Fungal Phytotoxin Research Articles

Biotransformation of the Fungal Phytotoxin Fomannoxin by Soil Streptomycetes

Rhizosphere-associated Streptomyces sp. AcH 505 (AcH 505) promotes infection of Norway spruce (Picea abies) with the pathogenic fungus Heterobasidion abietinum 331, while Streptomyces sp. GB 4-2 (GB 4-2) enhances spruce defense against the fungus. To identify whether these bacteria influence the availability of the fungal phytotoxin fomannoxin and hence spruce infection, we analyzed the fomannoxin yield in H. abietinum 331-AcH 505 dual cultures. Further, the fate of fomannoxin was studied by adding the compound to cultures of AcH 505, GB 4-2 and nine other soil streptomycetes. Culture filtrates were extracted with ethyl acetate and analyzed by HPLC. Structures of novel compounds were elucidated by HPLC-HR-ESI-Orbitrap-MS and NMR spectroscopy. Phytotoxicity of the compounds was determined by in vivo measurement of maximum photosystem II efficiency of Arabidopsis thaliana seedlings. The amount of fomannoxin in H. abietinum 331-AcH 505 dual cultures was reduced compared to axenic fungus cultures and fungus-plant dual cultures. Following addition of fomannoxin to AcH 505 cultures, the compound disappeared and three novel fomannoxin derivatives without phytotoxic activity were detected. Another novel compound, fomannoxin amide, was discovered following fomannoxin addition to GB 4-2 cultures. Nine other streptomycetes converted fomannoxin into fomannoxin acid or fomannoxin amide. Both compounds exhibit the same phytotoxicity as fomannoxin. We, thus, conclude that the streptomycete-mediated modulation of spruce infection with H. abietinum 331 does not depend on the availability of fomannoxin. We further add evidence to the observation that the lipophilic side chain of fomannoxin is an important structural element for its phytotoxicity.

The phytotoxin fusicoccin, a selective stabilizer of 14‐3‐3 interactions?

The study of the structure and dynamics of protein-protein interaction networks has become overwhelming in all biological systems. Most of the biological events are the consequence of several protein-protein interactions finely regulated by covalent modifications and physiological effectors. Moreover, several studies have shown that the complex interactome responsible for the progress and control of vital processes is disturbed in diseases. Besides the basic information on the mechanisms involved in the processes driven by protein-protein interactions, it appears nowadays extremely challenging to study possible regulators of the lifespan of protein networks. Small molecules able to stabilize or to inhibit protein complexes could easily find applications as potential innovative drugs. In this article, we hypothesize that a natural product, the fungal phytotoxin fusicoccin, can play a role as a stabilizer of interactions between 14-3-3 proteins and specific natural targets. A very specific stabilizer molecule is the ideal starting point for the development of a family of structurally related drugs able to selectively tune 14-3-3 interaction with their targets.

Polyphenols as fungal phytotoxins, seed germination stimulants and phytoalexins

This review deals with the sources and chemical and biological characterization of phytotoxic polyphenols produced essentially by pathogenic fungi of forest and crop plants and of weeds. Their potential use as natural herbicides and fungicides is discussed. The use of some polyphenols which could be applied as an alternative method to control parasitic weeds, the so called “suicidal germination”, will be covered. The sources and the isolation and identification of polyphenols produced by some crop plants in consequence of the attack of pathogenic fungi as plant defence compounds (phytoalexins), are also described.

Characterization of the Biosynthetic Genes for 10,11-Dehydrocurvularin, a Heat Shock Response-Modulating Anticancer Fungal Polyketide from Aspergillus terreus

10,11-Dehydrocurvularin is a prevalent fungal phytotoxin with heat shock response and immune-modulatory activities. It features a dihydroxyphenylacetic acid lactone polyketide framework with structural similarities to resorcylic acid lactones like radicicol or zearalenone. A genomic locus was identified from the dehydrocurvularin producer strain Aspergillus terreus AH-02-30-F7 to reveal genes encoding a pair of iterative polyketide synthases (A. terreus CURS1 [AtCURS1] and AtCURS2) that are predicted to collaborate in the biosynthesis of 10,11-dehydrocurvularin. Additional genes in this locus encode putative proteins that may be involved in the export of the compound from the cell and in the transcriptional regulation of the cluster. 10,11-Dehydrocurvularin biosynthesis was reconstituted in Saccharomyces cerevisiae by heterologous expression of the polyketide synthases. Bioinformatic analysis of the highly reducing polyketide synthase AtCURS1 and the nonreducing polyketide synthase AtCURS2 highlights crucial biosynthetic programming differences compared to similar synthases involved in resorcylic acid lactone biosynthesis. These differences lead to the synthesis of a predicted tetraketide starter unit that forms part of the 12-membered lactone ring of dehydrocurvularin, as opposed to the penta- or hexaketide starters in the 14-membered rings of resorcylic acid lactones. Tetraketide N-acetylcysteamine thioester analogues of the starter unit were shown to support the biosynthesis of dehydrocurvularin and its analogues, with yeast expressing AtCURS2 alone. Differential programming of the product template domain of the nonreducing polyketide synthase AtCURS2 results in an aldol condensation with a different regiospecificity than that of resorcylic acid lactones, yielding the dihydroxyphenylacetic acid scaffold characterized by an S-type cyclization pattern atypical for fungal polyketides.

Changes of phenolic secondary metabolite profiles in the reaction of narrow leaf lupin (Lupinus angustifolius) plants to infections with Colletotrichum lupini fungus or treatment with its toxin

Plant interactions with environmental factors cause changes in the metabolism and regulation of biochemical and physiological processes. Plant defense against pathogenic microorganisms depends on an innate immunity system that is activated as a result of infection. There are two mechanisms of triggering this system: basal immunity activated as a result of a perception of microbe-associated molecular patterns through pattern recognition receptors situated on the cell surface and effector-triggered immunity (ETI). An induced biosynthesis of bioactive secondary metabolites, in particular phytoalexins, is one of the mechanisms of plant defense to fungal infection. Results of the study on narrow leaf lupin (Lupinus angustifolius L.) plants infected with the anthracnose fungus Colletotrichum lupini and treated with fungal phytotoxic metabolites are described in the paper. The C. lupini phytotoxins were isolated from liquid cultures, purified and partially characterized with physicochemical methods. Accumulation of secondary metabolites on leaf surface and within the tissues of plants either infected, treated with the fungal phytotoxin or submitted to both treatments was studied using GC-MS and LC-MS, respectively. Substantial differences in isoflavone aglycones and glycoconjugate profiles occurred in response to different ways of plant treatment.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-012-0475-8) contains supplementary material, which is available to authorized users.

Evaluation of in vitro anticancer activity of sphaeropsidins A–C, fungal rearranged pimarane diterpenes, and semisynthetic derivatives

Sphaeropsidins A (1), B (7) and C (10), three fungal phytotoxins, unrearranged pimarane diterpenes produced by Diplodia cupressi and 10 semisynthetic derivatives were evaluated for their in vitro anticancer activities. Among these 13 compounds, sphaeropsidin A and two derivatives (2 and 6) thereof display 50% growth-inhibitory concentration in the low micromolar range for all cell lines analyzed. Structure activity relationship paralleled the phytopathogenic and antimicrobial ones except regarding the vinyl group at C-13 that does not seems to be required as it is for their antipathogenic activity.

Relationships between the stereochemistry and biological activity of fungal phytotoxins

Toxins produced by phytopathogenic fungi assume great importance because of their involvement in several plant diseases. Although such pathogens are known to have seriously damaged crops, forest, and environmental resources, they represent a very important tool to develop new environmentally friendly herbicides and fungicides. This review deals with the relationships between the biological activity of some phytotoxins produced by pathogenic fungi for major forest plants and for damaging weeds and their stereochemistry. In particular, the methods used to determine their relative and/or absolute configuration will be illustrated. These include the application of Mosher's and Murata's methods, X-ray diffractometric analysis, circular dichroism, and the use of computational methods to determine the theoretical optical rotatory power as well as the CD spectrum. The importance of determining the absolute configuration to achieve the total synthesis of some phytotoxins, interesting for their potential practical application, is also discussed.

Evaluation of the anticancer activities of two fungal polycyclic ethanones, alternethanoxins A and B, and two of their derivatives

Alternethanoxins A (1) and B (2) are fungal phytotoxins that are produced by Alternaria sonchi and have been recently characterized as new polycyclic ethanones. Triacetyl (3) and dimethyl (4) derivatives of compound 1 were evaluated together with alternethanoxins for their in vitro growth inhibitory activities in five human and one mouse cancer cell lines in comparison to the reference compound temozolomide (TMZ). Compounds 1-4 and TMZ displayed similar growth inhibitory activities, and these anticancer activities were equivalent in cancer cell lines that display certain levels of resistance to pro-apoptotic stimuli and those that are sensitive to pro-apoptotic stimuli. Of the six cancer cell lines under study, the human esophageal cancer cell line OE21 was the most sensitive to the four polycyclic ethanones. Computer-assisted phase-contrast microscopy (quantitative videomicroscopy) revealed that compounds 1, 2 and 4 displayed cytostatic rather than cytotoxic growth inhibitory effects, while compound 3 appeared to have cytotoxic effects. Thus, this study creates a stimulus for further structure-activity investigations with respect to the anticancer activities of compounds belonging to the alternethanoxin group. The observed toxicity does not seem to be affected by the stereochemistry of C-6 of the B ring, the presence of a hydroxy group at C-1 or the presence of a furan ring joining rings A and C in alternethanoxin B. The anticancer activity (cytostatic versus cytotoxic) of this type of compound could be affected by the chemical moieties present at the hydroxy groups at C-4 and C-6, as was observed for the cytostatic and cytotoxic activities of derivatives 4 and 3, respectively.

Fungal phytotoxins as mediators of virulence

Many phytopathogenic fungi exert their destructive effects by producing and secreting toxic low molecular weight compounds. In the past years a large number of novel fungal virulence factors and their modes of action have been identified. This review highlights effective phytotoxin-mediated strategies to distress, weaken or kill the plant host.

A review of fungal phytotoxins: from basic studies to practical use

Recent materials are summarized, pertaining to classification of fungal phytotoxins, methods of their isolation, and assays for biological activity. Producers of phytotoxic substances have been characterized, and the chemical nature of phytotoxins has been subjected to analysis. The review gives consideration to the mechanisms of action of phytotoxins on susceptible plants and the mechanisms of plant resistance to such agents. Other matters discussed include prospects of utilizing basic knowledge of the nature and mechanisms of action of phytotoxins for (1) developing means of plant protection against diseases and weeds and (2) identifying or classifying fungi (chemosystematics).

Stress-driven discovery of metabolites from the phytopathogenic fungus Leptosphaeria maculans: Structure and activity of leptomaculins A–E

A search for stress inducing metabolites produced by the plant pathogenic fungus Leptosphaeria maculans led to the isolation and structure elucidation of eight new metabolites, the leptomaculins and deacetylleptomaculins A–E. The chemical structures and absolute stereochemistry of the new metabolites were deduced by detailed analysis of 1D and 2D NMR spectroscopic data and chemical degradation of the toxin sirodesmin PL. Leptomaculins A and B are the first examples of naturally occurring 2,3-oxopiperazinethione and 2,3-dioxopiperazine, respectively. Stress inducing activity was found in the fungal phytotoxins sirodesmin PL and deacetylsirodesmin PL but not in any of the new leptomaculins, phomalide or phomamide. A metabolic pathway for biosynthesis of the first 2,3-(di)oxopiperazine(thione) from sirodesmin PL is proposed.

Effect of fusaric acid on biomass and photosynthesis of watermelon seedlings leaves

Wilt disease of watermelon caused by Fusarium oxysporum f. sp. niveum in long-term monoculture system has been much often found particularly in protected land cultivation system worldwide. Fusaric acid (FA), a fungal phytotoxin, was extracted from F. oxysporum f. sp. niveum isolated from diseased watermelon plant. The influence of FA on the biomass and leaves photosynthesis of watermelon seedlings was investigated in a labora- tory study. Results obtained are listed as follows. Plant height, fresh weight, main root length and lateral roots of watermelon seedling exposed to FA (400 mg·L -1 ) for 12 hours and then grown in ordinary condition for nine days were decreased by 23.0%, 23.1%, 23.6% and 33.6% compared with control respectively. Cotyledons were wilted to death completely and necroses occurred on the first true leaf and the upper leaves had become crinkled and chlorosis, which was a typical symptom of wilting disease of watermelon. And thus physiological functions were inhibited and damaged strongly. Net photosynthesis rate, stomatal conductance, intercellular CO2 concentration, and chlorophyll (SPAD reading) of the seedlings decreased with the increasing of concentrations of FA and dura- tion of exposure to FA. Compared with control, stomatal conductances of leaves treated with FA were declined by 87.4-98.4% and net photosynthesis rates of leaves were decreased by 51.8-92.1%. Intercellular CO2 concentration of the seedlings treated with FA was reduced by 66.6 -94.8%. Chlorophyll content in the leaves of the seedlings was decreased by 42.7-72.3%. Transpiration rate fell down to 86.8-96.3%. It was concluded that FA strongly reduced the chlorophyll content of watermelon seedlings leaves resulting to heavy suppression of leaf photosynthesis, which therefore affected seedlings growth finally leading to leaf wilting and necrosis.

Comparative Toxicity of the Phytotoxins (8R,16R)-(−)-Pyrenophorin and (5S,8R,13S,16R)-(−)-Pyrenophorol on Aquatic Organisms

The acute toxicities of the fungal phytotoxins (8R,16R)-(-)-pyrenophorin and (5S,8R,13S,16R)-(-)-pyrenophorol on Vibrio fischeri, Oscillatoria perornata, Pseudokirchneriella subcapitata, Lemna minor and Artemia fransiscana were evaluated. (8R,16R)-(-)-pyrenophorin was more toxic than (5S,8R,13S,16R)-(-)-pyrenophorol to V. fischeri, O. perornata, L. minor and A. fransiscana. The highest acute toxicity of (8R,16R)-(-)-pyrenophorin was exhibited on V. fischeri (5 min median effective concentration of 3.57 M 10(-5)) whereas the corresponding value for (5S,8R,13S,16R)-(-)-pyrenophorol was 801 M 10(-5). P. subcapitata exhibited a lack of sensitivity (median inhibitory concentration of >10 M 10(-5)) to both phytotoxins.

Structural view of a fungal toxin acting on a 14-3-3 regulatory complex.

The fungal phytotoxin fusicoccin stabilizes the interaction between the C-terminus of the plant plasma membrane H(+)-ATPase and 14-3-3 proteins, thus leading to permanent activation of the proton pump. This results in an irreversible opening of the stomatal pore, followed by wilting of plants. Here, we report the crystal structure of the ternary complex between a plant 14-3-3 protein, fusicoccin and a phosphopeptide derived from the C-terminus of the H(+)-ATPase. Comparison with the corresponding binary 14-3-3 complexes indicates no major conformational change induced by fusicoccin. The compound rather fills a cavity in the protein-phosphopeptide interaction surface. Isothermal titration calorimetry indicates that the toxin alone binds only weakly to 14-3-3 and that peptide and toxin mutually increase each others' binding affinity approximately 90-fold. These results are important for herbicide development but might have general implications for drug development, since rather than inhibiting protein-protein interactions, which is difficult to accomplish, it might be easier to reverse the strategy and stabilize protein-protein complexes. As the fusicoccin interaction shows, only low-affinity interactions would be required for this strategy.

Bioactivation of the Fungal Phytotoxin 2,5-Anhydro-D-glucitol by Glycolytic Enzymesisan Essential Component of itsMechanism of Action

An isolate of Fusarium solani, NRRL 18883, produces the natural phytotoxin 2,5-anhydro-D-glucitol (AhG). This fungal metabolite inhibited the growth of roots (150 of 1.6 mM), but it did not have any in vitro inhibitory activity. The mechanism of action of AhG requires enzymatic phosphorylation by plant glycolytic kinases to yield AhG-1,6-bisphosphate (AhG-1,6-bisP), an inhibitor of Fru-1,6-bisP aldolase. AhG-1,6-bisP had an I50 value of 570 microM on aldolase activity, and it competed with Fru-1,6-bisP for the catalytic site on the enzyme, with a Ki value of 103 microm. The hydroxyl group on the anomeric carbon of Fru-1,6-bisP is required for the formation of an essential covalent bond to zeta amino functionality of lysine 225. The absence of this hydroxyl group on AhG-1,6-bisP prevents the normal catalytic function of aldolase. Nonetheless, modeling of the binding of AhG-1,6-bisP to the catalytic pocket shows that the inhibitor interacts with the amino acid residues of the binding site in a manner similar to that of Fru-1,6-bisP. The ability of F. solani to produce a fructose analog that is bioactivated by enzymes of the host plant in order to inhibit a major metabolic pathway illustrates the intricate biochemical processes involved in plant-pathogen interactions.

Phosphorylation-independent interaction between 14-3-3 protein and the plant plasma membrane H+-ATPase.

14-3-3 proteins interact with a novel phosphothreonine motif (Y(946)pTV) at the extreme C-terminal end of the plant plasma membrane H(+)-ATPase molecule. Phosphorylation-independent binding of 14-3-3 protein to the YTV motif can be induced by the fungal phytotoxin fusicoccin. The molecular basis for the phosphorylation-independent interaction between 14-3-3 and H(+)-ATPase in the presence of fusicoccin has been investigated in more detail. Fusicoccin binds to a heteromeric receptor that involves both 14-3-3 protein and H(+)-ATPase. Binding of fusicoccin is dependent upon the YTV motif in the H(+)-ATPase and, in addition, requires residues further upstream of this motif. Apparently, 14-3-3 proteins interact with the unusual epitope in H(+)-ATPase via its conserved amphipathic groove. This implies that very diverse epitopes bind to a common structure in the 14-3-3 protein.

Analysis of the phosphorylation level in guard-cell plasma membrane H+-ATPase in response to fusicoccin.

A fungal phytotoxin fusicoccin (FC) causes irreversible opening of stomata by activation of the plasma membrane H+-ATPase in guard cells. However, the mechanism by which FC activates the H+-ATPase is not fully understood with respect to the event of phosphorylation. In this study, we provide quantitative evidence that FC-dependent activation of H+-ATPase requires the phosphorylation of the C-terminus, and that FC maintains the activated state by preventing the dephosphorylation. The plasma membrane H+-ATPase in guard cells was phosphorylated on serine and threonine residues in the C-termini of both VHA1 and VHA2 by FC, and the phosphorylation level paralleled the rates of H+-pumping and ATP hydrolysis. An endogenous 14-3-3 protein was co-precipitated with the H+-ATPase, and the amount of 14-3-3 protein was proportional to the phosphorylation level of H+-ATPASE: The recombinant 14-3-3 protein bound to the C-terminus only when it was phosphorylated, even in the presence of FC. The phosphorylated C-terminus was dephosphorylated by alkaline phosphatase, and the dephosphorylation was completely prevented when the C-terminus had been incubated with both FC and 14-3-3 protein. The results suggest that FC activates the H+-ATPase by accumulating the complex of phosphorylated H+-ATPase and 14-3-3 protein through inhibition of the dephosphorylation in guard cells.

In planta sequential hydroxylation and glycosylation of a fungal phytotoxin: Avoiding cell death and overcoming the fungal invader

To facilitate plant colonization, some pathogenic fungi produce phytotoxic metabolites that damage tissues; plants may be resistant to a particular pathogen if they produce an enzyme(s) that catalyzes detoxification of this metabolite(s). Alternaria blackspot is one of the most damaging and significant fungal diseases of brassica crops, with no source of resistance known within the Brassica species. Destruxin B is the major phytotoxin produced by the blackspot-causing fungus, Alternaria brassicae (Berkley) Saccardo. We have established that a blackspot-resistant species (Sinapis alba) metabolized (14)C-labeled destruxin B to a less toxic product substantially faster than any of the susceptible species. The first metabolite, hydroxydestruxin B ((14)C-labeled), was further biotransformed to the beta-d-glucosyl derivative at a slower rate. The structures of hydroxydestruxin B and beta-d-glucosyl hydroxydestruxin B were deduced from their spectroscopic data [NMR, high resolution (HR)-MS, Fourier transform infrared (FTIR)] and confirmed by total chemical synthesis. Although these hydroxylation and glucosylation reactions occurred in both resistant (S. alba) and susceptible (Brassica napus, Brassica juncea, and Brassica rapa) species, hydroxylation was the rate limiting step in the susceptible species, whereas glucosylation was the rate limiting step in the resistant species. Remarkably, it was observed that the hydroxydestruxin B induced the biosynthesis of phytoalexins in blackspot-resistant species but not in susceptible species. This appears to be a unique example of phytotoxin detoxification and simultaneous phytoalexin elicitation by the detoxification product. Our studies suggest that S. alba can overcome the fungal invader through detoxification of destruxin B coupled with production of phytoalexins.

Changes in soluble carbohydrate level in Hordeum vulgare (L.) and Festuca pratensis (Huds.) calli treated with metabolites of Bipolaris sorokiniana (Sacc.) Shoem

Calli of spring barley (Hordeum vulgare L.) and meadow fescue (Festuca pratensis Huds.) were treated with metabolites of Bipolaris sorokiniana and then the level of soluble carbohydrates was estimated. Fructose and glucose occurred in the greatest amount in non-treated calli (control). Control tissue of both species responded to a change in culture conditions with fluctuation in the sugar level. Calli treated with fungal phytotoxins demonstrated rapid decrease in sugar content 1, 3 and 24 hrs after elicitation. Fescue calli, as less susceptible, showed moderate increase in carbohydrate level, yet it was still significantly lower than that in control. In barley very small amount of carbohydrates was observed as soon as 24 hrs after elicitation. In the elicited tissue of both species rapid increase in soluble carbohydrate level was noted in the 10th hour. It is suggested that a defence response of barley and fescue takes place in two phases. The 1st phase occurred between the 1st and the 10th hour after elicitation with phytotoxins and it seems to be an adaptation time to this stress factor. This stage is typical for both studied species. The 2nd phase was observed after 10 hrs of pathogenesis. Its course may reflect a various sensitivity degree of both species to B. sorokiniana metabolites.

Complementation of the Saccharomyces cerevisiaePlasma Membrane H+-ATPase by a Plant H+-ATPase Generates a Highly Abundant Fusicoccin Binding Site

Accumulating evidence suggests that the H+-ATPase of the plant plasma membrane is activated by a direct, reversible interaction with 14-3-3 proteins involving the displacement of the C-terminal autoinhibitory domain of the enzyme. The fungal phytotoxin fusicoccin (FC) appears to stabilize this H+-ATPase.14-3-3 complex, thus leading to a persistent activation of the H+-ATPase in vivo. In this study we show that functional replacement of the Saccharomyces cerevisiae H+-ATPase genes by a Nicotiana plumbaginifolia H+-ATPase (pma2) results in the generation of a high affinity fusicoccin binding site that is exceptionally abundant. Acquisition of FC binding capacity is accompanied by a significant increase in the amount of plasma membrane-associated yeast 14-3-3 homologs. The existence of a (plant) PMA2.(yeast)14-3-3 complex was demonstrated using two-dimensional gel systems (native/denaturing). After expression of PMA2 lacking most of its C-terminal region, neither H+-ATPase.14-3-3 complex formation nor FC binding activity could be observed. Furthermore, we obtained direct biochemical evidence for a minimal FC binding complex consisting of the C-terminal PMA2 domain and yeast 14-3-3 homologs. Thus we demonstrated unambiguously the relevance of this regulatory ATPase domain for 14-3-3 interaction as well as its requirement for FC binding.