Protoplast Fusion of Trichoderma reesei, Using Immature Conidia

Protoplast fusion of strains derived from Trichoderma reesei QM9414 and QM9136 and the segregation of the resulting fusants were studied. Combinations of protoplasts prepared from young conidia with double amino acid requirements, one of which was a common requirement and the other uncommon, were fused in the presence of polyethylene glycol 6000. Fusants were selected as regenerant colonies requiring only the commonly deficient amino acid. The frequency of fusion was 0.9 × 10⁻⁴ to 4.0 × 10⁻⁴ for the starting conidia and 3.0 × 10⁻² to 4.9 × 10⁻² for the regenerated protoplasts, which was significantly higher than the expected reversion frequencies by mutation. Conidia generated on the fusant colonies showed diverse phenotypes, i.e., parental types (40 to 80%) and nonparental types (20 to 60%). Colonies developed from single conidia of the nonparental phenotype contained special spots called “knobs” that have a higher density of mycelia. The phenotype of the knobs was again varied among prototrophs, parental types, and recombinant types; and their traits were inherited stably. The phenotype of the mycelia in the nonknob part was essentially the same as that of the original conidia and again formed knobs in colonies upon transfer of a piece of mycelia to a fresh medium. The conidial DNA content of the knob clone was almost the same as that of the parents, but that of the fusants was 1.2 to 2.0 times higher than that of the parents. From these results, we conclude that knobs are the segregants from the fusants. One knob clone showed twice the carboxymethyl cellulose hydrolyzing activity of the parents, suggesting the possibility of breeding T. reesei cells by the protoplast fusion technique.     

Comparison of peptidase activities in some fungi pathogenic to arthropods

Peptidases, highly specific toward several synthetic chromogenic peptides, were found in the mycelia of four arthropod pathogenic fungi: Aphanomyces astaci, Beauveria bassiana, Metarrhizium anisopliae, and Paecilomyces farinosus. A. astaci peptidases had high hydrolyzing activities toward most of the peptides, especially those with arginine in the P1 position, while those of B. bassiana and P. farinosus readily hydrolyzed peptides with valine and arginine, as well as proline and tyrosine in the P2 and P1 positions, respectively. The hydrolyzing capacities of M. anisopliae peptidases were similar to A. astaci, but showed lower specific activities. Casein or azocoll was only hydrolyzed by A. astaci peptidases. B. bassiana and M. anisopliae had a very low hydrolyzing capacity toward casein and could not degrade azocoll. P. farinosus had no hydrolyzing activity toward casein or azocoll. Only peptidases from the crayfish pathogen A. astaci could degrade the crayfish cuticle. The peptidase preparations of A. astaci and B. bassiana hydrolyzing MeO-Suc-Arg-Pro-Tyr-pNA or Bz-Phe-Val-Arg-pNA were of the serine type. The possible importance of peptidase activity of arthropod pathogenic fungi in the infection process is discussed.     

The Density of Knobs on Plasmodium falciparum-Infected Erythrocytes Depends on Developmental Age and Varies among Isolates

Background

The virulence of Plasmodium falciparum malaria is related to the parasite’s ability to evade host immunity through clonal antigenic variation and tissue-specific adhesion of infected erythrocytes (IEs). The P. falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on dome-shaped protrusions called knobs on the IE surface is central to both. Differences in receptor specificity and affinity of expressed PfEMP1 are important for IE adhesiveness, but it is not known whether differences in the number and size of the knobs on which the PfEMP1 proteins are expressed also play a role. Therefore, the aim of this study was to provide detailed information on isolate- and time-dependent differences in knob size and density.

Methodology/Principal Findings

We used atomic force microscopy to characterize knobs on the surface of P. falciparum-infected erythrocytes. Fourteen ex vivo isolates from Ghanaian children with malaria and 10 P. falciparum isolates selected in vitro for expression of a particular PfEMP1 protein (VAR2CSA) were examined. Knob density increased from ∼20 h to ∼35 h post-invasion, with significant variation among isolates. The knob density ex vivo, which was about five-fold higher than following long-term in vitro culture, started to decline within a few months of culture. Although knob diameter and height varied among isolates, we did not observe significant time-dependent variation in these dimensions.

Conclusions/Significance

The density of knobs on the P. falciparum-IE surface depends on time since invasion, but is also determined by the infecting isolate in a time-independent manner. This is the first study to quantitatively evaluate knob densities and dimensions on different P. falciparum isolates, to examine ex vivo isolates from humans, and to compare ex vivo and long-term in vitro-cultured isolates. Our findings contribute to the understanding of the interaction between P. falciparum parasites and the infected host.     

iTRAQ-based quantitative proteomic analysis of conidia and mycelium in the filamentous fungus Metarhizium robertsii

Metarhizium robertsii is widely applied in biological control via conidia application. To clarify the proteomic differences between conidia and mycelia and explore the underlying mechanisms of conidia as a unit responsible for dispersal and environmental stress, we carried out an iTRAQ (isobaric tags for relative and absolute quantitation)-based quantitative proteomic analysis for two developmental stages from M. robertsii. A total of 2052 proteins were detected, and 90 showed differential protein abundance between the conidia and mycelia. These 90 proteins were primarily associated with stress resistance, amino acid and protein metabolism, and energy metabolism. Further bioinformatics analysis showed that these proteins could be mapped to 52 pathways, five of which were significantly enriched after mapping to KEGG pathways. Interestingly, many proteins involved in the significantly enriched pathway of peroxisome, biosynthesis of secondary metabolites and glyoxylate and dicarboxylate metabolism, including catalase, peroxisomal membrane anchor protein, formate dehydrogenase and isocitrate lyase, were identified with higher abundance in conidia. The results deepened our understanding of the conidia proteome in M. robertsii and provide a basis for further exploration for improving the efficiency of the fungi as biocontrol agents.     

Plasmodium falciparum: loss of knobs on the infected erythrocyte surface after long-term cultivation.

After long-term in vitro cultivation in human erythrocytes, variants of three strains of the malaria parasite Plasmodium falciparum no longer produce the “knob” alterations on the host erythrocyte surface. The time in continuous culture before knobs failed to appear ranged from 18 months for the Gambian strain FCR-4 to 33 months for the Vietnamese strain FCR-1. The loss of knobs is correlated with the inability to concentrate trophozoites, schizonts, and segmenters from these variant lines by the use of gelatin-containing media. This is the first report of a change in Plasmodium falciparum or its host cell as a consequence of long-term culture.     

Estimating Impact Forces of Tail Club Strikes by Ankylosaurid Dinosaurs

Background

It has been assumed that the unusual tail club of ankylosaurid dinosaurs was used actively as a weapon, but the biological feasibility of this behaviour has not been examined in detail. Ankylosaurid tail clubs are composed of interlocking vertebrae, which form the handle, and large terminal osteoderms, which form the knob.

Methodology/Principal Findings

Computed tomographic (CT) scans of several ankylosaurid tail clubs referred to Dyoplosaurus and Euoplocephalus, combined with measurements of free caudal vertebrae, provide information used to estimate the impact force of tail clubs of various sizes. Ankylosaurid tails are modeled as a series of segments for which mass, muscle cross-sectional area, torque, and angular acceleration are calculated. Free caudal vertebrae segments had limited vertical flexibility, but the tail could have swung through approximately 100° laterally. Muscle scars on the pelvis record the presence of a large M. longissimus caudae, and ossified tendons alongside the handle represent M. spinalis. CT scans showed that knob osteoderms were predominantly cancellous, which would have lowered the rotational inertia of the tail club and made it easier to wield as a weapon.

Conclusions/Significance

Large knobs could generate sufficient force to break bone during impacts, but average and small knobs could not. Tail swinging behaviour is feasible in ankylosaurids, but it remains unknown whether the tail was used for interspecific defense, intraspecific combat, or both.     

Assessment of germination and carnivorous activities of a nematode-trapping fungus Arthrobotrys dactyloides in fungistatic and fungicidal soil environment

Carnivorism is the ability of nematode-trapping fungi to trap and digest the nematodes by sophisticated devices called traps. Delivery of nematode-trapping fungi in soil for bio-control of pest nematodes often fails or gives inconsistent results. Possible reasons for failure could be the effect of soil fungistasis on germination of nematode-trapping fungi in soil environment, use of avirulent species and sensitivity of these fungi to fungicidal residues in soil. Exploitation of nematode-trapping fungi for nematode control demands that it be compatible with fungicides applied in soil or crops and proliferate in soil. This investigation represents is one of the first to evaluate the effect of fungicides on the nematode-trapping fungus Arthrobotrys dactyloides. A. dactyloides showed in vitro carnivorous potential against Meloidogyne incognita, Meloidogyne javanica, Meloidogyne graminicola, Helicotylenchus dihystera and Heterodera cajani. Conidia of A. dactyloides exposed to agricultural soils showed poor germination but formed conidial traps, which captured and killed the soil nematodes. Conidial traps, which trapped the nematodes, grew well in all soils after killing and nutrient absorption from nematode body. Soil amended with 20 mg ai kg⁻¹ of carbendazim and thiram, 30 mg ai kg⁻¹ of mancozeb, 50 mg ai kg⁻¹ of captan, and 100 mg ai kg⁻¹ of carboxin completely checked the conidial trap formation and nematode capturing. 30, 50 and 100 mg ai kg⁻¹ of metalaxyl adversely affected the conidial trap formation and nematode capturing in soil. Propiconazole inhibited 15.2% conidial trap formation up to 50 mg ai kg⁻¹ but caused 93.3% inhibition of conidial traps formation and complete inhibition of nematode capturing at 100 mg ai kg⁻¹. Sulphur, triademefon, and tricyclazole showed least toxic effect on conidial trap formation and nematode capturing activities of A. dactyloides in soil up to 100 mg ai kg⁻¹.     

Feeding on fungi: genomic and proteomic analysis of the enzymatic machinery of bacteria decomposing fungal biomass

Dead fungal biomass is an abundant source of nutrition in both litter and soil of temperate forests largely decomposed by bacteria. Here, we have examined the utilization of dead fungal biomass by the five dominant bacteria isolated from the in situ decomposition of fungal mycelia using a multiOMIC approach. The genomes of the isolates encoded a broad suite of carbohydrate-active enzymes, peptidases and transporters. In the extracellular proteome, only Ewingella americana expressed chitinases while the two Pseudomonas isolates attacked chitin by lytic chitin monooxygenase, deacetylation and deamination. Variovorax sp. expressed enzymes acting on the side-chains of various glucans and the chitin backbone. Surprisingly, despite its genomic potential, Pedobacter sp. did not produce extracellular proteins to decompose fungal mycelia but presumably feeds on simple substrates. The ecological roles of the five individual strains exhibited complementary features for a fast and efficient decomposition of dead fungal biomass by the entire bacterial community.     

The predicted secretomes of Monosiga brevicollis and Capsaspora owczarzaki,close unicellular relatives of metazoans,reveal new insights into the evolution of the metazoan extracellular matrix

The extracellular matrix (ECM) is a major mediator of multi-cellularity in the metazoa. Multiple ECM proteins are conserved from sponges to human, raising questions about the evolutionary origin of ECM. Choanoflagellates are the closest unicellular relatives of the metazoa and proteins with domains characteristic of metazoan ECM proteins have been identified from the genome-predicted proteome of the choanoflagellate Monosiga brevicollis. However, a systematic analysis of M. brevicollis secretory signal peptide-containing proteins with ECM domains has been lacking. We analysed all predicted secretory signal-peptide-containing proteins of M. brevicollis for ECM domains. Nine domains that are widespread in metazoan ECM proteins are represented, with EGF, fibronectin III, laminin G, and von Willebrand Factor_A domains being the most numerous. Three proteins contain more than one category of ECM domain, however, no proteins correspond to the domain architecture of metazoan ECM proteins. The fibronectin III domains are all present within glycoside hydrolases and none contain an integrin-binding motif. Glycosaminoglycan-binding motifs identified in animal thrombospondin type 1 domains are conserved in some M. brevicollis representatives of this domain, whereas there is little evidence of conservation of glycosaminoglycan-binding motifs in the laminin G domains. The identified proteins were compared with the predicted secretory ECM domain-containing proteins of the integrin-expressing filasterean, Capsaspora owczarzaki. C. owczarzaki encodes a smaller number of secretory, ECM domain-containing proteins and only EGF, fibronectin type III and laminin G domains are represented. The M. brevicollis and C. owczarzaki proteins have distinct domain architectures and all proteins differ in their domain architecture to metazoan ECM proteins. These identifications provide a basis for future experiments to validate the extracellular location of these proteins and uncover their functions in choanoflagellates and C. owczarzaki. The data strengthen the model that ECM proteins are metazoan-specific and evolved as innovations in the last common metazoan ancestor.     

The interaction of knobs and B chromosomes of maize in determining the level of recombination

Enhancement of recombination by B chromosomes is influenced by the kind of heterochromatic knob present in or near the tested region of the A chromosomes. In homomorphic chromosome 9 bivalents of K^s/K^s constitution, double exchanges were increased at the expense of singles, but in the K*/K^s heteromorphs there was a gain in both single and double exchanges at the expense of no-exchange tetrads. Modification of the B chromosome enhancement in different knob compounds was observed only in the megasporocytes.—Different frequencies of recombination are found in plants with odd and even numbers of B chromosomes; this effect is especially striking in the megasporocytes. The modification in recombination produced by an odd or even number of B chromosomes is a function of the interaction of a particular region and the knob constitution. Odd numbers of B chromosomes were more effective than even numbers in causing increased recombination.—It is concluded that heterochromatic knobs and the essentially heterochromatic supernumeraries may interact in the process of crossing over, with the level of recombination determined in part by knob constitution.     

Ultrastructure of Nematode-Trapping Fungi

Capture cells differ ultrastructurally from vegetative cells in the nematode-trapping fungi, Dactylella drechslerii, Monacrosporium rutgeriensis and Arthrobotrys dactyloides, which capture prey by means of adhesive knobs, adhesive hyphal networks, and constricting rings, respectively. Adhesive knobs and adhesive networks contain dense inclusions not found in hyphal tips or subapical regions of the vegetative hyphae, and rough- and smooth-surfaced membranes are abundant in these trap cells. The fine structure of constricting rings differs from that of adhesive traps, and it is altered by closure. In the open configuration, there are membrane-bound inclusions, labyrinthine networks, and electron-lucent regions between the protoplasts and cell wall, all localized on the luminal side of the ring cells. After closure, these features no longer are evident and the cytoplasm of trap cells stains less densely.     

Light trap transects – a field method for ascertaining the habitat preferences of night-flying Lepidoptera, using Mythimna turca (Linnaeus 1761) (Lepidoptera: Noctuidae) as an example

The habitat associations of the noctuid moth Double Line Mythimna turca (Linnaeus 1761) were studied in three areas in Britain by setting up light traps equidistant along transects which passed through different habitat types. Counts were made of M. turca at each trap station and the results compared with the habitat present in the vicinity of each trap. The results using chi-square analysis indicate that the preferred habitats for M. turca are woodland and Pteridium aquilinum scrub, especially where the grasses Agrostis capillaris and Holcus mollis occur. The environmental factors affecting moth catches are discussed and chi-squared expectations are adjusted to take account of the different efficiency of light traps in open grassland and woodland situations. The light trap transect method is shown to be useful for moth species where little is known of their ecology and can be used to provide a straightforward insight informing additional targeted autecological studies. The methodology may be less suited to those species which occur at low density, are poorly attracted to light or are known to migrate.     

Quantitative Proteomic Analysis Reveals Important Roles of the Acetylation of ER-Resident Molecular Chaperones for Conidiation in Fusarium oxysporum

Fusarium oxysporum is one of the most abundant and diverse fungal species found in soils and includes nonpathogenic, endophytic, and pathogenic strains affecting a broad range of plant and animal hosts. Conidiation is the major mode of reproduction in many filamentous fungi, but the regulation of this process is largely unknown. Lysine acetylation (Kac) is an evolutionarily conserved and widespread posttranslational modification implicated in regulation of multiple metabolic processes. A total of 62 upregulated and 49 downregulated Kac proteins were identified in sporulating mycelia versus nonsporulating mycelia of F. oxysporum. Diverse cellular proteins, including glycolytic enzymes, ribosomal proteins, and endoplasmic reticulum–resident molecular chaperones, were differentially acetylated in the sporulation process. Altered Kac levels of three endoplasmic reticulum–resident molecular chaperones, PDI^K70, HSP70^K604, and HSP40^K32 were identified that with important roles in F. oxysporum conidiation. Specifically, K70 acetylation (K70ac) was found to be crucial for maintaining stability and activity of protein disulphide isomerase and the K604ac of HSP70 and K32ac of HSP40 suppressed the detoxification ability of these heat shock proteins, resulting in higher levels of protein aggregation. During conidial formation, an increased level of PDI^K70ac and decreased levels of HSP70^K604ac and HSP40^K32ac contributed to the proper processing of unfolded proteins and eliminated protein aggregation, which is beneficial for dramatic cell biological remodeling during conidiation in F. oxysporum.     

Intracellular Proteolytic Activity in Mycelia of Arthrobotrys oligospora Bearing Mycoparasitic or Nematode Trapping Structures

Persson, Y., and Friman, E. 1993. Intracellular proteolytic activity in mycelia of Arthrobotrys oligospora bearing mycoparasitic or nematode trapping structures. Experimental Mycology 17, 182-190. The fungus Arthrobotrys oligospora parasitizes other fungi with the aid of coils and captures and digests nematodes by means of adhesive traps. We have compared proteolytic activities of mycelial extracts from coils and traps with those of vegetative hyphae. A. oligospora produced a number of proteases active at both alkaline and acidic pH. Coil extract had significantly higher proteolytic activity than extracts of vegetative hyphae. Several coil culture-specific bands were found after substrate gel electrophoresis. Pepstatin-sensitive proteolytic activity at acidic pH was higher in coil extract than in normal mycelial extracts, although the total proteolytic activity was the same. No proteolytic activity was connected solely to mycelial extracts with traps and no enhancement of proteolytic activity was observed during infection of nematodes.     

Aging of Utricularia traps and variability of microorganisms associated with that microhabitat

Various authors have described algae in aquatic Utricularia traps as commensals, as stress factors or as prey. This study examined the diversity and abundance of organisms (prey, algae, protozoa and bacteria) in the traps of aquatic Utricularia reflexa in relation to prey occurrence and trap age. The number of organisms increased with the trap age. In both young and old traps, phytoplankton dominated of all organisms found. In young traps, Scenedesmus spp. and Characiopsis sp. were the most abundant algae, while Scenedesmus spp. and the palmelloidal form of Euglena spp. dominated in the old traps. Most of the algal species found stayed alive in the trap environment. The number of living algae and ciliates inside the traps increased with the increasing trap age, too. As the number of Paramecium bursaria inside traps consistently increased with the trap age and number of bacteria, which serve as a food for them, ciliates can be regarded as commensals, but not as prey for the plant. The predominant organisms in the traps were those that can be considered either commensals or intruders, exceeding captured macroscopic prey.