Increased sterol biosynthesis in tobacco calli resistant to a triazole herbicide which inhibits demethylation of 14α-methyl sterols

The γ-keto triazole derivative 4,4-dimethyl-1-(2-methoxyphenyl)-1-(1,2,4-triazol-1-yl)-1-penten-3-one is toxic to Nicotiana tabacum L. cv. Xanthi plants or cell cultures. Analysis of the sterol composition of treated wild-type plant material demonstrates that this herbicide is an inhibitor of the C-14α-methyl demethylation process in sterol biosynthesis. Selection experiments, consisting of screening large populations of microcalli derived from UV-mutagenized tobacco protoplasts for resistance to a lethal dose (1 mg · 1^?1) of the γ-keto triazole, have resulted in the recovery of two groups of resistant calli. In the first group, selected calli show a sterol composition in the absence or presence of the inhibitor very similar to that of wild-type sensitive calli, whereas in the second group the main feature of the selected calli is a new sterol profile. These calli present an overproduction of sterols with a concomitant esterification of overproduced metaolites, just as it was demonstrated for calli previously selected in our laboratory for resistance to LAB 170250F, a triazole fungicide (Maillot-Vernier et al., 1991, Mol. Gen. Genet. 231, 33–40).     

Biochemical characterization of a sterol mutant plant regenerated from a tobacco callus resistant to a triazole cytochrome-P-450-obtusifoliol-14-demethylase inhibitor

We report here, for the first time, the biochemical characterization of a plant mutant impaired in sterol biosynthesis. A fertile plant was regenerated from a tobacco callus resistant to LAB170250F, a potent inhibitor of the cytochrome-P450-obtusifoliol-14-demthylase. The resistant callus and the leaves from the regenerated plant are characterized by profound qualitative and quantitative changes in their sterol content. Self-fertilization of this plant yielded seeds with the same biochemical features, indicating that the new phenotype is of mutational origin.     

Genetic study and further biochemical characterization of a tobacco mutant that overproduces sterols

Summary A genetic and biochemical characterization is presented of a tobacco mutant that was previously shown to have an increased sterol content with an accumulation of biosynthetic intermediates. We first show that a precise regulation of the membrane sterol composition occurs in this mutant, via a selective esterification process. Indeed, sterols representing the usual end-products of the biosynthetic pathway are preferably integrated into the membranes as free sterols, whereas most of the intermediates pool is esterified and stored in cytoplasmic lipid droplets. It is further demonstrated that overproduction of sterols by the LAB1-4 mutant is due to a single nuclear and semi-dominant mutation. Finally, increase of biosynthesis and esterification of unusual sterols are shown to be responsible for the resistance of LAB1-4 calli to LAB170 250F, the triazole pesticide used to select this mutant. However, differentiated LAB1-4 tissues do not express the resistance trait, suggesting that sterol biosynthesis might not be the only site of action for the triazole at the plant level.     

Functional expression of Saccharomyces cerevisiae CYP51A1 encoding lanosterol-14-demethylase in tobacco results in bypass of endogenous sterol biosynthetic pathway and resistance to an obtusifoliol-14-demethylase herbicide inhibitor

Nicotiana tabacum protoplasts have been transformed by Agrobacterium tumefaciens containing a T-DNA in which the gene CYP51A1 encoding lanosterol-14-demethylase (LAN14DM) from Saccharomyces cerevisiae is under the control of a cauliflower mosaic virus (CaMV) 35S promoter. Two transformants strongly expressed the LAN14DM as shown by Northern and Western experiments. These transgenic calli were killed by LAB 170250F (LAB) (a phytotoxic fungicide inhibiting both plant obtusifoliol-14-demethylase (OBT14DM) and LAN14DM) but were resistant to γ-ketotriazole (γ-kt), a herbicide which has been shown to inhibit OBT14DM but not LAN14DM at a concentration that was lethal to control calli. However, these transgenic calli were killed by mixtures of γ-kt plus fungicide inhibitors of LAN14DM such as ketoconazole, itraconazole or flusilazole which alone were not effective. Further analysis of the transgenic calli grown in the presence of γ-kt showed that their Δ⁵-sterol content was close to that of untreated control calli obtained from protoplasts transformed with control plasmid; this is in agreement with evidence that the LAN14DM expressed from the transgene could bypass the blocked OBT14DM by using the plant substrate obtusifoliol. In contrast, control calli when treated with γ-kt, displayed a sterol content strongly enriched in 14α-methyl sterols and depressed in physiological Δ⁵-sterols. When the transgenic calli were cultured in mixtures of γ-kt and LAN14DM inhibitors sterol compositions enriched in 14α-methyl sterols were obtained, reflecting a strong inhibition of both ‘endogenous’ OBT14DM and ‘exogenous’ LAN14DM. Taken together these results show that in tobacco calli transformed with CYP51A1, resistance to a triazole herbicide arises from expression of a functional LAN14DM enzyme; its activity in transgenic tissues creates a bypass of the sterol biosynthetic pathway at the 14-demethylase level when this latter is blocked by an OBT14DM herbicide inhibitor.     

Optimized expression and catalytic properties of a wheat obtusifoliol 14alpha-demethylase (CYP51) expressed in yeast. Complementation of erg11Delta yeast mutants by plant CYP51.

CYP51s form the only family of P450 proteins conserved in evolution from prokaryotes to fungi, plants and mammals. In all eukaryotes, CYP51s catalyse 14alpha-demethylation of sterols. We have recently isolated two CYP51 cDNAs from sorghum [Bak, S., Kahn, R.A., Olsen, C. E. & Halkier, B.A. (1997) Plant J. 11, 191-201] and wheat [Cabello-Hurtado, F., Zimmerlin, A., Rahier, A., Taton, M., DeRose, R., Nedelkina, S., Batard, Y., Durst, F., Pallett, K.E. & Werck-Reichhart, D. (1997) Biophys. Biochem. Res. Commun. 230, 381-385]. Wheat and sorghum CYP51 proteins show a high identity (92%) compared with their identity with their fungal and mammalian orthologues (32-39%). Data obtained with plant microsomes have previously suggested that differences in primary sequences reflect differences in sterol pathways and CYP51 substrate specificities between animals, fungi and plants. To investigate more thoroughly the properties of the plant CYP51, the wheat enzyme was expressed in yeast strains overexpressing different P450 reductases as a fusion with either yeast or plant (sorghum) membrane targeting sequences. The endogenous sterol demethylase gene (ERG11) was then disrupted. A sorghum-wheat fusion protein expressed with the Arabidopsis thaliana reductase ATR1 showed the highest level of expression and activity. The expression induced a marked proliferation of microsomal membranes so as to obtain 70 nmol P450.(L culture)-1, with CYP51 representing 1.5% of microsomal protein. Without disruption of the ERG11 gene, the expression level was fivefold reduced. CYP51 from wheat complemented the ERG11 disruption, as the modified yeasts did not need supplementation with exogenous ergosterol and grew normally under aerobic conditions. The fusion plant enzyme catalysed 14alpha-demethylation of obtusifoliol very actively (Km,app = 197 microm, kcat = 1.2 min-1) and with very strict substrate specificity. No metabolism of lanosterol and eburicol, the substrates of the fungal and mammalian CYP51s, nor metabolism of herbicides and fatty acids was detected in the recombinant yeast microsomes. Surprisingly lanosterol (Ks = 2.2 microM) and eburicol (Ks = 2.5 microm) were found to bind the active site of the plant enzyme with affinities higher than that for obtusifoliol (Ks = 289 microM), giving typical type-I spectra. The amplitudes of these spectra, however, suggested that lanosterol and eburicol were less favourably positioned to be metabolized than obtusifoliol. The recombinant enzyme was also used to test the relative binding constants of two azole compounds, LAB170250F and gamma-ketotriazole, which were previously reported to be potent inhibitors of the plant enzyme. The Ks of plant CYP51 for LAB170250F (0.29 microM) and gamma-ketotriazole (0.40 microM) calculated from the type-II sp2 nitrogen-binding spectra were in better agreement with their reported effects as plant CYP51 inhibitors than values previously determined with plant microsomes. This optimized expression system thus provides an excellent tool for detailed enzymological and mechanistic studies, and for improving the selectivity of inhibitory molecules.     

Regulation of Sterol Content in Membranes by Subcellular Compartmentation of Steryl-Esters Accumulating in a Sterol-Overproducing Tobacco Mutant

The study of sterol overproduction in tissues of LAB 1-4 mutant tobacco (Nicotiana tabacum L. cv Xanthi) (P. Maillot-Vernier, H. Schaller, P. Benveniste, G. Belliard [1989] Biochem Biophys Res Commun 165: 125-130) over several generations showed that the overproduction phenotype is stable in calli, with a 10-fold stimulation of sterol content when compared with wild-type calli. However, leaves of LAB 1-4 plants obtained after two steps of self-fertilization were characterized by a mere 3-fold stimulation, whereas calli obtained from these plants retained a typical sterol-overproducing mutant phenotype (i.e. a 10-fold increase of sterol content). These results suggest that the expression of the LAB 1-4 phenotype is dependent on the differentiation state of cells. Most of the sterols accumulating in the mutant tissues were present as steryl-esters, which were minor species in wild-type tissues. Subcellular fractionation showed that in both mutant and wild-type tissues, free sterols were associated mainly with microsomal membranes. In contrast, the bulk of steryl-esters present in mutant tissues was found in the soluble fraction of cells. Numerous lipid droplets were detected in the hyaloplasm of LAB 1-4 cells by cytochemical and cytological techniques. After isolation, these lipid granules were shown to contain steryl-esters. These results show that the overproduced sterols of mutant tissues accumulate as steryl-esters in hyaloplasmic bodies. The esterification process thus allows regulation of the amount of free sterols in membranes by subcellular compartmentation.     

The factors affecting the evolution of the anthocyanin biosynthesis pathway genes in monocot and dicot plant species

Background

The available data demonstrate that even in universal metabolic pathways, some species-specific regulatory features of structural genes are present. For instance, in the anthocyanin biosynthesis pathway (ABP), genes may be regulated by ABP-specific regulatory factors, and their expression levels may be strongly associated with anthocyanin pigmentation, or they may be expressed independently of pigmentation. A dataset of orthologous ABP genes (Chs, Chi, F3h, F3’h, Dfr, Ans) from monocot and dicot plant species that have distinct gene regulation patterns and different types of pollination was constructed to test whether these factors affect the evolution of the genes.

Results

Using a maximum likelihood approach, we demonstrated that although the whole set of the ABP genes is under purifying selection, with greater selection acting on the upstream genes than on the downstream genes, genes from distinct groups of plant species experienced different strengths of selective pressure. The selective pressure on the genes was higher in dicots than in monocots (F3h and further downstream genes) and in pollinator-dependent plants than in pollinator-independent species (Chi and further downstream genes), suggesting an important role of pollination type in the evolution of the anthocyanin biosynthesis gene network. Contrasting effects of the regulation patterns on evolution were detected for the F3h and Dfr genes, with greater selective pressure on the F3h gene in plant species where the gene expression was not strongly associated with pigmentation and greater selective pressure on Dfr in plant species where the gene expression was associated with pigmentation.

Conclusions

We demonstrated the effects of pollination type and patterns of regulation on the evolution of the ABP genes, but the evolution of some of the genes could not be explained in the framework of these factors, such as the weaker selective pressure acting on Chs in species that attract pollinators or the stronger selective pressure on F3h in plant species where the gene expression was not associated with pigmentation. The observations suggest that additional factors could affect the evolution of these genes. One such factor could be an effect of gene duplication with further division of functions among gene copies and relaxed selective pressure acting on them. Additional tests with an appropriate dataset combining data on duplicated gene sequences and their functions in the flavonoid biosynthesis pathway are required to test this hypothesis.

     

Agrobacterium-mediated transformation of Lolium rigidum Gaud.

Lolium rigidum Gaud. is an annual grass grown for forage but also an economically damaging crop weed. A single genotype somatic embryogenic callus line, VLR1-60, was identified from a herbicide susceptible L. rigidum population, VLR1, and proved to be amenable to Agrobacterium tumefaciens-mediated transformation. Somatic embryogenic calli were continuously induced from the meristematic region of VLR1-60 plants multiplied in vitro and the basic tolerance level of VLR1-60 to hygromycin B was determined. A hygromycin phosphotransferase gene was used as a selectable marker for hygromycin B selection. Somatic embryogenic calli derived from in vitro grown vegetative tillers were co-cultivated with the A. tumefaciens strain EHA105 harbouring binary vector carrying reporter genes and selectable marker in the presence of acetosyringone for 3 days. Inoculated calli were recovered on callus proliferation medium containing Timentin? but lacking hygromycin and were then subcultured onto media with hygromycin concentrations increased progressively through time for selection of transformed plant cells. Putative transgenic plants were recovered and integration of transgenes was confirmed by Southern hybridization analysis and by detection of DsRed or GUS activity in transgenic plants. The frequency of plant transformation was 1.3 %. The ability to transform L. rigidum will provide opportunities for functional characterization of genes to improve forage quality and increase our understanding of the evolution of herbicide resistance and of the basic genetics underlying traits that make L. rigidum a damaging crop weed.     

Albinism and cell viability in cycloartenol synthase deficient Arabidopsis

Phenotypes of Arabidopsis thaliana that carry mutations in CYCLOARTENOL SYNTHASE 1 (CAS1) which is required in sterol biosynthesis have been described. Knockout mutant alleles are responsible of a male-specific transmission defect. Plants carrying a weak mutant allele cas1-1 accumulate 2,3-oxidosqualene, the substrate of CAS1, in all analyzed organs. Mutant cas1-1 plants develop albino inflorescence shoots that contain low amount of carotenoids and chlorophylls. The extent of this albinism, which affects Arabidopsis stems late in development, may be modulated by the light/dark regime. The fact that chloroplast differentiation and pigment accumulation in inflorescence shoots are associated with a low CAS1 expression could suggest the involvement of 2,3-oxidosqualene in a yet unknown regulatory mechanism linking the sterol biosynthetic segment, located in the cytoplasm, and the chlorophyll and carotenoid biosynthetic segments, located in the plastids, in the highly complex terpenoid network. CAS1 loss of function in a mosaic analysis of seedlings further demonstrated that leaf albinism associated with an accumulation of 2,3-oxidosqualene is a novel phenotype for plant sterol deficient mutant.Key words: albinism, cell viability, sterol, terpenoid, light     

Light Inhibition of Shoot Regeneration Is Regulated by Endogenous Abscisic Acid Level in Calli Derived from Immature Barley Embryos

Shoot regeneration in calli derived from immature barley embryos is regulated by light conditions during the callus-induction period. Barley cultivars Kanto Nijo-5 (KN5) and K-3 (K3) showed lower efficiency of shoot regeneration in a 16-h photoperiod during callus-induction than those in continuous darkness, whereas shoot regeneration was enhanced in cultures under a 16-h photoperiod in Golden Promise (GP) and Lenins (LN). These cultivars were classified as photo-inhibition type (KN5 and K3) or photo-induction type (GP and LN) according to their response to light. Contents of endogenous plant hormones were determined in calli cultured under a 16-h photoperiod and continuous darkness. In photo-inhibition type, higher accumulation of abscisic acid (ABA) was detected in calli cultured under a 16-h photoperiod, whereas calli showed lower levels of endogenous ABA in continuous darkness. However, cultivars of photo-induction type showed lower levels of ABA in calli cultured under both light conditions, similarly to photo-inhibition type in continuous darkness. Exogenous ABA inhibited the callus growth and shoot regeneration independent of light conditions in all cultivars. In photo-inhibition type, lower levels of endogenous ABA induced by ABA biosynthesis inhibitor, fluridone, reduced the photo-inhibition of shoot regeneration. Expression of ABA biosynthesis gene, HvNCED1, in calli was regulated by the light conditions. Higher expression was observed in calli cultured under a 16-h photoperiod. These results indicate that ABA biosynthesis could be activated through the higher expression of HvNCED1 in a 16-h photoperiod and that the higher accumulations of ABA inhibit shoot regeneration in the photo-inhibition type cultivars.     

Synthesis and biological evaluation of novel azole derivatives as selective potent inhibitors of brassinosteroid biosynthesis

Brassinosteroids (BRs) are phytohormones that control several important agronomic traits, such as flowering, plant architecture, seed yield, and stress tolerance. To manipulate the BR levels in plant tissues using specific inhibitors of BR biosynthesis, a series of novel azole derivatives were synthesized and their inhibitory activity on BR biosynthesis was investigated. Structure–activity relationship studies revealed that 2RS, 4RS-1-[4-(2-allyloxyphenoxymethyl)-2-(4-chlorophenyl)-[1,3]dioxolan-2-ylmethyl]-1H-[1,2,4]triazole (G₂) is a highly selective inhibitor of BR biosynthesis, with an IC₅₀ value of approximately 46 ± 2 nM, which is the most potent BR biosynthesis inhibitor observed to date. Use of gibberellin (GA) biosynthesis mutants and BR signaling mutants to analyze the mechanism of action of this synthetic series indicated that the primary site of action is BR biosynthesis. Experiments feeding BR biosynthesis intermediates to chemically treated Arabidopsis seedlings suggested that the target sites of this synthetic series are CYP90s, which are responsible for the C-22 and/or C-23 hydroxylation of campesterol.     

Structural Insights into Inhibition of Sterol 14α-Demethylase in the Human Pathogen Trypanosoma cruzi

Trypanosoma cruzi causes Chagas disease (American trypanosomiasis), which threatens the lives of millions of people and remains incurable in its chronic stage. The antifungal drug posaconazole that blocks sterol biosynthesis in the parasite is the only compound entering clinical trials for the chronic form of this infection. Crystal structures of the drug target enzyme, Trypanosoma cruzi sterol 14α-demethylase (CYP51), complexed with posaconazole, another antifungal agent fluconazole and an experimental inhibitor, (R)-4′-chloro-N-(1-(2,4-dichlorophenyl)-2-(1H-imid-azol-1-yl)ethyl)biphenyl-4-carboxamide (VNF), allow prediction of important chemical features that enhance the drug potencies. Combined with comparative analysis of inhibitor binding parameters, influence on the catalytic activity of the trypanosomal enzyme and its human counterpart, and their cellular effects at different stages of the Trypanosoma cruzi life cycle, the structural data provide a molecular background to CYP51 inhibition and azole resistance and enlighten the path for directed design of new, more potent and selective drugs to develop an efficient treatment for Chagas disease.     

Cloning and characterization of the Saccharomyces cerevisiae C-22 sterol desaturase gene,encoding a second cytochrome P-450 involved in ergosterol biosynthesis

The ERG5 gene from Saccharomyces cerevisiae was cloned by complementation of an erg5-1 mutation using a negative selection protocol involving screening for nystatin-sensitive transformants. ERG5 is the putative gene encoding the C-22 sterol desaturase required in ergosterol biosynthesis. The functional gene was localized to a 2.15-kb SacI-EcoRI DNA fragment containing an open reading frame of 538 amino acids (aa). ERG5 contains a 10-aa motif consistent with its role as a cytochrome P-450 (CyP450) enzyme and is similar to a number of mammalian CyP450 enzymes. Gene disruption demonstrates that ERG5 is not essential for cell viability     

Viable somatic hybrids are obtained by direct current electrofusion of chemically aggregated plant protoplasts

Using low levels of polyethylene glycol as aggregating agent, protoplasts of Nicotiana glauca and N. langsdorffii were electropulsed (square wave direct current electric pulses) and on subsequent culture on a selective medium (without phytohormones), calli developed. The hybrid character of some of the calli was demonstrated by direct comparison of the isoelectrophoretic pattern of the ribulose-1, 5-bisphosphate carboxylase subunits with those of the parent plants. In optimum conditions, the electrofusion technique described is about 30-fold more efficient for hybrid production than the conventional polyethylene glycol method when applied to the same plant systems.     

Effects of the Calmodulin Antagonist W7 on Resveratrol Biosynthesis in Vitis amurensis Rupr.

The calmodulin antagonist N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) binds to calmodulzin and inhibits Ca²⁺/calmodulin-regulated enzyme activities. In plant cells, W7 inhibits the activity of calcium-dependent protein kinases (CDPKs)—the major calcium sensors in plants. In the present study, we examined the effect of W7 on increased resveratrol biosynthesis and expression of CDPK and stilbene synthase (STS) genes in a cell culture of Vitis amurensis Rupr. We used coumaric acid (CA), salicylic acid (SA), and phenylalanine (Phe) to increase the content of resveratrol in V. amurensis calli, since its content is low under standard conditions. W7 significantly decreased resveratrol production and expression of STS genes in CA-, SA-, and Phe-treated grape cells. Also, treatment of the V. amurensis calli with SA, Phe, or CA considerably increased expression of VaCDPK1a (with SA, Phe), VaCDPK1L (with SA, Phe), VaCDPK2a (with Phe) genes, and decreased expression of VaCDPK3a (with CA). Addition of W7 to CA-, SA-, and Phe-treated grape cells reversed this effect, resulting in increased VaCDPK3a expression and decreased VaCDPK1a, VaCDPK1L, and VaCDPK2a expression. The results obtained suggest that CDPK activities might play an important role in resveratrol biosynthesis.     

A Sulfonylurea Herbicide Resistance Gene from Arabidopsis thaliana as a New Selectable Marker for Production of Fertile Transgenic Rice Plants

A mutant acetolactate synthase (ALS) gene, csr1-1, isolated from sulfonylurea herbicide-resistant Arabidopsis thaliana, was placed under control of a cauliflower mosaic virus 35S promoter (35S). Rice protoplasts were transformed with the 35S/ALS chimeric gene and regenerated into fertile transgenic rice (Oryza sativa) plants. The 35S/ALS gene was expressed effectively as demonstrated by northern blot hybridization analysis, and conferred to transformed calli at least 200-fold greater chlorsulfuron resistance than nontransformed control calli. Effective selection of 35S/ALS-transformed protoplasts was achieved at extremely low chlorsulfuron concentrations of 10 nm. The results demonstrated that the 35S/ALS gene is an alternative selectable marker for rice protoplast transformation and fertile transgenic rice production. The results also suggest that the mutant form of Arabidopsis ALS enzyme operates normally in rice cells. Thus, the mechanism of protein transport to chloroplast and ALS inhibition by chlorsulfuron is apparently conserved among plant species as diverse as Arabidopsis (dicotyledon) and rice (monocotyledon).     

Cytostatic effects of structurally different ginsenosides on yeast cells with altered sterol biosynthesis and transport

Triterpene glycosides are a diverse group of plant secondary metabolites, consisting of a sterol-like aglycon and one or several sugar groups. A number of triterpene glycosides show membranolytic activity, and, therefore, are considered to be promising antimicrobial drugs. However, the interrelation between their structure, biological activities, and target membrane lipid composition remains elusive. Here we studied the antifungal effects of four Panax triterpene glycosides (ginsenosides) with sugar moieties at the C-3 (ginsenosides Rg3, Rh2), C-20 (compound K), and both (ginsenoside F2) positions in Saccharomyces cerevisiae mutants with altered sterol plasma membrane composition. We observed reduced cytostatic activity of the Rg3 and compound K in the UPC2-1 strain with high membrane sterol content. Moreover, LAM gene deletion reduced yeast resistance to Rg3 and digitonin, another saponin with glycosylated aglycon in the C-3 position. LAM genes encode plasma membrane-anchored StARkin superfamily-member sterol transporters. We also showed that the deletion of the ERG6 gene that inhibits ergosterol biosynthesis at the stage of zymosterol increased the cytostatic effects of Rg3 and Rh2, but not the other two tested ginsenosides. At the same time, in silico simulation revealed that the substitution of ergosterol with zymosterol in the membrane changes the spatial orientation of Rg3 and Rh2 in the membranes. These results imply that the plasma membrane sterol composition defines its interaction with triterpene glycoside depending on their glycoside group position. Our results also suggest that the biological role of membrane-anchored StARkin family protein is to protect eukaryotic cells from triterpenes glycosylated at the C-3 position.     

Effects of Alteria alternata f.sp. lycopersici toxins at different levels of tomato plant cell development

Since the host-specific toxins of Alternaria alternata f. sp. lycopersici play an important role in pathogenesis, they potentially could be applied as selective agents in in vitro selection at the cellular level for disease resistance. Prerequisite for this is that sensitivity to the Alternaria alternata f.sp. lycopersici pathotoxins is manifest at the cellular level. To gain insight into cellular effects of AAL-toxins and into the mechanisms of plant insensitivity to AAL-toxins, effects of AAL-toxins on leaves, leaf discs, roots, calli, suspension cells, minicalli and protoplasts of susceptible and resistant tomato genotypes were studied. In leaves of susceptible genotypes, toxins cause severe necrosis, while in leaves of resistant genotypes necrosis was never observed. Inhibition effects of toxins were observed at all other levels in susceptible and resistant genotypes: toxins inhibited shoot induction on leaf discs, root growth and growth of calli, suspension cells and protoplasts. This indicates a cellular site for AAL-toxins. Differences in sensitivity to AAL-toxins between susceptible and resistant genotypes were observed in leaves and roots, but were not observed during shoot induction on leaf discs, in calli, suspension cells and protoplasts. However, differences in sensitivity to AAL-toxins in roots were at least 20 times less than in leaves. Therefore insensitivity seems related to a higher level of tomato plant differentiation and is most pronounced in leaves.     

Sterol methyltransferase a target for anti-amoeba therapy: towards transition state analog and suicide substrate drug design

Ergosterol biosynthesis pathways essential to pathogenic protozoa growth and absent from the human host offer new chokepoint targets. Here, we present characterization and cell-based interference of Acanthamoeba spp sterol 24-/28-methylases (SMTs) that catalyze the committed step in C₂₈- and C₂₉-sterol synthesis. Intriguingly, our kinetic analyses suggest that 24-SMT prefers plant cycloartenol whereas 28-SMT prefers 24(28)-methylene lophenol in similar fashion to the substrate preferences of land plant SMT1 and SMT2. Transition state analog-24(R,S),25-epiminolanosterol (EL) and suicide substrate 26,27-dehydrolanosterol (DHL) differentially inhibited trophozoite growth with IC₅₀ values of 7 nM and 6 µM, respectively, and EL yielded 20-fold higher activity than reference drug voriconazole. Against either SMT assayed with native substrate, EL exhibited tight binding ∼K_i 9 nM. Alternatively, DHL is methylated at C26 by 24-SMT that thereby, generates intermediates that complex and inactivate the enzyme, whereas DHL is not productively bound to 28-SMT. Steroidal inhibitors had no effect on human epithelial kidney cell growth or cholesterol biosynthesis at minimum amoebicidal concentrations. We hypothesize the selective inhibition of Acanthamoeba by steroidal inhibitors representing distinct chemotypes may be an efficient strategy for the development of promising compounds to combat amoeba diseases.