A unique one-dimensional (1D) legume-like structure of cobalt nanoparticles was prepared by a simple magnetic-field-induced assembly approach with the assistance of polyvinylpyrrolidone (PVP). In each ‘legume’, cobalt nanoparticles were regularly aligned along the lines of magnetic force in a row with visible spacing between adjacent nanoparticles and permanently linked with PVP molecule layers in order to maintain the ordered shape after the removal of the external magnetic field. Magnetic measurement showed that these legume-like structures were superparamagnetic at room temperature, while appearing somewhat ferromagnetic at 10 K, obviously indicating magnetostatic coupling between nanoparticles at low temperature. This novel legume-like structure would provide a new model system for the study of magnetization properties of one-dimensionally ordered magnetic nanostructures.

The nematophagous fungus Lecanicillium psalliotae (syn. Verticillium psalliotae) is a well-known biocontrol agent. In this study, a chitinase gene Lpchi1 was isolated for the first time from L. psalliotae using degenerate primers and DNA-walking technique. The cloned gene Lpchi1 encoding 423 amino acid residues shares a high degree of homology with other pathogenicity-related chitinases from entomopathogenic and mycoparasitic fungi. The complementary DNA sequence of the mature chitinase was amplified via reverse transcription polymerase chain reaction and expressed well in Pichia pastoris GS115. Through gel filtration, the recombinant chitinase was purified as a protein of ca. 45 kDa with an optimal activity at pH 7.0 and 37.6 degrees C. The purified chitinase LPCHI1 was found degrading chitinous components of eggs of the root-knot nematode Meloidogyne incognita and significantly influence its development. Moreover, our results also demonstrate that the protease Ver112 and the chitinase LPCHI1 from the same fungus interacted on the egg infection.

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Nematophagous fungi are commonly used as biological control agents of plant and animal parasitic nematodes. However, relatively little is known of the environmental attributes conferring pathogenicity in these fungi. In this report, we investigated the role of PacC-mediated pH response in the pathogenesis of the nematophagous fungus Clonostachys rosea. We identified a pacC orthologue from this fungus and found that its transcript was elevated in C. rosea during the early stage of its infection of nematode. Disruption of pacC resulted in slowed growth at alkaline pH, altered filamentation, reduced conidiation and attenuated virulence to nematodes. The expression of an extracellular serine protease PrC, a putative virulence factor, was downregulated in the pacC mutants. The PrC transcript levels were significantly higher under alkaline growth conditions than under acidic growth conditions. Promoter activity analysis and electrophoretic mobility shift assay indicated that the regulation of PrC by pH via the PacC pathway occurred at the transcriptional level. In conclusion, PacC functions as a positive regulator of virulence to nematodes in C. rosea.

Micrometer scaled nickel dendritic crystals with distinctive style were synthesized via a self-assembled solution route without any surfactant. Structure characterizations suggest that the dendritic crystal shows first preferential orientation along [111] which is the magnetic easy axis of cubic nickel crystal and subsequently along [100], which differs from the prevalent dendritic fractals synthesized in nonequilibrium system. A formation process is proposed to illustrate the growth of ferromagnetic nickel microleaves. Additionally, the magnetic properties of nickel microleaf have been observed, which demonstrates that the saturation magnetization (Ms) and the coercivity (Hc) are 48 emu/cm−3 and 77 Oe, respectively. The relatively high Ms and low Hc for as-prepared product could be attributed to its special structure.

Nematophagous fungi have been used as biological control agents against nematodes parasitic to plants and animals. These fungi can secret subtilisin-like extracellular serine proteases during the infection of nematodes. The expression of these subtilisin-like serine proteases is regulated by nitrogen sources, including nematode cuticle. However, the mechanisms underlying the nitrogen sources-induced expression of these serine proteases is not well understood. In this study, we investigated the effect of nitrogen sources on the expression of a subtilisin-like extracellular protease, prC, in the nematophagous fungus Clonostachys rosea. Disruption of prC attenuated infection of the fungus to nematodes, indicating that this gene functions as a virulence factor. The inhibition of basal expression of prC by the preferred nitrogen sources (glutamine, ammonia) occurred at the transcriptional level. In contrast, nematode cuticle induced the expression of prC at the post-transcriptional level. The inducible expression of prC by nematode cuticle was significantly suppressed by glutamine, ammonia and phenylmethylsulfonyl fluoride (an inhibitor of serine protease). Thus, the existence of active PrC, albeit at a low level in the medium, is probably essential for further induction of this gene by nematode cuticle. Moreover, the low molecule weight (< 3 kD) degradation products of nematode cuticle could significantly induce the expression of prC. Ammonia suppresses the virulence of C. rosea against nematodes, probably by inhibiting prC expression. Thus, the nematophagous fungi probably could not function well as biocontrol agents in fields fertilized with a large amount of ammonium salt.

Chitinases are a group of enzymes capable of hydrolysing the b-(1,4)-glycosidic bonds of chitin, an essential component of the fungal cell wall, the shells of nematode eggs, and arthropod exoskeletons. Chitinases from pathogenic fungi have been shown to be putative virulence factors, and can play important roles in infecting hosts. However, very limited information is available on the structure of chitinases from nematophagous fungi. Here, we present the 1.8 Å resolution of the first structure of a Family 18 chitinase from this group of fungi, that of Clonostachys rosea CrChi1, and the 1.6 Å resolution of CrChi1 in complex with a potent inhibitor, caffeine. Like other Family 18 chitinases, CrChi1 has the DXDXE motif at the end of strand b5, with Glu174 as the catalytic residue in the middle of the open end of the (b/a) 8 barrel. Two caffeine molecules were shown to bind to CrChi1 in subsites "1 to +1 in the substrate-binding domain. Moreover, sitedirected mutagenesis of the amino acid residues forming hydrogen bonds with caffeine molecules suggests that these residues are important for substrate binding and the hydrolytic process. Our results provide a foundation for elucidating the catalytic mechanism of chitinases from nematophagous fungi and for improving the pathogenicity of nematophagous fungi against agricultural pest hosts. INTRODUCTIONChitin, a polymer of b-(1,4)-linked N-acetylglucosamine (GlcNAc), is an essential structural component of fungal cell walls, the shells of nematode eggs, and the exoskeletons of arthropods. Family 18 chitinases (CAZY GH 18), which degrade this polymer, play key roles in the life cycles of pathogenic fungi (Lorito et al., 1996). Fungi can produce chitinases throughout their growth cycle, and these enzymes are believed to contribute to morphogenetic and pathogenic processes, including spore germination, hyphal branching and mycoparasitic interaction (Gooday et al., 1992;Kuranda & Robbins, 1991;Seidl et al., 2005). For many pathogenic fungi, their chitinases are important virulence factors and promising antifungal targets.Structural studies of chitinase-inhibitor complexes have provided crucial information on the modes of binding, the specificity of chitinase inhibitors, and the mechanism of the hydrolysis reaction (Terwisscha van Scheltinga et al., 1995; van Aalten et al., 2001). Several chitinase inhibitors have been identified, including allosamidin (Bortone et al., 2002), the cyclic pentapeptides argifin and argadin (Arai et al., 2000;Omura et al., 2000), and 8-chlorotheophylline, kinetin and acetazolamide (Hurtado-Guerrero & van Aalten, 2007 (Rao et al., 2005a, b). These xanthine derivatives have low molecular masses and are commercially available. These properties suggest that they might be ideal for developing specific inhibitors of Family 18 chitinases. In addition, site-directed mutagenesis provides a tool for studying the function of amino acid residues and domains. For example, substitution of loop 2 residue N372 with Ala or Gly in d-endotoxin increased the toxicit...

BackgroundSoil fungi face a variety of environmental stresses such as UV light, high temperature, and heavy metals. Adaptation of gene expression through transcriptional regulation is a key mechanism in fungal response to environmental stress. In Saccharomyces cerevisiae, the transcription factors Msn2/4 induce stress-mediated gene expression by binding to the stress response element. Previous studies have demonstrated that the expression of extracellular proteases is up-regulated in response to heat shock in fungi. However, the physiological significance of regulation of these extracellular proteases by heat shock remains unclear. The nematophagous fungus Clonostachys rosea can secret an extracellular serine protease PrC during the infection of nematodes. Since the promoter of prC has three copies of the stress response element, we investigated the effect of environmental stress on the expression of prC.Methodology/Principal FindingsOur results demonstrated that the expression of prC was up-regulated by oxidants (H2O2 or menadione) and heat shock, most likely through the stress response element. After oxidant treatment or heat shock, the germination of conidia in the wild type strain was significantly higher than that in the prC mutant strain in the presence of nematode cuticle. Interestingly, the addition of nematode cuticle significantly attenuated the production of reactive oxygen species (ROS) induced by oxidants and heat shock in the wild type strain, but not in prC mutant strain. Moreover, low molecule weight (<3 kD) degradation products of nematode cuticle suppressed the inhibitory effect of conidial germination induced by oxidants and heat shock.Conclusions/SignificanceThese results indicate that PrC plays a protective role in oxidative stress in C. rosea. PrC degrades the nematode cuticle to produce degradation products, which in turn offer a protective effect against oxidative stress by scavenging ROS. Our study reveals a novel strategy for fungi to adapt to environmental stress.