Phytoalexins from Thlaspi arvense, a wild crucifer resistant to virulent Leptosphaeria maculans: structures, syntheses and antifungal activity

Phytoalexins are inducible chemical defenses produced by plants in response to diverse forms of stress, including microbial attack. Our search for phytoalexins from cruciferous plants resistant to economically important fungal diseases led us to examine stinkweed or pennycress (Thlaspi arvense), a potential source of disease resistance to blackleg. We have investigated phytoalexin production in leaves of T. arvense under abiotic (copper chloride) and biotic elicitation by Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.], and report here two phytoalexins, wasalexin A and arvelexin (4-methoxyindolyl-3-acetonitrile), their syntheses and antifungal activity against isolates of P. lingam/L. maculans, as well as the isolation of isovitexin, a constitutive glycosyl flavonoid of stinkweed, having antioxidant properties but devoid of antifungal activity.     

Methoxycamalexins and related compounds: Syntheses,antifungal activity and inhibition of brassinin oxidase

The phytoalexin camalexin is a competitive inhibitor of brassinin oxidase, an enzyme that detoxifies the phytoalexin brassinin and is produced by an economically important plant pathogen. For this reason, the camalexin scaffold has guided the design of inhibitors of brassinin detoxification. To further understand the structure–activity relationships of camalexin related compounds, the syntheses of monomethoxy and dimethoxycamalexins were undertaken. Four monomethoxy camalexins together with 4,6-dimethoxy and 5,7-dimethoxy camalexins were prepared from the corresponding methoxyindoles using the Ayer's method. The dimethoxy derivatives were prepared from the corresponding dimethoxyindole-3-thiocarboxamides using the Hantzsch reaction; however, this method did not work for the syntheses of 4,6-dimethoxy and 5,7-dimethoxycamalexins due to the lower reactivities of the corresponding indole-3-thiocarboxamides. The antifungal activity and brassinin oxidase inhibitory activity of all methoxycamalexins and ten camalexin related compounds were investigated. Among the 20 compounds evaluated, monomethoxycamalexins were stronger antifungals than the dimethoxy derivatives. However, remarkably, 5,6-dimethoxycamalexin, 6,7-dimethoxycamalexin and 5-methoxycamalexin displayed the strongest inhibitory activity against brassinin oxidase, while 4,5-dimethoxycamalexin displayed no inhibitory effect. Altogether the structure–activity relationships of camalexin related compounds suggest that the targets for fungal growth inhibition and brassinin oxidase inhibition are unrelated and emphasize that brassinin oxidase inhibitors do not need to be antifungal.     

Tenualexin,Other Phytoalexins and Indole Glucosinolates from Wild Cruciferous Species

In general, the chemodiversity of phytoalexins, elicited metabolites involved in plant defense mechanisms against microbial pathogens, correlates with the biodiversity of their sources. In this work, the phytoalexins produced by four wild cruciferous species (Brassica tournefortii, Crambe abyssinica (crambe), Diplotaxis tenuifolia (sand rocket), and Diplotaxis tenuisiliqua (wall rocket)) were identified and quantified by HPLC with photodioarray and electrospray mass detectors. In addition, the production of indole glucosinolates, biosynthetic precursors of cruciferous phytoalexins, was evaluated. Tenualexin, (=2‐(1,4‐dimethoxy‐1H‐indol‐3‐yl)acetonitrile), the first cruciferous phytoalexin containing two MeO substituents in the indole ring, was isolated from D. tenuisiliqua, synthesized, and evaluated for antifungal activity. The phytoalexins cyclobrassinin and spirobrassinin were detected in B. tournefortii and C. abyssinica, whereas rutalexin and 4‐methoxybrassinin were only found in B. tournefortii. D. tenuifolia, and D. tenuisiliqua produced 2‐(1H‐indol‐3‐yl)acetonitriles as phytoalexins. Because tenualexin appears to be one of the broad‐range antifungals occurring in crucifers, it is suggested that D. tenuisiliqua may have disease resistance traits important to be incorporated in commercial breeding programs.     

Metabolism of the phytoalexins camalexins,their bioisosteres and analogues in the plant pathogenic fungus Alternaria brassicicola

The metabolism of the phytoalexins camalexin (1), 1-methylcamalexin (10) and 6-methoxycamalexin (11) by Alternaria brassicicola and their antifungal activity is reported. This work establishes that camalexins are slowly biotransformed (ca. six days) to the corresponding indole-3-thiocarboxamides, which are further transformed to the indole-3-carboxylic acids. These metabolites are substantially less inhibitory to A. brassicicola than the parent camalexins, indicating that these enzyme-mediated transformations are detoxifications. In addition, analyses of the metabolism of synthetic isomers and bioisosteres of camalexin (1) indicate that isomers of camalexin in the thiazole ring are not metabolized. Based on these results, the potential intermediates that lead to formation of indole-3-thiocarboxamides are proposed.     

Phytoalexins and phytoanticipins from the wild crucifers Thellungiella halophila and Arabidopsis thaliana: rapalexin A, wasalexins and camalexin

Investigation of phytoalexin production using abiotic elicitation showed that the phytoalexin rapalexin A was produced by both Thellungiella halophila and Arabidopsis thaliana, but while A. thaliana produced camalexin, T. halophila produced wasalexins A and B and methoxybrassenin B. Considering that the genome of T. halophila is being sequenced currently and that the wasalexin pathway present in T. halophila is expected to involve a number of genes also present in Brassica species, our discovery should facilitate the isolation of genes involved in biosynthetic pathways of phytoalexins of the most economically important crucifer species.     

Procedures for identifying S-allele genotypes of Brassica

Summary Procedures are described for efficient selection of: (1) homozygous and heterozygous S-allele genotypes; (2) homozygous inbreds with the strong self- and sib-incompatibility required for effective seed production of single-cross F₁ hybrids; (3) heterozygous genotypes with the high self- and sib-incompatibility required for effective seed production of 3- and 4-way hybrids.From reciprocal crosses between two first generation inbred (I₁) plants there are three potential results: both crosses are incompatible; one is incompatible and the other compatible; and both are compatible. Incompatibility of both crosses is useful information only when combined with data from other reciprocal crosses. Each compatible cross, depending on whether its reciprocal is incompatible or compatible, dictates alternative reasoning and additional reciprocal crosses for efficiently and simultaneously identifying: (A) the S-allele genotype of all individual I₁ plants, and (B) the expressions of dominance or codominance in pollen and stigma (sexual organs) of an S-allele heterozygous genotype. Reciprocal crosses provide the only efficient means of identifying S-allele genotypes and also the sexual-organ x S-allele-interaction types.Fluorescent microscope assay of pollen tube penetration into the style facilitates quantitation within 24–48 hours of incompatibility and compatibility of the reciprocal crosses. A procedure for quantitating the reciprocal difference is described that maximizes informational content of the data about interactions between S alleles in pollen and stigma of the S-allele-heterozygous genotype.Use of the non-inbred I_o generation parent as a known heterozygous S-allele genotype in crosses with its first generation selfed (I₁) progeny usually reduces at least 7 fold the effort required for achieving objectives 1, 2, and 3, compared to the method of making reciprocal crosses only among I₁ plants.Identifying the heterozygous and both homozygous S-allele genotypes during the I₁ generation facilitates, during subsequent inbred generations, strong selection for or against modifier genes that influence the intensity of self- and sib-incompatibility. Selection for strong self and sib incompatibility can be effective for both homozygous inbreds and also for the S-allele heterozygote, thus facilitating production of single-cross F₁ hybrids and also of 3-and 4-way hybrids.Department of Plant Breeding and Biometry paper No. 690     

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.     

Transformation of the host-selective toxin destruxin B by wild crucifers: probing a detoxification pathway

The destruxin B detoxification pathway present in Sinapis alba is also present in three unrelated species, Camelina sativa, Capsella bursa-pastoris, and Eruca sativa, suggesting a conservation of this pathway across crucifers. The chemical structure of a destruxin B metabolite, (6'-O-malonyl)hydroxydestruxin B beta-D-glucopyranoside, was also establised. Considering that Camelina sativa and Capsella bursa-pastoris detoxify destruxin B and produce the phytoalexins camalexins, these wild crucifers appear to represent unique and perhaps useful sources of blackleg resistance in strategic plant breeding.     

Interaction of cruciferous phytoanticipins with plant fungal pathogens: indole glucosinolates are not metabolized but the corresponding desulfo-derivatives and nitriles are

Glucosinolates represent a large group of plant natural products long known for diverse and fascinating physiological functions and activities. Despite the relevance and huge interest on the roles of indole glucosinolates in plant defense, little is known about their direct interaction with microbial plant pathogens. Toward this end, the metabolism of indolyl glucosinolates, their corresponding desulfo-derivatives, and derived metabolites, by three fungal species pathogenic on crucifers was investigated. While glucobrassicin, 1-methoxyglucobrassicin, 4-methoxyglucobrassicin were not metabolized by the pathogenic fungi Alternaria brassicicola, Rhizoctonia solani and Sclerotinia sclerotiorum, the corresponding desulfo-derivatives were metabolized to indolyl-3-acetonitrile, caulilexin C (1-methoxyindolyl-3-acetonitrile) and arvelexin (4-methoxyindolyl-3-acetonitrile) by R. solani and S. sclerotiorum, but not by A. brassicicola. That is, desulfo-glucosinolates were metabolized by two non-host-selective pathogens, but not by a host-selective. Indolyl-3-acetonitrile, caulilexin C and arvelexin were metabolized to the corresponding indole-3-carboxylic acids. Indolyl-3-acetonitriles displayed higher inhibitory activity than indole desulfo-glucosinolates. Indolyl-3-methanol displayed antifungal activity and was metabolized by A. brassicicola and R. solani to the less antifungal compounds indole-3-carboxaldehyde and indole-3-carboxylic acid. Diindolyl-3-methane was strongly antifungal and stable in fungal cultures, but ascorbigen was not stable in solution and displayed low antifungal activity; neither compound appeared to be metabolized by any of the three fungal species. The cell-free extracts of mycelia of A. brassicicola displayed low myrosinase activity using glucobrassicin as substrate, but myrosinase activity was not detectable in mycelia of either R. solani or S. sclerotiorum.     

The phytoalexins brassilexin and camalexin inhibit cyclobrassinin hydrolase,a unique enzyme from the fungal pathogen Alternaria brassicicola

Alternaria brassicicola is a fungal pathogen of many agriculturally important cruciferous crops. Cyclobrassinin hydrolase (CH) is an enzyme produced by A. brassicicola that catalyzes the transformation of the cruciferous phytoalexin cyclobrassinin into S-methyl[(2-sulfanyl-1H-indolyl-3)methyl]carbamothioate. The purification and characterization of CH was performed using a four-step chromatography method. SDS–PAGE and gel exclusion chromatography indicated that CH is a tetrameric protein with molecular mass of 330 kDa. Sequence analysis and chemical modification of CH with selective reagents suggested that the enzyme mediates hydrolysis of cyclobrassinin using a catalytic amino acid triad. Enzyme kinetic studies using cyclobrassinin and 1-methylcyclobrassinin as substrates revealed that CH displayed positive substrate cooperativity. Investigation of the effect of nine phytoalexins and two derivatives on the activity of CH indicated that six compounds displayed inhibitory activity: brassilexin, 1-methylbrassilexin, dioxibrassinin, camalexin, brassicanal A and sinalexin. The enzyme kinetics of CH strongly suggested that brassilexin and 1-methylbrassilexin are noncompetitive inhibitors of CH activity, and that camalexin is a competitive inhibitor while dioxibrassinin inhibits CH through a mixed mechanism. The phytoalexin brassilexin is the most effective inhibitor of CH (K_i = 32 ± 9 μM). These results suggest that crops able to accumulate higher concentration of brassilexin would display higher resistance levels to the fungus.