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.

Impact of temperature and species interaction on filamentous cyanobacteria may be more important than salinity and increased pCO2 levels

A future business-as-usual scenario (A1FI) was tested on two bloom-forming cyanobacteria of the Baltic Proper, Nodularia spumigena and Aphanizomenon sp., growing separately and together. The projected scenario was tested in two laboratory experiments where (a) interactive effects of increased temperature and decreased salinity and (b) interactive effects of increased temperature and elevated levels of pCO2 were tested. Increased temperature, from 12 to 16 °C, had a positive effect on the biovolume and photosynthetic activity (F v/F m) of both species. Compared when growing separately, the biovolume of each species was lower when grown together. Decreased salinity, from 7 to 4, and elevated levels of pCO2, from 380 to 960 ppm, had no effect on the biovolume, but on F v/F m of N. spumigena with higher F v/F m in salinity 7. Our results suggest that the projected A1FI scenario might be beneficial for the two species dominating the extensive summer blooms in the Baltic Proper. However, our results further stress the importance of studying interactions between species.

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

Evidence for balancing selection operating at the het-c heterokaryon incompatibility locus in a group of filamentous fungi

In filamentous fungi, het loci (for heterokaryon incompatibility) are believed to regulate self/nonself-recognition during vegetative growth. As filamentous fungi grow, hyphal fusion occurs within an individual colony to form a network. Hyphal fusion can occur also between different individuals to form a heterokaryon, in which genetically distinct nuclei occupy a common cytoplasm. However, heterokaryotic cells are viable only if the individuals involved have identical alleles at all het loci. One het locus, het-c, has been characterized at the molecular level in Neurospora crassa and encodes a glycine-rich protein. In an effort to understand the role of this locus in filamentous fungi, we chose to study its evolution by analyzing het-c sequence variability in species within Neurospora and related genera. We determined that the het-c locus was polymorphic in a field population of N. crassa with close to equal frequency of each of the three allelic types. Different species and even genera within the Sordariaceae shared het-c polymorphisms, indicating that these polymorphisms originated in an ancestral species. Finally, an analysis of the het-c specificity region shows a high occurrence of nonsynonymous substitution. The persistence of allelic lineages, the nearly equal allelic distribution within populations, and the high frequency of nonsynonymous substitutions in the het-c specificity region suggest that balancing selection has operated to maintain allelic diversity at het-c. Het-c shares this particular evolutionary characteristic of departing from neutrality with other self/nonself-recognition systems such as major histocompatibility complex loci in mammals and the S (self-incompatibility) locus in angiosperms.

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.

A mitogen-activated protein kinase of the corn leaf pathogen Cochliobolus heterostrophus is involved in conidiation, appressorium formation, and pathogenicity: Diverse roles for mitogen-activated protein kinase homologs in foliar pathogens

Fungal pathogens perceive and respond to molecules from the plant, triggering pathogenic development. Transduction of these signals may use heterotrimeric G proteins, and it is thought that protein phosphorylation cascades are also important. We have isolated a mitogen-activated protein kinase homolog from the corn pathogen Cochliobolus heterostrophus to test its role as a component of the transduction pathways. The new gene, CHK1, has a deduced amino acid sequence 90% identical to Pmk1 of the rice blast fungus Magnaporthe grisea and 59% identical to Fus3 of Saccharomyces cerevisiae. A series of chk1 deletion mutants has poorly developed aerial hyphae, autolysis, and no conidia. No pseudothecia are formed when a cross between two Δchk1 mutants is attempted. The ability of Δchk1 mutants to infect corn plants is reduced severely. The growth pattern of hyphae on a glass surface is strikingly altered from that of the wild type, forming coils or loops, but no appressoria. This set of phenotypes overlaps only partially with that of pmk1 mutants, the homologous gene of the rice blast fungus. In particular, sexual and asexual sporulation both require Chk1 function in Cochliobolus heterostrophus, in contrast to Pmk1, but perhaps more similar to yeast, where Fus3 transmits the mating signal. Chk1 is required for efficient colonization of leaf tissue, which can be compared with filamentous invasive growth of yeast, modulated through another closely related mitogen-activated protein kinase, Kss1. Ubiquitous signaling elements thus are used in diverse ways in different plant pathogens, perhaps the result of coevolution of the transducers and their targets.

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.