UV-C irradiation compromises conidial germination, formation of appressoria, and induces transcription of three putative photolyase genes in the barley powdery mildew fungus, Blumeria graminis f. sp. hordei.

UV-C irradiation is known to compromise germination of Blumeria graminis conidia and to reduce powdery mildew infestation. However, only scarce information is available on the effects of UV-C irradiation on B. graminis appressorium formation. Applying a Formvar® resin-based in vitro system allowed for analyzing B. graminis germination and appressorium formation in absence of plant defense. UV-C irradiation more strongly affected the differentiation of appressoria than conidial germination. In vivo and in vitro, a single dose of 100 J m-2 UV-C was sufficient to reduce germination to less than 20 % and decrease appressorium formation to values below 5 %. UV-C irradiation negatively affected pustule size and conidiation. White light-mediated photoreactivation was most effective immediately after UV-C irradiation, indicating that a prolonged phase of darkness after UV-C treatment increases the efficacy of B. graminis control. UV-C irradiation increased transcript levels of three putative B. graminis photolyase genes, while mere white light or blue light irradiation did not contribute to the transcriptional up-regulation. Thus, UV-C irradiation effectively controls B. graminis infestation and proliferation by restricting prepenetration processes. Nevertheless, photoreactivation plays an important role in UV-C-based powdery mildew control in crops and hence has to be considered for planning specific irradiation schedules.

Direct Effects of Physcion, Chrysophanol, Emodin, and Pachybasin on Germination and Appressorium Formation of the Barley ( Hordeum vulgare L.) Powdery Mildew Fungus Blumeria graminis f. sp. hordei (DC.) Speer.

Several anthraquinone derivatives are active components of fungicidal formulations particularly effective against powdery mildew fungi. The antimildew effect of compounds such as physcion and chrysophanol is largely attributed to host plant defense induction. However, so far a direct fungistatic/fungicidal effect of anthraquinone derivatives on powdery mildew fungi has not been unequivocally demonstrated. By applying a Formvar-based in vitro system we demonstrate a direct, dose-dependent effect of physcion, chrysophanol, emodin, and pachybasin on conidial germination and appressorium formation of Blumeria graminis f. sp. hordei (DC.) Speer, the causative agent of barley ( Hordeum vulgare L.) powdery mildew. Physcion was the most effective among the tested compounds. At higher doses, physcion mainly inhibited conidial germination. At lower rates, however, a distinct interference with appressorium formation became discernible. Physcion and others may act by modulating both the infection capacity of the powdery mildew pathogen and host plant defense. Our results suggest a specific arrangement of substituents at the anthraquinone backbone structure being crucial for the direct antimildew effect.

Very-long-chain aldehydes induce appressorium formation in ascospores of the wheat powdery mildew fungus Blumeria graminis.

Asexually produced conidia of the wheat powdery mildew fungus Blumeria graminis f. sp. tritici (Bgt) are known to perceive cuticular very-long-chain aldehydes as signal substances strongly stimulating germination and differentiation of infection structures in a concentration- and chain-length-dependent manner. Conidial germination and appressorium formation are widely prevented by the presence of free water on the host surface. However, sexually produced ascospores can differentiate immersed in water. Applying a Formvar®-based in vitro-system showed that ascospore appressorium formation was strongly induced by the presence of wheat leaf cuticular wax. Similar to conidia, ascospore appressorium formation is triggered by the presence of very-long-chain aldehydes in a chain-length-dependent manner with n-octacosanal as the most inducing aldehyde. Surface hydrophobicity positively affected ascospore germination but not appressorium formation. Ascospores required significantly more time to complete the differentiation of appressoria and exhibited a more distinct dependence on the availability of free water than their conidial counterparts. Unlike conidia, ascospores showed a more variable germination and differentiation pattern even with a single germ tube differentiating an appressorium. Despite these differences our results demonstrate that a host surface recognition principle based on cuticular very-long-chain aldehydes is a common feature of B. graminis f. sp. tritici ascospores and conidia.

Draft genome of the Arabidopsis thaliana phyllosphere bacterium, Williamsia sp. ARP1.

The Gram-positive actinomycete Williamsia sp. ARP1 was originally isolated from the Arabidopsis thaliana phyllosphere. Here we describe the general physiological features of this microorganism together with the draft genome sequence and annotation. The 4,745,080 bp long genome contains 4434 protein-coding genes and 70 RNA genes. To our knowledge, this is only the second reported genome from the genus Williamsia and the first sequenced strain from the phyllosphere. The presented genomic information is interpreted in the context of an adaptation to the phyllosphere habitat.

CYP83A1 is required for metabolic compatibility of Arabidopsis with the adapted powdery mildew fungus Erysiphe cruciferarum.

Aliphatic glucosinolates function in the chemical defense of Capparales. The cytochrome P450 83A1 monooxygenase (CYP83A1) catalyzes the initial conversion of methionine-derived aldoximes to thiohydroximates in the biosynthesis of glucosinolates, and thus cyp83a1 mutants have reduced levels of aliphatic glucosinolates. Loss of CYP83A1 function leads to dramatically reduced parasitic growth of the biotrophic powdery mildew fungus Erysiphe cruciferarum on Arabidopsis thaliana. The cyp83a1 mutants support less well the germination and appressorium formation of E. cruciferarum on the leaf surface and post-penetration conidiophore formation by the fungus. By contrast, a myb28-1 myb29-1 double mutant, which totally lacks aliphatic glucosinolates, shows a wild-type level of susceptibility to E. cruciferarum. The cyp83a1 mutants also lack very-long-chain aldehydes on their leaf surface. Such aldehydes support appressorium formation by E. cruciferarum in vitro. In addition, when chemically complemented with the C26 aldehyde n-hexacosanal, cyp83a1 mutants can again support appressorium formation. The mutants further accumulate 5-methylthiopentanaldoxime, the potentially toxic substrate of CYP83A1. Loss of powdery mildew susceptibility by cyp83a1 may be explained by a reduced supply of the fungus with inductive signals from the host and an accumulation of potentially fungitoxic metabolites.

Distinct phyllosphere bacterial communities on Arabidopsis wax mutant leaves.

The phyllosphere of plants is inhabited by diverse microorganisms, however, the factors shaping their community composition are not fully elucidated. The plant cuticle represents the initial contact surface between microorganisms and the plant. We thus aimed to investigate whether mutations in the cuticular wax biosynthesis would affect the diversity of the phyllosphere microbiota. A set of four Arabidopsis thaliana eceriferum mutants (cer1, cer6, cer9, cer16) and their respective wild type (Landsberg erecta) were subjected to an outdoor growth period and analysed towards this purpose. The chemical distinctness of the mutant wax phenotypes was confirmed by gas chromatographic measurements. Next generation amplicon pyrosequencing of the bacterial communities showed distinct community patterns. This observation was supported by denaturing gradient gel electrophoresis experiments. Microbial community analyses revealed bacterial phylotypes that were ubiquitously present on all plant lines (termed "core" community) while others were positively or negatively affected by the wax mutant phenotype (termed "plant line-specific" community). We conclude from this study that plant cuticular wax composition can affect the community composition of phyllosphere bacteria.

Appressorium morphogenesis and cell cycle progression are linked in the grass powdery mildew fungus Blumeria graminis.

Conidial germination and differentiation - the so-called prepenetration processes - of the barley powdery mildew fungus (Blumeria graminis f. sp. hordei) are essential prerequisites for facilitating penetration of the host cuticle. Although the cell cycle is known to be pivotal to cellular differentiation in several phytopathogenic fungi there is as yet no information available concerning the relationship between cell cycle and infection structure development in the obligate biotroph B. graminis. The timing of specific developmental events with respect to nuclear division and morphogenesis was followed on artificial and host leaf surfaces by 4',6-diamidino-2-phenylindole (DAPI) staining in combination with a pharmacological approach applying specific cell cycle inhibitors. It was found that the uninucleate conidia germinated and then underwent a single round of mitosis 5-6 h after inoculation. During primary germ tube formation the nucleus frequently migrated close to the site of primary germ tube emergence. This nuclear repositioning was distinctly promoted by very-long-chain aldehydes that are common host cuticular wax constituents known to induce conidial differentiation. The subsequent morphogenesis of the appressorial germ tube preceded mitosis that was spatially uncoupled from subsequent cytokinesis. Blocking of S-phase with hydroxyurea did not inhibit formation of the appressorial germ tube but prevented cytokinesis and appressorium maturation. Benomyl treatment that arrests the cell cycle in mitosis inhibited nuclear separation, cytokinesis, and formation of mature appressoria. Thus, we conclude that a completed mitosis is not a prerequisite for the formation and swelling of the appressorial germ tube, which normally provides the destination for one of the daughter nuclei, while appressorium maturation depends on mitosis.

Abscisic acid mediates the formation of a suberized stem scar tissue in tomato fruits.

During harvest, fleshy berry tomato fruits (Solanum lycopersicum) were wounded at their stem scar. Within 3 d, this wound was rapidly sealed by a process covering the wound site with a membranous layer which effectively protects the tomato fruit from excessive water loss, nutrient elution and the entry of pathogens. Chemical analysis of the de novo synthesized stem scar tissue revealed the presence of aromatic and aliphatic components characteristic of the biopolyester suberin. Gene expression patterns associated with suberization were identified at the stem scar region. Changes in the relative abundance of different transcripts suggested a potential involvement of the plant hormone abscisic acid (ABA) in the wound-healing processes. The amount of ABA present in the stem scar tissue showed a significantly increased level during wound healing, whereas ABA-deficient mutants notabilis, flacca and sitiens were largely devoid of this rise in ABA levels. The mutant fruits showed a retarded and less efficient suberization response at the stem scar wound, whereas the rate and strength of this response were positively correlated with ABA content. These results clearly indicate in vivo the involvement of ABA in the suberization-based wound-healing processes at the stem scar tissue of tomato fruits.

Phyllosphere bacterial communities of trichome-bearing and trichomeless Arabidopsis thaliana leaves.

This study aimed to investigate whether the presence of trichomes as conspicuous physical attributes of the leaf surface affects the microbial community composition on Arabidopsis thaliana leaves. The A. thaliana ecotype Col-0 and its trichomeless gl1 mutant were grown in growth cabinets under climate-controlled conditions. The gl1 mutant showed a similar wax composition as the Col-0 wild type with slightly reduced amounts of C(29), C(31) and C(33) alkanes by GC/MS and GC/FID analyses. 120 bacterial isolates representing 39 bacterial genera were obtained from A. thaliana Col-0 leaf surfaces. Phylogenetic analysis of nearly full-length 16S rRNA sequences from 29 selected isolates confirmed their affiliation to the Proteobacteria (Alpha-, Beta-, Gamma-), Actinobacteria, Bacteroidetes and Firmicutes. The bacterial diversity on A. thaliana ecotype Col-0 and its gl1 mutant, devoid of trichomes, were further compared by denaturing gradient gel electrophoresis (DGGE). Banding patterns and sequencing of representative DGGE bands revealed the presence of phylotypes related to Sphingomonas (Alphaproteobacteria), Methylophilus (Betaproteobacteria) and Dyadobacter (Bacteroidetes) which are common phyllosphere inhabitants. Furthermore, wildtype and trichomeless mutant plants were exposed to outdoor conditions for 4-5 weeks. The DGGE gels showed only minor differences between the two plant lines, thus suggesting that trichomes per se do not affect bacterial diversity on Arabidopsis leaves under the experimental conditions tested.

The positional sterile (ps) mutation affects cuticular transpiration and wax biosynthesis of tomato fruits.

Cuticular waxes are known to play a pivotal role in limiting transpirational water loss across primary plant surfaces. The astomatous tomato fruit is an ideal model system that permits the functional characterization of intact cuticular membranes and therefore allows direct correlation of their permeance for water with their qualitative and quantitative composition. The recessive positional sterile (ps) mutation, which occurred spontaneously in tomato (Solanum lycopersicum L.), is characterized by floral organ fusion and positional sterility. Because of a striking phenotypical similarity with the lecer6 wax mutant of tomato, which is defective in very-long-chain fatty acid elongation, ps mutant fruits were analyzed for their cuticular wax and cutin composition. We also examined their cuticular permeance for water following the developmental course of fruit ripening. Wild type and ps mutant fruits showed considerable differences in their cuticular permeance for water, while exhibiting similar quantitative wax accumulation. The ps mutant fruits showed a five- to eightfold increase in water loss per unit time and surface area when compared to the corresponding wild type fruits. The cuticular waxes of ps mutant fruits were characterized by an almost complete absence of n-alkanes and aldehydes, with a concomitant increase in triterpenoids and sterol derivatives. We also noted the occurrence of alkyl esters not present in the wild type. Quantitative and qualitative cutin monomer composition remained largely unaffected. The significant differences in the cuticular wax composition of ps mutant fruits induced a distinct increase of cuticular water permeance. The fruit wax compositional phenotype indicates the ps mutation is responsible for effectively blocking the decarbonylation pathway of wax biosynthesis in epidermal cells of tomato fruits.

Very-long-chain aldehydes promote in vitro prepenetration processes of Blumeria graminis in a dose- and chain length-dependent manner.

Surface properties of aerial plant organs have been shown to affect the interaction of fungal plant pathogens and their hosts. Conidial germination and differentiation - the so-called prepenetration processes - of the barley powdery mildew fungus (Blumeria graminis f. sp. hordei) are known to be triggered by n-hexacosanal (C(26)-aldehyde), a minor constituent of barley leaf wax. In order to analyze the differentiation-inducing capabilities of typical aldehyde wax constituents on conidia of wheat and barley powdery mildew, synthetic even-numbered very-long-chain aldehydes (C(22)-C(30)) were assayed, applying an in vitro system based on Formvar(®)/n-hexacosane-coated glass slides. n-Hexacosanal was the most effective aldehyde tested. Germination and differentiation rates of powdery mildew conidia increased with increasing concentrations of very-long-chain aldehydes. Relative to n-hexacosanal, the other aldehyde compounds showed a gradual decrease in germination- and differentiation-inducing capabilities with both decreasing and increasing chain length. In addition to n-hexacosanal, several other ubiquitous very-long-chain aldehyde wax constituents were capable of effectively stimulating B. graminis prepenetration processes in a dose- and chain length-dependent manner. Other wax constituents, such as n-alkanes, primary alcohols (with the exception of n-hexacosanol), fatty acids and alkyl esters, did not affect fungal prepenetration.

Two sides of a leaf blade: Blumeria graminis needs chemical cues in cuticular waxes of Lolium perenne for germination and differentiation.

Plant surface characteristics were repeatedly shown to play a pivotal role in plant-pathogen interactions. The abaxial leaf surface of perennial ryegrass (Lolium perenne) is extremely glossy and wettable compared to the glaucous and more hydrophobic adaxial surface. Earlier investigations have demonstrated that the abaxial leaf surface was rarely infected by powdery mildew (Blumeria graminis), even when the adaxial surface was densely colonized. This led to the assumption that components of the abaxial epicuticular leaf wax might contribute to the observed impairment of growth and development of B. graminis conidia on abaxial surfaces of L. perenne. To re-assess this hypothesis, we analyzed abundance and chemical composition of L. perenne ab- and adaxial epicuticular wax fractions. While the adaxial epicuticular waxes were dominated by primary alcohols and esters, the abaxial fraction was mainly composed of n-alkanes and aldehydes. However, the major germination and differentiation inducing compound, the C26-aldehyde n-hexacosanal, was not present in the abaxial epicuticular waxes. Spiking of isolated abaxial epicuticular Lolium waxes with synthetically produced n-hexacosanal allowed reconstituting germination and differentiation rates of B. graminis in an in vitro germination assay using wax-coated glass slides. Hence, the absence of the C26-aldehyde from the abaxial surface in combination with a distinctly reduced surface hydrophobicity appears to be primarily responsible for the failure of normal germling development of B. graminis on the abaxial leaf surfaces of L. perenne.

Biodiversity of arbuscular mycorrhizal fungi in roots and soils of two salt marshes.

The occurrence of arbuscular mycorrhizal fungi (AMF) was assessed by both morphological and molecular criteria in two salt marshes: (i) a NaCl site of the island Terschelling, Atlantic Coast, the Netherlands and (ii) a K(2)CO(3) marsh at Schreyahn, Northern Germany. The overall biodiversity of AMF, based on sequence analysis, was comparably low in roots at both sites. However, the morphological spore analyses from soil samples of both sites exhibited a higher AMF biodiversity. Glomus geosporum was the only fungus of the Glomerales that was detected both as spores in soil samples and in roots of the AMF-colonized salt plants Aster tripolium and Puccinellia sp. at both saline sites and on all sampling dates (one exception). In roots, sequences of Glomus intraradices prevailed, but this fungus could not be identified unambiguously from DNA of soil spores. Likewise, Glomus sp. uncultured, only deposited as sequence in the database, was widely detected by DNA sequencing in root samples. All attempts to obtain the corresponding sequences from spores isolated from soil samples failed consistently. A small sized Archaeospora sp. was detected, either/or by morphological and molecular analyses, in roots or soil spores, in dead AMF spores or orobatid mites. The study noted inconsistencies between morphological characterization and identification by DNA sequencing of the 5.8S rDNA-ITS2 region or part of the 18S rDNA gene. The distribution of AMF unlikely followed the salt gradient at both sites, in contrast to the zone formation of plant species. Zygotes of the alga Vaucheria erythrospora (Xanthophyceae) were retrieved and should not be misidentified with AMF spores.

Plant surface wax affects parasitoid's response to host footprints.

The plant surface is the substrate upon which herbivorous insects and natural enemies meet and thus represents the stage for interactions between the three trophic levels. Plant surfaces are covered by an epicuticular wax layer which is highly variable depending on species, cultivar or plant part. Differences in wax chemistry may modulate ecological interactions. We explored whether caterpillars of Spodoptera frugiperda, when walking over a plant surface, leave a chemical trail (kairomones) that can be detected by the parasitoid Cotesia marginiventris. Chemistry and micromorphology of cuticular waxes of two barley eceriferum wax mutants (cer-za.126, cer-yp.949) and wild-type cv. Bonus (wt) were assessed. The plants were then used to investigate potential surface effects on the detectability of caterpillar kairomones. Here we provide evidence that C. marginiventris responds to chemical footprints of its host. Parasitoids were able to detect the kairomone on wild-type plants and on both cer mutants but the response to cer-yp.949 (reduced wax, high aldehyde fraction) was less pronounced. Experiments with caterpillar-treated wt and mutant leaves offered simultaneously, confirmed this observation: no difference in wasp response was found when wt was tested against cer-za.126 (reduced wax, wt-like chemical composition) but wt was significantly more attractive than cer-yp.949. This demonstrates for the first time that the wax layer can modulate the detectability of host kairomones.

Drought, but not salinity, determines the apparent effectiveness of halophytes colonized by arbuscular mycorrhizal fungi.

The halophytes Plantago maritima, Aster tripolium, Artemisia santonicum, Puccinellia limosa, Festuca pseudovina and Lepidium crassifolium from two different saline soils of the Hungarian steppe were examined for colonization by arbuscular mycorrhizal fungi (AMF). The salt aster (A. tripolium) and the sea plantain (P. maritima) were examined more thoroughly by recording root colonization parameters, the salt content in the soil and monthly precipitations in 2001 and 2002. Mycorrhizal colonization was maximal in late spring to early summer and had a second peak later in the autumn. Arbuscule formation and overall mycorrhizal colonization appeared to be inversely correlated with the intensity of rainfall at the investigated sites. The results suggest that, in addition to seasonality, drought may play an important role in governing mycorrhizal activity in saline habitats. In greenhouse experiments, conditions in which AMF could overcome the inhibitory effects of sodium chloride on establishing plant-mycorrhizal symbiosis were not met.

Host surface properties affect prepenetration processes in the barley powdery mildew fungus.

The initial contact between Blumeria graminis f.sp. hordei and its host barley (Hordeum vulgare) takes place on epicuticular waxes at the surfaces of aerial plant organs. Here, the extent to which chemical composition, crystal structure and hydrophobicity of cuticular waxes affect fungal prepenetration processes was explored. The leaf surface properties of barley eceriferum (cer) wax mutants were characterized in detail. Barley leaves and artificial surfaces were used to investigate the early events of fungal infection. Even after epicuticular waxes had been stripped away, cer mutant leaf surfaces did not affect fungal prepenetration properties. Removal of total leaf cuticular waxes, however, resulted in a 20% reduction in conidial germination and differentiation. Two major components of barley leaf wax, hexacosanol and hexacosanal, differed considerably in their ability to effectively trigger conidial differentiation on glass surfaces. While hexacosanol, attaining a maximum hydrophobicity with contact angles of no more than 80 degrees, proved to be noninductive, hexacosanal significantly stimulated differentiation in c. 50% of B. graminis conidia, but only at contact angles > 80 degrees. These results, together with an observed inductive effect of highly hydrophobic, wax-free artificial surfaces, provide new insights into the interplay of physical and chemical surface cues involved in triggering prepenetration processes in B. graminis.

The developmental pattern of tomato fruit wax accumulation and its impact on cuticular transpiration barrier properties: effects of a deficiency in a beta-ketoacyl-coenzyme A synthase (LeCER6).

Cuticular waxes play a pivotal role in limiting transpirational water loss across the primary plant surface. The astomatous fruits of the tomato (Lycopersicon esculentum) 'MicroTom' and its lecer6 mutant, defective in a beta-ketoacyl-coenzyme A synthase, which is involved in very-long-chain fatty acid elongation, were analyzed with respect to cuticular wax load and composition. The developmental course of fruit ripening was followed. Both the 'MicroTom' wild type and lecer6 mutant showed similar patterns of quantitative wax accumulation, although exhibiting considerably different water permeances. With the exception of immature green fruits, the lecer6 mutant exhibited about 3- to 8-fold increased water loss per unit time and fruit surface area when compared to the wild type. This was not the case with immature green fruits. The differences in final cuticular barrier properties of tomato fruits in both lines were fully developed already in the mature green to early breaker stage of fruit development. When the qualitative chemical composition of fruit cuticular waxes during fruit ripening was investigated, the deficiency in a beta-ketoacyl-coenzyme A synthase in the lecer6 mutant became discernible in the stage of mature green fruits mainly by a distinct decrease in the proportion of n-alkanes of chain lengths > C(28) and a concomitant increase in cyclic triterpenoids. This shift in cuticular wax biosynthesis of the lecer6 mutant appears to be responsible for the simultaneously occurring increase of water permeance. Changes in cutin composition were also investigated as a function of developmental stage. This integrative functional approach demonstrates a direct relationship between cuticular transpiration barrier properties and distinct chemical modifications in cuticular wax composition during the course of tomato fruit development.

Arbuscular mycorrhiza and heavy metal tolerance.

Arbuscular mycorrhizal fungi (AMF) have repeatedly been demonstrated to alleviate heavy metal stress of plants. The current manuscript summarizes results obtained to date on the colonization of plants by AMF in heavy metal soils, the depositions of heavy metals in plant and fungal structures and the potential to use AMF-plant combinations in phytoremediation, with emphasis on pennycresses (Thlaspi ssp.). The focus of this manuscript is to describe and discuss studies on the expression of genes in plants and fungi under heavy metal stress. The summary is followed by data on differential gene expression in extraradical mycelia (ERM) of in vitro cultured Glomus intraradices Sy167 supplemented with different heavy metals (Cd, Cu or Zn). The expression of several genes encoding proteins potentially involved in heavy metal tolerance varied in their response to different heavy metals. Such proteins included a Zn transporter, a metallothionein, a 90 kD heat shock protein and a glutathione S-transferase (all assignments of protein function are putative). Studies on the expression of the selected genes were also performed with roots of Medicago truncatula grown in either a natural, Zn-rich heavy metal "Breinigerberg" soil or in a non-polluted soil supplemented with 100 microM ZnSO(4). The transcript levels of the genes analyzed were enhanced up to eight fold in roots grown in the heavy metal-containing soils. The data obtained demonstrate the heavy metal-dependent expression of different AMF genes in the intra- and extraradical mycelium. The distinct induction of genes coding for proteins possibly involved in the alleviation of damage caused by reactive oxygen species (a 90 kD heat shock protein and a glutathione S-transferase) might indicate that heavy metal-derived oxidative stress is the primary concern of the fungal partner in the symbiosis.

Development of bioinformatic tools to support EST-sequencing, in silico- and microarray-based transcriptome profiling in mycorrhizal symbioses.

The great majority of terrestrial plants enters a beneficial arbuscular mycorrhiza (AM) or ectomycorrhiza (ECM) symbiosis with soil fungi. In the SPP 1084 "MolMyk: Molecular Basics of Mycorrhizal Symbioses", high-throughput EST-sequencing was performed to obtain snapshots of the plant and fungal transcriptome in mycorrhizal roots and in extraradical hyphae. To focus activities, the interactions between Medicago truncatula and Glomus intraradices as well as Populus tremula and Amanita muscaria were selected as models for AM and ECM symbioses, respectively. Together, almost, 20.000 expressed sequence tags (ESTs) were generated from different random and suppressive subtractive hybridization (SSH) cDNA libraries, providing a comprehensive overview of the mycorrhizal transcriptome. To automatically cluster and annotate EST-sequences, the BioMake and SAMS software tools were developed. In connection with the eNorthern software SteN, plant genes with a predicted mycorrhiza-induced expression were identified. To support experimental transcriptome profiling, macro- and microarray tools have been constructed for the two model mycorrhizae, based either on PCR-amplified cDNAs or 70mer oligonucleotides. These arrays were used to profile the transcriptome of AM and ECM roots under different conditions, and the data obtained were uploaded to the ArrayLIMS and EMMA databases that are designed to store and evaluate expression profiles from DNA arrays. Together, the EST- and transcriptome databases can be mined to identify candidate genes for targeted functional studies.

The bacterium Paenibacillus validus stimulates growth of the arbuscular mycorrhizal fungus Glomus intraradices up to the formation of fertile spores.

Two isolates of Paenibacillus validus (DSM ID617 and ID618) stimulated growth of the arbuscular mycorrhizal fungus Glomus intraradices Sy167 up to the formation of fertile spores, which recolonize carrot roots. Thus, the fungus was capable of completing its life cycle in the absence of plant roots, but relied instead on the simultaneous growth of bacteria. The supernatant of a mixed batch culture of the two P. validus isolates contained raffinose and another, unidentified trisaccharide. Among the oligosaccharides tested, raffinose was most effective in stimulating hyphal mass formation on plates but could not promote growth to produce fertile spores. A suppressive subtractive hybridization library followed by reverse Northern analyses indicated that several genes with products involved in signal transduction are differentially expressed in G. intraradices SY 167 when grown in coculture with P. validus (DSM 3037). The present investigation, while likely representing a significant step forward in understanding the arbuscular mycorrhizal fungus symbioses, also confirms that its optimal establishing and functioning might rely on many, as yet unidentified factors.