Ecological Roles of Tryptanthrin, Indirubin and N-Formylanthranilic Acid in Isatis indigotica: Phytoalexins or Phytoanticipins?

Leaves of the plant species Isatis indigotica Fortune ex Lindl. (Chinese woad) produce the metabolites tryptanthrin, indirubin and N-formylanthranilic acid upon spraying with an aqueous solution of copper chloride but not after spraying with water. The antifungal activities of these metabolites against the phytopathogens Alternaria brassicicola, Leptosphaeria maculans and Sclerotinia sclerotiorum established that tryptanthrin is a much stronger growth inhibitor of L. maculans than the phytoalexin camalexin. The biosynthetic precursors of tryptanthrin and N-formylanthranilic acid are proposed based on the deuterium incorporations of isotopically labeled compounds. The overall results suggest that tryptanthrin is a phytoalexin and indirubin and N-formylanthranilic acid are phytoanticipins in the plant species I. indigotica and that chemical diversity and biodiversity are intimately connected.

Plant chemical defenses: are all constitutive antimicrobial metabolites phytoanticipins?

A critical perspective on phytoanticipins, constitutive plant secondary metabolites with defensive roles against microbes is presented. This mini-review focuses on the chemical groups and structural types of defensive plant metabolites thus far not reviewed from the phytoanticipin perspective: i) fatty acid derivatives and polyketides, ii) terpenoids, iii) shikimates, phenylpropanoids and derivatives, and iv) benzylisoquinoline and pyrrolizidine alkaloids. The more traditional groups of phytoanticipins are briefly summarized, with particular focus on the latest results: i) benzoxazinoids, ii) cyanogenic glycosides, iii) glucosinolates and their metabolic products, and iv) saponins. Current evidence suggests that a better understanding of the functions of plant metabolites will drive their application to protect crops against microbial diseases.

Discovery of inhibitors and substrates of brassinin hydrolase: Probing selectivity with dithiocarbamate bioisosteres.

Brassinin hydrolase (BHAb), an inducible enzyme produced by the plant pathogen Alternaria brassicicola under stress conditions, catalyzes the hydrolysis of the methyl dithiocarbamate group of the phytoalexin brassinin, to indolyl-3-methanamine, methane thiol and carbonyl sulfide. Thirty four substrate inspired compounds, bioisosteres of brassinin and a range of related compounds, were evaluated as potential substrates and inhibitors of BHAb for the first time. While six compounds containing thiocarbamate, carbamate and carbonate groups displayed inhibitory activity against BHAb, only two were found to be substrates (thionecarbamate and dithiocarbamate). Methyl naphthalen-1-yl-methyl carbamate, the most potent inhibitor of the six, and methyl N'-(1-methyl-3-indolylmethyl)carbamate inhibited BHAb through a reversible noncompetitive mechanism (K(i)=89±9 and 695±60µM, respectively). Importantly, these carbamate inhibitors were resistant to degradation by A. brassicicola. Carbonates were also inhibitory of BHAb, but a quick degradation by A. brassicicola makes their potential use as crop protectants less likely. Overall, these results indicate that indolyl and naphthalenyl carbamates are excellent lead structures to design new paldoxins that could inhibit the detoxification of brassinin by A. brassicicola.

Maculansins, cryptic phytotoxins from blackleg fungi.

The phytotoxins and other metabolites produced by isolates L2/M2 of the fungal species Leptosphaeria maculans under different culture conditions, together with those of two new, but related isolates are disclosed. The common metabolic characteristics suggest a phylogenetic similarity between these isolates with potential to become widespread in mustard growing areas.

Impact of cruciferous phytoalexins on the detoxification of brassilexin by the blackleg fungus pathogenic to brown mustard.

The biotransformation of brassilexin, a potent phytoalexin produced by brown mustard (Brassica juncea L.), in the presence of various cruciferous phytoalexins was investigated. An important group of isolates of the fungal species Leptosphaeria maculans (Laird 2 and Mayfair 2), which is virulent to brown mustard, but not to canola, was used in this investigation. Brassilexin was detoxified by the fungus, but none of the phytoalexins seemed to affect substantially the rate of brassilexin detoxification; after 12 h of incubation, the amounts of brassilexin remaining in culture were as low as in controls, except in co-incubations with cyclobrassinin and sinalexin, which afforded intermediates that in solution oxidized spontaneously to brassilexin.

Phytotoxins, elicitors and other secondary metabolites from phytopathogenic "blackleg" fungi: structure, phytotoxicity and biosynthesis.

The metabolites produced by the fungal species Leptosphaeria maculans and L. biglobosa under different culture conditions, together with their phytotoxic activities are reviewed. In addition, the biosynthetic studies of blackleg metabolites carried out to date are described and suggestions for species reclassification are provided.

Salt stress induces production of melanin related metabolites in the phytopathogenic fungus Leptosphaeria maculans.

A search for stress metabolites produced by the plant pathogenic fungus Leptosphaeria maculans (asexual stage Phoma lingam) cultured in high salt medium led to the isolation and structure elucidation of two metabolites associated with melanin biosynthesis and cell melanization, a self-protection mechanism against salt stress. The chemical structures of the metabolites were deduced by detailed analysis of 1D and 2D NMR spectroscopic data and chemical transformations.

Remarkable incorporation of the first sulfur containing indole derivative: another piece in the biosynthetic puzzle of crucifer phytoalexins.

The first sulfur labelled compound, [(2)H(4),(34)S]indolyl-3-acetothiohydroxamic acid, is incorporated into the phytoalexins cyclobrassinin and spirobrassinin and the indole glucosinolate glucobrassicin, indicating that both biosynthetic pathways are closely related.

Isosteric probes provide structural requirements essential for detoxification of the phytoalexin brassinin by the fungal pathogen Leptosphaeria maculans.

Brassinin is a plant defense metabolite with antimicrobial activity produced de novo by a variety of Brassica species in response to stress, that is, a phytoalexin. The inhibition of brassinin oxidase (BO), a brassinin-detoxifying enzyme produced by the phytopathogenic fungus Leptosphaeria maculans, is a target in our continuing search for novel crop protection agents. To probe the substrate specificity of BO, in particular the mechanism of the detoxification step, several analogues of brassinin, including functional group isosteres ((mono/dithio)carbamate, urea, and thiourea) and homologue methyl tryptaminedithiocarbamate, were investigated using fungal cultures and purified BO. It was concluded that the essential structural features of substrates of BO were: (i) an -NH at the (mono/dithio)carbamate, urea or thiourea group; (ii) a methylene bridge between indole and the functional group; (iii) a methyl or ethyl group attached to the thiol moiety of the (mono/di)thiocarbamate group. A general stepwise pathway for the oxidation of brassinin was proposed that accounts for the structural requirements of detoxification of brassinin analogues in L. maculans. All compounds that were BO substrates appeared to be oxidized in mycelial cultures to aldehydes, except for the two most polar compounds N'-(3-indolylmethyl)-N''-methylurea and methyl N'-(3-indolylmethyl)carbamate. The substrate specificity of BO suggests that selective inhibitors can be designed for the potential control of L. maculans.

The phytopathogenic fungi Leptosphaeria maculans and Leptosphaeria biglobosa: chemotaxonomical characterization of isolates and metabolite production in different culture media.

Previous molecular chemotaxonomic analyses of isolates of the plant pathogenic fungus Leptosphaeria maculans (Desm.) Ces. et de Not. (asexual stage Phoma lingam (Tode ex Fr.) Desm.) in a chemically defined medium suggested that this species complex was composed of at least three distinct groups. Subsequently, a group within L. maculans was classified as Leptosphaeria biglobosa, on the basis of morphologic characteristics and the lack of sexual crossing. To obtain clarification regarding the metabolite profiles of the various groups or species of blackleg fungi, the objectives of this work were (i) to determine the chemical structures of metabolites produced by Canadian V isolates and Polish-type isolates in potato dextrose broth (PDB) and (ii) to determine the chemotaxonomic relationship among French isolates of L. biglobosa and among Canadian W isolates and Thlaspi isolates of L. maculans. Here, we report for the first time that Canadian V isolates grown in PDB produced 2,4-dihydroxy-3,6-dimethylbenzaldehyde, a metabolite never reported from L. maculans, but none of the usual phytotoxins (sirodesmins). In addition, we report a new metabolite, 2-[2-(5-hydroxybenzofuranyl)]-3-(4-hydroxyphenyl)propanenitrile, from Polish-type isolates of L. maculans grown in PDB and the metabolite profiles of 16 Thlaspi isolates. The metabolite profiles of Thlaspi isolates indicate that these are part of two distinct groups, the Polish W group and the Canadian W group, i.e., L. biglobosa. Finally, we demonstrate that the metabolite profiles of the French isolates classified as L. biglobosa are similar to those of Canadian W isolates.

Metabolism of crucifer phytoalexins in Sclerotinia sclerotiorum: detoxification of strongly antifungal compounds involves glucosylation.

The strongly antifungal phytoalexins brassilexin and sinalexin were metabolized by the stem rot fungus Sclerotinia sclerotiorum to glucosyl derivatives, whereas the phytoalexins brassicanal A, spirobrassinin and 1-methoxyspirobrassinin, displaying lower antifungal activity, were transformed via non-glucosylating pathways. Significantly, these transformations led to metabolites displaying no detectable antifungal activity. The chemical characterization of all new metabolites as well as the chemistry of these processes and a facile chemical synthesis of 1-beta-D-glucopyranosylbrassilexin are reported. Overall, our results indicate that phytoalexins, strongly antifungal against S. sclerotiorum, are detoxified via glucosylation, which in turn suggests that S. sclerotiorum has acquired efficient glucosyltransferase(s) that can disarm some of the most active plant chemical defenses. Consequently, we suggest that these glucosylation reactions are potential metabolic targets to control S. sclerotiorum.

Toward the control of Leptosphaeria maculans: design, syntheses, biological activity, and metabolism of potential detoxification inhibitors of the crucifer phytoalexin brassinin.

Brassinin (1), a crucial plant defense produced by crucifers, is detoxified by the phytopathogenic fungus Leptosphaeria maculans (Phoma lingam) to indole-3-carboxaldehyde using a putative brassinin oxidase. Potential inhibitors of brassinin detoxification were designed by replacement of its dithiocarbamate group (toxophore) with carbamate, dithiocarbonate, urea, thiourea, sulfamide, sulfonamide, dithiocarbazate, amide, and ester functional groups. In addition, the indolyl moiety was substituted for naphthalenyl and phenyl. The syntheses and chemical characterization of these potential detoxification inhibitors, along with their antifungal and cytotoxic activity, as well as screening using cultures of L. maculans are reported. Overall, three types of interaction were observed in cultures of L. maculans co-incubated with the potential inhibitors and brassinin: (1) a decrease on the rate of brassinin detoxification due to the strong inhibitory activity of the compound on fungal growth, (2) a decrease on the rate of brassinin detoxification due to the inhibitory activity of the compound on the putative brassinin oxidase, and (3) a low to no detectable effect on the rate of brassinin detoxification. A noticeable decrease in the rate of brassinin detoxification was observed in the presence of N'-methylbrassinin, methyl N-methyl-N-(naphthalen-2-ylmethyl) dithiocarbamate, tryptophol dithiocarbonate, and methyl 3-phenyldithiocarbazate. Tryptophol dithiocarbonate appeared to be the best inhibitor among the designed compounds, representing the first inhibitor of brassinin detoxification and potentially the first selective protecting agent of oilseed crucifers against L. maculans infestation.

Unprecedented chemical structure and biomimetic synthesis of erucalexin, a phytoalexin from the wild crucifer Erucastrum gallicum.

The isolation, structure determination, total synthesis and antifungal activity of erucalexin, a novel phytoalexin produced by the wild crucifer dog mustard are described. Erucalexin is a structurally unique plant alkaloid, representing the first example of a spiro[2H-indole-2,5'(4'H)-thiazol]-3-one, likely derived from a C-3-C-2 carbon migration in a 3-substituted indolyl nucleus.

Design, synthesis, and antifungal activity of inhibitors of brassilexin detoxification in the plant pathogenic fungus Leptosphaeria maculans.

Potential inhibitors of Leptosphaeria maculans mediated detoxification of the phytoalexin brassilexin were designed and synthesized based on the planar heteroaromatic structure of isothiazolo[5,4-b]indole. Screening of these compounds for inhibition of brassilexin detoxification in cultures of L. maculans indicated that 4-(2-chlorophenyl)isothiazole had the largest effect on the rate of brassilexin detoxification. However, the most antifungal compound among the potential inhibitors, isothiazolo[5,4-b]quinoline, did not appear to affect the metabolism of brassilexin noticeably, suggesting that growth inhibition is not sufficient to slow down the rate of brassilexin detoxification. Furthermore, it was determined that 4-arylisothiazoles as well as isothiazolo[5,4-b]thianaphthene displayed antifungal activity against L. maculans.

Metabolism and detoxification of phytoalexins and analogs by phytopathogenic fungi.

To date, the many examples reporting that fungal pathogens can efficiently detoxify phytoalexins provide strong evidence that the pathogenicity and/or virulence of some fungi is linked to their ability to detoxify their hosts' phytoalexins. The pathways used by plant pathogenic fungi to metabolize and detoxify phytoalexins are reviewed. Prospects for application of recent findings are discussed.

Concise syntheses of the cruciferous phytoalexins brassilexin, sinalexin, wasalexins, and analogues: expanding the scope of the vilsmeier formylation.

Efficient syntheses of the phytoalexins brassilexin, sinalexin, and analogues are demonstrated through the application of the Vilsmeier formylation to indoline-2-thiones followed by a new aqueous ammonia workup procedure. Similarly, a very concise two-pot synthesis of the phytoalexins wasalexins using sequential formylation-amination of indolin-2-ones is described. Remarkably, this novel aqueous ammonia workup allows the sequential one-pot formylation-amination, expanding substantially the scope of the Vilsmeier formylation of both indoline-2-thiones and indolin-2-ones. The examination of the formylation-amination reaction and optimization of conditions, as well as the syntheses and antifungal activities of several brassilexin analogues, are reported.

Detoxification of the cruciferous phytoalexin brassinin in Sclerotinia sclerotiorum requires an inducible glucosyltransferase.

The phytoalexins, brassinin, 1-methoxybrassinin and cyclobrassinin, were metabolized by the stem rot fungus Sclerotinia sclerotiorum into their corresponding glucosyl derivatives displaying no detectable antifungal activity. Importantly, co-incubation of S. sclerotiorum with camalexins, various phytoalexin analogs, and brassinin indicated that a synthetic camalexin derivative could slow down substantially the rate of brassinin detoxification. Furthermore, inducible brassinin glucosyltransferase (BGT) activity was detected in crude cell-free extracts of S. sclerotiorum. BGT activity was induced by the phytoalexin camalexin, and the brassinin analogs methyl tryptamine dithiocarbamate and methyl 1-methyltryptamine dithiocarbamate. The overall results suggest that the fungus S. sclerotiorum in its continuous adaptation and co-evolution with brassinin producing plants, has acquired efficient glucosyltransferase(s) that can disarm some of the most active plant chemical defenses.

Phytoalexins from the crucifer rutabaga: structures, syntheses, biosyntheses, and antifungal activity.

Phytoalexins are inducible chemical defenses produced de novo by plants in response to diverse forms of stress, including microbial attack. Our search for phytoalexins from economically important crucifers lead us to examine rutabaga tubers (Brassica napus L. ssp. rapifera). Three new phytoalexins, named isalexin (9), brassicanate A (10), and rutalexin (11), were isolated together with five known phytoalexins, brassinin (4), 1-methoxybrassinin (5), spirobrassinin (13), brassicanal A (14), and brassilexin (15). The chemical structures of the new phytoalexins were proven by syntheses, and their biological activity against four plant pathogens were determined. Biosynthetic studies using tetra- and pentadeuterated precursors established that indolyl-3-acetaldoxime (22) and brassinin (4) are precursors of brassicanate A (10) and rutalexin (11) and that cyclobrassinin (23) is a biosynthetic precursor of rutalexin (11), whereas tryptamine (24) is not a precursor of rutabaga phytoalexins.

Designer phytoalexins: probing camalexin detoxification pathways in the phytopathogen Rhizoctonia solani.

To probe the specificity of a camalexin detoxifying enzyme(s) produced by Rhizoctonia solani, the putative 5-camalexin hydroxylase (5-CAHY), the naturally occurring phytoalexin 1-methylcamalexin and designer phytoalexins in which the H-5 of camalexin was replaced with either a methyl group or a fluorine atom were synthesised. This investigation showed that biotransformation of 5-fluorocamalexin by R. solani was substantially slower than that of camalexin (12 days vs. six to eight hours), 5-methylcamalexin (5-6 days) or 1-methylcamalexin (5-6 days). Antifungal bioassays showed that 5-fluorocamalexin, 5-methylcamalexin and 1-methylcamalexin were more inhibitory to R. solani than camalexin, whereas their metabolic products displayed substantially lower inhibitory activity. It was concluded that detoxification via oxidation of the indole moiety of camalexins is predominant in the biotransformation of both camalexin and 5-methylcamalexin and likely catalysed by a specific 5-CAHY. By contrast, the pathways for detoxification of 1-methylcamalexin and 5-fluorocamalexin are likely catalysed by non-specific "house-keeping" enzymes. Most importantly, because 1- methylcamalexin showed stronger antifungal activity and was metabolised at substantially slower rate than camalexin this work suggested that, from a plant's perspective 1-methylcamalexin could be a more effective antifungal defence than camalexin.

Phytotoxin production and phytoalexin elicitation by the phytopathogenic fungus Sclerotinia sclerotiorum.

The fungus Sclerotinia sclerotiorum (Lib.) de Bary causes rot disease in a vast range of plant families, including Cruciferae (Brassicaceae). We investigated the production of phytotoxins by S. sclerotiorum by using a bioassay-guided isolation, as well as the phytoalexins produced by the resistant wild crucifer Erucastrum gallicum under elicitation by S. sclerotiorum and other agents. We established for the first time that S. sclerotiorum produces a somewhat selective phytotoxin, sclerin, which is phytotoxic to three cruciferous species (Brassica napus, B. juncea, and Sinapis alba) susceptible to Sclerotinia stem rot disease, causing severe necrosis and chlorosis, but not to a resistant species (Erucastrum gallicum). In addition, we have shown that oleic acid, the major fatty acid isolated from sclerotia of S. sclerotiorum is responsible for the toxic activity of extracts of sclerotia to brine shrimp larvae (Artemia salina). Phytoalexin elicitation in leaves of E. gallicum led to the isolation of three known phytoalexins: indole-3-acetonitrile, arvelexin, and 1-methoxyspirobrassinin. Considering that resistance of E. gallicum to S. sclerotiorum is potentially transferable to B. rapa, a susceptible canola species, and that arvelexin, and 1-methoxyspirobrassinin are not produced by B. rapa, these phytoalexins may become useful markers for resistance against S. sclerotiorum.