Patterns of functional enzyme activity in fungus farming ambrosia beetles

Introduction In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae), wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. Results We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Conclusion Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose components of the ray-parenchyma cells in the wood xylem. Furthermore, the detection of xylanolytic enzymes exclusively in larvae (which feed on fungus colonized wood) and not in adults (which feed only on fungi) indicates that only larvae (pre-) digest plant cell wall structures. This implies that in X. saxesenii and likely also in many other ambrosia beetles, adults and larvae do not compete for the same food within their nests - in contrast, larvae increase colony fitness by facilitating enzymatic wood degradation and fungus cultivation.

Accumulation of the pathogenic fungus Batrachochytrium dendrobatidis on the regressing tail of midwife toads Alytes obstetricans undergoing metamorphosis

Batrachochytrium dendrobatidis (Bd) is a fungus infecting the skin of amphibians. On metamorphosing animals the infection is difficult to detect because of the limited information concerning the location of Bd on the animals during this stage. Histological investigation revealed that Bd accumulated on the reabsorbing tail of metamorphosing animals. This observation may facilitate the detection of Bd in metamorphosing amphibians.

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. ^

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.

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