Phytotoxicity of nitrobenzene bioaccumulation in rice seedlings: Nitrobenzene inhibits growth, induces oxidative stress, and reduces photosynthetic pigment synthesis.

Nitrobenzene (NB) has been used in numerous industrial and agricultural fields as an organic compound intermediate. NB has mutagenicity and acute toxicity, and is typically a toxic pollutant in industrial wastewater worldwide. To evaluate its phytotoxicity, we treated rice (Oryza sativa) with different concentrations of NB (0, 5, 25, 50, 75, and 100 mg L-1). NB inhibited growth indices of rice (shoot and root length, fresh shoot and root weight, and dry shoot and root weight) as NB treatment concentrations increased. High concentrations (>25 mg L-1) of NB significantly inhibited rice root and shoot growth; root growth was more susceptible to NB. NB treatment could damage the structure and reduce the activity of rice seedling roots. The result of high performance liquid chromatography (HPLC) indicated that the bioaccumulation of NB in rice seedlings had a dose-dependent effect on the growth inhibition. NB reduced the photosynthetic pigment content and the expression levels of chlorophyll synthesis genes. NB treatment increased active oxygen radicals, electrical conductivity, malondialdehyde (MDA), proline, and soluble sugar contents. The expressions of antioxidant enzyme genes were induced by NB stress, and exhibited a phenomenon of initial increase followed by decrease. When the NB concentration was higher than 50 mg L-1, the gene expression levels decreased rapidly. This study provides insight into the association between exposure to NB and its phytotoxic effects on rice seedlings, and assesses the potential risk of NB bioaccumulation for crops that require a large amount of irrigation water.

Overexpression of the Capebp2 Gene Encoding the PEBP-like Protein Promotes the Cap Redifferentiation in Cyclocybe aegerita.

Phosphatidylethanolamine-binding protein (PEBP) is widely involved in various physiological behaviors, such as the transition from vegetative growth to reproductive growth in plants, tumorigenesis in the human, etc. However, few functional studies have examined pebp genes affecting the development of fungi. In this study, Capebp2 was cloned from Cyclocybe aegerita AC0007 strains based on the genome sequence and gene prediction, and the sequence alignment of CaPEBP2 with other PEBP proteins from other biological sources including plant, animal, fungi, and bacteria indicated that PEBP had low sequence similarity in fungi, whereas all protein sequences had some conserved motifs such as DPDAP and HRY. Expression analysis showed the transcription level of Capebp2 increased approximately 20-fold in fruiting bodies compared with mycelia. To uncover the function of Capebp2 in C. aegetita development, Capebp2 was cloned into a pATH vector driven by the actin promoter for obtaining overexpression transformant lines. Fruiting experiments showed the transformed strains overexpressing Capebp2 exhibited redifferentiation of the cap on their surface, including intact fruiting bodies or partial lamella during fruiting development stage, and the longitudinal section indicated that all regenerated bodies or lamella sprouted from the flesh and shared the epidermis with the mother fruiting bodies. In summary, the sequence characterization of Capebp2, expression level during different development stages, and function on fruiting body development were documented in this study, and these findings provided a reference to study the role of pebp in the development process of basidiomycetes. Importantly, gene mining of pebp, function characterization, and the regulating pathways involved need to be uncovered in further studies.

Organization and Unconventional Integration of the Mating-Type Loci in Morchella Species.

True morels (Morchella spp.) are a group of delicious fungi in high demand worldwide, and some species of morels have been successfully cultivated in recent years. To better understand the sexual reproductive mechanisms of these fungi, we characterized the structure of the mating-type loci from ten morel species, and seven of them were obtained using long-range PCR amplification. Among the studied species, eight were heterothallic, two were homothallic, and four types of composition were observed in the MAT loci. In three of the five black morel species, the MAT1-1-1, MAT1-1-10, and MAT1-1-11 genes were in the MAT1-1 idiomorph, and only the MAT1-2-1 gene was in the MAT1-2 idiomorph, while an integration event occurred in the other two species and resulted in the importation of the MAT1-1-11 gene into the MAT1-2 idiomorph and survival as a truncated fragment in the MAT1-1 idiomorph. However, the MAT1-1-11 gene was not available in the four yellow morels and one blushing morel species. M. rufobrunnea, a representative species of the earliest diverging branch of true morels, along with another yellow morel Mes-15, were confirmed to be homothallic, and the MAT1-1-1, MAT1-1-10, and MAT1-2-1 genes were arranged in a tandem array. Therefore, we hypothesized that homothallism should be the ancestral reproductive state in Morchella. RT-PCR analyses revealed that four mating genes could be constitutively expressed, while the MAT1-1-10 gene underwent alternative splicing to produce different splice variants.

A Putative Guanosine Triphosphate Cyclohydrolase I Named CaGCH1 Is Involved in Hyphal Branching and Fruiting Development in Cyclocybe aegerita.

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) is the limiting enzyme of the tetrahydrobiopterin (BH4) synthesis pathway. The disruption of gch1 gene may cause conditional lethality due to folic acid auxotrophy in microorganisms, although the function of gch1 in basidiomycetes has not been deciphered so far. In the present study, gch1 expression in Cyclocybe aegerita (cagch1) was downregulated using the RNAi method, which resulted in growth retardation in both solid and liquid medium, with the hyphal tips exhibiting increased branching compared to that in the wild strain. The development of fruiting bodies in the mutant strains was significantly blocked, and there were short and bottle-shaped stipes. The transcriptional profile revealed that the genes of the MAPK pathway may be involved in the regulation of these effects caused by cagch1 knockdown, which provided an opportunity to study the role of gch1 in the development process of basidiomycetes.

Characterization of Vegetative Incompatibility in Morchella importuna and Location of the Related-Genes by Bulk Segregant Analysis.

Vegetative incompatibility (VI) is a widespread phenomenon developed in Morchella importuna, a species of ascomycete fungus that is cultivated on a rapidly expanding scale in China. Understanding the genetic bases of this nonself-recognition phenomenon is beneficial for resolving some problems that are associated with the production of this highly prized edible fungus, such as crossbreeding, strain classification, and pathogen transmission. VI is genetically controlled by het genes, organized in two different systems, namely allelic and nonallelic. These het genes have been well characterized in Podospora anserina and Neurospora crassa. In this work, putative het-homologs were identified in the genome of M. importuna, but their low allelic polymorphism in different vegetative compatibility groups (VCGs) suggested that VI in this fungus might not be regulated by these het genes. The progeny derived from vegetative compatible parents became a VCG, while the single-ascospore strains from vegetative incompatible parents were divided into four VCGs, and the interaction between the inter-group strains led to the formation of two types of barrages, viz., thin dark line and raised aggregate of hyphae. The Bulk Segregant Analysis confirmed that the genes mimpvic32 and mimpvic33 were linked to VI reactions in M. importuna; nevertheless, the formation of barrages also occurred between the pairs carrying the same allele of these two genes. In sum, the VI control system in M. importuna was complicated, and there were more other allelic or non-allelic VI-related genes.

Systematic Investigation of the Effects of Macro-elements and Iron on Soybean Plant Response to Fusarium oxysporum Infection.

Nutrient manipulation is a promising strategy for controlling plant diseases in sustainable agriculture. Although many studies have investigated the relationships between certain elements and plant diseases, few have comprehensively explored how differing mineral nutrition levels might affect plant-fungal pathogen interactions, namely plant susceptibility and resistance. Here, we systematically explored the effects of the seven mineral elements that plants require in the greatest amounts for normal development on the susceptibility of soybean plants (Glycine max) to Fusarium oxysporum infection in controlled greenhouse conditions. Nitrogen (N) negligibly affected plant susceptibility to infection in the range 4 to 24 mM for both tested soybean cultivars. At relatively high concentrations, phosphorus (P) increased plant susceptibility to infection, which led to severely reduced shoot and root dry weights. Potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), and iron (Fe) induced plant resistance to infection as their concentrations were increased. For K and Ca, moderate concentrations had a positive effect on plant resistance to the pathogen, whereas relatively high doses of either element adversely affected plant growth and promoted disease symptoms. Further experiments were conducted, assessing disease suppression by selected combinations of macro-elements and Fe at screened concentrations, i.e., K (9 mM) plus Fe (0.2 mM), and S (4 mM) plus Fe (0.2 mM). The disease index was significantly reduced by the combination of K plus Fe. In conclusion, this systematic investigation of soybean plant responses to F. oxysporum infection provides a solid basis for future environmentally-friendly choices for application in soybean disease control programs.

Genotoxicity and growth inhibition effects of aniline on wheat.

Aniline is a synthetic compound widely used in industrial and pesticide production, which can lead to environmental pollution. Its high concentration in rivers and lakes is hazardous to aquatic species. Although the mechanism of aniline toxicity has been studied extensively in animals and algae, little is known about its genotoxicity in plants. In this study, we investigated the genotoxicity effects of aniline on wheat root tip cells. The mitotic index of wheat root tip cells decreased when the aniline test concentration was higher than 10 mg L-1. The frequency of micronucleus and chromosomal aberrations increased at aniline concentrations ranging between 5 and 100 mg L-1, and reached 23.3‰ ± 0.3‰ and 8.9‰ ± 0.68‰, respectively, at an aniline concentration of 100 mg L-1. These values were sevenfold higher than those in the control group. The wheat seedlings showed various growth toxicity effects under different concentrations of aniline. The shoot height, root length, fresh weight, and dry weight of wheat seedlings decreased at aniline test concentrations ranging between 25 and 200 mg L-1. At 200 mg L-1 aniline, the dry weight was only one-third that of the control group. Overall, the findings of this study provide evidence that aniline is a serious environmental pollutant causing deleterious genotoxic effects on wheat root tip cells and growth toxic effects on wheat seedlings. However, understanding the mechanisms that underlie aniline genotoxicity in plants needs further study.

[Progress on nitrogen regulation gene expression of plant pathogenic fungi under nitrogen starvation].

It has been confirmed that the occurrence of plant disease is caused by the effector molecules secreted by plant pathogens. The regulation effector gene expression is an important aspect in understanding of the infection process. The nutritional status of cells has been postulated to be a vital role for effector gene expression. Studies have indicated that the induction of the same effecter genes during growth in vitro as those during growth in planta under nitrogen-starved conditions. This showed that the nitrogen poor environment existed in the early time of plant evolution. This paper describes the system in the pathogenesis of several fungal pathogens and nitrogen in the process of gene expression effects from the results of several species by comparing and contrasting the function of nitrogen regulatory genes, as well as by studying plants in vivo and in vitro gene under nitrogen limitation inductive effect in order to reveal the effectiveness of nitrogen in the development process of host plant disease is an important factor.

[Genome-wide analysis of the secreted proteins of phytophthora infestans].

Based on the Phytophthora infestans genome sequence, we used bioinformatics and computer-based prediction algorithms to predict the secreted proteins of P infestans in detail, which would help us to elucidate the molecular mechanism underlying the interaction between the host plants and the P infestans. In this study, the signal peptide prediction algorithms SignalP v3.0 and PSORT, transmembrane helix prediction algorithms TMHMM-2.0 and THUMBUP, GPI-anchoring site prediction algorithm big-PI Predictor, and subcellular protein location distribution algorithm TargetP v1.01 were used to analyze the 22658 protein sequences of P infestans published. Our results suggested that there might be 671 secreted pro- teins, accounting for 3.0% of the total proteins. Among them, the functions of the 45 secreted proteins had been described previously. Their functions involved cellular metabolism and signal transduction etc. In addition, some of the secreted proteins were functionally similar to elicitin, which were likely to be associated with the virulence of P. infestans.

Expression of a serine protease gene prC is up-regulated by oxidative stress in the fungus Clonostachys rosea: implications for fungal survival.

BACKGROUND: Soil 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 FINDINGS: Our results demonstrated that the expression of prC was up-regulated by oxidants (H(2)O(2) 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/SIGNIFICANCE: These 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.

Crystal structure and mutagenesis analysis of chitinase CrChi1 from the nematophagous fungus Clonostachys rosea in complex with the inhibitor caffeine.

Chitinases are a group of enzymes capable of hydrolysing the ß-(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 ß5, with Glu174 as the catalytic residue in the middle of the open end of the (ß/α)(8) barrel. Two caffeine molecules were shown to bind to CrChi1 in subsites -1 to +1 in the substrate-binding domain. Moreover, site-directed 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.

Regulation of subtilisin-like protease prC expression by nematode cuticle in the nematophagous fungus Clonostachys rosea.

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.

PacC in the nematophagous fungus Clonostachys rosea controls virulence to nematodes.

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.

Crystallization and preliminary crystallographic analysis of a chitinase from Clonostachys rosea.

CrChi1 is a chitinase from the nematophagous fungus Clonostachys rosea that plays a role in the infection of nematodes. In order to resolve the crystal structure of CrChi1 and to gain a better understanding of its biological functions, recombinant CrChi1 was crystallized at 291 K using PEG 3350 and ammonium dihydrogen phosphate as precipitant and a 1.8 A resolution X-ray data set was collected from a single flash-cooled crystal (100 K). The crystals belonged to space group P2(1), with unit-cell parameters a = 44.1, b = 71.7, c = 59.1 A, alpha = gamma = 90, beta = 91.3 degrees. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient and solvent content were calculated to be 2.45 A(3) Da(-1) and 40%, respectively. To our knowledge, this is the first structure determination study of a chitinase from a nematophagous fungus.

Cloning and expression analysis of a chitinase gene Crchi1 from the mycoparasitic fungus Clonostachys rosea (syn. Gliocladium roseum).

Clonostachys rosea (syn. Gliocladium roseum) is a well-known biocontrol agent and widely distributed around the world. In this study, an endochitinase gene Crchi1 was isolated from the mycoparasitic fungus C. rosea using the DNA walking strategy. The Crchi1 ORF is 1,746 bp long and interrupted by three introns. The cloned gene Crchi1 encodes 426 amino acid residues and shares a high degree of similarity with other chitinases from entomopathogenic and mycoparasitic fungi. Several putative binding sites for transcriptional regulation of Crchi1 in response to carbon (5'-SYGGRG-3') and nitrogen (5'-GATA-3') were identified in the upstream of Crchi1. Expression of Crchi1 gene in different carbon sources was analyzed using real-time PCR (RT-PCR). We found that the Crchi1 expression was suppressed by glucose but strongly stimulated by chitin or solubilized components of the cell wall from Rhizoctonia solani. Phylogenetic analysis of chitinases from entomopathogenic and mycoparasitic fungi suggests that these chitinases have probably evolved from a common ancestor.

Cloning of the gene Lecanicillium psalliotae chitinase Lpchi1 and identification of its potential role in the biocontrol of root-knot nematode Meloidogyne incognita.

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.