Transcriptional changes in two types of pre-mycorrhizal roots and in ectomycorrhizas of oak microcuttings inoculated with Piloderma croceum.

The formation of the ectomycorrhiza implies an alteration in gene expression of both the plant and fungal partners, a process which starts before the formation of any symbiotic interface. However, little is known on the regulation pattern occurring in different parts of the root system. Our experimental system consisting of a micropropagated oak with a hierarchical root system was shown to exhibit symbiosis functional traits prior to any mycorrhizal tissue differentiation after the inoculation with the basidiomycete Piloderma croceum. Using a cDNA array, the plant gene regulation was analyzed in the pre-mycorrhizal phase. Seventy-five transcripts showed differential expression in pre-mycorrhizal lateral and principal roots, and both root types exhibited different sets of responsive genes. For transcripts selected according to a statistical analysis, the alteration in gene expression was confirmed by RT-PCR and quantitative real-time PCR. Genes regulated in pre-mycorrhizal lateral roots displayed an almost identical expression in mycorrhizas. In contrast, genes regulated in pre-mycorrhizal principal roots were often regulated differently in ectomycorrhizas. Down-regulation affected most of the regulated genes involved in metabolism, whereas most of the regulated genes related to cell rescue functions, water regulation and defence response were up-regulated. Regulation of such genes could explain the increase of global resistance observed in mycorrhizal plants.

Differential expression of two class III chitinases in two types of roots of Quercus robur during pre-mycorrhizal interactions with Piloderma croceum.

Expression of two plant chitinase genes, representing members of class III chitinases, was studied in Quercus robur roots during interactions in a pre-mycorrhizal stage with the ectomycorrhizal fungus Piloderma croceum. Chitinase gene expression was compared in lateral roots destined to form ectomycorrhiza, and in principal roots that are not directly involved in mycorrhizal interactions. The transcript level of the first chitinase (QrchitIII-1) was upregulated in lateral roots, whereas no significant differential expression was observed in principal roots. The second chitinase (QrchitIII-2) was regulated neither in lateral nor in principal roots in presence of the fungus. Because P. croceum did not induce significant chitinase responses in principal roots, the enhanced expression of QrchitIII-1 in lateral roots after inoculation may be related to some steps in symbiosis ontogenesis.

Effect of coal ash on growth and metal uptake by some selected ectomycorrhizal fungi in vitro.

Six isolates of ectomycorrhizal fungi namely, Laccaria fraterna (EM-1083), Pisolithus tinctorius (EM-1081), Pisolithus tinctorius (EM-1290), Pisolithus tinctorius (EM-1293), Scleroderma verucosum (EM-1283), and Scleroderma cepa (EM-1233), were grown on three variants of coal ash, namely electrostatically precipitated (ESP) ash, pond ash, and bottom ash moistened with Modified Melin-Norkans (MMN) medium in vitro The colony diameter reflected the growth of the isolates on the coal ash. Metal accumulation in the mycelia was assayed by atomic absorption spectrophotometry. Six metals, namely aluminum, cadmium, chromium, iron, lead, and nickel, were selected on the basis of their abundance in coal ash and toxicity potential for the present work. Growth of vegetative mycelium on fly ash variants and metal accumulation data indicated that Pisolithus tinctorius (EM-1290) was the most tolerant among the isolates tested for most of the metals. Since this isolate is known to be mycorrhizal with Eucalyptus, it could be used for the reclamation of coal ash over burdened sites.

Successful inoculation of mature pine with Tricholoma matsutake.

Our finding demonstrates, for the first time, that the roots of mature pine trees can be successfully inoculated with a symbiotic ectomycorrhizal fungus, the valuable matsutake mushroom. Long root segments (ca. 5-10 mm in diameter, ca. 50 cm in length) of 50-year-old Pinus densiflora trees were excavated, washed, auxin-treated (2-5 mg indole butyric acid, IBA, per root) and incubated in moist Spagnum moss. Twelve months later, short roots were regenerated, of which approximately 90% were free of mycorrhizae. Mycorrhiza-free short roots were inoculated with mycelial pieces of Tricholoma matsutake and incubated further in a sterilized substrate. Four-and-a-half months later, roots putatively colonized by Matsutake were sampled near the inoculation points. A T. matsutake-specific ITS-rDNA fragment was amplified by nested PCR from approximately 80% of the root samples analyzed, whereas approximately 66% of the root samples processed for staining with Chlorazol black E displayed characteristic T. matsutake Hartig net structures. These results confirm the symbiotic infection of mature P. densiflora roots by matsutake.

Aluminum as a specific inhibitor of plant TPC1 Ca2+ channels.

In plant cells, Al ion plays dual roles as an inducer and an inhibitor of Ca(2+) influx depending on the concentration. Here, the effects of Al on Ca(2+) signaling were assessed in tobacco BY-2 cells expressing aequorin and a putative plant Ca(2+) channel from Arabidopsis thaliana, AtTPC1 (two-pore channel 1). In wild-type cells (expressing only aequorin), Al treatment induced the generation of superoxide, and Ca(2+) influx was secondarily induced by superoxide. Higher Al concentrations inhibited the Al-stimulated and superoxide-mediated Ca(2+) influx, indicating that Ca(2+) channels responsive to reactive oxygen species (ROS) are blocked by high concentration of Al. H(2)O(2)-induced Ca(2+) influx was also inhibited by Al. Thus, inhibitory action of Al against ROS-induced Ca(2+) influx was confirmed. Similarly, known Ca(2+) channel blockers such as ions of La and Gd inhibited the H(2)O(2)-induced Ca(2+) influx. While La also inhibited the hypoosmotically induced Ca(2+) influx, Al showed no inhibitory effect against the hypoosmotic Ca(2+) influx. The effects of Al and La on Ca(2+) influx were also tested in the cell line overexpressing AtTPC1 and the cell line AtTPC1-dependently cosuppressing the endogenous TPC1 equivalents. Notably, responsiveness to H(2)O(2) was lost in the cosuppression cell line, thus TPC1 channels are required for ROS-responsive Ca(2+) influx. Data also suggested that hypoosmotic shock induces TPC1-independent Ca(2+) influx and Al shows no inhibitory action against the TPC1-independent event. In addition, AtTPC1 overexpression resulted in a marked increase in Al-sensitive Ca(2+) influx, indicating that TPC1 channels participate in osmotic Ca(2+) influx only when overexpressed. We concluded that members of TPC1 channel family are the only ROS-responsive Ca(2+) channels and are the possible targets of Al-dependent inhibition.

The mycorrhizal fungus Tricholoma matsutake stimulates Pinus densiflora seedling growth in vitro.

While it has been suggested that Matsutake mycorrhizae might not be functional and that Matsutake may behave as a saprobic fungus in soil or even have some pathogenic activity on seedlings, we investigated the consequences of Matsutake inoculation on Pinus densiflora growth. Seventy-five days after inoculation, hyphae were anchored on short roots and well-developed Hartig net palmettis were observed. Compared to both control treatments--seedlings treated with distilled water and seedlings treated with autoclaved mycelium--inoculation significantly stimulated seedling total dry weight by 70.9% and 98.0%, respectively. These findings attest that some type of symbiotic relationship must be functional and favour host growth, ruling out claims of pathogenicity under the sterile conditions used here.

The indole alkaloids brucine, yohimbine, and hypaphorine are indole-3-acetic acid-specific competitors which do not alter auxin transport.

The indole alkaloids brucine and yohimbine, just like hypaphorine, counteract indole-3-acetic acid (IAA) activity in seedling roots, root hairs and shoots, but do not appear to alter auxin transport in roots or in cultured cells. In roots, the interactions between IAA and these three alkaloids appear competitive and specific since these molecules interact with IAA but with neither 1-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D), two synthetic auxins. The data reported further support the hypothesis that hypaphorine brucine and yohimbine compete with IAA on some auxin-binding proteins likely to be auxin receptors and that 2,4-D and NAA are not always perceived by the same receptor as IAA or the same component of that receptor. At certain steps of plant development and in certain cells, endogenous indole alkaloids could be involved in IAA activity regulation together with other well-described mechanisms such as conjugation or degradation. Hypaphorine with other active indole alkaloids remaining to be identified, might be regarded as a new class of IAA antagonists.

Hypaphorine, an indole-3-acetic acid antagonist delivered by the ectomycorrhizal fungus Pisolithus tinctorius, induces reorganisation of actin and the microtubule cytoskeleton in Eucalyptus globulus ssp bicostata root hairs.

Hypaphorine, an indole alkaloid from the ectomycorrhizal fungus Pisolithus tinctorius Coker & Couch., counteracts indole-3-acetic acid (IAA) activity and controls the rate of root hair elongation in Eucalyptus globulus ssp. bicostata. The present investigation shows that hypaphorine changes cytoskeletal organisation in elongating root hairs of the host. The actin cytoskeleton was investigated by two different fixation and labelling procedures, which gave similar results. In control root hairs, actin organisation was characterised by (i) an actin cap at the very tip region, (ii) a subapical region with reduced labelling and containing fine actin filaments, and (iii) axial bundles of actin filaments running from the subapical part to the base of the root hair. In the hypaphorine-treated root hairs no actin cap was distinguished. The fine actin filaments occurring in the subapical region were replaced by a few thick actin filament bundles that extended from the subapical region toward the root hair tip. In the hypaphorine-treated hairs the total number of actin filament bundles along most of the root hair length was significantly reduced, presumably due to aggregation of pre-existing actin filaments. The first signs of alteration to the cytoskeleton could be detected as soon as 15 min after hypaphorine treatment. In hypaphorine-treated, but not in control root hairs, a patch of aggregated microtubules regularly occurred at a distance of approximately 10 microm from the tip, possibly as a consequence of changes induced by hypaphorine in the actin cytoskeleton. The hypaphorine-induced aggregations in the actin and microtubule cytoskeletons could stabilise the structure of cytoskeletal elements, which in turn could hinder the vesicle delivery at the tip necessary for elongation. Such cytoskeletal alterations may be a consequence of the antagonism between IAA and hypaphorine. The latter view was supported by restoration of the actin cytoskeleton in hypaphorine-treated root hairs by IAA application.

Aluminum-induced distortion in calcium signaling involving oxidative bursts and channel regulation in tobacco BY-2 cells.

Trivalent cations such as those of Al, La, and Gd are phytotoxic. Our previous works showed that addition of LaCl(3) or GdCl(3) to tobacco cells triggers the generation of superoxide (O(2)*-). Here, we show that AlCl(3) at normal physiological pH (5.8) induces much greater production of O(2)*- (detected with a specific chemiluminescence probe), indicating that these trivalent cations similarly induce the oxidative bursts. It was shown that NADPH oxidase is involved in the generation of O(2)*- and the yield of O(2)*- was dose-dependent (ca. 6mM Al, optimal). Following the acute spike of O(2)*-, a gradual increase in cytosolic-free Ca(2+) concentration ([Ca(2+)](c)) was detected with the luminescence of recombinant aequorin over-expressed in the cytosol. Interestingly, a O(2)*- scavenger and a Ca(2+) chelator significantly lowered the level of [Ca(2+)](c) increase, indicating that the Al-induced O(2)*- stimulates the influx of Ca(2+). Compared to the induction of O(2)*- generation, the [Ca(2+)](c) elevation was shown to be maximal (340 nM) at relatively lower Al concentrations (ca. 1.25 mM). Thus, the Al concentration optimal for O(2)*- is too much (inhibitory) for [Ca(2+)](c). In addition, high concentrations of Al were shown to be inhibitory to the H(2)O(2)-induced Ca(2+) influx. This explains the ineffectiveness of high Al concentration in the oxidative burst-mediated induction of [Ca(2+)](c) increase. It is likely that Al-induced [Ca(2+)](c) elevation is manifested from the finely geared balance between the O(2)*- -mediated driving force and the channel inhibition-mediated brake. Furthermore, it is note-worthy that Al (< or =10mM) showed no inhibitory effect on the hypo-osmolarity-induced Ca(2+) influx, implying that Al may be a selective inhibitor of redox-responsive Ca(2+) channels. Possible target channels of Al actions are discussed.

Inhibition of the indole-3-acetic acid-induced epinastic curvature in tobacco leaf strips by 2,4-dichlorophenoxyacetic acid.

It has been reported that auxin induces an epinastic growth response in plant leaf tissues. Leaf strips of tobacco (Nicotiana tabacum L. 'Bright Yellow 2') were used to study the effects of indole-3-acetic acid (IAA), the principal form of auxin in higher plants, and a synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), on epinastic leaf curvature. Incubation of leaf strips with 10 micro M IAA resulted in a marked epinastic curvature response. Unexpectedly, 2,4-D showed only a weak IAA-like activity in inducing epinasty. Interestingly, the presence of 2,4-D resulted in inhibition of the IAA-dependent epinastic curvature. In vivo Lineweaver-Burk kinetic analysis clearly indicated that the interaction between IAA and 2,4-D reported here is not a result of competitive inhibition. Using kinetic analysis, it was not possible to determine whether the mode of interaction between IAA and 2,4-D was non-competitive or uncompetitive. 2,4-D inhibits the IAA-dependent epinasty via complex and as yet unidentified mechanisms.

The indolic compound hypaphorine produced by ectomycorrhizal fungus interferes with auxin action and evokes early responses in nonhost Arabidopsis thaliana.

Signals leading to mycorrhizal differentiation are largely unknown. We have studied the sensitivity of the root system from plant model Arabidopsis thaliana to hypaphorine, the major indolic compound isolated from the basidiomycetous fungus Pisolithus tinctorius. This fungi establishes ectomycorrhizas with Eucalyptus globulus. Hypaphorine controls root hair elongation and counteracts the activity of indole-3-acetic acid on root elongation on A. thaliana, as previously reported for the host plant. In addition, we show that hypaphorine counteracts the rapid upregulation by indole-3-acetic acid and 1-naphthalenic-acetic acid of the primary auxin-responsive gene IAA1 and induces a rapid, transient membrane depolarization in root hairs and suspension cells, due to the modulation of anion and K+ currents. These early responses indicate that components necessary for symbiosis-related differentiation events are present in the nonhost plant A. thaliana and provide tools for the dissection of the hypaphorine-auxin interaction.

A fungal auxin antagonist, hypaphorine prevents the indole-3-acetic acid-dependent irreversible inactivation of horseradish peroxidase: inhibition of Compound III-mediated formation of P-670.

Hypaphorine, an indolic alkaloid from an ectomycorrhizal fungus is a putative antagonist of indole-3-acetic acid (IAA) known to inhibit the effect of IAA in growing roots of Eucalyptus seedling. Previously we have used horseradish peroxidase-C (HRP) as a sensitive reporter of IAA-binding to the IAA-binding domain, and reported that hypaphorine specifically inhibits the HRP-catalyzed superoxide generation coupled to oxidation of IAA [Kawano et al., Biochem. Biophys. Res. Commun. 288]. Since binding of IAA to the auxin-binding domain is the key step required for IAA oxidation by HRP, it was assumed that the inhibitory effect of hypaphorine is due to its competitive binding to the auxin-binding domain in HRP. Here, we obtained further evidence in support of our assumption that hypaphorine specifically inhibits binding of IAA to HRP. In this study, HRP arrested at the temporal inactive form known as Compound III was used as a sensitive indicator for binding of IAA to HRP. Addition of IAA to the preformed Compound III resulted in rapid decreases in absorption maxima at 415, 545, and 578 nm characteristic to Compound III, and in turn a rapid increase in absorption maximum at 670 nm representing the formation of P-670, the irreversibly inactivated form of hemoproteins, was induced. In contrast, the IAA-dependent irreversible inactivation of HRP was inhibited in the presence of hypaphorine. In addition, the mode of interaction between IAA and hypaphorine was determined to be competitive inhibition, further confirming that hypaphorine is an IAA antagonist which specifically compete with IAA in binding to the IAA-binding site in plant peroxidases.

Retardation and inhibition of the cation-induced superoxide generation in BY-2 tobacco cell suspension culture by Zn2+ and Mn2+

Salts at high concentrations may cause oxidative damage to plant cells since many studies indicated the involvement of reactive oxygen species in salt-stress response. Recently, we have demonstrated that treatment of tobacco (Nicotiana tabacum) cell suspension culture with various salts result in an immediate burst of superoxide production via activation of NADPH oxidase by ions of alkali metals (Li+, Na+, K+), alkali earth metals (Mg2+, Ca2+) or lanthanides (La3+, Gd3+). In this study, we tested the effect of extracellular supplementation of Zn2+ and Mn2+ on the cation-induced oxidative burst in tobacco cell suspension culture, measured with a superoxide-specific Cypridina luciferin-derived chemiluminescent reagent. Extracellular supplementation of Zn2+ and Mn2+ inhibited the generation of superoxide in response to addition of salts. Although both Zn2+ and Mn2+ inhibited the salt-induced generation of superoxide, the modes of inhibition by those ions seemed to be different since Mn2+ simply inhibited total production of superoxide while Zn2+ inhibited the early phase of superoxide production and induced the slow release of superoxide. Roles of Mn2+ and Zn2+ in protection of plant cells from salt stress, as an effective superoxide scavenger and an effective inhibitor of plasma membrane-bound NADPH oxidase, respectively, are discussed.

Spectroscopic evidence that salicylic acid converts a temporally inactivated form of horseradish peroxidase (compound III) to the irreversibly inactivated verdohemoprotein (P-670).

We obtained spectroscopic evidence in support of salicylate-dependent inactivation of horseradish peroxidase-C. Addition of salicylate to the enzyme arrested at a temporal inactive state (Compound III) in the presence of H2O2, resulted in rapid and irreversible inactivation of the enzyme yielding verdohemoproteins (P-670). Multiple roles for salicylate in peroxidase-catalyzed reactions are discussed.

Spectroscopic evidence in support of horseradish peroxidase compound II-catalyzed oxidation of salicylic acid but not of phenylethylamine.

Salicylic acid and phenylethylamine are putative substrates for naturally occurring reactions for generation of reactive oxygen species, which are catalyzed by plant peroxidases. Here, we used commercially available highly purified horseradish peroxidase-C (HRP-C) as a model enzyme for spectroscopic analysis, and obtained data suggesting that the Compound II form of HRP-C does not utilize phenylethylamine as substrate. In contrast, addition of salicylic acid to Compound II resulted in rapid conversion of Compound II to the native form.

Saprobic potential of Tricholoma matsutake: growth over pine bark treated with surfactants.

Saprotrophic growth of Tricholoma matsutake isolates was investigated over Pinus densiflora bark fragments either on soil or on agar media. Preferential colonization of pine bark fragments by hyphae, in glucose-deprived environments suggested that Matsutake was able to extract some nutrients to sustain its growth. This was confirmed in glucose-free liquid nutrient medium, where bark as sole carbon source significantly stimulated (up to two-fold) growth of T. matsutake isolates. The addition of surfactants (Tween 80 and Tween 40) in liquid medium further stimulated mycelium growth over pine bark by up to 55%. Such growth stimulation was associated with a sharp increase in protein and beta-glucosidase excretion by hyphae in culture filtrates. As T. matsutake has some saprotrophic ability, the initiation and extension of Matsutake Shiro in forest soil might require simultaneously nutrients derived from the host plant and from soil organic compounds. Data reported here may contribute to the formulation of new culture substrates adapted to the co-culture of T. matsutake and its host plant under controlled conditions.

Developmental cross talking in the ectomycorrhizal symbiosis: signals and communication genes.

Development of ectomycorrhizas involves multiple genes that are implicated in a complex series of interdependent, sequential steps. Current research into ectomycorrhiza development and functioning is aimed at understanding this plant-microbe interaction in a framework of the developmental and physiological processes that underlie colonization and morphogenesis. After a brief introduction to the ectomycorrhizal symbiosis, the present article highlights recent work on the early signal exchange taking place between symbionts, and sketches the way functional genomics is altering our thinking about changes in gene expression during the early steps of the ectomycorrhiza development.

Rutin, the phenolglycoside from eucalyptus root exudates, stimulates Pisolithus hyphal growth at picomolar concentrations.

• Ectomycorrhizal hyphal growth is shown to be stimulated by a phenol compound isolated from Eucalyptus globulus ssp. bicostata root exudates, highlighting the importance of phenolics in host-fungal interaction. • HPLC analysis allowed separation and identification of phenolic compounds from Eucalyptus seedling tissues and root exudates. The activity of the flavonol, rutin, was tested on a range of mycorrhizal and saprophytic fungi. • Rutin stimulated Pisolithus hyphal growth by more than twofold, and the fungus responded significantly to concentrations as low as 1 pM; only a few strains responded. • Rutin from Eucalyptus globulus ssp. bicostata root exudates is a flavonoid signal for Pisolithus, and is the first such flavonoid signal identified. A rutin gradient could contribute to orientating hyphal elongation toward the root tip thereby favouring mycorrhizal infection, and might also influence the interaction between fungi in the rhizosphere.

Endomycorrhizae and rhizobial Nod factors both require SYM8 to induce the expression of the early nodulin genes PsENOD5 and PsENOD12A.

We report here that the pea early nodulin genes PsENOD5 and PsENOD12A are induced during the interaction of pea roots and the endomycorrhizal fungus Gigaspora margarita. Using the pea nodulation mutant Sparkle-R25, which is mutated in SYM8, it is shown that SYM8 is essential for the induction of PsENOD5 and PsENOD12Ain pea roots interacting either with Rhizobium or the endomycorrhizal fungus Gigaspora margarita. Our results suggest that mycorrhizal signals activate a signal transduction cascade sharing at least one common step with the Nod factor-activated signal transduction cascade.