DNA metabolism. We found that the aseptate phenotype of bimA9 and bimA10 strains was substantially relieved by mutations in uvsB(MEC1/rad3) or uvsD(+), suggesting that the presence of a functional DNA damage checkpoint inhibits septation in these bimA(APC3) strains. Our results demonstrate that mutations in bimA(APC3) lead to errors in DNA metabolism that indirectly block septation.

Alleles↗
PubMed2000

The Colletotrichum lagenarium MAP kinase gene CMK1 regulates diverse aspects of fungal pathogenesis.

The infection process of Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, involves several key steps: germination; formation of melanized appressoria; appressorial penetration; and subsequent invasive growth in host plants. Here we report that the C. lagenarium CMK1 gene encoding a mitogen-activated protein (MAP) kinase plays a central role in these infection steps. CMK1 can complement appressorium formation of the Pmk1 MAP kinase mutant of Magnaporthe grisea. Deletion of CMK1 causes reduction of conidiation and complete lack of pathogenicity to the host plant. Surprisingly, in contrast to M. grisea pmk1 mutants, conidia of cmk1 mutants fail to germinate on both host plant and glass surfaces, demonstrating that the CMK1 MAP kinase regulates conidial germination. However, addition of yeast extract rescues germination, indicating the presence of a CMK1-independent pathway for regulation of conidial germination. Germinating conidia of cmk1 mutants fail to form appressoria and the mutants are unable to grow invasively in the host plant. This strongly suggests that MAP kinase signaling pathways have general significance for infection structure formation and pathogenic growth in phytopathogenic fungi. Furthermore, three melanin genes show no or slight expression in the cmk1 mutant when conidia fail to germinate, suggesting that CMK1 plays a role in gene expression required for appressorial melanization.

Amino Acid Sequence↗
PubMed1999

Interaction between developmental and cell cycle regulators is required for morphogenesis in Aspergillus nidulans.

In Aspergillus nidulans, mutation of the transcriptional regulator brlA arrests formation of asexual spore-forming structures called conidiophores but does not hinder vegetative hyphal growth. During conidiophore development a 6-fold, brlA-dependent increase in the kinase activities of NIMX(cdc2) and NIMA occurs. A similar level of kinase induction was promoted by ectopic expression of brlA. Northern and Western analysis revealed marked induction of nimX(cdc2) mRNA after ectopic expression of brlA and increased amounts of NIMX(cdc2). Therefore, nimX(cdc2) is developmentally regulated by brlA indicating a direct role for brlA in the regulation of cell cycle genes. That correct regulation of nimX(cdc2) is important for normal development was further supported by analysis of conidiophore development and septation in cell cycle specific mutants. Most noticeably, the nimX(cdc2AF) mutation promoted inappropriate septation and hindered the switch from filamentous growth to budding growth seen during conidiophore development. Therefore, in contrast to the situation previously reported for other multicellular eukaryotes, interaction between developmental regulators and cell cycle regulators is essential for normal morphogenesis in A.nidulans.

Aspergillus nidulans↗
PubMed1999

An ATP-driven efflux pump is a novel pathogenicity factor in rice blast disease.

Cells tolerate exposure to cytotoxic compounds through the action of ATP-driven efflux pumps belonging to the ATP-binding cassette (ABC) superfamily of membrane transporters. Phytopathogenic fungi encounter toxic environments during plant invasion as a result of the plant defense response. Here we demonstrate the requirement for an ABC transporter during host infection by the fungal plant pathogen Magnaporthe grisea. The ABC1 gene was identified in an insertional mutagenesis screen for pathogenicity mutants. The ABC1 insertional mutant and a gene-replacement mutant arrest growth and die shortly after penetrating either rice or barley epidermal cells. The ABC1-encoded protein is similar to yeast ABC transporters implicated in multidrug resistance, and ABC1 gene transcripts are inducible by toxic drugs and a rice phytoalexin. However, abc1 mutants are not hypersensitive to antifungal compounds. The non-pathogenic, insertional mutation in ABC1 occurs in the promoter region and dramatically reduces transcript induction by metabolic poisons. These data strongly suggest that M.grisea requires the up-regulation of specific ABC transporters for pathogenesis; most likely to protect itself against plant defense mechanisms.

ATP Binding Cassette Transporter 1↗
PubMed1998

Inactivation of the mitogen-activated protein kinase Mps1 from the rice blast fungus prevents penetration of host cells but allows activation of plant defense responses.

The rice blast fungus, Magnaporthe grisea, generates enormous turgor pressure within a specialized cell called the appressorium to breach the surface of host plant cells. Here, we show that a mitogen-activated protein kinase, Mps1, is essential for appressorium penetration. Mps1 is 85% similar to yeast Slt2 mitogen-activated protein kinase and can rescue the thermosensitive growth of slt2 null mutants. The mps1-1Delta mutants of M. grisea have some phenotypes in common with slt2 mutants of yeast, including sensitivity to cell-wall-digesting enzymes, but display additional phenotypes, including reduced sporulation and fertility. Interestingly, mps1-1Delta mutants are completely nonpathogenic because of the inability of appressoria to penetrate plant cell surfaces, suggesting that penetration requires remodeling of the appressorium wall through an Mps1-dependent signaling pathway. Although mps1-1Delta mutants are unable to cause disease, they are able to trigger early plant-cell defense responses, including the accumulation of autofluorescent compounds and the rearrangement of the actin cytoskeleton. We conclude that MPS1 is essential for pathogen penetration; however, penetration is not required for induction of some plant defense responses.

Amino Acid Sequence↗
PubMed1998

Infection-related development in the rice blast fungus Magnaporthe grisea.

Recent developments have been made in the identification of signal transduction pathways and gene products involved in the infection-related development of the rice blast fungus, Magnaporthe grisea. It has been established that cAMP-dependent and MAP kinase-mediated signaling are both critical for appressorium morphogenesis and function. These signaling pathways may act downstream of hydrophobin-mediated surface sensing by the growing germ tube. Several genes have been identified that are required for invasive growth of M. grisea including genes that allow adaptation of fungal metabolism to growth within plant tissues.

Gene Expression Regulation, Fungal↗
PubMed1998

hyp loci control cell pattern formation in the vegetative mycelium of Aspergillus nidulans.

Aspergillus nidulans grows by apical extension of multinucleate cells called hyphae that are subdivided by the insertion of crosswalls called septa. Apical cells vary in length and number of nuclei, whereas subapical cells are typically 40 microm long with three to four nuclei. Apical cells have active mitotic cycles, whereas subapical cells are arrested for growth and mitosis until branch formation reinitiates tip growth and nuclear divisions. This multicellular growth pattern requires coordination between localized growth, nuclear division, and septation. We searched a temperature-sensitive mutant collection for strains with conditional defects in growth patterning and identified six mutants (designated hyp for hypercellular). The identified hyp mutations are nonlethal, recessive defects in five unlinked genes (hypA-hypE). Phenotypic analyses showed that these hyp mutants have aberrant patterns of septation and show defects in polarity establishment and tip growth, but they have normal nuclear division cycles and can complete the asexual growth cycle at restrictive temperature. Temperature shift analysis revealed that hypD and hypE play general roles in hyphal morphogenesis, since inactivation of these genes resulted in a general widening of apical and subapical cells. Interestingly, loss of hypA or hypB function lead to a cessation of apical cell growth but activated isotropic growth and mitosis in subapical cells. The inferred functions of hypA and hypB suggest a mechanism for coordinating apical growth, subapical cell arrest, and mitosis in A. nidulans.

Aspergillus nidulans↗
PubMed1998

Divergent cAMP signaling pathways regulate growth and pathogenesis in the rice blast fungus Magnaporthe grisea.

cAMP is involved in signaling appressorium formation in the rice blast fungus Magnaporthe grisea. However, null mutations in a protein kinase A (PKA) catalytic subunit gene, CPKA, do not block appressorium formation, and mutations in the adenylate cyclase gene have pleiotropic effects on growth, conidiation, sexual development, and appressorium formation. Thus, cAMP signaling plays roles in both growth and morphogenesis as well as in appressorium formation. To clarify cAMP signaling in M. grisea, we have identified strains in which a null mutation in the adenylate cyclase gene (MAC1) has an unstable phenotype such that the bypass suppressors of the Mac1(-) phenotype (sum) could be identified. sum mutations completely restore growth and sexual and asexual morphogenesis and lead to an ability to form appressoria under conditions inhibitory to the wild type. PKA assays and molecular cloning showed that one suppressor mutation (sum1-99) alters a conserved amino acid in cAMP binding domain A of the regulatory subunit gene of PKA (SUM1), whereas other suppressor mutations act independently of PKA activity. PKA assays demonstrated that the catalytic subunit gene, CPKA, encodes the only detectable PKA activity in M. grisea. Because CPKA is dispensable for growth, morphogenesis, and appressorium formation, divergent catalytic subunit genes must play roles in these processes. These results suggest a model in which both saprophytic and pathogenic growth of M. grisea is regulated by adenylate cyclase but different effectors of cAMP mediate downstream effects specific for either cell morphogenesis or pathogenesis.

Alleles↗
PubMed1997

The Aspergillus nidulans sepA gene encodes an FH1/2 protein involved in cytokinesis and the maintenance of cellular polarity.

Cytokinesis (septation) in the fungus Aspergillus nidulans occurs through the formation of a transient actin ring at the incipient division site. Temperature-sensitive mutations in the sepA gene prevent septation and cause defects in the maintenance of cellular polarity, without affecting growth and nuclear division. The sepA gene encodes a member of the growing family of FH1/2 proteins, which appear to have roles in morphogenesis and cytokinesis in organisms such as yeast and Drosophila. Results from temperature shift and immunofluorescence microscopy experiments strongly suggest that sepA function requires a preceding mitosis and that sepA acts prior to actin ring formation. Deletion mutants of sepA exhibit temperature-sensitive growth and severe delays in septation at the permissive temperature, indicating that expression of another gene may compensate for the loss of sepA. Conidiophores formed by sepA mutants exhibit abnormal branching of the stalk and vesicle. These results suggest that sepA interacts with the actin cytoskeleton to promote formation of the actin ring during cytokinesis and that sepA is also required for maintenance of cellular polarity during hyphal growth and asexual morphogenesis.

Alleles↗
PubMed1997

Relationship of actin, microtubules, and crosswall synthesis during septation in Aspergillus nidulans.

Studies of cytokinesis in animal cells demonstrate that microtubules play an important role in signaling the position of the actin-containing contractile ring and subsequent formation of the cleavage furrow. Septation in several fungi closely resembles animal cell cytokinesis in that a circumferential ring of actin is visible at the incipient division site. However, this does not necessarily mean that division is contractile since actin may also serve to localize septal wall synthesis. In addition, several studies in fission yeast have suggested that microtubules are dispensable for actin ring formation. We have used synchronized cells and fluorescence microscopy to follow actin structures, nuclear division and septal wall synthesis during septation in Aspergillus nidulans. Our data suggest that actin first appears at the septum site as a circumferential ring and that it later broadens and invaginates, forming an hourglass-shaped structure coincident with septal cell wall synthesis. Depolymerization of microtubules early in septation prevents circumferential actin ring formation. Depolymerization of microtubules after circumferential actin ring formation blocks both the progression to invaginating bands and septal wall synthesis. In contrast to studies in yeast cells, our data suggest that microtubules are required for both the initiation and progression of septation in A. nidulans.

Actins↗
PubMed1997

The Aspergillus nidulans septin encoding gene, aspB, is essential for growth.

Septins are a highly conserved family of presumed cytoskeletal proteins involved in cytokinesis and morphogenesis in Saccharomyces cerevisiae and Drosophila melanogaster. To investigate the role of septins in filamentous fungi, we have identified presumptive septin homologues in Aspergillus nidulans. One of these septins, aspB, is expressed during vegetative growth and asexual sporulation. Based on cDNA and genomic sequences, the predicted aspB protein shares a P-loop motif and coiled coil regions with septins from other organisms. Antibodies generated against an aspB fusion protein recognized an A. nidulans protein of approximately 50 kDa, but could not localize the septin product in cells by immunofluorescence. Hybridization to a chromosome-specific ordered cosmid library placed the aspB gene on the right arm of chromosome I. Disruption of aspB showed that it is an essential gene.

Amino Acid Sequence↗
PubMed1997

Linking approaches in the study of fungal pathogenesis: a commentary.

Thus far, it has not been possible to demonstrate that fungal pathogens possess specific extracellular enzymes with important roles in pathogenesis. In contrast, candidate gene knock-out experiments have revealed regulatory networks that appear to be specific to pathogenesis. The tracings of these networks may have already been described in the well-known cell differentiation pathways of many nonpathogens. The challenge for fungal pathologists will be to link these signaling pathways to the host environment and to the expression of determinants that advance the disease state.

Animals↗
PubMed1996

MAP kinase and cAMP signaling regulate infection structure formation and pathogenic growth in the rice blast fungus Magnaporthe grisea.

Many fungal pathogens invade plants using specialized infection structures called appressoria that differentiate from the tips of fungal hyphae contacting the plant surface. We demonstrate a role for a MAP kinase that is essential for appressorium formation and infectious growth in Magnaporthe grisea, the fungal pathogen responsible for rice blast disease. The PMK1 gene of M. grisea is homologous to the Saccharomyces cerevisiae MAP kinases FUS3/KSS1, and a GST-Pmk1 fusion protein has kinase activity in vitro. pmk1 mutants of M. grisea fail to form appressoria and fail to grow invasively in rice plants. pmk1 mutants are still responsive to cAMP for early stages of appressorium formation, which suggests Pmk1 acts downstream of a cAMP signal for infection structure formation. PMK1 is nonessential for vegetative growth and sexual and asexual reproduction in culture. Surprisingly, when expressed behind the GAL1 promoter in yeast, PMK1 can rescue the mating defect in a fus3 kss1 double mutant. These results demonstrate that PMK1 is part of a highly conserved MAP kinase signal transduction pathway that acts cooperatively with a cAMP signaling pathway for fungal pathogenesis.

Amino Acid Sequence↗
PubMed1996

Genetic differentiation in the rice blast fungus revealed by the distribution of the Fosbury retrotransposon.

Repetitive DNA sequences genetically differentiate certain host-specific forms of the plant pathogenic fungus Pyricularia grisea. For example, high copy numbers of a sequence designated MGR586 are conserved in isolates that infect rice. In this report, we describe the molecular characterization of another repetitive DNA sequence designated fosbury. Restriction mapping and DNA sequence analysis show that fosbury is a member of a long terminalrepeat (LTR)-containing retrotransposon family, and gel blot hybridization analysis suggests that, like MGR586, fosbury is preferentially found in isolates that infect rice. This supports the view that rice pathogens comprise a genetically distinct form of P. grisea. We also investigated the distribution of fosbury and MGR586 and found that these elements are sometimes associated in the genome, and gel blot hybridization analysis shows that restriction sites flanking both of these elements resolve rice pathogens into similar clonal lineage groups. We conclude that the transposition of repetitive DNA sequences plays an important role in generating DNA fingerprint variation in the rice blast fungus.

Ascomycota↗