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.     

Partial purification of pisatin demethylase,a cytochrome P-450 from the pathogenic fungus Nectria haematococca

The plant pathogen Nectria haematococca can demethylate pisatin, a phytoalexin from pea. Demethylation is apparently necessary for virulence on pea and is catalyzed by a microsomal cytochrome P-450 monooxygenase system. The cytochrome P-450 and NADPH-cytochrome P-450 reductase of this system were solubilized with sodium cholate and partially purified by chromatography on blue A-agarose and -aminohexyl-agarose. The reductase was further purified by chromatography on 2,5-ADP-agarose to a specific activity of about 16 moles cytochrome c reduced per min per mg protein. Upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the reductase fraction contained one major band of molecular weight 84,000. The partially purified cytochrome P-450 fraction contained a number of minor bands and three major bands of molecular weights 52,000, 56,000 and 58,000. This fraction lost all demethylase activity during concentration after -aminohexyl-agarose chromatography, so it could not be purified further. The purified reductase could reconstitute demethylase activity of cytochrome P-450 fractions and appeared to be rate-limiting for demethylase activity in microsomal extracts.     

The biosynthetic pathway of crucifer phytoalexins and phytoanticipins: De novo incorporation of deuterated tryptophans and quasi-natural compounds

Although several biosynthetic intermediates in pathways to cruciferous phytoalexins and phytoanticipins are common, questions regarding the introduction of substituents at N-1 of the indole moiety remain unanswered. Toward this end, we investigated the potential incorporations of several perdeuterated d- and l-1′-methoxytryptophans, d- and l-tryptophans and other indol-3-yl derivatives into pertinent phytoalexins and phytoanticipins (indolyl glucosinolates) produced in rutabaga (Brassica napus L. ssp. rapifera) roots. In addition, we probed the potential transformations of quasi-natural compounds, these being analogues of biosynthetic intermediates that might lead to “quasi-natural” products (products similar to natural products but not produced under natural conditions). No detectable incorporations of deuterium labeled 1′-methoxytryptophans into phytoalexins or glucobrassicin were detected. l-tryptophan was incorporated in a higher percentage than d-tryptophan into both phytoalexins and phytoanticipins. However, in the case of the phytoalexin rapalexin A, both d- and l-tryptophan were incorporated to the same extent. Furthermore, the transformations of both 1′-methylindolyl-3′-acetaldoxime and 1′-methylindolyl-3′-acetothiohydroxamic acid (quasi-natural products) into 1′-methylglucobrassicin but not into phytoalexins suggested that post-aldoxime enzymes in the biosynthetic pathway of indolyl glucosinolates are not substrate-specific. Hence, it would appear that the 1-methoxy substituent of the indole moiety is introduced downstream from tryptophan and that the post-aldoxime enzymes of the glucosinolate pathway are different from the enzymes of the phytoalexin pathway. A higher substrate specificity of some enzymes of the phytoalexin pathway might explain the relatively lower structural diversity among phytoalexins than among glucosinolates.     

Production of phytotoxic spore germination liquids by Alternaria brassicae and A. brassicicola and their effect on species of the family Brassicaceae

Experiments assessed the susceptibility of Brassica spp. and non-Brassica spp. in the family Brassicaceae to infection by Alternaria brassicae and A. brassicicola, and determined the sensitivity of the host species to spore germination liquids (SGLs) produced by the pathogens on B. napus leaves. There was a wide range of sensitivity to the pathogens. Brassica spp. were generally more susceptible, and some non-Brassica spp. (Barbarea vulgaris and Capsella bursa-pastoris) were immune to A. brassicicola. Measurable damage was caused by SGLs but with significant variation between host species. Non-hosts and weak hosts also showed necrosis. It was concluded that, in the case of both pathogens, the toxic factors in these SGLs were host-selective. Selectivity in toxin production was also demonstrated in relation to the host surface or growing medium in which spores germinated. A substantial amount of toxin was produced on all Brassicaceae tested but not on unrelated species (Triticum aestivum, Pisum sativum and Lycopersicon esculentum). Neither pathogen produced measurable amounts of toxin when cultured in Czapek (Dox) broth.     

Molecular cloning and characterization of an amidase from Arabidopsis thaliana capable of converting indole-3-acetamide into the plant growth hormone,indole-3-acetic acid

Acylamidohydrolases from higher plants have not been characterized or cloned so far. AtAMI1 is the first member of this enzyme family from a higher plant and was identified in the genome of Arabidopsis thaliana based on sequence homology with the catalytic-domain sequence of bacterial acylamidohydrolases, particularly those that exhibit indole-3-acetamide amidohydrolase activity. AtAMI1 polypeptide and mRNA are present in leaf tissues, as shown by immunoblotting and RT-PCR, respectively. AtAMI1 was expressed from its cDNA in enzymatically active form and exhibits substrate specificity for indole-3-acetamide, but also some activity against L-asparagine. The recombinant enzyme was characterized further. The results show that higher plants have acylamidohydrolases with properties similar to the enzymes of certain plant-associated bacteria such as Agrobacterium-, Pseudomonas- and Rhodococcus-species, in which these enzymes serve to synthesize the plant growth hormone, indole-3-acetic acid, utilized by the bacteria to colonize their host plants. As indole-3-acetamide is a native metabolite in Arabidopsis thaliana, it can no longer be ruled out that one pathway for the biosynthesis of indole-3-acetic acid involves indole-3-acetamide-hydrolysis by AtAMI1.     

New insight into the biosynthesis and regulation of indole compounds in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In spite of their silent and sessile life, plants are dynamic organisms that have developed advanced defence strategies in their adaptation to the pressure of herbivores and pathogens. Natural plant products play an important role as chemical weapons in this warfare. Characteristic of cruciferous plants is the synthesis of nitrogen- and sulphur-rich compounds, such as glucosinolates (Mikkelsen et al. 2002) and indole alkaloids (Pedras et al. 2000). Glucosinolates are believed to be largely non-toxic, but upon tissue disruption, they are hydrolyzed by endogenous -thioglucosidases (myrosinases) (Rask et al. 2000) to primarily isothiocyanates and nitriles, which have many biological activities. These include not only important roles as repellents against herbivorous insects and microorganisms, but also as volatile attraction of specialized insects (Wittstock and Halkier 2002). For humans, these compounds serve as cancer-preventive agents, biopesticides, and flavor compounds (Talalay and Fahey 2001). Indole alkaloids are phytoalexins and production of specific alkaloids is usually limited to only a few species. Cruciferous plants include the model plant Arabidopsis, which produces the indole alkaloid camalexin. This review will focus on the central role of indole-3-acetaldoxime (IAOx) in the biosynthesis of indole glucosinolates, camalexin, and the phytohormone IAA.     

Structure and biological activity of maculansin A, a phytotoxin from the phytopathogenic fungus Leptosphaeria maculans

During a search for elicitors and phytotoxins produced by virulent isolates of the phytopathogenic fungus Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phomalingam (Tode ex Fr.) Desm.], the selective phytotoxin maculansin A was isolated and its structure determined by analysis of spectroscopic data and chemical degradation. Maculansin A, a unique derivative of mannitol containing the unusual chromophore 2-isocyano-3-methyl-2-butenoyl, was isolated from potato dextrose cultures of L. maculans virulent on canola (Brassica napus L. cv. Westar). Surprisingly, maculansin A was more toxic to resistant plants (B. juncea L. cv. Cutlass, brown mustard) than to susceptible plants (canola). Maculansin A, however, did not elicit the production of phytoalexins either in resistant or susceptible plants. In addition, other maculansin type structures and the metabolite 2,4-dihydroxy-3,6-dimethylbenzaldehyde were isolated and the latter was found to be a strong inhibitor of root growth of both brown mustard and canola. Considering that L. maculans seems to be expanding its host range to infect brown mustard as well, maculansins could assist in chemotaxonomic studies to group the diverse isolates.     

The phytoalexins from cauliflower, caulilexins A, B and C: isolation, structure determination, syntheses and antifungal activity

Our continuous search for phytoalexins from crucifers led us to examine phytoalexin production in florets of cauliflower (Brassica oleracea var. botrytis) under abiotic (UV light) elicitation. Four known (isalexin, S-(-)-spirobrassinin, 1-methoxybrassitin, brassicanal C) and three new (caulilexins A-C) phytoalexins were isolated. The syntheses and antifungal activity of caulilexins A-C against the economically important pathogenic fungi Leptosphaeria maculans, Rhizoctonia solani and Sclerotinia sclerotiorum, and the first synthesis of brassicanal C are reported.     

Metabolism of the phytoalexins medicarpin and maackiain by Fusarium solani

Non-inhibitory concentrations of the pterocarpan phytoalexin medicarpin were completely metabolized by isolates of Fusarium solani f. sp. pisi, f. sp. cucurbitae, f. sp. phaseoli and two other F. solani isolates genetically related to f. sp. pisi during 24 hr of growth in liquid medium. The major metabolic products accumulated without significant further degradation. Medicarpin was modified at one of three adjacent carbon atoms to form either an isoflavanone derivative, a 1a-hydroxydienone derivative or 6a-hydroxymedicarpin. Whereas each isolate degraded medicarpin to one or more metabolises, the isolates varied as to which metabolise they produced. Maackiain, another pterocarpan phytoalexin, was also metabolized by all the isolates to products analogous to those formed from medicarpin. The ability to metabolize medicarpin and maackiain was not always associated with the ability to metabolize pisatin and phaseollin, two other pterocarpan phytoalexins that were degraded by several of the isolates. Tolerance of medicarpin and maackiain was similarly not always associated with tolerance to pisatin.     

In silico analysis of RNA interference components and miRNAs-like RNAs in the seed-borne necrotrophic fungus Alternaria brassicicola

RNA interference is a mechanism of suppressing gene expression in plants, animals and fungi. This regulation mechanism involves three main enzymes, Dicers (Dcr), Argonautes (Ago) and RNA Dependent RNA Polymerases (Rdrp) allowing to produce smallRNAs. RNA interference and smallRNAs have a role in the plant–microorganisms interaction, either in a pathogenic or in a symbiotic relationships. Alternaria brassicicola is a pathogenic fungus of the Brassicaceae plants. During plant infection, it is able to transmit itself vertically and horizontally, giving advantages for new infection and dissemination. To investigate RNA interference and the presence of smallRNAs in A. brassicicola, an in silico analysis was achieved. Two DCR, 4 AGO and 3 RDRP genes were identified comforting the presence of smallRNAs in A. brassicicola. SmallRNA sequencing from wild-type strain and DCR deleted mutants allowed the identifcation of 17 miRNAs in A. brassicicola. The synthesis of these miRNAs is only weakly influenced by the inactivation of DCR genes suggesting the possible existence of an alternative Dicer-independent miRNA synthesis pathway. Target's prediction of A. brassicicola miRNAs identified genes in the fungus and in the plant model Arabidopsis thaliana. Some miRNAs were predicted to target A. thaliana genes involved in the methylation of histone and in the disease resistance.     

Further analogues of plant peptide hormone phytosulfokine-alpha (PSK-alpha) and their biological evaluation.

Phytosulfokine-alpha (PSK-alpha), a sulfated growth factor of structure H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH universally found in both monocotyledons and dicotyledons, strongly promotes proliferation of plant cells in culture. In studies on the structure/activity relationship of PSK-alpha the synthesis was performed of a series of a further 23 analogues modified in position 1, 3 or 4 as well as simultaneously in positions 1 and 3 of the peptide chain. Peptides were synthesized by the solid phase method according to the Fmoc procedure on a Wang-resin. Free peptides were released from the resin by 95% TFA in the presence of EDT. All peptides were tested by competitive binding assay to the carrot membrane using 3H-labelled PSK-alpha according to the test of Matsubayashi et al. Among these peptide analogues, [H-Phe(4-Cl)1]-PSK-alpha (IV), [H-Phe(4-I)1]-PSK-alpha (VII), and [Phe(4-Cl)3]-PSK-alpha (XI) retained 30% PSK-alpha activity. Analogue [Tyr(PO3H2)3]-PSK-alpha (IX) showed 10% of PSK-alpha activity.     

Clonal dispersal and spatial mixing in populations of the plant pathogenic fungus, Sclerotinia sclerotiorum

Two thousand seven hundred and forty-seven isolates of Sclerotinia sclerotiorum were sampled from four field populations of canola in western Canada. Each field was sampled in a grid of 128 50-m 50-m quadrats plus four intensive quadrats each sampled in a diagonal transect. Sampling was done at two phases of the disease cycle: (1) from ascospore inoculum on petals and (2) from disease lesions in stems. A total of 594 unique genotypes was identified by DNA fingerprinting. In each field, a small group of clones represented the majority of the sample, with a large group of clones or genotypes sampled once or twice. Clone frequencies were compared by χ² tests. The difference in profiles of clone frequencies for the two fields sampled in 1991 was not significant, but in 1992 the difference in profiles was marginally significant, indicating some local population substructure. The difference in profiles of clone frequencies for petals and lesions was not significant in each of the two fields sampled in 1991. In each of the two fields sampled in 1992, however, the difference was highly significant, consistent either with selection for some clones or with waves of immigration during the disease cycle. Nine of the 30 most frequently sampled clones from this study were previously recovered in a macrogeographical sample from western Canada in 1990. For spatial analyses, randomization tests indicated no significant spatial aggregation of either clones on petals or clones from lesions. Also, isolates of a clone on petals were not closer to isolates of the same clone from lesions than could be predicted by chance. Both observations suggest spatial mixing of ascospore inoculum from resident or immigrant sources.     

Replication of double-stranded RNA in mycovirus from the plant pathogenic fungus, Fusarium solani

Abstract A mycovirus (named FusoV) from the plant pathogenic fungus Fusarium solani possessed two types of double-stranded (ds) RNA genome, designated Ml and M2. RNA-dependent RNA polymerase activity was detected in FusoV particle fractions. An in vitro RNA polymerase reaction using purified FusoV particles that was supplemented with NTPs revealed the synthesis of single-stranded (ss) RNA species and a subsequent formation of dsRNAs having the same size as Ml and M2. The ssRNA species synthesized in the first stage were proved to be of positive polarity (coding strand) for both M1 and M2 by dot blot hybridization analysis. These results suggest that FusoV genomic dsRNA replicates in a conservative manner.     

Bldesin,the first functionally characterized pathogenic fungus defensin with Kv1.3 channel and chymotrypsin inhibitory activities

Fungus defensin is a kind of important natural peptide resource, such as plectasin from the soil fungus Pseudoplectania nigrella with potential application in the antimicrobial peptide lead drug discovery. Here, a fungus defensin named Bldesin with Kv1.3 channel and serine protease inhibitory activities was first explored. By GST‐Bldesin fusion expression and enterokinase cleaving strategy, recombinant Bldesin was obtained successfully. Antimicrobial assays showed that Bldesin had potent activity against Gram‐positive Staphylococcus aureus, but had no effect on Gram‐negative Escherichia coli. Electrophysiological experiments showed that Bldesin had Kv1.3 channel inhibitory activity. Serine protease inhibitory associated experiments showed that Bldesin had unique chymotrypsin protease inhibitory, elastase protease inhibitory, and serine protease‐associated coagulation inhibitory activities. To the best of our knowledge, Bldesin is the first functionally characterized pathogenic fungus defensin with Kv1.3 channel and chymotrypsin inhibitory activities and highlighted novel pharmacological effects of fungus‐derived defensin peptides.     

Known and estimated distribution in Mexico of Batrachochytrium dendrobatidis,a pathogenic fungus of amphibians

Chytridiomycosis caused by fungus Batrachochytrium dendrobatidis (Bd) is one of the decline global causes of amphibians. Currently, it is distributed throughout a broad range of climates and ecosystems around the world. An epidemic wave of chytridiomycosis began in North America, resulting in population decline and local extinction of many species, reconfiguring species composition of amphibian communities in the Americas. In Mexico, Bd has caused an amphibian population decrease, and its potential distribution area has not been determined. We reviewed the number of species infected, obtaining Bd frequency of infection by land use and vegetation type, and by elevation range. We examined the known distribution of Bd, estimated the potential distribution, and obtained the bioclimate variables relevant for Bd. Our results indicate that in Mexico, Bd has been detected in 78 species of amphibians in 10 families, from 29 different land use and vegetation types, with cloud forest having the highest number of cases (139) and infected species (15). Bd occurs over an elevation range of 1–3,300 m asl and is most frequent at 1,200–1,500 m asl (36%). In addition to the regions previously described as suitable for Bd, our model included desert, coastal, and tropical forest regions, revealing an increase in the area where amphibians could be at risk of infection. Distribution of Bd is mainly associated with temperature of the wettest quarter and potential evapotranspiration of the warmer quarter. We offer an estimate of the ideal conditions for Bd in Mexico, also information for future studies on Bd and the conservation of amphibians. Abstract in Spanish is available with online material.     

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 m 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.     

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.     

Genetic discontinuities and disequilibria in recently established populations of the plant pathogenic fungus Mycosphaerella fijiensis

Dispersal processes of fungal plant pathogens can be inferred from analysis of spatial genetic structures resulting from recent range expansion. The relative importance of long‐distance dispersal (LDD) events vs. gradual dispersal in shaping population structures depends on the geographical scale considered. The fungus Mycosphaerella fijiensis, pathogenic on banana, is an example of a recent worldwide epidemic. Founder effects in this species were detected at both global and continental scale, suggesting stochastic spread of the disease through LDD events. In this study, we analysed the structure of M. fijiensis populations in two recently (∼1979–1980) colonized areas in Costa Rica and Cameroon. Isolates collected in 10–15 sites distributed along a ∼250‐ to 300‐ km‐long transect in each country were analysed using 19 microsatellite markers. We detected low‐to‐moderate genetic differentiation among populations in both countries and isolation by distance in Cameroon. Combined with historical data, these observations suggest continuous range expansion at the scale of banana‐production area through gradual dispersal of spores. However, both countries displayed specific additional signatures of colonization: a sharp discontinuity in gene frequencies was observed along the Cameroon transect, while the Costa Rican populations seemed not yet to have reached genetic equilibrium. These differences in the genetic characteristics of M. fijiensis populations in two recently colonized areas are discussed in the light of historical data on disease spread and ecological data on landscape features.