Functional analysis of GNA -1, a Galphai superfamily member found in the filamentous fungus Neurospora crassa

Heterotrimeric GTP-binding proteins, G proteins, are integral components of eukaryotic signaling systems linking extracellular signals to intracellular responses. Through coupling to seven-transmembrane helix receptors, G proteins convey primary signaling events into multi-leveled cascades of intracellular activity by regulating downstream enzymes, collectively called effectors. The effector enzymes regulated by G proteins include adenylyl cyclase, cAMP phosphodiesterase, phospolipase C-β, mitogen-activated protein kinases, and ion channels. ^ Neurospora crassa is a multicellular, filamentous fungus that is capable of both asexual and sexual reproduction by elaboration of specialized, developmentally controlled structures that give rise to either asexual or sexual spores, respectively. N. crassa possesses at least three heterotrimeric Gα proteins (GNA-1–3) and one Gβ subunit (GNB-1). GNA-1 was the first microbial protein that could be classified in the Gαi superfamily based on its amino acid identity and demonstration that it is a substrate for ADP-ribosylation by pertussis toxin. ^ Experiments were designed to identify the signal transduction pathways and the effector enzymes regulated by GNA-1. Targeted gene-replacement of gna-1 revealed that GNA-1 controls multiple developmental pathways including both asexual and sexual reproduction, maintenance of growth, and resistance to osmotic stress. The Gαi and Gαz members of the Gαi superfamily negatively regulate adenylyl cyclase activity in mammalian cells; therefore, adenylyl cyclase and cAMP levels were measured in Δgna-1 strains and also in strains that were deleted for both gna-1 and gna-2, a second Gα in N. crassa shown to have overlapping functions with GNA-1. Direct measurements of adenylyl cyclase activity revealed that GNA-1, but not GNA-2, was responsible for GTP-stimulated adenylyl cyclase activity in N. crassa. Furthermore, anti-GNA-1 IgG could specifically inhibit GTP-stimulated adenylyl cyclase activity in wild-type strain extracts. These studies also provided evidence that N. crassa possesses feedback mechanisms that control steady-state cAMP levels through indirect regulation of cAMP-phosphodiesterase activity; mutations in gna-1 and gna-2 were additive in their effect on lowering cAMP-phosphodiesterase activity under growth conditions where steady-state cAMP levels were normal but GTP-stimulated adenylyl cyclase activity was reduced 90% in comparison to control strains. ^ Genetic and biochemical epistasis experiments utilizing a Δ gna-1 cr-1 mutant suggest that GNA-1 is essential for female fertility in a cAMP-independent pathway. Furthermore, deletion of gna-1 in a cr-1 background exacerbated many of the defects already observed in the cr-1 strain including more severe growth restriction and developmental defects. However, deletion of gna-1 had no effect on the increased thermotolerance of cr-1, which has been attributed to loss of cAMP. cr-1 possesses GNA-1 protein, and crude membrane fractions from this strain reconstituted GTP-stimulated adenylyl cyclase activity in Δgna-1 membrane fractions. These studies provide direct evidence for the involvement of Gα proteins in the regulation of adenylyl cyclase activity in eukaryotic microbes. ^

Characterization of the NOP -1 opsin photoreceptor in the filamentous fungus Neurospora crassa

Light absorption is an important process for energy production and sensory perception in many organisms. In the filamentous fungus, Neurospora crassa, blue-light is an important regulator of both asexual and sexual development, but the identity of the blue-light receptor is unknown. The work presented in this dissertation initiated the characterization of the putative N. crassa opsin photoreceptor, NOP-1. Opsins were thought to exist only in the archaea and mammals until the discovery of nop-1. All opsins have the same conserved structure of seven transmembrane helical domains with a lysine residue in the seventh helix specific for forming a Schiff-base linkage with retinal. The predicted NOP-1 protein sequence is equally similar to archaeal rhodopsins and a newly identified fungal opsin-related protein group (ORPs). ORPs maintain the seven transmembrane helical structure of opsins, but lack the conserved lysine residue for binding retinal. An ORP gene, orp-1 was identified in N. crassa and this work includes the cloning and sequence analysis of this gene. Characterization of NOP-1 function in N. crassa development began with the construction of a Δnop-1 deletion mutant. Extensive phenotypic analysis of Δnop-1 mutants revealed only subtle defects during development primarily under environmental conditions that induce a stress response. NOP-1 was overexpressed in the heterologous system Pichia pastoris, and it was demonstrated that NOP-1 protein bound all-trans retinal to form a green-light absorbing pigment (λmax = 534 nm) with a photochemical reaction cycle similar to archaeal sensory rhodopsins. nop-1 gene expression was monitored during N. crassa development. nop-1 transcript is highly expressed during asexual sporulation (conidiation) and transcript levels are abundant in the later stages of conidial development. nop-1 expression is not regulated by blue-light or elevated temperatures. Potential functions for NOP-1 were discovered through the transcriptional analysis of conidiation-associated genes in Δnop-1 mutants. NOP-1 exhibits antagonistic transcriptional regulation of conidiation-associated genes late in conidial development, by enhancing the carotenogenic gene, al-2 and repressing the conidiation-specific genes, con-10 and con-13. ^

Effects of Seed Treatments and a Soil-applied Nematicide on Corn Yields and Nematode Population Densities

Plant-parasitic nematodes are microscopic worms that feed on plants. Almost every nematode that feeds on corn is capable of feeding on many other plants. These nematode parasites are thought to be native to most Iowa soils and to have fed upon native plants before corn was grown as a cultivated crop. Population densities (numbers) of most species of plant-parasitic nematodes that feed on corn have to increase to damaging levels (called damage thresholds) before yield loss occurs.

Evaluation of Soybean Varieties Resistant to Soybean Cyst Nematode

Use of resistant soybean varieties is a very effective strategy for managing soybean cyst nematode (SCN), and numerous SCN-resistant soybean varieties are available for Iowa soybean growers. Each year, public and private SCN-resistant soybean varieties are evaluated in SCN-infested fields in Iowa by Iowa State University personnel. The research described in this report was performed to assess the agronomic performance of SCNresistant soybean varieties and to determine the effects of the varieties on SCN numbers or population densities.

Effects of Seed Treatments and a Soil-applied Nematicide on Corn Yields and Nematode Population Densities

Plant-parasitic nematodes are microscopic worms that feed on plants. Almost every nematode that feeds on corn is capable of feeding on many other plants. These nematode parasites are thought to be native to most Iowa soils and to have fed upon native plants before corn was grown as a cultivated crop. Population densities (numbers) of most species of plant-parasitic nematodes that feed on corn have to increase to damaging levels (called damage thresholds) before yield loss occurs.

Effects of Seed Treatments and a Soil-applied Nematicide on Corn Yields and Nematode Population Densities

Plant-parasitic nematodes are microscopic worms that feed on plants. Almost every nematode that feeds on corn is capable of feeding on many other plants. These nematode parasites are thought to be native to most Iowa soils and to have fed upon native plants before corn was grown as a cultivated crop. Population densities (numbers) of most species of plant-parasitic nematodes that feed on corn have to increase to damaging levels (called damage thresholds) before yield loss occurs. Products that are currently available to manage plant-parasitic nematodes on corn in the state include the soil-applied insecticide/nematicide Counter® and two relatively new protectant seed treatments, Avicta® and Votivo®. Counter® is a contact and systematic nematicide with the active ingredient terbufos. Avicta® is a contact nematicide (active ingredient abamectin) that moves on the surface of the root, and Votivo® is a special strain of the natural soil bacterium Bacillus firmus that grows on the root. Counter® is available from AMVAC, Avicta® from Syngenta Seedcare, and Votivo® from Bayer CropScience. The objective of this experiment was to assess and compare the nematode population densities and yields of corn growing in plots with and without the seed-treatment nematode protectants and the soil-applied nematicide Counter®.

Effects of Seed Treatments and a Soil-applied Nematicide on Corn Yields and Nematode Population Densities

Plant-parasitic nematodes are microscopic worms that feed on plants. Almost every nematode that feeds on corn is capable of feeding on many other plants. These nematode parasites are thought to be native to most Iowa soils and to have fed on native plants before corn was grown as a cultivated crop. Population densities (numbers) of most species of plant-parasitic nematodes that feed on corn have to increase to damaging levels (called damage thresholds) before yield loss occurs.

Meiofauna densities, nematode biomass, nematode genus diversity, sediment grain size, organic matter, and pigments for the surface layers of two sites in summer (November 2009) and winter (august 2010)

The meiobenthic community of Potter Cove (King George Island, west Antarctic Peninsula) was investigated, focusing on responses to summer/winter conditions in two study sites contrasting in terms of organic matter inputs. Meiofaunal densities were found to be higher in summer and lower in winter, although this result was not significantly related to the in situ availability of organic matter in each season. The combination of food quality and competition for food amongst higher trophic levels may have played a role in determining the standing stocks at the two sites. Meiobenthic winter abundances were sufficiently high to infer that energy sources were not limiting during winter, supporting observations from other studies for both shallow water and continental shelf Antarctic ecosystems. Recruitment within meiofaunal communities was coupled to the seasonal input of fresh detritus for harpacticoid copepods but not for nematodes, suggesting that species-specific life history or trophic features form an important element of the responses observed.

NMR spectrum of lindgomycin from a Marine Fungus of the Lindgomycetaceae

An unusual polyketide with a new carbon skeleton, lindgomycin (1), and the recently described ascosetin (2) were extracted from mycelia and culture broth of different Lindgomycetaceae strains, which were isolated from a sponge of the Kiel Fjord in the Baltic Sea (Germany) and from the Antarctic. Their structures were established by spectroscopic means. In the new polyketide, two distinct domains, a bicyclic hydrocarbon and a tetramic acid, are connected by a bridging carbonyl. The tetramic acid substructure of compound 1 was proved to possess a unique 5-benzylpyrrolidine-2,4-dione unit. The combination of 5-benzylpyrrolidine-2,4-dione of compound 1 in its tetramic acid half and 3-methylbut-3-enoic acid pendant in its decalin half allow the assignment of a new carbon skeleton. The new compound 1 and ascosetin showed antibiotic activities with IC50 value of 5.1 (±0.2) µM and 3.2 (±0.4) µM, respectively, against methicillin-resistant Staphylococcus aureus.

Experiment on marine fungus Microascus brevicaulis strain

The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularide A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing besides higher production levels faster growth and differences in pellet formation. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of this fungus and its mutant. For this purpose, an optimised protein extraction protocol was established. Here, we show the first proteome study of a marine fungus. In total, 4759 proteins were identified. The central metabolic pathway of LF580 could be mapped by using KEGG pathway analysis and GO annotation. Using iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to a limited nutrient availability in wild type strain due to a strong pellet formation. This information can be applied to optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum.

Nematode abundance in cold seep sediments of the Darwin mud volcano

During the JC-10 cruise (2007), we sampled the Darwin mud volcano (MV) for meiofaunal community and trophic structure in relation of pore-water geochemistry along a 10 m transect from a seep site on the rim of the crater towards the MV slope. Sediment samples were retrieved by the ROV Isis using push cores. On board and after the pore water extraction, the top 10 cm of the cores were sliced into 1 cm sections and fixed them in 4% formaldehyde for meiofaunal community analysis. In the home laboratory, the formaldehyde-fixed samples were washed over a 32 µm mesh sieve and extracted the meiofauna from the sediment by Ludox centrifugation (Heip et al. 1985). Meiofauna was then sorted, enumerated and identified at coarse taxonomic level. From each slice, ca. 100 nematodes were identified to genus level. Afterwards, abundance of Nematoda were depth integrated over the top 5 cm to gain individual abundances per 10 cm**2. Overall, total nematode biomass in the top 5 cm of the seep sediment core was ~10x higher than that in the core taken 1100 m away. Nematode genus composition varied little among cores and was mainly dominated by Sabatieria.