Rüdiger Hampp

Plant surface microbiology

Plant surface microbiology , Plant surface microbiology , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

Carbon allocation in ectomycorrhizas : Identification and expression analysis of an Amanita muscaria monosaccharide transporter

Ectomycorrhizas are formed between certain soil fungi and fine roots of predominantly woody plants. An important feature of this symbiosis is the supply of plant-derived carbohydrates to the fungus. As a first step toward a better understanding of the molecular basis of this process, we cloned a monosaccharide transporter from the ectomycorrhizal fungus Amanita muscaria. Degenerate oligonucleotide primers were designed to match conserved regions from known fungal sugar transporters. A cDNA fragment of the transporter was obtained from mycorrhizal mRNA by reverse transcription-polymerase chain reaction. This fragment was used to identify a clone (AmMst1) encoding the entire monosaccharide transporter in a Picea abies/A. muscaria mycorrhizal cDNA library. The cDNA codes for an open reading frame of 520 amino acids, showing best homology to a Neurospora crassa monosaccharide transporter. The function of AmMST1 as monosaccharide transporter was confirmed by heterologous expression of the cDNA in a Schizosaccharomyces pombe mutant lacking a monosaccharide uptake system. AmMst1 was constitutively expressed in fungal hyphae under all growth conditions. Nevertheless, in mycorrhizas as well as in hyphae grown at monosaccharide concentrations above 5 mM, the amount of AmMst1 transcript increased fourfold. We therefore suggest that AmMst1 is upregulated in ectomycorrhizas by a monosaccharide-controlled mechanism.

An in vitro method for establishing mycorrhizae on coniferous tree seedlings

SummaryA method for in vitro synthesis of mycorrhizae on coniferous tree seedlings is described. Tree seedlings (Larix decidua Mill., Picea abies (L.) Karst, and Pinus sylvestris L.) and fungi Amanita muscaria (L. ex Fr.) Hooker, Piloderma croceum Erikss. et Hjorst., Pisolithus tinctorius (Pers.) Coker et Couch, and Suillus grevillei (Klotzsch) Singer were maintained under sterile conditions in petri dishes. Typical ectomycorrhizae were established within 2–3 weeks after inoculation and within 2 months after germination of seedlings. Eventually a high percentage of mycorrhizal root tips was obtained.

Auxofuran, a Novel Metabolite That Stimulates the Growth of Fly Agaric, Is Produced by the Mycorrhiza Helper Bacterium Streptomyces Strain AcH 505

ABSTRACT The mycorrhiza helper bacterium Streptomyces strain AcH 505 improves mycelial growth of ectomycorrhizal fungi and formation of ectomycorrhizas between Amanita muscaria and spruce but suppresses the growth of plant-pathogenic fungi, suggesting that it produces both fungal growth-stimulating and -suppressing compounds. The dominant fungal-growth-promoting substance produced by strain AcH 505, auxofuran, was isolated, and its effect on the levels of gene expression of A. muscaria was investigated. Auxofuran and its synthetic analogue 7-dehydroxy-auxofuran were most effective at a concentration of 15 μM, and application of these compounds led to increased lipid metabolism-related gene expression. Cocultivation of strain AcH 505 and A. muscaria stimulated auxofuran production by the streptomycete. The antifungal substances produced by strain AcH 505 were identified as the antibiotics WS-5995 B and C. WS-5995 B completely blocked mycelial growth at a concentration of 60 μM and caused a cell stress-related gene expression response in A. muscaria. Characterization of these compounds provides the foundation for molecular analysis of the fungus-bacterium interaction in the ectomycorrhizal symbiosis between fly agaric and spruce.

Aquaporins in poplar: What a difference a symbiont makes!

The formation of ectomycorrhizas, a tight association between fine roots of trees and certain soil fungi, improves plant nutrition in a nutrient-limited environment and may increase plant survival under water stress conditions. To investigate the impact of mycorrhiza formation on plant water uptake, seven genes coding for putative water channel proteins (aquaporins) were isolated from a poplar ectomycorrhizal cDNA library. Four out of the seven genes were preferentially expressed in roots. Mycorrhiza formation resulted in an increased transcript level for three of these genes, two of which are the most prominently expressed aquaporins in roots. When expressed in Xenopus laevis oocytes, the corresponding proteins of both genes were able to transport water. Together, these data indicate, that the water transport capacity of the plasma membrane of root cells is strongly increased in mycorrhized plants. Measurements of the hydraulic conductance of intact root systems revealed an increased water transport capacity of mycorrhized poplar roots. These data, however, also indicate that changes in the properties of the plasma membrane as well as those of the apoplast are responsible for the increased root hydraulic conductance in ectomycorrhizal symbiosis.

The effects of sublethal concentrations of zinc, cadmium and mercury on Euglena

Results on the effect of sub-lethal concentrations of zinc chloride (ZnCl2), cadmium chloride (CdCl2), and mercuric chloride (HgCl2) on Euglena are presented. During the growth cycle respiratory oxygen uptake and photosynthetic oxygen evolution in the light are initially strongly inhibited by Zn, Cd and Hg. The effects of the three metals on photosynthesis, using oxygen evolution as a criterion was confirmed by carbon fixation techniques.Photosystem I (PSI) associated electron transport 2,6-dichlorophenol indophenol (DCPIP)red. → methyl viologen (MV) → O2, in contrast to total photosynthetic capacity, was only slightly inhibited by Zn, Cd and Hg, whereas the levels of activity of NADP-oxidoreductase in cells untreated or treated with heavy metals showed development like total photosynthesis. Metals strongly inhibited this enzyme which means that the supply of NADPH is lowered due to the action of Zn, Cd and Hg. Photosystem II (PSII) associated electron transport (H2O → dibromothymoquinone/2,3-dimethyl-5,6-methylenedioxy-D-benzoquinone → O2), however, was severely inhibited in a way similar to total photosynthesis. Effects on the cooperation of PSI + II showed patterns similar to PSII alone, i.e., heavy metals strongly reduced PSI + II dependent activities.

Sulfate and sulfite translocation via the phosphate translocator of the inner envelope membrane of chloroplasts.

The permeability of the inner envelope membranes of spinach (Spinacia oleracea) chloroplasts to sulfite and sulfate was investigated in vitro, using the technique of silicone oil centrifugal filtration. The results show that there is a permeability towards both ions, resulting in rates of uptake of about 1.0 (SO32-) and 0.7 (SO42-) μmol mg chlorophyll-1 h-1 respectively (external concentration 2 mmol l-1). The rates depend on the external concentration of the anions. Anion exchange experiments with 35S-preloaded chloroplasts indicate that sulfite and sulfate are exchanged for inorganic phosphate, phosphoglyceric acid, and dihydroxyacetone phosphate with rates up to 14 nmol mg chlorophyll-1 min-1. There is no exchange for glucose-6-phosphate and malate. Because of the similarities to the transport of inorganic phosphate and triose phosphates the results give evidence that the phosphate translocator of the inner envelope membrane of chloroplasts is also involved in sulfite and sulfate transport — at least in part.

Effects of Zinc and Cadmium on Photosynthetic CO2-Fixation and Hill Activity of Isolated Spinach Chloroplasts

Summary 14 CO 2 -fixation of isolated chloroplasts of spinach ( Spinacia oleracea L.) is inhibited by zinc and calcium at all concentrations investigated (1.0 to 1000 μM). The concentrations causing half maximum decrease of photosynthetic CO 2 -fixation when compared to the control were found to be 22.5 μM (zinc) and 3.5 μM (cadmium). The inhibition of both is noncompetitive in nature. Non-cyclic electron transport as measured by dichlorophenolindophenol (DCIP) reduction is also affected. Clearly defined effects are shown at concentrations higher than 100 μM. Mixtures of both metals show additive effects on CO 2 -fixation and the nature of the inhibitions is possibly synergistic rather than additive. The results are discussed in the light of the in vivo concentrations.

Early developmentally regulated genes in the arbuscular mycorrhizal fungus Glomus mosseae: identification of GmGIN1, a novel gene with homology to the C-terminus of metazoan hedgehog proteins

The life cycle of the obligate biotrophic arbuscular mycorrhizal fungi comprises several well-defined developmental stages whose genetic determinants are still unknown. With the aim of understanding the molecular processes governing the early developmental phase of the AM fungal life cycle, a subtractive cDNA library was constructed using a suppressive subtractive hybridization technique. The library contains more than 600 clones with an average size of 500 bp. The isolated cDNAs correspond to genes up-regulated during the early development of the AM fungus Glomus mosseae versus genes expressed in extraradical hyphae. The expression of several of the isolated genes was further confirmed by RT-PCR analysis. Among the isolated clones, a novel gene named GmGIN1 only expressed during early development in G. mosseae was found. The full-length GmGIN1 cDNA codes for a protein of 429 amino acids. The most interesting feature of the deduced protein is its two-domain structure with a putative self-splicing activity. The N-terminal domain shares sequence similarity with a novel family of GTP binding proteins while the C-terminus has a striking homology to the C-terminal part of the hedgehog protein family from metazoa. The C-terminal part of hedgehog proteins is known to participate in the covalent modification of the N-terminus by cholesterol, and in the self-splicing activity which renders the active form of the protein with signalling function. We speculate that the N-terminal part of GmGIN1, activated through a similar mechanism to the hedgehog proteins, has GTP-binding activity and participates in the signalling events prior to symbiosis formation.

Time-course of changes in amounts of specific proteins upon exposure to hyper-g, 2-D clinorotation, and 3-D random positioning of Arabidopsis cell cultures

In previous studies it has been shown that callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by altered gene expression. In this study an investigation was carried out into how different g conditions affect the proteome of such cells. For this purpose, callus cells were exposed to 8 g (centrifugation) and simulated microgravity (2-D clinorotation: fast rotating clinostat, yielding 0.0016 g at maximum; and 3-D random positioning) for up to 16 h. Extracts containing total soluble protein were subjected to 2-D SDS-PAGE. Image analysis of Sypro Ruby-stained gels showed that approximately 28 spots reproducibly and significantly (P <0.05) changed in amount after 2 h of hypergravity (18 up- and 10 down-regulated). These spots were analysed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). In the case of 2-D clinorotation, 19 proteins changed in a manner similar to hypergravity, while random positioning affected only eight spots. Identified proteins were mainly stress related, and are involved in detoxification of reactive oxygen species, signalling, and calcium binding. Surprisingly, centrifugation and clinorotation showed homologies which were not detected for random positioning. The data indicate that simulation of weightlessness is different between clinorotation and random positioning.