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. ^

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.

The interactive effects of temperature and osmotic potential on the growth of marines isolates of Fusarium solani

The mycelial growth of 18 Fusarium solani strains isolated from sea beds of the south-eastern coast of Spain was tested on potato-dextrose agar adjusted to different osmotic potentials with either KCl or NACl (-1.50 to -144.54 bars) in 10ºC intervals ranging from 15 to 35ºC. Fungal growth was determined by measuring colony diameter after 4 days incubation. Mycelial growth was maximal at 25ºC. The quantity and frequency pattern of mycelial growth of F. solani differ significantly at 15 and 25ºC, with maximal occurring at the highest water potential tested (-1.50 bars); and at 35ºC, with a maximal mycelial growth at -13.79 bars. The effect of water potential was independent of salt composition. The general growth pattern of F. solani showed declining growth at potentials below -41.79 bars. Fungal growth at 35ºC was always higher than that growth at 15ºC, of all the water potentials tested. Significant differences observed in the response of mycelia to water potential and temperature as main and interactive effects. The viability of cultures was increasingly inhibited as the water potential dropped, but some growth was still observed at -99.56 bars. These findings could indicate that marine strains of F. solani have a physiological mechanism that permits survival in environments with low water potential. The observed differences in viability and the magnitude growth could indicate that the biological factors governing potential and actual growth are affected by osmotic potential in different ways.

Disruption of abscisic acid signaling constitutively activates Arabidopsis resistance to the necrotrophic fungus Plectosphaerella cucumerina.

Plant resistance to necrotrophic fungi is regulated by a complex set of signaling pathways that includes those mediated by the hormones salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and abscisic acid (ABA). The role of ABA in plant resistance remains controversial, as positive and negative regulatory functions have been described depending on the plant-pathogen interaction analyzed. Here, we show that ABA signaling negatively regulates Arabidopsis (Arabidopsis thaliana) resistance to the necrotrophic fungus Plectosphaerella cucumerina. Arabidopsis plants impaired in ABA biosynthesis, such as the aba1-6 mutant, or in ABA signaling, like the quadruple pyr/pyl mutant (pyr1pyl1pyl2pyl4), were more resistant to P. cucumerina than wild-type plants. In contrast, the hab1-1abi1-2abi2-2 mutant impaired in three phosphatases that negatively regulate ABA signaling displayed an enhanced susceptibility phenotype to this fungus. Comparative transcriptomic analyses of aba1-6 and wild-type plants revealed that the ABA pathway negatively regulates defense genes, many of which are controlled by the SA, JA, or ET pathway. In line with these data, we found that aba1-6 resistance to P. cucumerina was partially compromised when the SA, JA, or ET pathway was disrupted in this mutant. Additionally, in the aba1-6 plants, some genes encoding cell wall-related proteins were misregulated. Fourier transform infrared spectroscopy and biochemical analyses of cell walls from aba1-6 and wild-type plants revealed significant differences in their Fourier transform infrared spectratypes and uronic acid and cellulose contents. All these data suggest that ABA signaling has a complex function in Arabidopsis basal resistance, negatively regulating SA/JA/ET-mediated resistance to necrotrophic fungi.

Functional genomics tools to decipher the pathogenicity mechanisms of the necrotrophic fungus Plectosphaerella cucumerina in Arabidopsis thaliana

The analysis of the interaction between Arabidopsis thaliana and adapted (PcBMM) and nonadapted (Pc2127) isolates of the necrotrophic fungus Plectosphaerella cucumerina has contributed to the identification of molecular mechanisms controlling plant resistance to necrotrophs.To characterize the pathogenicity bases of the virulence of necrotrophic fungi in Arabidopsis, we developed P. cucumerina functional genomics tools using Agrobacterium tumefaciens-mediated transformation.We generated PcBMM-GFP and Pc2127-GFP transformants constitutively expressing the green fluorescence protein (GFP), and a collection of random T-DNA insertional PcBMM transformants. Confocal microscopy analyses of the initial stages of PcBMM-GFP infection revealed that this pathogen, like other necrotrophic fungi, does not form an appressorium or penetrate into plant cells, but causes successive degradation of leaf cell layers

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 mps1–1Δ 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, mps1–1Δ 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 mps1–1Δ 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.

A rhodopsin-guanylyl cyclase gene fusion functions in visual perception in a fungus

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

The protein product of the het-s heterokaryon incompatibility gene of the fungus Podospora anserina behaves as a prion analog

The het-s locus of Podospora anserina is a heterokaryon incompatibility locus. The coexpression of the antagonistic het-s and het-S alleles triggers a lethal reaction that prevents the formation of viable heterokaryons. Strains that contain the het-s allele can display two different phenotypes, [Het-s] or [Het-s*], according to their reactivity in incompatibility. The detection in these phenotypically distinct strains of a protein expressed from the het-s gene indicates that the difference in reactivity depends on a posttranslational difference between two forms of the polypeptide encoded by the het-s gene. This posttranslational modification does not affect the electrophoretic mobility of the protein in SDS/PAGE. Several results suggest a similarity of behavior between the protein encoded by the het-s gene and prions. The [Het-s] character can propagate in [Het-s*] strains as an infectious agent, producing a [Het-s*] → [Het-s] transition, independently of protein synthesis. Expression of the [Het-s] character requires a functional het-s gene. The protein present in [Het-s] strains is more resistant to proteinase K than that present in [Het-s*] mycelium. Furthermore, overexpression of the het-s gene increases the frequency of the transition from [Het-s*] to [Het-s]. We propose that this transition is the consequence of a self-propagating conformational modification of the protein mediated by the formation of complexes between the two different forms of the polypeptide.

Isolation of human and mouse genes based on homology to REC2, a recombinational repair gene from the fungus Ustilago maydis

A human and a mouse gene have been isolated based on homology to a recombinational repair gene from the corn smut Ustilago maydis. The new human (h) gene, termed hREC2, bears striking resemblance to several others, including hRAD51 and hLIM15. hREC2 is located on human chromosome 14 at q23–24. The overall amino acid sequence reveals characteristic elements of a RECA-like gene yet harbors an src-like phosphorylation site curiously absent from hRAD51 and hLIM15. Unlike these two relatives, hREC2 is expressed in a wide range of tissues including lung, liver, placenta, pancreas, leukocytes, colon, small intestine, brain, and heart, as well as thymus, prostate, spleen, and uterus. Of greatest interest is that hREC2 is undetectable by reverse transcription-coupled PCR in tissue culture unless the cells are treated by ionizing radiation.

Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans

The CST20 gene of Candida albicans was cloned by functional complementation of a deletion of the STE20 gene in Saccharomyces cerevisiae. CST20 encodes a homolog of the Ste20p/p65PAK family of protein kinases. Colonies of C. albicans cells deleted for CST20 revealed defects in the lateral formation of mycelia on synthetic solid “Spider” media. However, hyphal development was not impaired in some other media. A similar phenotype was caused by deletion of HST7, encoding a functional homolog of the S. cerevisiae Ste7p protein kinase. Overexpression of HST7 partially complemented the deletion of CST20. Cells deleted for CST20 were less virulent in a mouse model for systemic candidiasis. Our results suggest that more than one signaling pathway can trigger hyphal development in C. albicans, one of which has a protein kinase cascade that is analogous to the mating response pathway in S. cerevisiae and might have become adapted to the control of mycelial formation in asexual C. albicans.

Phenotypic switching in the human pathogenic fungus Cryptococcus neoformans is associated with changes in virulence and pulmonary inflammatory response in rodents

High-frequency reversible changes in colony morphology were observed in three strains of Cryptococcus neoformans. For one strain (SB4, serotype A), this process produced three colony types: smooth (S), wrinkled (W), and serrated (C). The frequency of switching between colony types varied for the individual colony transitions and was as high as 10−3. Mice infected with colony type W died faster than those infected with other colony types. The rat inflammatory response to infection with colony types S, W, and C was C > S > W and ranged from intense granulomatous inflammation with caseous necrosis for infection with type C to minimal inflammation for infection with type W. Infection with the various colony types was associated with different antibody responses to cryptococcal proteins in rats. Analysis of cellular characteristics revealed differences between the three colony types. High-frequency changes in colony morphology were also observed in two additional strains of C. neoformans. For one strain (24067A, serotype D) the switching occurred between smooth and wrinkled colonies. For the other strain (J32A, serotype A), the switching occurred between mucoid and nonmucoid colonies. The findings indicate that C. neoformans undergoes phenotypic switching and that this process can affect virulence and host inflammatory and immune responses. Phenotypic switching may play a role in the ability of this fungus to escape host defenses and establish chronic infections.

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.