Genomic and proteomic analyses of the fungus Arthrobotrys oligospora provide insights into nematode-trap formation

Nematode-trapping fungi are "carnivorous" and attack their hosts using specialized trapping devices. The morphological development of these traps is the key indicator of their switch from saprophytic to predacious lifestyles. Here, the genome of the nematode-trapping fungus Arthrobotrys oligospora Fres. (ATCC24927) was reported. The genome contains 40.07 Mb assembled sequence with 11,479 predicted genes. Comparative analysis showed that A. oligospora shared many more genes with pathogenic fungi than with non-pathogenic fungi. Specifically, compared to several sequenced ascomycete fungi, the A. oligospora genome has a larger number of pathogenicity-related genes in the subtilisin, cellulase, cellobiohydrolase, and pectinesterase gene families. Searching against the pathogen-host interaction gene database identified 398 homologous genes involved in pathogenicity in other fungi. The analysis of repetitive sequences provided evidence for repeat-induced point mutations in A. oligospora. Proteomic and quantitative PCR (qPCR) analyses revealed that 90 genes were significantly up-regulated at the early stage of trap-formation by nematode extracts and most of these genes were involved in translation, amino acid metabolism, carbohydrate metabolism, cell wall and membrane biogenesis. Based on the combined genomic, proteomic and qPCR data, a model for the formation of nematode trapping device in this fungus was proposed. In this model, multiple fungal signal transduction pathways are activated by its nematode prey to further regulate downstream genes associated with diverse cellular processes such as energy metabolism, biosynthesis of the cell wall and adhesive proteins, cell division, glycerol accumulation and peroxisome biogenesis. This study will facilitate the identification of pathogenicity-related genes and provide a broad foundation for understanding the molecular and evolutionary mechanisms underlying fungi-nematodes interactions.     

Development of ToxA and ToxB promoter-driven fluorescent protein expression vectors for use in filamentous ascomycetes

The green fluorescent protein (GFP) has been established as the premier in vivo reporter for investigations of gene expression, protein localization, and cell and organism dynamics. The fungal transformation vector pCT74, with sGFP under the control of the ToxA promoter from Pyrenophora tritici-repentis, effectively expresses GFP in a diverse group of filamentous ascomycetes. Due to the versatility of ToxA promoter-driven expression of GFP, we constructed an additional set of fluorescent protein expression vectors to expand the color palette of fluorescent markers for use in filamentous fungi. EYFP, ECFP and mRFP1 were successfully expressed from the ToxA promoter in its fungus of origin, P. tritici-repentis, and a distant relative, Verticillium dahliae. Additionally the ToxB promoter from P. tritici-repentis drove expression of sGFP in V. dahliae, suggesting a similar potential to the ToxA promoter for heterologous expression in ascomycetes. The suite of fungal transformation vectors presented here promise to be useful for a variety of fungal research applications.     

Extracellular proteases of mycelial fungi as participants of pathogenic processes

The interest in proteases secreted by mycelial fungi is due to several reasons of which one of the most important is their involvement in the initiation and development of the pathogenic process. A comparison of saprophytic and phytopathogenic mycelial fungi revealed one characteristic feature, namely, the appearance of a new trypsin-like activity in phytopathogens that is absent in saprophytes. To clear up the question of whether the degree of pathogenicity of a fungus is related to the activity of secreted trypsin-like protease, several species of Fusarium of various pathogenicity were compared. In two species, F. sporotrichioides (which causes ear fusa-riosis of rye) and F. heterosporum (the causative agent of root rot in wheat), a clear correlation between the activity and pathogenicity was revealed: the more pathogenetic F. sporotrichioides exhibited a higher extracellular trypsin-like activity than the less pathogenetic species F. heterosporum. Thus, the presence of trypsin-like activity in a saprotroph-pathogen pair may be an indicator of the pathogenicity of a fungus; in some cases, the value of this activity may indicate the degree of its pathogenicity. This suggests that trypsin-like proteases specific to phytopathogens are directly involved in the pathogenetic process, probably, through interaction with the "sentry" protein or the product of the resistance gene.     

Transformation of the mycorrhizal fungus Laccaria bicolor using Agrobacterium tumefaciens

Most boreal and temperate forest trees form a mutualistic symbiosis with soil borne fungi called ectomycorrhiza (ECM). In this association both partners benefit due to nutrient exchange at the symbiotic interface. Laccaria bicolor is the first mycorrhizal fungus with its genome sequenced thus making possible for the first time to analyze genome scale gene expression profiles of a mutualistic fungus. However, in order to be able to take full advantage of the genome sequence, reverse genetic tools are needed. Among them a high throughput transformation system is crucial. Herein we present a detailed protocol for genetic transformation of L. bicolor by means of Agrobacterium tumefaciens with emphasis on critical steps affecting the success and efficiency of the approach.     

Selection of an effective red-pigment producing Monascus pilosus by efficient transformation with aurintricarboxylic acid

The filamentous fungus Monascus pilosus was genetically transformed with a reporter plasmid, pMS-1.5hp, by aurintricarboxylic acid (ATA) treatment to obtain an efficient red-pigment producing mutant. The transformation efficiency of Monascus pilosus was higher with the ATA-treatment than with either a non-restriction-enzyme-mediated integration (REMI) or a REMI method. This valid and convenient random mutagenesis method shows that ATA can be applied in fungi for efficient genetic transformation.     

Effect of a saprophytic fungus on the growth and the lead uptake, translocation and immobilization in Dodonaea viscosa

Phytoremediation is a feasible alternative to remediate soils polluted with toxic elements, which can be enhanced by manipulating plant-microbe interactions. Regarding this, free-living saprophytic fungi that interact beneficially with roots have been scarcely studied. Thus, the aim of this study was to assess the effect of a saprophytic fungus, Lewia sp., on the plant growth and the ability of Dodonaea viscosa to phytoaccumulate or phytostabilize soluble and insoluble sources of lead in a solid support. The growth of D. viscosa was influenced by both Pb and Lewia sp. While seedlings exposed to Pb showed a decrease in biomass production, in seedlings grown without Pb the biomass was stimulated by Lewia sp. The fungus strongly stimulated the weight-to-length ratio in roots. Regardless of the treatment, D. viscosa accumulated 4.4-6.5 times more Pb in roots than in shoots, conducting to low translocation factors (< 0.2). The presence of Lewia sp. significantly improved Pb accumulation, achieving high bioconcentration factors (> 22), which was attributed to an increased bioavailability and uptake of Pb due to the fungus. This study demonstrated that Lewia sp. could improve Pb-phytostabilization by D. viscosa in soils polluted with soluble and insoluble forms of Pb.     

Molecular characterization of GmFOX2, an evolutionarily highly conserved gene from the mycorrhizal fungus Glomus mosseae, down-regulated during interaction with rhizobacteria.

Arbuscular mycorrhizal (AM) fungi form the most wide-spread symbiosis of the plant kingdom. More than 80% of vascular plants are susceptible to colonization by the zygomycetous fungi from the order Glomales, and profit significantly by the nutrient exchange between plant and fungus. However, knowledge of the biology of these fungi still remains elusive because of their obligate biotrophism and, up to now, unculturability. The molecular mechanisms underlying the pre-symbiotic stages and the cell-to-cell communication between AM fungi and other soil microorganisms are, particularly, unknown. Here, we study these aspects by means of a molecular approach to monitor changes in the gene expression of the fungus Glomus mosseae (BEG12) in response to the rhizobacterium Bacillus subtilis NR1. The bacterium was found to induce specific increases in mycelial growth as well as changes in expression of GmFOX2, a highly conserved gene encoding a multifunctional protein of the peroxisomal beta-oxidation. We determined the gene structure and studied its expression in response to rhizobacteria at two time points. The results show that the fungus is able to change its gene expression in response to stimuli other than the plant.     

Expression of effector gene SIX1 of Fusarium oxysporum requires living plant cells

Fusarium oxysporum is an asexual, soil inhabiting fungus that comprises many different formae speciales, each pathogenic towards a different host plant. In absence of a suitable host all F. oxysporum isolates appear to have a very similar lifestyle, feeding on plant debris and colonizing the rhizosphere of living plants. Upon infection F. oxysporum switches from a saprophytic to an infectious lifestyle, which probably includes the reprogramming of gene expression. In this work we show that the expression of the known effector gene SIX1 of F. oxysporum f. sp. lycopersici is strongly upregulated during colonization of the host plant. Using GFP (green fluorescent protein) as reporter, we show that induction of SIX1 expression starts immediately upon penetration of the root cortex. Induction requires living plant cells, but is not host specific and does not depend on morphological features of roots, since plant cells in culture can also induce SIX1 expression. Taken together, F. oxysporum seems to be able to distinguish between living and dead plant material, preventing unnecessary switches from a saprophytic to an infectious lifestyle.     

Green fluorescent protein as a reporter for gene expression in the mucoralean fungus Absidia glauca

Mucoralean fungi (Zygomycota) are used for many industrial processes and also as important model organisms for investigating basic biological problems. Their genetic analysis is severely hampered by low transformation frequencies, by their strong tendency towards autonomous replication of plasmids instead of stable integration, and by the lack of reliable genetic reporter systems. We constructed plasmids for transforming the model zygomycete Absidia glauca that carry the versatile reporter gene coding for green fluorescent protein (GFP). gfp expression is controlled either by the homologous actin promoter or the promoter for the elongation factor of translation, EF1alpha. These plasmids also confer neomycin resistance and carry one of two genetic elements (rag1, seg1) that improve mitotic stability of the plasmid. The gfp constructs were replicated extrachromosomally and could be recovered from retransformed Escherichia coli cells. gfp expression was monitored by epifluorescence microscopy. The gfp reporter gene plasmids presented here for the model zygomycete A. glauca constitute the first reliable system that allows the monitoring of gene expression in this important group of fungi.     

Stable transformation of erysiphe graminis an obligate biotrophic pathogen of barley

Barley powdery mildew, Erysiphe graminis f.sp. hordei, is an obligate biotrophic pathogen and as such cannot complete its life cycle without a living host. The inability to transform this fungus and manipulate its genome has constrained research towards understanding its life cycle and pathogenicity. Here we describe an in planta transformation system based on delivery of DNA using a gold-particle gun and selection using benomyl or bialaphos. Using this method, we consistently obtained stable transformants with efficiencies comparable to other filamentous fungi. Stable expression of the beta-glucuronidase in E. graminis was demonstrated by co-transforming the uidA gene with the selectable markers.     

Staurosporine,a Prolyl Endopeptidase Inhibitor

The filamentous fungus Monascus pilosus was genetically transformed with a reporter plasmid, pMS-1.5hp, by aurintricarboxylic acid (ATA) treatment to obtain an efficient red-pigment producing mutant. The transformation efficiency of Monascus pilosus was higher with the ATA-treatment than with either a non-restriction-enzyme-mediated integration (REMI) or a REMI method. This valid and convenient random mutagenesis method shows that ATA can be applied in fungi for efficient genetic transformation.     

Fungal Parasitism of Heterodera glycines Eggs as Influenced by Egg Age and Pre-colonization of Cysts by Other Fungi

The objective of this study was to determine the effect of egg age and pre-colonization of cysts by a saprophytic or parasitic fungus on parasitism of Heterodera glycines eggs by other parasitic fungi. In agar and in soil tests, fungi generally parasitized more eggs in early developmental stages than eggs containing a juvenile. The effect of pre-colonization of cysts by a fungus on parasitism of eggs by other fungi depended on the fungi involved. In most cases, pre-colonization of cysts by an unidentified, saprophytic fungal isolate (A-1-24) did not affect parasitism of eggs in the cysts subsequently treated with other fungi. However, pre-colonization of cysts by A-1-24 reduced fungal parasitism of eggs in cysts subsequently treated with Cylindrocarpon destructans isolate 3. In agar tests, pre-colonization of cysts by Chaetomium cochliodes, a saprophytic or weakly parasitic fungus, reduced parasitism of eggs in cysts subsequently treated with Verticillium chlamydosporium Florida isolate, Fusarium oxysporum, Fusarium solani, ARF18, and another sterile fungus. However, in soil tests, pre-colonization of cysts by C. cochliodes had no effect on parasitism of eggs by subsequent fungal parasites. In another test, parasitism of eggs by V. chlamydosporium in cysts was not affected by pre-colonizing fungi C. destructans, F. oxysporum, and F. solani but was reduced by Mortierella sp., Pyrenochaeta terrestris, and C. cochliodes. Parasitism of eggs in cysts by ARF18 was reduced by pre-colonizing fungi C. destructans, F. oxysporum, F. solani, P. terrestris, and C. cochliodes but not Mortierella sp.     

Efficient expressions of reporter genes in the industrial filamentous fungus Sclerotium rolfsii mediated by Agrobacterium tumefaciens

Sclerotium rolfsii (teleomorph Athelia rolfsii) is one of the plant pathogenic basidiomycetes, which causes severe stem-rot disease in hundreds of plants and produces important metabolites, such as scleroglucan and TF-specific lectin. However, further molecular biological research on this filamentous fungus is severely plateaued out due to the lack of genetic methods. In this study, the A. tumefaciens strain LBA4404 harboring a binary vector containing the basta resistance gene fused with three reporters (DsRed, tdTomato, and GUSPlus) respectively, driven by the SrGPD promoter, was used for genetic transformation of S. rolfsii. The results showed that the three reporter genes were all effectively expressed in S. rolfsii. This study also showed that the intron of the SrGPD promoter is not necessary for transgene expression in this fungus. Besides, we showed that these reporters’ signals could be observed easily but in a short time window. The efficient Agrobacterium-mediated transformation system and the three reporter gene plasmids for S. rolfsii developed in this study are of significance in overcoming current limitations of no available transformation and genetic manipulation techniques in S. rolfsii, facilitating further genetic manipulations and gene function exploration.     

Hyphal growth and mycorrhiza formation by the arbuscular mycorrhizal fungus Glomus claroideum BEG 23 is stimulated by humic substances

Effects of humic substances (humic acid or fulvic soil extract) or saprophytic microorganisms (Paecilomyces lilacinus and an unidentified actinomycete) on growth of mycelium and mycorrhiza formation by Glomus claroideum BEG23 were studied in a hydroponic system. Humic substances stimulated root colonization and production of extraradical mycelium by the mycorrhizal fungus. Both humic and fulvic acids tended to decrease populations of culturable bacteria and fungi in the cultivation system, indicating a moderately antibiotic activity. The addition of saprophytic microorganisms able to use humic substances to the cultivation system further stimulated the development of the mycorrhizal fungus. However, stimulation of G. claroideum was also observed when the saprophytic microorganisms were heat-killed, suggesting that their effect was not linked to a specific action on humic substances. The results indicate that humic substances may represent a stimulatory component of the soil environment with respect to arbuscular mycorrhizal fungi.     

A novel selectable marker based on Aspergillus niger arginase expression

Selectable markers are valuable tools in transforming asexual fungi like Aspergillus niger. An arginase (agaA) expression vector and a suitable arginase-disrupted host would define a novel nutritional marker/selection for transformation. The development of such a marker was successfully achieved in two steps. The single genomic copy of A. niger arginase gene was disrupted by homologous integration of the bar marker. The agaA disruptant was subsequently complemented by transforming it with agaA expression vectors. Both citA and trpC promoters were able to drive the expression of arginase cDNA. Such agaA+ transformants displayed arginase expression pattern distinct from that of the parent strain. The results are also consistent with a single catabolic route for arginine in this fungus. A simple yet novel arginine-based selection for filamentous fungal transformation is thus described.     

Spermidine and the ectomycorrhizal fungus Pisolithus tinctorius synergistically induce maturation of Scots pine embryogenic cultures

Exogenous spermidine (Spd) and the ectomycorrhizal (ECM) fungus Pisolithus tinctorius (Pers.) Coker and Couch had a synergistic effect on the maturation of Scots pine (Pinus sylvestris L.) somatic embryos. Induced maturation was expressed as a higher number of cell masses able to form embryos and a greater number of embryos formed per cell mass. In contrast, treatment with P. tinctorius alone on the hormone-free medium resulted in the lowest embryo-forming capacity. Retarded proliferation growth appeared to be required for maturation, but did not explain the synergistic effect of the fungus and exogenous Spd. Simultaneous treatment did not result in lower concentrations of putrescine (Put), Spd or spermine (Spm) in the embryogenic cell masses relative to the separate treatments. Our study is the first report on the use of a specific ECM fungus to induce maturation of somatic embryos, and it indicates that P. tinctorius was able to modify the maturation media in a way that, together with exogenous Spd, positively affected embryogenic cultures of Scots pine. Our study also shows that it is possible to enhance plant development other than root formation by using specific ECM fungi.