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Research Article
Engineering Plant Synthetic Pathways for the Biosynthesis of Novel AntifungalsClick to copy article linkArticle link copied!
- Amy Calgaro-KozinaAmy Calgaro-KozinaDepartment of Chemical Engineering, Stanford University, Stanford, California 94305, United StatesMore by Amy Calgaro-Kozina
- Khanh M. VuuKhanh M. VuuJoint BioEnergy Institute, Emeryville, California 94608, United StatesEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, United StatesMore by Khanh M. Vuu
- Jay D. KeaslingJay D. KeaslingDepartment of Chemical and Biomolecular Engineering, UC Berkeley, Berkeley, California 94720, United StatesDepartment of Bioengineering, UC Berkeley, Berkeley, California 94720, United StatesBiological Systems and Engineering Division, Lawrence Berkeley National Laboratory, One Cyclotron Road, Berkeley, California 94720, United StatesJoint BioEnergy Institute, Emeryville, California 94608, United StatesMore by Jay D. Keasling
- Dominique LoquéDominique LoquéJoint BioEnergy Institute, Emeryville, California 94608, United StatesEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, United StatesMore by Dominique Loqué
- Elizabeth S. Sattely*Elizabeth S. Sattely*E-mail: [email protected]Department of Chemical Engineering, Stanford University, Stanford, California 94305, United StatesHoward Hughes Medical Institute, Stanford University, Stanford, California 94305, United StatesMore by Elizabeth S. Sattely
- Patrick M. Shih*Patrick M. Shih*E-mail: [email protected]Joint BioEnergy Institute, Emeryville, California 94608, United StatesEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, United StatesDepartment of Plant Biology, University of California, Davis, Davis, California 95616, United StatesGenome Center, University of California, Davis, Davis, California 95616, United StatesMore by Patrick M. Shih
Abstract
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Plants produce a wealth of biologically active compounds, many of which are used to defend themselves from various pests and pathogens. We explore the possibility of expanding upon the natural chemical diversity of plants and create molecules that have enhanced properties, by engineering metabolic pathways new to nature. We rationally broaden the set of primary metabolites that can be utilized by the core biosynthetic pathway of the natural biopesticide, brassinin, producing in planta a novel class of compounds that we call crucifalexins. Two of our new-to-nature crucifalexins are more potent antifungals than brassinin and, in some instances, comparable to commercially used fungicides. Our findings highlight the potential to push the boundaries of plant metabolism for the biosynthesis of new biopesticides.
Copyright © 2020 American Chemical Society
Synopsis
We have designed and engineered new synthetic metabolic pathways to create new-to-nature biopesticides with novel anti-fungal activity.
Introduction
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Engineering of metabolic pathways in plants holds great promise for moving specialized metabolism from one plant to another. (1) Beyond direct pathway transfer there is the opportunity to engineer these pathways to generate new-to-nature compounds that are optimized for a particular application. (2) This pathway engineering approach is inspired by the success of natural product analogues and/or derivatives that have been generated for improved activity and/or efficacy in the clinic. Notably, many natural compounds are biosynthesized by plants as biopesticides, protecting them from various pathogens, albeit many times at much lower activity than traditional synthetic pesticides. (3) To explore the limitations of naturally occurring secondary metabolites, we sought to engineer novel biosynthetic pathways for new-to-nature biopesticides, inspired in structure by the natural metabolite brassinin.
The antipathogenic properties of cruciferous phytoalexins often have toxicity on plant cells, and so they are only produced in response to stress. However, phytoalexin pathways can be long, and de novo biosynthesis of a phytoalexin from primary metabolism may be too slow to stop pathogens. (4) To circumvent this problem, many plants constitutively accumulate phytoanticipins, nontoxic phytoalexin precursors which are quickly converted to phytoalexins as part of the stress response. In crucifers, the most common phytoanticipins are glucosinolates, biologically inert metabolites with a variable side chain on an imidothioic acid derivative, with the sulfur bound to glucose and the nitrogen bound to sulfate. (5) When the plant is under pathogen stress, or is physically damaged, the sugar of the glucosinolate is cleaved by a specific β-glucosidase enzyme, called myrosinase. At neutral pH, the aglucone forms a thiocyanate (pH > 7) or, more commonly, an isothiocyanate. (6) In Brassica species and a handful of others, indole isothiocyanates can be further modified into the dithiocarbamate containing molecule brassinin. (4,7)
Brassinin is an indole-sulfur phytoalexin found in several cruciferous crop species (e.g., Brassica rapa, Brassica oleracea) involved in plant disease resistance against various plant pathogens. (8) As with many plant natural products, brassinin has long been studied for its anticancer properties and has been found to inhibit the immune checkpoint enzyme indoleamine 2,3-dioxygenase. (9) Given its potential application in both agriculture and human health, the minimal set of enzymes necessary to engineer brassinin biosynthesis in planta has recently been elucidated. (4) In this pathway, tryptophan is first oxidized to its oxime via CYP79B2, and then tailored to indole glucosinolate. Subsequently, indole glucosinolate is activated to indole isothiocyanate and ultimately converted to the methyldithiocarbamate, brassinin (Figure 1a, Figure S1). Notably, chemically synthesized variations of brassinin have increased efficacy against cancer cell lines, suggesting that slight structural modifications may enhance the bioactive properties of the molecule. (9) In order to biologically produce brassinin alternatives, we aimed to expand the diversity of substrates used by the brassinin biosynthetic machinery. To unlock the full biochemical diversity of the pathway, we tested various CYP79 homologs known to accept alternative amino acid substrates as the first committed enzyme in the pathway, effectively acting as the gatekeeper for pathway specificity. (10,11) Importantly, other CYP79 homologues have been characterized from disparate plant species for the oxidation of a range of amino acids. (12−14)
Figure 1
Figure 1. Engineering of crucifalexins from canonical amino acids. (a) Schematic of metabolic engineering for new-to-nature crucifalexins. For 1-methylpropyl crucifalexin, select enzymes in the crucifalexin pathway were taken from aliphatic ITC biosynthesis (purple arrows) rather than brassinin biosynthesis. There are several enzymes involved in the conversion of the isothiocyanate to brassinin; notably, there is a S-methyltransferase specific to this pathway. (b) LC/MS extracted ion chromatograph for crucifalexins accumulating in N. benthamiana plants engineered with the core crucifalexin pathway and one of several CYP79 enzymes or no heterologous enzymes (wild-type). Each chromatograph is normalized to its largest peak, with the exception of wild-type which is at the same scale as CYP79D3. The following ions were extracted for all chromatographs: m/z 130.0651, brassinin (dedithiocarbamate ion, molecule fragments in source, parent ion not detectable); 164.0562, 1-methylpropyl crucifalexin; 198.0406, benzyl crucifalexin; 214.0355, 4-hydroxybenzyl crucifalexin. (c) Accumulation of multiple crucifalexins in N. benthamiana expressing the core crucifalexin pathway along with CYP79A1, CYP79A2, or CYP79B2 individually, in pairs, or all together. Molecules were detected via LC/MS and quantified using the curve of synthetic standards.
Since the discovery of glucosinolate genes, researchers have engineered their biosynthesis in the native plant and transferred the biosynthesis to heterologous plant hosts with the eventual goal of improving crop nutrition and defense. The first efforts were in 1999, when Bak et al. engineered Arabidopsis thaliana to produce 4-hyroxybenzyl glucosinolate by expressing CYP79A1 from Sorghum bicolor in the plant, demonstrating the native promiscuity of the glucosinolate pathway. (10) CYP79A1 oxidizes tyrosine to 4-hydroxyphenylaldoxime, which is tailored through the A. thaliana glucosinolate pathway, even though this substrate is not naturally present in the plant. CYP79D2 was later introduced into A. thaliana, causing the plant to produce leucine- and isoleucine-derived glucosinolates. (11) While all of these glucosinolates are found in nature, they were previously unreported in A. thaliana. Researchers soon found that expression of these glucosinolates altered the immunity of A. thaliana. Plants overexpressing CYP79A1/2 were more resistant to common bacterial pathogen Pseudomonas syringae, but more susceptible to the crucifer specialist fungi Alternaria brassicicola. Similarly, isopropyl and methylpropyl glucosinolates produced by expression of CYP79D2 increased resistance to soft rot fungus Erwinia carotovora. (15) Although there has been significant research demonstrating the potential in engineering various glucosinolates, these approaches have not been well translated to downstream hydrolysis products of glucosinolates, which have novel bioactive properties.
Results and Discussion
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To test the capacity of the brassinin pathway for the biosynthesis of new-to-nature methyldithiocarbamates, we heterologously expressed the B. rapa pathway in Nicotiana benthamiana leaves, swapping tryptophan-specific CYP79B2 with phenylalanine-oxidizing CYP79A2 from Setaria italica, tyrosine-oxidizing CYP79A1 from Sorghum bicolor, or isoleucine- and valine-oxidizing CYP79D3 from Lotus japonicus. (12−14) Leaves expressing the engineered biosynthesis accumulated the intended new-to-nature molecules, indicating substantial substrate promiscuity across all the enzymes in the pathway (Figure 1b).
For simplicity, we will refer to this expanded class of cruciferous phytoalexins as “crucifalexins” and describe the crucifalexin pathway as the brassinin pathway without the R-group determining CYP79 enzyme. We have accordingly named these new-to-nature molecules benzyl crucifalexin, 4-hydroxybenzyl crucifalexin, and 1-methylpropyl crucifalexin. We also observed accumulation of a putative glycosylated version of 4-hydroxybenzyl crucifalexin with expression of CYP79A1 (Figure S2). Interestingly, we did not observe accumulation of valine-derived propyl crucifalexin with expression of CYP79D3, although we did observe the corresponding intermediate propyl isothiocyanate (Figure S3). In order to optimize metabolic flux through our various crucifalexin pathways, we tested the effect of various enzymes on overall product titers and found that TGG4, a promiscuous myrosinase from A. thaliana, had higher activity for new-to-nature products than the B. rapa myrosinase BABGLU.A (Figure S4).
As these novel molecules likely have different activities, it could be beneficial to engineer a suite of them into a single plant, mimicking the cocktail of phytoalexins produced simultaneously in crucifers like B. rapa and Nasturtium officinale. (7,8) Furthermore, the production of multiple compounds simultaneously may not only provide a defense against different pathogens but also potentially decrease the chances of evolving disease resistance to a single compound. Thus, we attempted to heterologously produce multiple new-to-nature crucifalexins in the same plant by transiently expressing the core pathway with pairwise combinations of BrCYP79B2, SbCYP79A2, and SiCYP79A1, as well as all three together. When two or more CYP79s are expressed, all expected crucifalexins are detected, although their abundance drastically differs from when they are expressed individually (Figure 1c). This is especially apparent in combinations with CYP79A1. Surprisingly, although brassinin is the only “natural” molecule of the three, when all CYP79s were expressed together, 4-hydroxybenzyl crucifalexin production remained relatively fixed, while brassinin and benzyl crucifalexin levels dropped an order of magnitude. This finding suggests that enzyme promiscuity may be high enough to accept non-native substrate at significant rates. However, it is possible that production of all crucifalexins may be limited in this case, especially if the phenol group competitively inhibits one or more enzymes in the pathway.
With the inherent promiscuity of the downstream pathway, it is surprising that more crucifalexins are not naturally made. The lack of CYP79A1 and CYP79D3 homologues in sequenced genomes of brassinin-accumulating plants makes natural 4-hydroxycrucifalexin and 1-methylpropyl crucifalexin unlikely. (16,17) However, CYP79A2 homologues are found in several Brassica genomes, including B. rapa. Our previous work shows that the gene is expressed at low levels in B. rapa leaves accumulating brassinin, but no benzyl glucosinolate or benzyl crucifalexin could be detected in these tissues. (18) Furthermore, intermediate benzyl isothiocyanate does not accumulate in B. rapa, perhaps because it increases susceptibility to the crucifer pathogen Alternaria brassicicola. (15) The innovation of new metabolic pathways has largely been limited to evolving the existing set of genes found in a given plant genome. Thus, synthetic biology provides a means to bypass these natural constraints and engineer novel biosynthetic pathways that might have never evolved on their own.
Given the promiscuity of the crucifalexin pathway, we expanded our efforts to push the capacity of this pathway beyond canonical amino acids. Notably, synthetically prepared halogenated brassinin analogues have been shown to be more bioactive than brassinin and, thus, may have enhanced antifungal properties. (9) To produce halogenated brassinins in planta, we expressed the brassinin pathway in N. benthamiana along with a combination of bacterial halogenases/reductases: PyrH/RebF which produces 5-chlorotryptophan or RebH/RebF, which produces 7-chloro and 7-bromotryptophan (19) (Figure 2a). In these plants we observed that halogenated tryptophan was incorporated into the brassinin pathway. Leaves expressing the halogenated PyrH/RebF pathways accumulate 5-chlorobrassinin. Product formed from the RebH/RebF pathway is proposed to be 7-chlorobrassinin based on the precedence for RebH specificity. Notably, no bromobrassinin was observed in leaves. We reasoned that low levels of bromine in the plant would limit incorporation of this halogen onto tryptophan, and eventually brassinin. Since addition of exogenous bromine in hairy root cultures expressing RebH/RebF resulted in 7-bromotryptophan, (19) we watered RebH/RebF + brassinin expressing plants with potassium bromide (KBr), resulting in detectable levels of 7-bromobrassinin (Figure 2b). Although in planta production of chlorinated brassinins is clearly more practical, the accumulation of 7-bromobrassinin demonstrates the versatility of this pathway. The demonstration of engineering halogenated variants opens the door to future studies exploring the broader use of other newly discovered halogenases, such as the highly promiscuous RadH halogenase. (20)
Figure 2
Figure 2. Engineering of halogenated brassinin derivatives. (a) Proposed halogenated brassinin pathway. Addition of halogenase reductase pairs PyrH/RebH or RebF/RebH produces 5-chlorotryptophan and 7-chlorotryptophan/7-bromotryptophan, respectively. (b) Accumulation of halogenated brassinin in engineered N. benthamiana plants. Selected ion chromatographs from LC/MS analysis of N. benthamiana leaf extracts expressing halogenases PyrH or RebH along with the reductase RebF and the brassinin pathway for production of chlorobrassinin or bromobrassinin (methylindole ions detected, m/z = 164.0262, m/z = 207.9756). Standard is mixture of 1 μM chemically synthesized 5-chlorobrassinin and 10 μM synthesized 7-bromobrassinin. Both RebH and PyrH produce a peak that matches an authentic synthetic standard of 5-chlorobrassinin mass and retention time. Given the specificity of RebH in the production of 7-chlorotryptophan previously reported in the literature [ref (19)], we anticipate that the RebH product is likely the 7-chloro isomer that coelutes with the 5-chlorobrassinin standard.
Several cruciferous plants that produce brassinin often further tailor it to other bioactive phytoalexins, such as spirobrassinin and cyclobrassinin. There are two known enzymes that tailor brassinin: BrCYP71CR1, which makes spirobrassinin, and BrCYP71CR2, which produces cyclobrassinin. (18) Expression of CYP71CR1 or CYP71CR2 in plants engineered with the halogenated brassinin pathway yielded compounds with masses, MS-MS spectra, and isotope ratios matching 5- and 7-chlorospirobrassinin, 7-bromospirobrassinin (Figure S5), 5- and 7-chlorocyclobrassinin, and 7-bromocyclobrassinin (Figure S6). Production of engineered halogenated crucifalexins indicates that halogenated substrates do not severely inhibit any enzymes in the brassinin pathway. The expanded diversity of new substrates that can be utilized by the brassinin pathway underscores the potential to exploit enzyme promiscuity as a means to diversify and engineer novel molecules not found in nature.
Given the expansion of our new engineered crucifalexins, we sought to determine if any of our novel compounds could act as biopesticides against agriculturally relevant fungal pathogens. We chemically synthesized standards of benzyl crucifalexin, 4-hydroxybenzyl crucifalexin, 1-methylpropyl crucifalexin, and 5-chlorobrassinin (Figure S7) and assayed their inhibitory activity against the agriculturally relevant generalist pathogen Botrytis cinerea. (21) Benzyl crucifalexin, 4-hydroxybenzyl crucifalexin, and 1-methylpropyl crucifalexin have a lower inhibitory effect than brassinin (Figure S8). However, 5-chlorobrassinin outperformed brassinin and is comparable to pyrimethanil (p < 0.05)—an active ingredient in commercial pesticides—in rich media. (22) Furthermore, this IC50 is comparable to estimated levels of 5-chlorobrassinin produced heterologously, even without optimization of the pathway for this product (Figure 3). We estimate ∼20 μM production of 5-chlorobrassinin based on dry weight accumulation of 35 ± 13 μg/g as detected on LC/MS. Furthermore, we are producing similar levels of 7-chlorobrassinin (42 ± 4 μg/g dry weight, based on 5-chlorobrassinin standard) and 7-bromobrassin (37 ± 17 μg/g dry weight). It should be noted that the activity of pyrimethanil has been observed to be reduced in rich media given its function as an anilinopyrimidine inhibitor. (23) For additional context, another antifungal, fludioxonil, inhibits the growth of Botrytis isolates with IC50s on the order of 2.5 μM for many isolates. (24)
Figure 3
Figure 3. Inhibition of plant pathogenic fungi. IC50 of brassinin, new-to-nature crucifalexins, and commercial pesticide pyrimethanil against (a) generalist pathogen Botrytis cinerea and (b) crucifer specific pathogen Alternaria brassicicola. Inhibition determined by mycelial growth assay against DMSO control. Error bars represent standard deviation from a minimum of 11 biological replicates measured over two experimental trials. ns, not significant; * p < 0.05, **** p < 0.0001, one-way ANOVA.
To demonstrate broad applicability of these novel metabolites, we also assayed their activity against the crucifer specialist Alternaria brassicicola (Figure S9). Both benzyl crucifalexin and 5-chlorobrassinin outperformed brassinin, with benzyl crucifalexin having the greatest inhibitory effect. Our findings demonstrate a means to produce new-to-nature crucifalexins that have improved efficacies over their natural analogue. Future efforts may optimize the large-scale production of these new compounds or directly engineer our synthetic metabolic pathways into crop plants as a complete or partial substitute to externally applied pesticides. Here, we present proof of concept that these new synthetic pathways can be ported and modified to produce new-to-nature compounds; however, there are several challenges that will be faced in the ultimate goal of being deployed in stable transgenic lines. Specifically, there are a minimum of eight genes that would need to be introduced into heterologous plant systems, requiring optimization of expression levels and accumulation. Moreover, there are several hurdles associated with the production of such glucosinolate-derived compounds which may necessitate proper compartmentalization. Our findings provide new targets for such long-term stable engineering efforts in the future.
Modern agricultural practices are heavily dependent on the use of external pesticides, as three billion kilograms of pesticide are utilized annually. (25) Pesticide runoff can result in environmental and human health impacts, (26) which might be addressed by the direct production of biopesticides in crops. By combining pathways from different organisms, we rationally designed and reconstituted synthetic metabolic pathways for the production of a suite of new-to-nature small molecules. The antifungal activity of the engineered crucifalexins highlights the bioactivity of their shared dithiocarbamate group, which is similar to chemical moieties in many synthetic commercial pesticides like mancozeb and metam sodium; (8) however, their differences in activity levels demonstrate that the R-group is extremely important for their potency and has varied effects from pathogen to pathogen. By expanding the structural diversity of this class of molecules, we have engineered new derivatives with improved bioactivity over the natural metabolite, brassinin, with efficacies comparable to commercially used synthetic pesticides. Notably, specialized metabolic pathways differ in plants across phylogeny; our engineering approach enables the combination of diverse mechanisms for pathogen defense that have evolved independently in a way not accessible through breeding.
There are still notable challenges beyond our first proof-of-principle biosynthesis of novel compounds, such as how such engineered plants may interact with the environment and how to avoid resistance in pathogen populations, if engineered into stable transgenic plants. Related issues are encountered with the engineering of resistance genes for pathogen defense; here it is thought that stacking multiple genes can help prevent the development of resistance. (27) The development of tissue-specific and targeted approaches in engineered plants may also help address a number of these issues. Moreover, the use of the myrosinase introduces a natural trigger to produce the final molecule on demand in response to pathogen exposure or environmental stimuli. Nonetheless, the biological production of such compounds may provide a cost-effective means to scale the biosynthesis and extraction of new-to-nature products. Many of the halogenated derivatives we have biosynthesized may enhance the bioactive properties of brassinin, not only for disease resistance in plants but also against cancer cell lines, as has been shown in previous work. (9) Thus, our findings also provide a biological means to produce such compounds biologically, avoiding chemical synthesis. This work demonstrates the potential in harnessing synthetic biology to create new compounds with novel or improved bioactive properties in order to enhance the chemical arsenal of crop species for plant disease resistance.
Methods
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Materials and General Methods
Synthetic standards were prepared according to procedures documented in the literature: benzyl crucifalexin was synthesized from benzyl amine, 4-hydroxybenzyl crucifalexin from 4-hydroxybenzyl amine, and 1-methylpropyl crucifalexin from sec-butyl amine via a procedure from Pedras et al. (7)
5-Chlorobrassinin was synthesized from 5-chloroindole carboxaldehyde using a combined protocol from Griffieon et al. and Pedras et al. (7,28) 5-Chloroindole carboxaldehyde (690 mg, 3.76 mmol) was added to a mixture of hydroxylamine hydrochloride (366 mg, 5.27 mmol) and sodium acetate (463 mg, 5.65 mmol) in ethanol (10 mL) and stirred at 40 °C with reflux for 3.5 h. The product was concentrated under vacuum, extracted with ethyl acetate and water, and concentrated again under vacuum. The dried product was dissolved in glacial acetic acid (30 mL) with zinc dust (1.48 g) and stirred overnight at room temperature. The resulting suspension was filtered, washed and extracted with ethyl acetate, dried with sodium sulfate, and concentrated under vacuum to yield crude 5-chloroindole-3-methylamine (462 mg, 68% yield). The procedure was repeated to produce additional crude 5-chloroindole-3-methylamine (322 mg, 47% yield). 595 mg of crude product was added to trimethylamine (505 μL, 3.62 mmol) in pyridine (1 mL). Carbon disulfide (220 μL, 3.62 mmol) was added dropwise, and the solution was stirred at 0 °C for 20 min, at which point methyl iodide (226 μL, 3.62 mmol) was added dropwise, and the solution was stirred at 0 °C for another 30 min. The reaction was quenched with 1.5 M sulfuric acid (3 mL), extracted with diethyl ether, dried with sodium sulfate, and concentrated under vacuum. The product was purified via a flash silica column with ethyl acetate, and with another silica column (1:1 ethyl acetate:hexanes) to yield 5-chlorobrassinin (51.9 mg, 6% yield). 7-Bromobrassinin was synthesized via the same procedure using 7-bromoindole carboxaldehyde as the starting material.
All the synthesized compounds showed NMR and high-resolution mass spectrometry (HRMS) spectral data consistent with that reported in the literature. 1H NMR spectra were acquired on a Varian 400 MHz spectrometer. HRMS data were obtained using an Agilent 6520 Q-TOF instrument, as described below. All chemicals were obtained from Sigma-Aldrich or Thermo Fisher Scientific, unless otherwise stated. Ultrapure water was generated by a Milli-Q system (EMD Millipore).
Cloning of the B. rapa Brassinin Pathway and CYP79 Genes
Phusion High-Fidelity DNA polymerase (Thermo Scientific) was used for all PCR amplification steps according to the manufacturer’s instructions. All other enzymes used for cloning were purchased from New England Biolabs. Oligonucleotide primers were purchased from Integrated DNA Technologies. DNA excised from agarose gels was purified using the Zymoclean Gel DNA recovery kit (Zymo Research). Escherichia coli TOP10 cells (New England Biolabs) were used for plasmid isolation prior to transformation into other heterologous hosts. Plasmid DNA was isolated from E. coli cultures using the QIAprep Spin Miniprep kit (Qiagen). For N. benthamiana transient expression, all gene sequences were amplified from the B. rapa cDNA template with the exception of AtTGG4 which was synthesized by Integrated DNA Technologies. Fusion CDS were generated using 2A peptides to express the core glucosinolate pathway. A BrSOT16–2A-BrUGT74B1–2A-BrSUR1 was expressed on the same plasmid under a tobacco mosaic virus 35S promotor. Similarly, a BrGGP1–2A-BrCYP83B1 fusion CDS was also expressed in the same way. BrAPK, AtTGG4, and BrDTCMT.a were expressed individually in pEAQ-HT. CYP79A2 was synthesized, inserted into pEAQ-HT (29) (KanR), and sequence verified by Gen9 based on its sequence in the S. italica genome. CYP79A1 (NCBI: XP_002466099.1), CYP79D2 (NCBI: AAF27290.1), CYP79D3 (NCBI: AY599895.1), RebH (NCBI: CAC93722.1), PyrH (NCBI: AY623051.1), and RebF (NCBI: BAC15756.1) were synthesized by Integrated DNA Technologies from sequences from S. italica, S. bicolor, Morchella esculenta, L. japonicus, Streptomyces rugosporus, and Lechevalieria aerocolonigenes.
Transient Expression in N. benthamiana
pEAQ-HT and constructs were transformed into Agrobacterium tumefaciens (GV3101). Transformants were grown on LB plates containing 50 μg/mL kanamycin and 30 μg/mL gentamicin at 30 °C. Cells were removed with a sterile inoculating loop and resuspended in 1 mL of LB medium and centrifuged at 5000g for 5 min, and the supernatant was removed. The pellet was resuspended in 10 mM MES buffer, pH = 5.6, 10 mM MgCl2, 150 μM acetosyringone, and incubated at room temperature for 1 h. Agrobacterium suspensions (OD600 = 0.1 for each strain) were infiltrated into the underside of N. benthamiana leaves with a needleless 1 mL syringe. Plants were grown 5–6 weeks under a 16 h light cycle prior to infiltration. Leaves were harvested 5 days postinfiltration, flash frozen, and stored at −80 °C for later processing. Three leaves per plant were infiltrated; a single biological replicate consisted of three pooled leaves from a single tobacco plant. Infiltrated leaf areas typically showed some signs of chlorosis or yellowing; leaves expressing GFP as a control also showed a similar phenotype.
Metabolite Extraction and LC/MS Analysis
For metabolite extraction, frozen samples were crushed with a pestle and lyophilized to dryness. The samples were subsequently homogenized on a ball mill (Retsch MM400) using 5 mm diameter steel beads, shaking at 25 Hz for 2 min. To the dry tissue, an 80:20 MeOH/H2O (v/v) solution was added (20 μL/mg N. benthamiana tissue). Tissue extracts were heat treated at 65 °C for 10 min, spun at 15 000g for 10 min, passed through 0.45 μm PTFE filters, and analyzed (20 μL injection volume) by LC/MS.
LC/MS was performed on an Agilent 1260 HPLC instrument using a Gemini NX-C18 column (Phenomenex, 5 μm, 2 × 100 mm). Water and acetonitrile, each supplemented with 0.1% formic acid, were used as the mobile phase components, with a flow rate of 0.4 mL/min. For metabolite analysis of plant extracts, the following 43 min gradient was used (percentages indicate acetonitrile concentration): 3–50% over 30 min; 50–97% over 2 min; 97% for 3 min; 97–3% over 1 min; 3% for 5 min. Column eluent was monitored by an Agilent 1260 diode array detector followed by an Agilent 6520 Accurate-Mass Q-TOF mass spectrometer with an ESI source (parameters: mass range, 50–1200 m/z; drying gas, 350 °C, 11 L/min; nebulizer, 35 psig; capillary, 3000 V; fragmentor, 150 V; skimmer, 65 V; octupole 1 RF Vpp, 750 V; 1000 ms per spectrum). MS data were collected in either positive or negative ion mode (in the case of glucosinolates) and analyzed using MassHunter Qualitative Analysis software (Agilent). Phytoalexin content was calculated from the integrated peak area of a selected ion chromatogram (extracted using a ± 100 ppm window around the theoretical exact mass) and comparison to a standard curve. The first minute of each run was discarded to avoid salt contamination of the MS apparatus. For tandem mass spectrometry (MS/MS) analysis, 5, 10, 20, and 40 V collision energies were used with an m/z window of 4 centered on the m/z analyzed. Standard curves for brassinin, benzyl crucifalexin, 4-hydroxybenzyl crucifalexin, 1-methylpropyl crucifalexin, 5-chlorobrassinin, and 7-chlorobrassinin based on ion abundance were constructed and used to quantify amounts produced in N. benthamiana.
Fungal Mycelia Growth Inhibition Assay
Fungal growth inhibition assays were performed following a similar protocol to Pedras et al. (7) Spores of Botrytis cinerea SF1 or Alternaria brassicicola FSU218 were grown on PDA at room temperature in the dark for 3 days (B. cinerea) or 7 days (A. brassicicola). Serial dilutions of phytoalexins were made in DMSO and added to warm potato dextrose agar to final concentrations of 20, 50, 100, 200, and 500 μM and poured into 6-well plates (2 mL/well). The same procedure was used for pyrimethanil containing media, but with concentrations of 0.1, 1, 10, 25, 50, and 100 μM. 5 cm circular plugs of growing fungal mycelia were transferred to cooled plates and grown at room temperature in the dark for 24 h (B. cinerea) or 75 h (A. brassicicola). At this time the longest diameter of fungal growth was measured and compared to plugs grown on PDA with 1% DMSO to calculate percent growth inhibition. IC50 values and significance were calculated from percent growth inhibition of all replicates using a nonlinear regression model in GraphPad Prism.
Safety Statement
No unexpected or unusually high safety hazards were encountered with the reported work.
Supporting Information
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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.0c00241.
Additional data and figures including LC/MS data, NMR spectra, and inhibitory assay data (PDF)
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Author Information
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- Elizabeth S. Sattely - Department of Chemical Engineering, Stanford University, Stanford, California 94305, United States; Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, United States;
http://orcid.org/0000-0002-7352-859X;
- Dominique Loqué - Joint BioEnergy Institute, Emeryville, California 94608, United States; Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, United States;http://orcid.org/0000-0002-2060-6611
Acknowledgments
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P.M.S. was supported by Grant R00AT009573 from the National Center for Complementary and Integrative Health (NCCIH) at the National Institutes of Health, and the Joint BioEnergy Institute, which is supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DEAC02-05CH11231. E.S.S. was supported by grant NIH R01GM121527.
References
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- 1Owen, C.; Patron, N. J.; Huang, A.; Osbourn, A. Harnessing plant metabolic diversity. Curr. Opin. Chem. Biol. 2017, 40, 24– 30, DOI: 10.1016/j.cbpa.2017.04.015Google Scholar1Harnessing plant metabolic diversityOwen, Charlie; Patron, Nicola J.; Huang, Ancheng; Osbourn, AnneCurrent Opinion in Chemical Biology (2017), 40 (), 24-30CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. Advances in DNA sequencing and synthesis technologies in the twenty-first century are now making it possible to build large-scale pipelines for engineering plant natural product pathways into heterologous prodn. species using synthetic biol. approaches. The ability to decode the chem. potential of plants by sequencing their transcriptomes and/or genomes and to then use this information as an instruction manual to make drugs and other high-value chems. is opening up new routes to harness the vast chem. diversity of the Plant Kingdom. Here we describe recent progress in methods for pathway discovery, DNA synthesis and assembly, and expression of engineered pathways in heterologous hosts. We also highlight the importance of standardization and the challenges assocd. with dataset integration in the drive to build a systematic framework for effective harnessing of plant metabolic diversity.
- 2Reed, J. A translational synthetic biology platform for rapid access to gram-scale quantities of novel drug-like molecules. Metab. Eng. 2017, 42, 185– 193, DOI: 10.1016/j.ymben.2017.06.012Google Scholar2A translational synthetic biology platform for rapid access to gram-scale quantities of novel drug-like moleculesReed, James; Stephenson, Michael J.; Miettinen, Karel; Brouwer, Bastiaan; Leveau, Aymeric; Brett, Paul; Goss, Rebecca J. M.; Goossens, Alain; O'Connell, Maria A.; Osbourn, AnneMetabolic Engineering (2017), 42 (), 185-193CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)Plants are an excellent source of drug leads. However availability is limited by access to source species, low abundance and recalcitrance to chem. synthesis. Although plant genomics is yielding a wealth of genes for natural product biosynthesis, the translation of this genetic information into small mols. for evaluation as drug leads represents a major bottleneck. For example, the yeast platform for artemisinic acid prodn. is estd. to have taken >150 person years to develop. Here we demonstrate the power of plant transient transfection technol. for rapid, scalable biosynthesis and isolation of triterpenes, one of the largest and most structurally diverse families of plant natural products. Using pathway engineering and improved agro-infiltration methodol. we are able to generate gram-scale quantities of purified triterpene in just a few weeks. In contrast to heterologous expression in microbes, this system does not depend on re-engineering of the host. We next exploit agro-infection for quick and easy combinatorial biosynthesis without the need for generation of multi-gene constructs, so affording an easy entre´e to suites of mols., some new-to-nature, that are recalcitrant to chem. synthesis. We use this platform to purify a suite of bespoke triterpene analogs and demonstrate differences in anti-proliferative and anti-inflammatory activity in bioassays, providing proof of concept of this system for accessing and evaluating medicinally important bioactives. Together with new genome mining algorithms for plant pathway discovery and advances in plant synthetic biol., this advance provides new routes to synthesize and access previously inaccessible natural products and analogs and has the potential to reinvigorate drug discovery pipelines.
- 3Pierce, M. L.; Cover, E. C.; Richardson, P. E.; Scholes, V. E.; Essenberg, M. Adequacy of cellular phytoalexin concentrations in hypersensitively responding cotton leaves. Physiol. Mol. Plant Pathol. 1996, 48, 305– 324, DOI: 10.1006/pmpp.1996.0025Google Scholar3Adequacy of cellular phytoalexin concentrations in hypersensitively responding cotton leavesPierce, M. L.; Cover, E. C.; Richardson, P. E.; Scholes, V. E.; Essenberg, M.Physiological and Molecular Plant Pathology (1996), 48 (5), 305-324CODEN: PMPPEZ; ISSN:0885-5765. (Academic)Av. cellular phytoalexin concns. at infection sites were detd. in Xanthomonas campestris pv. malvacearum infected cotton leaves to test the hypothesis that sesquiterpenoid phytoalexins play a major role in heritable resistance of cotton to bacterial blight. Bacteriostasis was achieved in leaves of resistant cotton line WbM(0.0) by 5-7 days after inoculation. Between days 3 and 7, phytoalexin amts. per g of tissue were above basal levels but were far below their peak values. Nevertheless, when the phytoalexin quantities present on day 7 were divided by the vol. of water contained in the hypersensitively responding cells defining the scattered infection centers, the concns. computed were ones previously found to be highly inhibitory in in vitro bioassays. In necrotic cells of susceptible line WbM(4.0), the values were ≤ 3% of those in the resistant line. In a comparison of highly resistant lines OK 1.2 and Im216, concns. of phytoalexins far exceeding inhibitory levels in vitro were calcd. for hypersensitively necrotic cells in both lines on days 3 and 4 post-inoculation. Anal. of the clustering of necrotic cells indicated that in OK1.2, only 25% of the infection sites had mounted a reaction by day 3 and nearly all had responded by day 4, while in Im216, 50% or more had responded hypersensitively by day 3 and all sites had shown a reaction by day 4. Bacterial inhibition occurred on the day following appearance of HR-cell clusters at all sites. The phytoalexins present in the HR cells had access to the bacteria, as indicated by loss of plasmalemma integrity of the yellow-green fluorescent, hypersensitively responding cells and by diffusion of the phytoalexins into the bathing medium.
- 4Klein, A. P.; Sattely, E. S. Biosynthesis of cabbage phytoalexins from indole glucosinolate. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 1910, DOI: 10.1073/pnas.1615625114Google Scholar4Biosynthesis of cabbage phytoalexins from indole glucosinolateKlein, Andrew P.; Sattely, Elizabeth S.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (8), 1910-1915CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Brassica crop species are prolific producers of indole-sulfur phytoalexins that are thought to have an important role in plant disease resistance. These mols. are conspicuously absent in the model plant Arabidopsis thaliana, and little is known about the enzymic steps that assemble the key precursor brassinin. Here, we report the min. set of biosynthetic genes required to generate cruciferous phytoalexins starting from the well-studied glucosinolate pathway. In vitro biochem. characterization revealed an addnl. role for the previously described carbon-sulfur lyase SUR1 in processing cysteine-isothiocyanate conjugates, as well as the S-methyltransferase DTCMT that methylates the resulting dithiocarbamate, together completing a pathway to brassinin. Addnl., the β-glucosidase BABG that is present in Brassica rapa but absent in Arabidopsis was shown to act as a myrosinase and may be a determinant of plants that synthesize phytoalexins from indole glucosinolate. Transient expression of the entire pathway in Nicotiana benthamiana yields brassinin, demonstrating that the biosynthesis of indole-sulfur phytoalexins can be engineered into noncruciferous plants. The identification of these biosynthetic enzymes and the heterologous reconstitution of the indole-sulfur phytoalexin pathway sheds light on an important pathway in an edible plant and opens the door to using metabolic engineering to systematically quantify the impact of cruciferous phytoalexins on plant disease resistance and human health.
- 5Halkier, B. A.; Gershenzon, J. Biology and biochemistry of glucosinolates. Annu. Rev. Plant Biol. 2006, 57, 303– 333, DOI: 10.1146/annurev.arplant.57.032905.105228Google Scholar5Biology and biochemistry of glucosinolatesHalkier, Barbara Ann; Gershenzon, JonathanAnnual Review of Plant Biology (2006), 57 (), 303-333CODEN: ARPBDW ISSN:. (Annual Reviews Inc.)A review. Glucosinolates are sulfur-rich, anionic natural products that upon hydrolysis by endogenous thioglucosidases called myrosinases produce several different products (e.g., isothiocyanates, thiocyanates, and nitriles). The hydrolysis products have many different biol. activities, e.g., as defense compds. and attractants. For humans these compds. function as cancer-preventing agents, biopesticides, and flavor compds. Since the completion of the Arabidopsis genome, glucosinolate research has made significant progress, resulting in near-complete elucidation of the core biosynthetic pathway, identification of the first regulators of the pathway, metabolic engineering of specific glucosinolate profiles to study function, as well as identification of evolutionary links to related pathways. Although much was learned in recent years, much more awaits discovery before the authors fully understand how and why plants synthesize glucosinolates. This may enable us to more fully exploit the potential of these compds. in agriculture and medicine.
- 6Wittstock, U.; Halkier, B. A. Glucosinolate research in the Arabidopsis era. Trends Plant Sci. 2002, 7, 263– 270, DOI: 10.1016/S1360-1385(02)02273-2Google Scholar6Glucosinolate research in the Arabidopsis eraWittstock, Ute; Halkier, Barbara A.Trends in Plant Science (2002), 7 (6), 263-270CODEN: TPSCF9; ISSN:1360-1385. (Elsevier Science Ltd.)A review. The wide range of biol. activities of products derived from the glucosinolate-myrosinase system is biol. and economically important. On the one hand, the degrdn. products of glucosinolates play an important role in the defense of plants against herbivores. On the other hand, these compds. have toxic (e.g. goitrogenic) as well as protective (e.g. cancer-preventing) effects in higher animals and humans. There is a strong interest in the ability to regulate and optimize the levels of individual glucosinolates tissue-specifically to improve the nutritional value and pest resistance of crops. Recent advances in our understanding of glucosinolate biosynthesis have brought us closer to this goal. Genetic engineering of glucosinolate profiles is now a realistic possibility. Individual glucosinolates could improve nutritional value and pest resistance of important crops.
- 7Pedras, M. S. C.; To, Q. H. Non-indolyl cruciferous phytoalexins: Nasturlexins and tridentatols, a striking convergent evolution of defenses in terrestrial plants and marine animals?. Phytochemistry 2015, 113, 57– 63, DOI: 10.1016/j.phytochem.2014.07.024Google Scholar7The first non-indolyl cruciferous phytoalexins: nasturlexins and tridentatols, a striking convergent evolution of defenses in terrestrial plants and marine animals?Pedras, M. Soledade C.; To, Q. HuyPhytochemistry (Elsevier) (2015), 113 (), 57-63CODEN: PYTCAS; ISSN:0031-9422. (Elsevier Ltd.)Highly specialized chem. defense pathways are a particularly noteworthy metabolic characteristic of sessile organisms, whether terrestrial or marine, providing protection against pests and diseases. For this reason, knowledge of the metabolites involved in these processes is crucial to producing ecol. fit crops. Toward this end, the elicited chem. defenses of the crucifer watercress (Nasturtium officinale R.Br.), i.e., phytoalexins, were investigated and are reported. Almost three decades after publication of cruciferous phytoalexins derived from (S)-Trp, phytoalexins derived from other arom. amino acids were isolated; their chem. structures were detd. by analyses of their spectroscopic data and confirmed by synthesis. Nasturlexin A, nasturlexin B, and tridentatol C are hitherto unknown Ph contg. cruciferous phytoalexins produced by watercress under abiotic stress; tridentatol C is also produced by a marine animal (Tridentata marginata), where it functions in chem. defense against predators. The biosynthesis of these metabolites in both a terrestrial plant and a marine animal suggests a convergent evolution of unique metabolic pathways recruited for defense.
- 8Pedras, M. S. C.; Yaya, E. E.; Glawischnig, E. The phytoalexins from cultivated and wild crucifers: Chemistry and biology. Nat. Prod. Rep. 2011, 28, 1381– 1405, DOI: 10.1039/c1np00020aGoogle Scholar8The phytoalexins from cultivated and wild crucifers: Chemistry and biologyPedras, M. Soledade C.; Yaya, Estifanos E.; Glawischnig, ErichNatural Product Reports (2011), 28 (8), 1381-1405CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)A review. Phytoalexins are antimicrobial secondary metabolites produced de novo by plants in response to stress, including microbial attack. In general, phytoalexins are important components of plant defenses against fungal and bacterial pathogens. The phytoalexins of crucifers are indole alkaloids derived from (S)-tryptophan, most of which contain a sulfur atom derived from cysteine. Beside their antimicrobial activity against different plant pathogenic species, cruciferous phytoalexins have shown anticarcinogenic effects on various human cell lines. This review focuses on the phytoalexins produced by cruciferous plants reported to date, with particular emphasis on their chem. synthesis, biosynthesis, metab. by plant fungal pathogens and biol. activities. A summary table contg. all phytoalexins, their cultivated and wild cruciferous sources, their synthetic starting materials, biotransformation products and biol. activities is provided.
- 9Banerjee, T. A key in vivo antitumor mechanism of action of natural product-based brassinins is inhibition of indoleamine 2,3-dioxygenase. Oncogene 2008, 27, 2851– 2857, DOI: 10.1038/sj.onc.1210939Google Scholar9A key in vivo antitumor mechanism of action of natural product-based brassinins is inhibition of indoleamine 2,3-dioxygenaseBanerjee, T.; DuHadaway, J. B.; Gaspari, P.; Sutanto-Ward, E.; Munn, D. H.; Mellor, A. L.; Malachowski, W. P.; Prendergast, G. C.; Muller, A. J.Oncogene (2008), 27 (20), 2851-2857CODEN: ONCNES; ISSN:0950-9232. (Nature Publishing Group)Agents that interfere with tumoral immune tolerance may be useful to prevent or treat cancer. Brassinin is a phytoalexin, a class of natural products derived from plants that includes the widely known compd. resveratrol. Brassinin has been demonstrated to have chemopreventive activity in preclin. models but the mechanisms underlying its anticancer properties are unknown. Here, we show that brassinin and a synthetic deriv. 5-bromo-brassinin (5-Br-brassinin) are bioavailable inhibitors of indoleamine 2,3-dioxygenase (IDO), a pro-tolerogenic enzyme that drives immune escape in cancer. Like other known IDO inhibitors, both of these compds. combined with chemotherapy to elicit regression of autochthonous mammary gland tumors in MMTV-Neu mice. Furthermore, growth of highly aggressive melanoma isograft tumors was suppressed by single agent treatment with 5-Br-brassinin. This response to treatment was lost in athymic mice, indicating a requirement for active host T-cell immunity, and in IDO-null knockout mice, providing direct genetic evidence that IDO inhibition is essential to the antitumor mechanism of action of 5-Br-brassinin. The natural product brassinin thus provides the structural basis for a new class of compds. with in vivo anticancer activity that is mediated through the inhibition of IDO.
- 10Bak, S.; Olsen, C. E.; Petersen, B. L.; Møller, B. L.; Halkier, B. A. Metabolic engineering of p-hydroxybenzylglucosinolate in Arabidopsis by expression of the cyanogenic CYP79A1 from Sorghum bicolor. Plant J. 1999, 20, 663– 671, DOI: 10.1046/j.1365-313X.1999.00642.xGoogle Scholar10Metabolic engineering of p-hydroxybenzylglucosinolate in Arabidopsis by expression of the cyanogenic CYP79A1 from Sorghum bicolorBak, Soren; Olsen, Carl Erik; Petersen, Bent Larsen; Moller, Birger Lindberg; Halkier, Barbara AnnPlant Journal (1999), 20 (6), 663-671CODEN: PLJUED; ISSN:0960-7412. (Blackwell Science Ltd.)Glucosinolates are natural products in cruciferous plants, including Arabidopsis thaliana. CYP79A1 is the cytochrome P 450 catalyzing the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of the cyanogenic glucoside dhurrin in sorghum. Both glucosinolates and cyanogenic glucosides have oximes as intermediates. Expression of CYP79A1 in A. thaliana results in the prodn. of high levels of the tyrosine-derived glucosinolate p-hydroxybenzylglucosinolate, which is not a natural constituent of A. thaliana. This provides further evidence that the enzymes have low substrate specificity with respect to the side chain. The ability of the cyanogenic CYP79A1 to integrate itself into the glucosinolate pathway has important implications for an evolutionary relationship between cyanogenic glucosides and glucosinolates, and for the possibility of genetic engineering of novel glucosinolates.
- 11Mikkelsen, M. D.; Halkier, B. A. Metabolic Engineering of Valine- and Isoleucine-Derived Glucosinolates in Arabidopsis Expressing CYP79D2 from Cassava. Plant Physiol. 2003, 131, 773, DOI: 10.1104/pp.013425Google Scholar11Metabolic engineering of valine- and isoleucine-derived glucosinolates in Arabidopsis expressing CYP79D2 from cassavaMikkelsen, Michael Dalgaard; Halkier, Barbara AnnPlant Physiology (2003), 131 (2), 773-779CODEN: PLPHAY; ISSN:0032-0889. (American Society of Plant Biologists)Glucosinolates are amino acid-derived natural products that, upon hydrolysis, typically release isothiocyanates with a wide range of biol. activities. Glucosinolates play a role in plant defense as attractants and deterrents against herbivores and pathogens. A key step in glucosinolate biosynthesis is the conversion of amino acids to the corresponding aldoximes, which is catalyzed by cytochromes P 450 belonging to the CYP79 family. Expression of CYP79D2 from cassava (Manihot esculenta Crantz.) in Arabidopsis resulted in the prodn. of valine (Val)- and isoleucine-derived glucosinolates not normally found in this ecotype. The transgenic lines showed no morphol. phenotype, and the level of endogenous glucosinolates was not affected. The novel glucosinolates were shown to constitute up to 35% of the total glucosinolate content in mature rosette leaves and up to 48% in old leaves. Furthermore, at increased concns. of these glucosinolates, the proportion of Val-derived glucosinolates decreased. As the isothiocyanates produced from the Val- and isoleucine-derived glucosinolates are volatile, metabolically engineered plants producing these glucosinolates have acquired novel properties with great potential for improvement of resistance to herbivorous insects and for biofumigation.
- 12Kahn, R. A.; Fahrendorf, T.; Halkier, B. A.; Møller, B. L. Substrate specificity of the cytochrome P450 enzymes CYP79A1 and CYP71E1 involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench. Arch. Biochem. Biophys. 1999, 363, 9– 18, DOI: 10.1006/abbi.1998.1068Google Scholar12Substrate Specificity of the Cytochrome P450 Enzymes CYP79A1 and CYP71E1 Involved in the Biosynthesis of the Cyanogenic Glucoside Dhurrin in Sorghum bicolor (L.) MoenchKahn, Rachel Alice; Fahrendorf, Theodor; Halkier, Barbara Ann; Moller, Birger LindbergArchives of Biochemistry and Biophysics (1999), 363 (1), 9-18CODEN: ABBIA4; ISSN:0003-9861. (Academic Press)The two multifunctional cytochrome P 450 enzymes, CYP79A1 and CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench have been characterized with respect to substrate specificity and cofactor requirements using reconstituted, recombinant enzymes and sorghum microsomes. CYP79A1 has a very high substrate specificity, tyrosine being the only substrate found. CYP71E1 has less stringent substrate requirements and metabolizes arom. oximes efficiently, whereas aliph. oximes are slowly metabolized. Neither CYP79A1 nor CYP71E1 catalyze the metab. of a range of different herbicides. The reported resistance of sorghum to bentazon is therefore not linked to the presence of CYP79A1 or CYP71E1. NADPH is a much better cofactor than NADH although NADH does support the entire catalytic cycle of both P 450 enzymes. Km and Vmax values for NADPH when supporting CYP71E1 activity are 0.013 mM and 111 nmol/mg protein/s. For NADH, the corresponding values are 0.3 mM and 42 nmol/mg protein/s. CYP79A1 is a fairly stable enzyme. In contrast, CYP71E1 is labile and prone to rapid denaturation at room temp. CYP71E1 is isolated in the low spin form. CYP71E1 catalyzes an unusual dehydration reaction of an oxime to the corresponding nitrile which subsequently is C-hydroxylated. The oxime forms a peculiar reverse Type I spectrum, whereas the nitrile forms a Type I spectrum. Several compds. which do not serve as substrates formed Type I substrate binding spectra with the two P 450 enzymes. (c) 1999 Academic Press.
- 13Forslund, K. Biosynthesis of the Nitrile Glucosides Rhodiocyanoside A and D and the Cyanogenic Glucosides Lotaustralin and Linamarin in Lotus japonicus. Plant Physiol. 2004, 135, 71, DOI: 10.1104/pp.103.038059Google Scholar13Biosynthesis of the nitrile glucosides rhodiocyanoside A and D and the cyanogenic glucosides lotaustralin and linamarin in Lotus japonicusForslund, Karin; Morant, Marc; Jorgensen, Bodil; Olsen, Carl Erik; Asamizu, Erika; Sato, Shusei; Tabata, Satoshi; Bak, SorenPlant Physiology (2004), 135 (1), 71-84CODEN: PLPHAY; ISSN:0032-0889. (American Society of Plant Biologists)Lotus japonicus was shown to contain the two nitrile glucosides rhodiocyanoside A and rhodiocyanoside D as well as the cyanogenic glucosides linamarin and lotaustralin. The content of cyanogenic and nitrile glucosides in L. japonicus depends on plant developmental stage and tissue. The cyanide potential is highest in young seedlings and in apical leaves of mature plants. Roots and seeds are acyanogenic. Biosynthetic studies using radioisotopes demonstrated that lotaustralin, rhodiocyanoside A, and rhodiocyanoside D are derived from the amino acid L-Ile, whereas linamarin is derived from Val. In silico homol. searches identified two cytochromes P 450 designated CYP79D3 and CYP79D4 in L. japonicus. The two cytochromes P 450 are 94% identical at the amino acid level and both catalyze the conversion of Val and Ile to the corresponding aldoximes in biosynthesis of cyanogenic glucosides and nitrile glucosides in L. japonicus. CYP79D3 and CYP79D4 are differentially expressed. CYP79D3 is exclusively expressed in aerial parts and CYP79D4 in roots. Recombinantly expressed CYP79D3 and CYP79D4 in yeast cells showed higher catalytic efficiency with L-Ile as substrate than with L-Val, in agreement with lotaustralin and rhodiocyanoside A and D being the major cyanogenic and nitrile glucosides in L. japonicus. Ectopic expression of CYP79D2 from cassava (Manihot esculenta Crantz.) in L. japonicus resulted in a 5- to 20-fold increase of linamarin content, whereas the relative amts. of lotaustralin and rhodiocyanoside A/D were unaltered.
- 14Wittstock, U.; Halkier, B. A. Cytochrome P450 CYP79A2 from Arabidopsis thaliana L. Catalyzes the Conversion of l-Phenylalanine to Phenylacetaldoxime in the Biosynthesis of Benzylglucosinolate. J. Biol. Chem. 2000, 275, 14659– 14666, DOI: 10.1074/jbc.275.19.14659Google Scholar14Cytochrome P450 CYP79A2 from Arabidopsis thaliana L. catalyzes the conversion of L-phenylalanine to phenylacetaldoxime in the biosynthesis of benzylglucosinolateWittstock, Ute; Halkier, Barbara AnnJournal of Biological Chemistry (2000), 275 (19), 14659-14666CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Glucosinolates are natural plant products gaining increasing interest as cancer-preventing agents and crop protectants. Similar to cyanogenic glucosides, glucosinolates are derived from amino acids and have aldoximes as intermediates. We report cloning and characterization of cytochrome P 450 CYP79A2 involved in aldoxime formation in the glucosinolate-producing Arabidopsis thaliana L. The CYP79A2 cDNA was cloned by polymerase chain reaction, and CYP79A2 was functionally expressed in Escherichia coli. Characterization of the recombinant protein shows that CYP79A2 is an N-hydroxylase converting L-phenylalanine into phenylacetaldoxime, the precursor of benzylglucosinolate. Transgenic A. thaliana constitutively expressing CYP79A2 accumulate high levels of benzylglucosinolate. CYP79A2 expressed in E. coli has a Km of 6.7 μmol liter-1 for L-phenylalanine. Neither L-tyrosine, L-tryptophan, L-methionine, nor DL-homophenylalanine are metabolized by CYP79A2, indicating that the enzyme has a narrow substrate specificity. CYP79A2 is the first enzyme shown to catalyze the conversion of an amino acid to the aldoxime in the biosynthesis of glucosinolates. Our data provide the first conclusive evidence that evolutionarily conserved cytochromes P 450 catalyze this step common for the biosynthetic pathways of glucosinolates and cyanogenic glucosides. This strongly indicates that the biosynthesis of glucosinolates has evolved based on a cyanogenic predisposition.
- 15Brader, G.; Mikkelsen, M. D.; Halkier, B. A.; Tapio Palva, E. Altering glucosinolate profiles modulates disease resistance in plants. Plant J. 2006, 46, 758– 767, DOI: 10.1111/j.1365-313X.2006.02743.xGoogle Scholar15Altering glucosinolate profiles modulates disease resistance in plantsBrader, Gunter; Mikkelsen, Michael Dalgaard; Halkier, Barbara Ann; Palva, E. TapioPlant Journal (2006), 46 (5), 758-767CODEN: PLJUED; ISSN:0960-7412. (Blackwell Publishing Ltd.)Plant diseases are major contributing factors for crop loss in agriculture. Here, the authors show that Arabidopsis plants with high levels of novel glucosinolates (GSs) as a result of the introduction of single CYP79 genes exhibit altered disease resistance. Arabidopsis expressing CYP79D2 from cassava accumulated aliph. iso-Pr and methylpropyl GS, and showed enhanced resistance against the bacterial soft-rot pathogen Erwinia carotovora, whereas Arabidopsis expressing the sorghum CYP79A1 or over-expressing the endogenous CYP79A2 accumulated p-hydroxybenzyl or benzyl GS, resp., and showed increased resistance towards the bacterial pathogen Pseudomonas syringae. In addn. to the direct toxic effects of GS breakdown products, increased accumulation of arom. GSs was shown to stimulate salicylic acid-mediated defenses while suppressing jasmonate-dependent defenses, as manifested in enhanced susceptibility to the fungus Alternaria brassicicola. Arabidopsis with modified GS profiles provide important tools for evaluating the biol. effects of individual GSs and thereby show potential as biotechnol. tools for the generation of plants with tailor-made disease resistance.
- 16Wang, X. The genome of the mesopolyploid crop species Brassica rapa. Nat. Genet. 2011, 43, 1035, DOI: 10.1038/ng.919Google Scholar16The genome of the mesopolyploid crop species Brassica rapaWang, Xiaowu; Wang, Hanzhong; Wang, Jun; Sun, Rifei; Wu, Jian; Liu, Shengyi; Bai, Yinqi; Mun, Jeong-Hwan; Bancroft, Ian; Cheng, Feng; Huang, Sanwen; Li, Xixiang; Hua, Wei; Wang, Junyi; Wang, Xiyin; Freeling, Michael; Pires, J. Chris; Paterson, Andrew H.; Chalhoub, Boulos; Wang, Bo; Hayward, Alice; Sharpe, Andrew G.; Park, Beom-Seok; Weisshaar, Bernd; Liu, Binghang; Li, Bo; Liu, Bo; Tong, Chaobo; Song, Chi; Duran, Christopher; Peng, Chunfang; Geng, Chunyu; Koh, Chushin; Lin, Chuyu; Edwards, David; Mu, Desheng; Shen, Di; Soumpourou, Eleni; Li, Fei; Fraser, Fiona; Conant, Gavin; Lassalle, Gilles; King, Graham J.; Bonnema, Guusje; Tang, Haibao; Wang, Haiping; Belcram, Harry; Zhou, Heling; Hirakawa, Hideki; Abe, Hiroshi; Guo, Hui; Wang, Hui; Jin, Huizhe; Parkin, Isobel A. P.; Batley, Jacqueline; Kim, Jeong-Sun; Just, Jeremy; Li, Jianwen; Xu, Jiaohui; Deng, Jie; Kim, Jin A.; Li, Jingping; Yu, Jingyin; Meng, Jinling; Wang, Jinpeng; Min, Jiumeng; Poulain, Julie; Wang, Jun; Hatakeyama, Katsunori; Wu, Kui; Wang, Li; Fang, Lu; Trick, Martin; Links, Matthew G.; Zhao, Meixia; Jin, Mina; Ramchiary, Nirala; Drou, Nizar; Berkman, Paul J.; Cai, Qingle; Huang, Quanfei; Li, Ruiqiang; Tabata, Satoshi; Cheng, Shifeng; Zhang, Shu; Zhang, Shujiang; Huang, Shunmou; Sato, Shusei; Sun, Silong; Kwon, Soo-Jin; Choi, Su-Ryun; Lee, Tae-Ho; Fan, Wei; Zhao, Xiang; Tan, Xu; Xu, Xun; Wang, Yan; Qiu, Yang; Yin, Ye; Li, Yingrui; Du, Yongchen; Liao, Yongcui; Lim, Yongpyo; Narusaka, Yoshihiro; Wang, Yupeng; Wang, Zhenyi; Li, Zhenyu; Wang, Zhiwen; Xiong, Zhiyong; Zhang, ZhonghuaNature Genetics (2011), 43 (10), 1035-1039CODEN: NGENEC; ISSN:1061-4036. (Nature Publishing Group)This report presents the annotation and anal. of the draft genome sequence of Brassica rapa accession Chiifu-401-42, a Chinese cabbage. The authors modeled 41,174 protein coding genes in the B. rapa genome, which has undergone genome triplication. Arabidopsis thaliana was used as an outgroup for investigating the consequences of genome triplication, such as structural and functional evolution. The extent of gene loss (fractionation) among triplicated genome segments varies, with one of the three copies consistently retaining a disproportionately large fraction of the genes expected to have been present in its ancestor. Variation in the no. of members of gene families present in the genome may contribute to the remarkable morphol. plasticity of Brassica species. The B. rapa genome sequence provides an important resource for studying the evolution of polyploid genomes and underpins the genetic improvement of Brassica oil and vegetable crops. The draft genome sequence is deposited in GenBank/EMBL/DDBJ with project accession nos. AENI01000001-AENI01051658.
- 17Liu, S. The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes. Nat. Commun. 2014, 5, 3930, DOI: 10.1038/ncomms4930Google Scholar17The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomesLiu, Shengyi; Liu, Yumei; Yang, Xinhua; Tong, Chaobo; Edwards, David; Parkin, Isobel A. P.; Zhao, Meixia; Ma, Jianxin; Yu, Jingyin; Huang, Shunmou; Wang, Xiyin; Wang, Junyi; Lu, Kun; Fang, Zhiyuan; Bancroft, Ian; Yang, Tae-Jin; Hu, Qiong; Wang, Xinfa; Yue, Zhen; Li, Haojie; Yang, Linfeng; Wu, Jian; Zhou, Qing; Wang, Wanxin; King, Graham J.; Pires, J. Chris; Lu, Changxin; Wu, Zhangyan; Sampath, Perumal; Wang, Zhuo; Guo, Hui; Pan, Shengkai; Yang, Limei; Min, Jiumeng; Zhang, Dong; Jin, Dianchuan; Li, Wanshun; Belcram, Harry; Tu, Jinxing; Guan, Mei; Qi, Cunkou; Du, Dezhi; Li, Jiana; Jiang, Liangcai; Batley, Jacqueline; Sharpe, Andrew G.; Park, Beom-Seok; Ruperao, Pradeep; Cheng, Feng; Waminal, Nomar Espinosa; Huang, Yin; Dong, Caihua; Wang, Li; Li, Jingping; Hu, Zhiyong; Zhuang, Mu; Huang, Yi; Huang, Junyan; Shi, Jiaqin; Mei, Desheng; Liu, Jing; Lee, Tae-Ho; Wang, Jinpeng; Jin, Huizhe; Li, Zaiyun; Li, Xun; Zhang, Jiefu; Xiao, Lu; Zhou, Yongming; Liu, Zhongsong; Liu, Xuequn; Qin, Rui; Tang, Xu; Liu, Wenbin; Wang, Yupeng; Zhang, Yangyong; Lee, Jonghoon; Kim, Hyun Hee; Denoeud, France; Xu, Xun; Liang, Xinming; Hua, Wei; Wang, Xiaowu; Wang, Jun; Chalhoub, Boulos; Paterson, Andrew H.Nature Communications (2014), 5 (), 3930CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Polyploidization has provided much genetic variation for plant adaptive evolution, but the mechanisms by which the mol. evolution of polyploid genomes establishes genetic architecture underlying species differentiation are unclear. Brassica is an ideal model to increase knowledge of polyploid evolution. Here we describe a draft genome sequence of Brassica oleracea, comparing it with that of its sister species B. rapa to reveal numerous chromosome rearrangements and asym. gene loss in duplicated genomic blocks, asym. amplification of transposable elements, differential gene co-retention for specific pathways and variation in gene expression, including alternative splicing, among a large no. of paralogous and orthologous genes. Genes related to the prodn. of anticancer phytochems. and morphol. variations illustrate consequences of genome duplication and gene divergence, imparting biochem. and morphol. variation to B. oleracea. This study provides insights into Brassica genome evolution and will underpin research into the many important crops in this genus.
- 18Klein, A. P.; Sattely, E. S. Two cytochromes P450 catalyze S-heterocyclizations in cabbage phytoalexin biosynthesis. Nat. Chem. Biol. 2015, 11, 837, DOI: 10.1038/nchembio.1914Google Scholar18Two cytochromes P450 catalyze S-heterocyclizations in cabbage phytoalexin biosynthesisKlein, Andrew P.; Sattely, Elizabeth S.Nature Chemical Biology (2015), 11 (11), 837-839CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Phytoalexins are abundant in edible crucifers and have important biol. activities, yet no dedicated gene for their biosynthesis is known. Here, we report two new cytochromes P 450 from Brassica rapa (Chinese cabbage) that catalyze unprecedented S-heterocyclizations in cyclobrassinin and spirobrassinin biosynthesis. Our results provide genetic and biochem. insights into the biosynthesis of a prominent pair of dietary metabolites and have implications for pathway discovery across >20 recently sequenced crucifers.
- 19Runguphan, W.; Qu, X.; O’Connor, S. E. Integrating carbon–halogen bond formation into medicinal plant metabolism. Nature 2010, 468, 461, DOI: 10.1038/nature09524Google Scholar19Integrating carbon-halogen bond formation into medicinal plant metabolismRunguphan, Weerawat; Qu, Xu-Dong; O'Connor, Sarah E.Nature (London, United Kingdom) (2010), 468 (7322), 461-464CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Halogenation, which was once considered a rare occurrence in nature, has now been obsd. in many natural product biosynthetic pathways. However, only a small fraction of halogenated compds. have been isolated from terrestrial plants. Given the impact that halogenation can have on the biol. activity of natural products, we reasoned that the introduction of halides into medicinal plant metab. would provide the opportunity to rationally bioengineer a broad variety of novel plant products with altered, and perhaps improved, pharmacol. properties. Here we report that chlorination biosynthetic machinery from soil bacteria can be successfully introduced into the medicinal plant Catharanthus roseus (Madagascar periwinkle). These prokaryotic halogenases function within the context of the plant cell to generate chlorinated tryptophan, which is then shuttled into monoterpene indole alkaloid metab. to yield chlorinated alkaloids. A new functional group-a halide-is thereby introduced into the complex metab. of C. roseus, and is incorporated in a predictable and regioselective manner onto the plant alkaloid products. Medicinal plants, despite their genetic and developmental complexity, therefore seem to be a viable platform for synthetic biol. efforts.
- 20Menon, B. R. K. RadH: A Versatile Halogenase for Integration into Synthetic Pathways. Angew. Chem., Int. Ed. 2017, 56, 11841– 11845, DOI: 10.1002/anie.201706342Google Scholar20RadH: A Versatile Halogenase for Integration into Synthetic PathwaysMenon, Binuraj R. K.; Brandenburger, Eileen; Sharif, Humera H.; Klemstein, Ulrike; Shepherd, Sarah A.; Greaney, Michael F.; Micklefield, JasonAngewandte Chemie, International Edition (2017), 56 (39), 11841-11845CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Flavin-dependent halogenases are useful enzymes for providing halogenated mols. with improved biol. activity, or intermediates for synthetic derivatization. We demonstrate how the fungal halogenase RadH can be used to regioselectively halogenate a range of bioactive arom. scaffolds. Site-directed mutagenesis of RadH was used to identify catalytic residues and provide insight into the mechanism of fungal halogenases. A high-throughput fluorescence screen was also developed, which enabled a RadH mutant to be evolved with improved properties. Finally we demonstrate how biosynthetic genes from fungi, bacteria, and plants can be combined to encode a new pathway to generate a novel chlorinated coumarin "non-natural" product in E. coli.
- 21Dean, R. The Top 10 fungal pathogens in molecular plant pathology. Mol. Plant Pathol. 2012, 13, 414– 430, DOI: 10.1111/j.1364-3703.2011.00783.xGoogle Scholar21The Top 10 fungal pathogens in molecular plant pathologyDean Ralph; Van Kan Jan A L; Pretorius Zacharias A; Hammond-Kosack Kim E; Di Pietro Antonio; Spanu Pietro D; Rudd Jason J; Dickman Marty; Kahmann Regine; Ellis Jeff; Foster Gary DMolecular plant pathology (2012), 13 (4), 414-30 ISSN:.The aim of this review was to survey all fungal pathologists with an association with the journal Molecular Plant Pathology and ask them to nominate which fungal pathogens they would place in a 'Top 10' based on scientific/economic importance. The survey generated 495 votes from the international community, and resulted in the generation of a Top 10 fungal plant pathogen list for Molecular Plant Pathology. The Top 10 list includes, in rank order, (1) Magnaporthe oryzae; (2) Botrytis cinerea; (3) Puccinia spp.; (4) Fusarium graminearum; (5) Fusarium oxysporum; (6) Blumeria graminis; (7) Mycosphaerella graminicola; (8) Colletotrichum spp.; (9) Ustilago maydis; (10) Melampsora lini, with honourable mentions for fungi just missing out on the Top 10, including Phakopsora pachyrhizi and Rhizoctonia solani. This article presents a short resume of each fungus in the Top 10 list and its importance, with the intent of initiating discussion and debate amongst the plant mycology community, as well as laying down a bench-mark. It will be interesting to see in future years how perceptions change and what fungi will comprise any future Top 10.
- 22Fritz, R.; Lanen, C.; Colas, V.; Leroux, P. Inhibition of Methionine Biosynthesis in Botrytis cinerea by the Anilinopyrimidine Fungicide Pyrimethanil. Pestic. Sci. 1997, 49, 40– 46, DOI: 10.1002/(SICI)1096-9063(199701)49:1<40::AID-PS470>3.0.CO;2-YGoogle Scholar22Inhibition of methionine biosynthesis in Botrytis cinerea by the anilinopyrimidine fungicide pyrimethanilFritz, Rene; Lanen, Catherine; Colas, Virginie; Leroux, PierrePesticide Science (1997), 49 (1), 40-46CODEN: PSSCBG; ISSN:0031-613X. (Wiley)When mycelium of B. cinerea was treated with low concns. of pyrimethanil, the total amt. of free amino acids increased. Qual. variations were also induced: alanine, glutamine, lysine, glycine, histidine, asparagine, arginine, threonine, α-aminobutyrate and β-alanine were accumulated; cyst(e)ine, valine, leucine and citrulline were reduced. When mycelium of B. cinerea was incubated with Na2[35S]O4, pyurimethanil, at 1·5 μM, induced a decrease of [35S]methionine and simultaneously an increase of [35S]cystathionine. Thus, pyrimethanil inhibits the biosynthesis of methionine and suggest that the primary target could be the cystathionine β-lyase.
- 23Milling, R. J.; Richardson, C. J. Mode of action of the anilino-pyrimidine fungicide Pyrimethanil. 2. Effects on enzyme secretion in Botrytis cinerea. Pestic. Sci. 1995, 45, 43– 48, DOI: 10.1002/ps.2780450107Google Scholar23Mode of action of the anilino-pyrimidine fungicide pyrimethanil. 2. Effects on enzyme secretion in Botrytis cinereaMilling, Richard J.; Richardson, Caroline J.Pesticide Science (1995), 45 (1), 43-8CODEN: PSSCBG; ISSN:0031-613X. (Wiley)The effect of pyrimethanil on the levels of cell wall degrading enzymes secreted by Botrytis cinerea Pers. was investigated in diseased plant tissues and in liq. B. cinerea cultures. Total proteinase activity isolated from infected carrot slices which were treated with 5·0 μM pyrimethanil was decreased by 76%, 3 d after inoculation. Polygalacturonase, cellulase, proteinase and laccase activities were all decreased in the medium of three day-old cultures grown in the presence of pyrimethanil. The pyrimethanil concns. resulting in 50% redn. in total enzyme activities (IC50) were approx. 0·25 μM for polygalacturonase, cellulase and proteinase, and approx. 1·0 μM for laccase. No significant growth inhibition was obsd. at these pyrimethanil concns. Pyrimethanil did not inhibit the enzymes directly, nor did it inhibit the synthesis of cytosolic proteins. Therefore, it was proposed that the fungicide inhibits protein secretion at a post-translational stage in the secretory pathway. Large differences were found in the effects of pyrimethanil on the growth of B. cinerea in liq. cultures and on agar plates, depending on the compn. of the medium. In liq. media contg. cellulose and protein as carbon and nitrogen sources, growth inhibition occurred at 5·0 μM pyrimethanil, while no growth inhibition was obsd. with 50 μM pyrimethanil in malt ext. Similarly, growth occurred on potato/dextrose agar (PDA) at 0·5 μM pyrimethanil, but no growth was seen at this concn. on agars contg. cellulose and protein. Thus it appears that pyrimethanil is most active in media where the fungus has to utilize extracellular enzymes to mobilize the nutrients it requires for growth.
- 24Li, X.; Fernández-Ortuño, D.; Grabke, A.; Schnabel, G. Resistance to Fludioxonil in Botrytis cinerea Isolates from Blackberry and Strawberry. Phytopathology 2014, 104, 724– 732, DOI: 10.1094/PHYTO-11-13-0308-RGoogle Scholar24Resistance to fludioxonil in Botrytis cinerea isolates from blackberry and strawberryLi, Xingpeng; Fernandez-Ortuno, Dolores; Grabke, Anja; Schnabel, GuidoPhytopathology (2014), 104 (7), 724-732CODEN: PHYTAJ; ISSN:0031-949X. (American Phytopathological Society)Site-specific fungicides, including the phenylpyrrole fludioxonil, are frequently used for gray mold control but are at risk for the development of resistance. In this study, field isolates that were low-resistant (LR) and moderately resistant (MR) to fludioxonil from blackberry and strawberry fields of North Carolina, South Carolina, and Virginia were characterized. Genes involved in osmoregulation, including bcsak1, BcOS4, bos5, and BRRG-1, were cloned and sequenced to detect potential target gene alterations; however, none were found. A previously described mutation (R632I) in transcription factor Mrr1, which is known to increase the expression of ATP-binding cassette transporter AtrB, was found in MR but not in sensitive (S) or LR isolates. Expression of atrB in MR isolates was ≈200-fold increased compared with an S isolate; however, 30- to 100-fold overexpression was also detected in LR isolates. Both MR isolates exhibited increased sensitivity to salt stress in the form of mycelial growth inhibition at 4% NaCl, indicating a disruption of osmoregulatory processes in those strains. However, the glycerol content was indistinguishable between S, LR, and MR isolates with and without exposure to fludioxonil, suggesting that the glycerol synthesis pathway may not be a part of the resistance mechanism in LR or MR strains. An investigation into the origin of LR and MR isolates from blackberry revealed two insertions in the mrr1 gene consistent with those found in the Botrytis clade group S. The emergence of strains overexpressing atrB in European and now in North American strawberry fields underscores the importance of this resistance mechanism for development of resistance to fludioxonil in Botrytis cinerea.
- 25Pimentel, D.; Burgess, M. In Integrated Pest Management; Peshin, R., Pimentel, D., Eds.; Springer: Dordrecht, 2014.Google ScholarThere is no corresponding record for this reference.
- 26van der Werf, H. M. G. Assessing the impact of pesticides on the environment. Agric., Ecosyst. Environ. 1996, 60, 81– 96, DOI: 10.1016/S0167-8809(96)01096-1Google Scholar26Assessing the impact of pesticides on the environmentvan der Werf, Hayo M. G.Agriculture, Ecosystems & Environment (1996), 60 (2,3), 81-96CODEN: AEENDO; ISSN:0167-8809. (Elsevier)A review with many refs. on factors which should be taken into consideration to assess pesticide environmental impact, and how can impact be quantified. Six recent approaches to assess the impact of pesticides on the environment are compared regarding choice, transformation and aggregation of input parameters. The use of simulation models to assess environmental impact is discussed.
- 27Dong, O. X.; Ronald, P. C. Genetic Engineering for Disease Resistance in Plants: Recent Progress and Future Perspectives. Plant Physiol. 2019, 180, 26, DOI: 10.1104/pp.18.01224Google Scholar27Genetic engineering for disease resistance in plants: recent progress and future perspectivesDong, Oliver Xiaoou; Ronald, Pamela C.Plant Physiology (2019), 180 (1), 26-38CODEN: PLPHAY; ISSN:1532-2548. (American Society of Plant Biologists)A review. This article discusses about the application of genetic engineering as powerful tools that allows new genes into vegetatively propagated crops including Musa, Manihot esculenta, and Solanum tuberosum for enhancing disease resistance against plant pathogens. First, it enables the introduction, removal, modification, or fine-tuning of specific genes of interest with minimal undesired changes to the rest of the crop genome. Second, genetic engineering allows for interchange of genetic material across species. Thus, the raw genetic materials that can be exploited for this process is not restricted to the genes available within the species. Third, plant transformation during genetic engineeringallows the introduction of new genes into vegetatively propagated crops such as banana (Musa sp.), cassava (Manihot esculenta), and potato (Solanum tuberosum). As a result, crops exhibiting desired agronomic traits can be obtained in fewer generations compared with conventional breeding.
- 28Griffioen, G. Indole amide derivatives and related compounds for use in the treatment of neurodegenerative diseases. US Patent US9434722B2, 2013.Google ScholarThere is no corresponding record for this reference.
- 29Sainsbury, F.; Thuenemann, E. C.; Lomonossoff, G. P. pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol. J. 2009, 7, 682– 693, DOI: 10.1111/j.1467-7652.2009.00434.xGoogle Scholar29pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plantsSainsbury, Frank; Thuenemann, Eva C.; Lomonossoff, George P.Plant Biotechnology Journal (2009), 7 (7), 682-693CODEN: PBJLAE; ISSN:1467-7644. (Wiley-Blackwell)Agro-infiltration of leaf tissue with binary vectors harbouring a sequence of interest is a rapid method of expressing proteins in plants. It has recently been shown that flanking the sequence to be expressed with a modified 5'-untranslated region (UTR) and the 3'-UTR from Cowpea mosaic virus (CPMV) RNA-2 (CPMV-HT) within the binary vector pBINPLUS greatly enhances the level of expression that can be achieved. To exploit this finding, a series of small binary vectors tailored for transient expression (termed the pEAQ vectors) has been created. In these, more than 7 kb of nonessential sequence was removed from the pBINPLUS backbone and T-DNA region, and unique restriction sites were introduced to allow for accommodation of multiple expression cassettes, including that for a suppressor of silencing, on the same plasmid. These vectors allow the high-level simultaneous expression of multiple polypeptides from a single plasmid within a few days. Furthermore, vectors have been developed which allow the direct cloning of genes into the binary plasmid by both restriction enzyme-based cloning and GATEWAY recombination. In both cases, N- or C-terminal histidine tags may be fused to the target sequence as required. These vectors provide an easy and quick tool for the prodn. of milligram quantities of recombinant proteins from plants with std. plant research techniques at a bench-top scale.
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Abstract
Figure 1
Figure 1. Engineering of crucifalexins from canonical amino acids. (a) Schematic of metabolic engineering for new-to-nature crucifalexins. For 1-methylpropyl crucifalexin, select enzymes in the crucifalexin pathway were taken from aliphatic ITC biosynthesis (purple arrows) rather than brassinin biosynthesis. There are several enzymes involved in the conversion of the isothiocyanate to brassinin; notably, there is a S-methyltransferase specific to this pathway. (b) LC/MS extracted ion chromatograph for crucifalexins accumulating in N. benthamiana plants engineered with the core crucifalexin pathway and one of several CYP79 enzymes or no heterologous enzymes (wild-type). Each chromatograph is normalized to its largest peak, with the exception of wild-type which is at the same scale as CYP79D3. The following ions were extracted for all chromatographs: m/z 130.0651, brassinin (dedithiocarbamate ion, molecule fragments in source, parent ion not detectable); 164.0562, 1-methylpropyl crucifalexin; 198.0406, benzyl crucifalexin; 214.0355, 4-hydroxybenzyl crucifalexin. (c) Accumulation of multiple crucifalexins in N. benthamiana expressing the core crucifalexin pathway along with CYP79A1, CYP79A2, or CYP79B2 individually, in pairs, or all together. Molecules were detected via LC/MS and quantified using the curve of synthetic standards.
Figure 2
Figure 2. Engineering of halogenated brassinin derivatives. (a) Proposed halogenated brassinin pathway. Addition of halogenase reductase pairs PyrH/RebH or RebF/RebH produces 5-chlorotryptophan and 7-chlorotryptophan/7-bromotryptophan, respectively. (b) Accumulation of halogenated brassinin in engineered N. benthamiana plants. Selected ion chromatographs from LC/MS analysis of N. benthamiana leaf extracts expressing halogenases PyrH or RebH along with the reductase RebF and the brassinin pathway for production of chlorobrassinin or bromobrassinin (methylindole ions detected, m/z = 164.0262, m/z = 207.9756). Standard is mixture of 1 μM chemically synthesized 5-chlorobrassinin and 10 μM synthesized 7-bromobrassinin. Both RebH and PyrH produce a peak that matches an authentic synthetic standard of 5-chlorobrassinin mass and retention time. Given the specificity of RebH in the production of 7-chlorotryptophan previously reported in the literature [ref (19)], we anticipate that the RebH product is likely the 7-chloro isomer that coelutes with the 5-chlorobrassinin standard.
Figure 3
Figure 3. Inhibition of plant pathogenic fungi. IC50 of brassinin, new-to-nature crucifalexins, and commercial pesticide pyrimethanil against (a) generalist pathogen Botrytis cinerea and (b) crucifer specific pathogen Alternaria brassicicola. Inhibition determined by mycelial growth assay against DMSO control. Error bars represent standard deviation from a minimum of 11 biological replicates measured over two experimental trials. ns, not significant; * p < 0.05, **** p < 0.0001, one-way ANOVA.
References
This article references 29 other publications.
- 1Owen, C.; Patron, N. J.; Huang, A.; Osbourn, A. Harnessing plant metabolic diversity. Curr. Opin. Chem. Biol. 2017, 40, 24– 30, DOI: 10.1016/j.cbpa.2017.04.0151Harnessing plant metabolic diversityOwen, Charlie; Patron, Nicola J.; Huang, Ancheng; Osbourn, AnneCurrent Opinion in Chemical Biology (2017), 40 (), 24-30CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. Advances in DNA sequencing and synthesis technologies in the twenty-first century are now making it possible to build large-scale pipelines for engineering plant natural product pathways into heterologous prodn. species using synthetic biol. approaches. The ability to decode the chem. potential of plants by sequencing their transcriptomes and/or genomes and to then use this information as an instruction manual to make drugs and other high-value chems. is opening up new routes to harness the vast chem. diversity of the Plant Kingdom. Here we describe recent progress in methods for pathway discovery, DNA synthesis and assembly, and expression of engineered pathways in heterologous hosts. We also highlight the importance of standardization and the challenges assocd. with dataset integration in the drive to build a systematic framework for effective harnessing of plant metabolic diversity.
- 2Reed, J. A translational synthetic biology platform for rapid access to gram-scale quantities of novel drug-like molecules. Metab. Eng. 2017, 42, 185– 193, DOI: 10.1016/j.ymben.2017.06.0122A translational synthetic biology platform for rapid access to gram-scale quantities of novel drug-like moleculesReed, James; Stephenson, Michael J.; Miettinen, Karel; Brouwer, Bastiaan; Leveau, Aymeric; Brett, Paul; Goss, Rebecca J. M.; Goossens, Alain; O'Connell, Maria A.; Osbourn, AnneMetabolic Engineering (2017), 42 (), 185-193CODEN: MEENFM; ISSN:1096-7176. (Elsevier B.V.)Plants are an excellent source of drug leads. However availability is limited by access to source species, low abundance and recalcitrance to chem. synthesis. Although plant genomics is yielding a wealth of genes for natural product biosynthesis, the translation of this genetic information into small mols. for evaluation as drug leads represents a major bottleneck. For example, the yeast platform for artemisinic acid prodn. is estd. to have taken >150 person years to develop. Here we demonstrate the power of plant transient transfection technol. for rapid, scalable biosynthesis and isolation of triterpenes, one of the largest and most structurally diverse families of plant natural products. Using pathway engineering and improved agro-infiltration methodol. we are able to generate gram-scale quantities of purified triterpene in just a few weeks. In contrast to heterologous expression in microbes, this system does not depend on re-engineering of the host. We next exploit agro-infection for quick and easy combinatorial biosynthesis without the need for generation of multi-gene constructs, so affording an easy entre´e to suites of mols., some new-to-nature, that are recalcitrant to chem. synthesis. We use this platform to purify a suite of bespoke triterpene analogs and demonstrate differences in anti-proliferative and anti-inflammatory activity in bioassays, providing proof of concept of this system for accessing and evaluating medicinally important bioactives. Together with new genome mining algorithms for plant pathway discovery and advances in plant synthetic biol., this advance provides new routes to synthesize and access previously inaccessible natural products and analogs and has the potential to reinvigorate drug discovery pipelines.
- 3Pierce, M. L.; Cover, E. C.; Richardson, P. E.; Scholes, V. E.; Essenberg, M. Adequacy of cellular phytoalexin concentrations in hypersensitively responding cotton leaves. Physiol. Mol. Plant Pathol. 1996, 48, 305– 324, DOI: 10.1006/pmpp.1996.00253Adequacy of cellular phytoalexin concentrations in hypersensitively responding cotton leavesPierce, M. L.; Cover, E. C.; Richardson, P. E.; Scholes, V. E.; Essenberg, M.Physiological and Molecular Plant Pathology (1996), 48 (5), 305-324CODEN: PMPPEZ; ISSN:0885-5765. (Academic)Av. cellular phytoalexin concns. at infection sites were detd. in Xanthomonas campestris pv. malvacearum infected cotton leaves to test the hypothesis that sesquiterpenoid phytoalexins play a major role in heritable resistance of cotton to bacterial blight. Bacteriostasis was achieved in leaves of resistant cotton line WbM(0.0) by 5-7 days after inoculation. Between days 3 and 7, phytoalexin amts. per g of tissue were above basal levels but were far below their peak values. Nevertheless, when the phytoalexin quantities present on day 7 were divided by the vol. of water contained in the hypersensitively responding cells defining the scattered infection centers, the concns. computed were ones previously found to be highly inhibitory in in vitro bioassays. In necrotic cells of susceptible line WbM(4.0), the values were ≤ 3% of those in the resistant line. In a comparison of highly resistant lines OK 1.2 and Im216, concns. of phytoalexins far exceeding inhibitory levels in vitro were calcd. for hypersensitively necrotic cells in both lines on days 3 and 4 post-inoculation. Anal. of the clustering of necrotic cells indicated that in OK1.2, only 25% of the infection sites had mounted a reaction by day 3 and nearly all had responded by day 4, while in Im216, 50% or more had responded hypersensitively by day 3 and all sites had shown a reaction by day 4. Bacterial inhibition occurred on the day following appearance of HR-cell clusters at all sites. The phytoalexins present in the HR cells had access to the bacteria, as indicated by loss of plasmalemma integrity of the yellow-green fluorescent, hypersensitively responding cells and by diffusion of the phytoalexins into the bathing medium.
- 4Klein, A. P.; Sattely, E. S. Biosynthesis of cabbage phytoalexins from indole glucosinolate. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 1910, DOI: 10.1073/pnas.16156251144Biosynthesis of cabbage phytoalexins from indole glucosinolateKlein, Andrew P.; Sattely, Elizabeth S.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (8), 1910-1915CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Brassica crop species are prolific producers of indole-sulfur phytoalexins that are thought to have an important role in plant disease resistance. These mols. are conspicuously absent in the model plant Arabidopsis thaliana, and little is known about the enzymic steps that assemble the key precursor brassinin. Here, we report the min. set of biosynthetic genes required to generate cruciferous phytoalexins starting from the well-studied glucosinolate pathway. In vitro biochem. characterization revealed an addnl. role for the previously described carbon-sulfur lyase SUR1 in processing cysteine-isothiocyanate conjugates, as well as the S-methyltransferase DTCMT that methylates the resulting dithiocarbamate, together completing a pathway to brassinin. Addnl., the β-glucosidase BABG that is present in Brassica rapa but absent in Arabidopsis was shown to act as a myrosinase and may be a determinant of plants that synthesize phytoalexins from indole glucosinolate. Transient expression of the entire pathway in Nicotiana benthamiana yields brassinin, demonstrating that the biosynthesis of indole-sulfur phytoalexins can be engineered into noncruciferous plants. The identification of these biosynthetic enzymes and the heterologous reconstitution of the indole-sulfur phytoalexin pathway sheds light on an important pathway in an edible plant and opens the door to using metabolic engineering to systematically quantify the impact of cruciferous phytoalexins on plant disease resistance and human health.
- 5Halkier, B. A.; Gershenzon, J. Biology and biochemistry of glucosinolates. Annu. Rev. Plant Biol. 2006, 57, 303– 333, DOI: 10.1146/annurev.arplant.57.032905.1052285Biology and biochemistry of glucosinolatesHalkier, Barbara Ann; Gershenzon, JonathanAnnual Review of Plant Biology (2006), 57 (), 303-333CODEN: ARPBDW ISSN:. (Annual Reviews Inc.)A review. Glucosinolates are sulfur-rich, anionic natural products that upon hydrolysis by endogenous thioglucosidases called myrosinases produce several different products (e.g., isothiocyanates, thiocyanates, and nitriles). The hydrolysis products have many different biol. activities, e.g., as defense compds. and attractants. For humans these compds. function as cancer-preventing agents, biopesticides, and flavor compds. Since the completion of the Arabidopsis genome, glucosinolate research has made significant progress, resulting in near-complete elucidation of the core biosynthetic pathway, identification of the first regulators of the pathway, metabolic engineering of specific glucosinolate profiles to study function, as well as identification of evolutionary links to related pathways. Although much was learned in recent years, much more awaits discovery before the authors fully understand how and why plants synthesize glucosinolates. This may enable us to more fully exploit the potential of these compds. in agriculture and medicine.
- 6Wittstock, U.; Halkier, B. A. Glucosinolate research in the Arabidopsis era. Trends Plant Sci. 2002, 7, 263– 270, DOI: 10.1016/S1360-1385(02)02273-26Glucosinolate research in the Arabidopsis eraWittstock, Ute; Halkier, Barbara A.Trends in Plant Science (2002), 7 (6), 263-270CODEN: TPSCF9; ISSN:1360-1385. (Elsevier Science Ltd.)A review. The wide range of biol. activities of products derived from the glucosinolate-myrosinase system is biol. and economically important. On the one hand, the degrdn. products of glucosinolates play an important role in the defense of plants against herbivores. On the other hand, these compds. have toxic (e.g. goitrogenic) as well as protective (e.g. cancer-preventing) effects in higher animals and humans. There is a strong interest in the ability to regulate and optimize the levels of individual glucosinolates tissue-specifically to improve the nutritional value and pest resistance of crops. Recent advances in our understanding of glucosinolate biosynthesis have brought us closer to this goal. Genetic engineering of glucosinolate profiles is now a realistic possibility. Individual glucosinolates could improve nutritional value and pest resistance of important crops.
- 7Pedras, M. S. C.; To, Q. H. Non-indolyl cruciferous phytoalexins: Nasturlexins and tridentatols, a striking convergent evolution of defenses in terrestrial plants and marine animals?. Phytochemistry 2015, 113, 57– 63, DOI: 10.1016/j.phytochem.2014.07.0247The first non-indolyl cruciferous phytoalexins: nasturlexins and tridentatols, a striking convergent evolution of defenses in terrestrial plants and marine animals?Pedras, M. Soledade C.; To, Q. HuyPhytochemistry (Elsevier) (2015), 113 (), 57-63CODEN: PYTCAS; ISSN:0031-9422. (Elsevier Ltd.)Highly specialized chem. defense pathways are a particularly noteworthy metabolic characteristic of sessile organisms, whether terrestrial or marine, providing protection against pests and diseases. For this reason, knowledge of the metabolites involved in these processes is crucial to producing ecol. fit crops. Toward this end, the elicited chem. defenses of the crucifer watercress (Nasturtium officinale R.Br.), i.e., phytoalexins, were investigated and are reported. Almost three decades after publication of cruciferous phytoalexins derived from (S)-Trp, phytoalexins derived from other arom. amino acids were isolated; their chem. structures were detd. by analyses of their spectroscopic data and confirmed by synthesis. Nasturlexin A, nasturlexin B, and tridentatol C are hitherto unknown Ph contg. cruciferous phytoalexins produced by watercress under abiotic stress; tridentatol C is also produced by a marine animal (Tridentata marginata), where it functions in chem. defense against predators. The biosynthesis of these metabolites in both a terrestrial plant and a marine animal suggests a convergent evolution of unique metabolic pathways recruited for defense.
- 8Pedras, M. S. C.; Yaya, E. E.; Glawischnig, E. The phytoalexins from cultivated and wild crucifers: Chemistry and biology. Nat. Prod. Rep. 2011, 28, 1381– 1405, DOI: 10.1039/c1np00020a8The phytoalexins from cultivated and wild crucifers: Chemistry and biologyPedras, M. Soledade C.; Yaya, Estifanos E.; Glawischnig, ErichNatural Product Reports (2011), 28 (8), 1381-1405CODEN: NPRRDF; ISSN:0265-0568. (Royal Society of Chemistry)A review. Phytoalexins are antimicrobial secondary metabolites produced de novo by plants in response to stress, including microbial attack. In general, phytoalexins are important components of plant defenses against fungal and bacterial pathogens. The phytoalexins of crucifers are indole alkaloids derived from (S)-tryptophan, most of which contain a sulfur atom derived from cysteine. Beside their antimicrobial activity against different plant pathogenic species, cruciferous phytoalexins have shown anticarcinogenic effects on various human cell lines. This review focuses on the phytoalexins produced by cruciferous plants reported to date, with particular emphasis on their chem. synthesis, biosynthesis, metab. by plant fungal pathogens and biol. activities. A summary table contg. all phytoalexins, their cultivated and wild cruciferous sources, their synthetic starting materials, biotransformation products and biol. activities is provided.
- 9Banerjee, T. A key in vivo antitumor mechanism of action of natural product-based brassinins is inhibition of indoleamine 2,3-dioxygenase. Oncogene 2008, 27, 2851– 2857, DOI: 10.1038/sj.onc.12109399A key in vivo antitumor mechanism of action of natural product-based brassinins is inhibition of indoleamine 2,3-dioxygenaseBanerjee, T.; DuHadaway, J. B.; Gaspari, P.; Sutanto-Ward, E.; Munn, D. H.; Mellor, A. L.; Malachowski, W. P.; Prendergast, G. C.; Muller, A. J.Oncogene (2008), 27 (20), 2851-2857CODEN: ONCNES; ISSN:0950-9232. (Nature Publishing Group)Agents that interfere with tumoral immune tolerance may be useful to prevent or treat cancer. Brassinin is a phytoalexin, a class of natural products derived from plants that includes the widely known compd. resveratrol. Brassinin has been demonstrated to have chemopreventive activity in preclin. models but the mechanisms underlying its anticancer properties are unknown. Here, we show that brassinin and a synthetic deriv. 5-bromo-brassinin (5-Br-brassinin) are bioavailable inhibitors of indoleamine 2,3-dioxygenase (IDO), a pro-tolerogenic enzyme that drives immune escape in cancer. Like other known IDO inhibitors, both of these compds. combined with chemotherapy to elicit regression of autochthonous mammary gland tumors in MMTV-Neu mice. Furthermore, growth of highly aggressive melanoma isograft tumors was suppressed by single agent treatment with 5-Br-brassinin. This response to treatment was lost in athymic mice, indicating a requirement for active host T-cell immunity, and in IDO-null knockout mice, providing direct genetic evidence that IDO inhibition is essential to the antitumor mechanism of action of 5-Br-brassinin. The natural product brassinin thus provides the structural basis for a new class of compds. with in vivo anticancer activity that is mediated through the inhibition of IDO.
- 10Bak, S.; Olsen, C. E.; Petersen, B. L.; Møller, B. L.; Halkier, B. A. Metabolic engineering of p-hydroxybenzylglucosinolate in Arabidopsis by expression of the cyanogenic CYP79A1 from Sorghum bicolor. Plant J. 1999, 20, 663– 671, DOI: 10.1046/j.1365-313X.1999.00642.x10Metabolic engineering of p-hydroxybenzylglucosinolate in Arabidopsis by expression of the cyanogenic CYP79A1 from Sorghum bicolorBak, Soren; Olsen, Carl Erik; Petersen, Bent Larsen; Moller, Birger Lindberg; Halkier, Barbara AnnPlant Journal (1999), 20 (6), 663-671CODEN: PLJUED; ISSN:0960-7412. (Blackwell Science Ltd.)Glucosinolates are natural products in cruciferous plants, including Arabidopsis thaliana. CYP79A1 is the cytochrome P 450 catalyzing the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of the cyanogenic glucoside dhurrin in sorghum. Both glucosinolates and cyanogenic glucosides have oximes as intermediates. Expression of CYP79A1 in A. thaliana results in the prodn. of high levels of the tyrosine-derived glucosinolate p-hydroxybenzylglucosinolate, which is not a natural constituent of A. thaliana. This provides further evidence that the enzymes have low substrate specificity with respect to the side chain. The ability of the cyanogenic CYP79A1 to integrate itself into the glucosinolate pathway has important implications for an evolutionary relationship between cyanogenic glucosides and glucosinolates, and for the possibility of genetic engineering of novel glucosinolates.
- 11Mikkelsen, M. D.; Halkier, B. A. Metabolic Engineering of Valine- and Isoleucine-Derived Glucosinolates in Arabidopsis Expressing CYP79D2 from Cassava. Plant Physiol. 2003, 131, 773, DOI: 10.1104/pp.01342511Metabolic engineering of valine- and isoleucine-derived glucosinolates in Arabidopsis expressing CYP79D2 from cassavaMikkelsen, Michael Dalgaard; Halkier, Barbara AnnPlant Physiology (2003), 131 (2), 773-779CODEN: PLPHAY; ISSN:0032-0889. (American Society of Plant Biologists)Glucosinolates are amino acid-derived natural products that, upon hydrolysis, typically release isothiocyanates with a wide range of biol. activities. Glucosinolates play a role in plant defense as attractants and deterrents against herbivores and pathogens. A key step in glucosinolate biosynthesis is the conversion of amino acids to the corresponding aldoximes, which is catalyzed by cytochromes P 450 belonging to the CYP79 family. Expression of CYP79D2 from cassava (Manihot esculenta Crantz.) in Arabidopsis resulted in the prodn. of valine (Val)- and isoleucine-derived glucosinolates not normally found in this ecotype. The transgenic lines showed no morphol. phenotype, and the level of endogenous glucosinolates was not affected. The novel glucosinolates were shown to constitute up to 35% of the total glucosinolate content in mature rosette leaves and up to 48% in old leaves. Furthermore, at increased concns. of these glucosinolates, the proportion of Val-derived glucosinolates decreased. As the isothiocyanates produced from the Val- and isoleucine-derived glucosinolates are volatile, metabolically engineered plants producing these glucosinolates have acquired novel properties with great potential for improvement of resistance to herbivorous insects and for biofumigation.
- 12Kahn, R. A.; Fahrendorf, T.; Halkier, B. A.; Møller, B. L. Substrate specificity of the cytochrome P450 enzymes CYP79A1 and CYP71E1 involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench. Arch. Biochem. Biophys. 1999, 363, 9– 18, DOI: 10.1006/abbi.1998.106812Substrate Specificity of the Cytochrome P450 Enzymes CYP79A1 and CYP71E1 Involved in the Biosynthesis of the Cyanogenic Glucoside Dhurrin in Sorghum bicolor (L.) MoenchKahn, Rachel Alice; Fahrendorf, Theodor; Halkier, Barbara Ann; Moller, Birger LindbergArchives of Biochemistry and Biophysics (1999), 363 (1), 9-18CODEN: ABBIA4; ISSN:0003-9861. (Academic Press)The two multifunctional cytochrome P 450 enzymes, CYP79A1 and CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench have been characterized with respect to substrate specificity and cofactor requirements using reconstituted, recombinant enzymes and sorghum microsomes. CYP79A1 has a very high substrate specificity, tyrosine being the only substrate found. CYP71E1 has less stringent substrate requirements and metabolizes arom. oximes efficiently, whereas aliph. oximes are slowly metabolized. Neither CYP79A1 nor CYP71E1 catalyze the metab. of a range of different herbicides. The reported resistance of sorghum to bentazon is therefore not linked to the presence of CYP79A1 or CYP71E1. NADPH is a much better cofactor than NADH although NADH does support the entire catalytic cycle of both P 450 enzymes. Km and Vmax values for NADPH when supporting CYP71E1 activity are 0.013 mM and 111 nmol/mg protein/s. For NADH, the corresponding values are 0.3 mM and 42 nmol/mg protein/s. CYP79A1 is a fairly stable enzyme. In contrast, CYP71E1 is labile and prone to rapid denaturation at room temp. CYP71E1 is isolated in the low spin form. CYP71E1 catalyzes an unusual dehydration reaction of an oxime to the corresponding nitrile which subsequently is C-hydroxylated. The oxime forms a peculiar reverse Type I spectrum, whereas the nitrile forms a Type I spectrum. Several compds. which do not serve as substrates formed Type I substrate binding spectra with the two P 450 enzymes. (c) 1999 Academic Press.
- 13Forslund, K. Biosynthesis of the Nitrile Glucosides Rhodiocyanoside A and D and the Cyanogenic Glucosides Lotaustralin and Linamarin in Lotus japonicus. Plant Physiol. 2004, 135, 71, DOI: 10.1104/pp.103.03805913Biosynthesis of the nitrile glucosides rhodiocyanoside A and D and the cyanogenic glucosides lotaustralin and linamarin in Lotus japonicusForslund, Karin; Morant, Marc; Jorgensen, Bodil; Olsen, Carl Erik; Asamizu, Erika; Sato, Shusei; Tabata, Satoshi; Bak, SorenPlant Physiology (2004), 135 (1), 71-84CODEN: PLPHAY; ISSN:0032-0889. (American Society of Plant Biologists)Lotus japonicus was shown to contain the two nitrile glucosides rhodiocyanoside A and rhodiocyanoside D as well as the cyanogenic glucosides linamarin and lotaustralin. The content of cyanogenic and nitrile glucosides in L. japonicus depends on plant developmental stage and tissue. The cyanide potential is highest in young seedlings and in apical leaves of mature plants. Roots and seeds are acyanogenic. Biosynthetic studies using radioisotopes demonstrated that lotaustralin, rhodiocyanoside A, and rhodiocyanoside D are derived from the amino acid L-Ile, whereas linamarin is derived from Val. In silico homol. searches identified two cytochromes P 450 designated CYP79D3 and CYP79D4 in L. japonicus. The two cytochromes P 450 are 94% identical at the amino acid level and both catalyze the conversion of Val and Ile to the corresponding aldoximes in biosynthesis of cyanogenic glucosides and nitrile glucosides in L. japonicus. CYP79D3 and CYP79D4 are differentially expressed. CYP79D3 is exclusively expressed in aerial parts and CYP79D4 in roots. Recombinantly expressed CYP79D3 and CYP79D4 in yeast cells showed higher catalytic efficiency with L-Ile as substrate than with L-Val, in agreement with lotaustralin and rhodiocyanoside A and D being the major cyanogenic and nitrile glucosides in L. japonicus. Ectopic expression of CYP79D2 from cassava (Manihot esculenta Crantz.) in L. japonicus resulted in a 5- to 20-fold increase of linamarin content, whereas the relative amts. of lotaustralin and rhodiocyanoside A/D were unaltered.
- 14Wittstock, U.; Halkier, B. A. Cytochrome P450 CYP79A2 from Arabidopsis thaliana L. Catalyzes the Conversion of l-Phenylalanine to Phenylacetaldoxime in the Biosynthesis of Benzylglucosinolate. J. Biol. Chem. 2000, 275, 14659– 14666, DOI: 10.1074/jbc.275.19.1465914Cytochrome P450 CYP79A2 from Arabidopsis thaliana L. catalyzes the conversion of L-phenylalanine to phenylacetaldoxime in the biosynthesis of benzylglucosinolateWittstock, Ute; Halkier, Barbara AnnJournal of Biological Chemistry (2000), 275 (19), 14659-14666CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Glucosinolates are natural plant products gaining increasing interest as cancer-preventing agents and crop protectants. Similar to cyanogenic glucosides, glucosinolates are derived from amino acids and have aldoximes as intermediates. We report cloning and characterization of cytochrome P 450 CYP79A2 involved in aldoxime formation in the glucosinolate-producing Arabidopsis thaliana L. The CYP79A2 cDNA was cloned by polymerase chain reaction, and CYP79A2 was functionally expressed in Escherichia coli. Characterization of the recombinant protein shows that CYP79A2 is an N-hydroxylase converting L-phenylalanine into phenylacetaldoxime, the precursor of benzylglucosinolate. Transgenic A. thaliana constitutively expressing CYP79A2 accumulate high levels of benzylglucosinolate. CYP79A2 expressed in E. coli has a Km of 6.7 μmol liter-1 for L-phenylalanine. Neither L-tyrosine, L-tryptophan, L-methionine, nor DL-homophenylalanine are metabolized by CYP79A2, indicating that the enzyme has a narrow substrate specificity. CYP79A2 is the first enzyme shown to catalyze the conversion of an amino acid to the aldoxime in the biosynthesis of glucosinolates. Our data provide the first conclusive evidence that evolutionarily conserved cytochromes P 450 catalyze this step common for the biosynthetic pathways of glucosinolates and cyanogenic glucosides. This strongly indicates that the biosynthesis of glucosinolates has evolved based on a cyanogenic predisposition.
- 15Brader, G.; Mikkelsen, M. D.; Halkier, B. A.; Tapio Palva, E. Altering glucosinolate profiles modulates disease resistance in plants. Plant J. 2006, 46, 758– 767, DOI: 10.1111/j.1365-313X.2006.02743.x15Altering glucosinolate profiles modulates disease resistance in plantsBrader, Gunter; Mikkelsen, Michael Dalgaard; Halkier, Barbara Ann; Palva, E. TapioPlant Journal (2006), 46 (5), 758-767CODEN: PLJUED; ISSN:0960-7412. (Blackwell Publishing Ltd.)Plant diseases are major contributing factors for crop loss in agriculture. Here, the authors show that Arabidopsis plants with high levels of novel glucosinolates (GSs) as a result of the introduction of single CYP79 genes exhibit altered disease resistance. Arabidopsis expressing CYP79D2 from cassava accumulated aliph. iso-Pr and methylpropyl GS, and showed enhanced resistance against the bacterial soft-rot pathogen Erwinia carotovora, whereas Arabidopsis expressing the sorghum CYP79A1 or over-expressing the endogenous CYP79A2 accumulated p-hydroxybenzyl or benzyl GS, resp., and showed increased resistance towards the bacterial pathogen Pseudomonas syringae. In addn. to the direct toxic effects of GS breakdown products, increased accumulation of arom. GSs was shown to stimulate salicylic acid-mediated defenses while suppressing jasmonate-dependent defenses, as manifested in enhanced susceptibility to the fungus Alternaria brassicicola. Arabidopsis with modified GS profiles provide important tools for evaluating the biol. effects of individual GSs and thereby show potential as biotechnol. tools for the generation of plants with tailor-made disease resistance.
- 16Wang, X. The genome of the mesopolyploid crop species Brassica rapa. Nat. Genet. 2011, 43, 1035, DOI: 10.1038/ng.91916The genome of the mesopolyploid crop species Brassica rapaWang, Xiaowu; Wang, Hanzhong; Wang, Jun; Sun, Rifei; Wu, Jian; Liu, Shengyi; Bai, Yinqi; Mun, Jeong-Hwan; Bancroft, Ian; Cheng, Feng; Huang, Sanwen; Li, Xixiang; Hua, Wei; Wang, Junyi; Wang, Xiyin; Freeling, Michael; Pires, J. Chris; Paterson, Andrew H.; Chalhoub, Boulos; Wang, Bo; Hayward, Alice; Sharpe, Andrew G.; Park, Beom-Seok; Weisshaar, Bernd; Liu, Binghang; Li, Bo; Liu, Bo; Tong, Chaobo; Song, Chi; Duran, Christopher; Peng, Chunfang; Geng, Chunyu; Koh, Chushin; Lin, Chuyu; Edwards, David; Mu, Desheng; Shen, Di; Soumpourou, Eleni; Li, Fei; Fraser, Fiona; Conant, Gavin; Lassalle, Gilles; King, Graham J.; Bonnema, Guusje; Tang, Haibao; Wang, Haiping; Belcram, Harry; Zhou, Heling; Hirakawa, Hideki; Abe, Hiroshi; Guo, Hui; Wang, Hui; Jin, Huizhe; Parkin, Isobel A. P.; Batley, Jacqueline; Kim, Jeong-Sun; Just, Jeremy; Li, Jianwen; Xu, Jiaohui; Deng, Jie; Kim, Jin A.; Li, Jingping; Yu, Jingyin; Meng, Jinling; Wang, Jinpeng; Min, Jiumeng; Poulain, Julie; Wang, Jun; Hatakeyama, Katsunori; Wu, Kui; Wang, Li; Fang, Lu; Trick, Martin; Links, Matthew G.; Zhao, Meixia; Jin, Mina; Ramchiary, Nirala; Drou, Nizar; Berkman, Paul J.; Cai, Qingle; Huang, Quanfei; Li, Ruiqiang; Tabata, Satoshi; Cheng, Shifeng; Zhang, Shu; Zhang, Shujiang; Huang, Shunmou; Sato, Shusei; Sun, Silong; Kwon, Soo-Jin; Choi, Su-Ryun; Lee, Tae-Ho; Fan, Wei; Zhao, Xiang; Tan, Xu; Xu, Xun; Wang, Yan; Qiu, Yang; Yin, Ye; Li, Yingrui; Du, Yongchen; Liao, Yongcui; Lim, Yongpyo; Narusaka, Yoshihiro; Wang, Yupeng; Wang, Zhenyi; Li, Zhenyu; Wang, Zhiwen; Xiong, Zhiyong; Zhang, ZhonghuaNature Genetics (2011), 43 (10), 1035-1039CODEN: NGENEC; ISSN:1061-4036. (Nature Publishing Group)This report presents the annotation and anal. of the draft genome sequence of Brassica rapa accession Chiifu-401-42, a Chinese cabbage. The authors modeled 41,174 protein coding genes in the B. rapa genome, which has undergone genome triplication. Arabidopsis thaliana was used as an outgroup for investigating the consequences of genome triplication, such as structural and functional evolution. The extent of gene loss (fractionation) among triplicated genome segments varies, with one of the three copies consistently retaining a disproportionately large fraction of the genes expected to have been present in its ancestor. Variation in the no. of members of gene families present in the genome may contribute to the remarkable morphol. plasticity of Brassica species. The B. rapa genome sequence provides an important resource for studying the evolution of polyploid genomes and underpins the genetic improvement of Brassica oil and vegetable crops. The draft genome sequence is deposited in GenBank/EMBL/DDBJ with project accession nos. AENI01000001-AENI01051658.
- 17Liu, S. The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes. Nat. Commun. 2014, 5, 3930, DOI: 10.1038/ncomms493017The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomesLiu, Shengyi; Liu, Yumei; Yang, Xinhua; Tong, Chaobo; Edwards, David; Parkin, Isobel A. P.; Zhao, Meixia; Ma, Jianxin; Yu, Jingyin; Huang, Shunmou; Wang, Xiyin; Wang, Junyi; Lu, Kun; Fang, Zhiyuan; Bancroft, Ian; Yang, Tae-Jin; Hu, Qiong; Wang, Xinfa; Yue, Zhen; Li, Haojie; Yang, Linfeng; Wu, Jian; Zhou, Qing; Wang, Wanxin; King, Graham J.; Pires, J. Chris; Lu, Changxin; Wu, Zhangyan; Sampath, Perumal; Wang, Zhuo; Guo, Hui; Pan, Shengkai; Yang, Limei; Min, Jiumeng; Zhang, Dong; Jin, Dianchuan; Li, Wanshun; Belcram, Harry; Tu, Jinxing; Guan, Mei; Qi, Cunkou; Du, Dezhi; Li, Jiana; Jiang, Liangcai; Batley, Jacqueline; Sharpe, Andrew G.; Park, Beom-Seok; Ruperao, Pradeep; Cheng, Feng; Waminal, Nomar Espinosa; Huang, Yin; Dong, Caihua; Wang, Li; Li, Jingping; Hu, Zhiyong; Zhuang, Mu; Huang, Yi; Huang, Junyan; Shi, Jiaqin; Mei, Desheng; Liu, Jing; Lee, Tae-Ho; Wang, Jinpeng; Jin, Huizhe; Li, Zaiyun; Li, Xun; Zhang, Jiefu; Xiao, Lu; Zhou, Yongming; Liu, Zhongsong; Liu, Xuequn; Qin, Rui; Tang, Xu; Liu, Wenbin; Wang, Yupeng; Zhang, Yangyong; Lee, Jonghoon; Kim, Hyun Hee; Denoeud, France; Xu, Xun; Liang, Xinming; Hua, Wei; Wang, Xiaowu; Wang, Jun; Chalhoub, Boulos; Paterson, Andrew H.Nature Communications (2014), 5 (), 3930CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Polyploidization has provided much genetic variation for plant adaptive evolution, but the mechanisms by which the mol. evolution of polyploid genomes establishes genetic architecture underlying species differentiation are unclear. Brassica is an ideal model to increase knowledge of polyploid evolution. Here we describe a draft genome sequence of Brassica oleracea, comparing it with that of its sister species B. rapa to reveal numerous chromosome rearrangements and asym. gene loss in duplicated genomic blocks, asym. amplification of transposable elements, differential gene co-retention for specific pathways and variation in gene expression, including alternative splicing, among a large no. of paralogous and orthologous genes. Genes related to the prodn. of anticancer phytochems. and morphol. variations illustrate consequences of genome duplication and gene divergence, imparting biochem. and morphol. variation to B. oleracea. This study provides insights into Brassica genome evolution and will underpin research into the many important crops in this genus.
- 18Klein, A. P.; Sattely, E. S. Two cytochromes P450 catalyze S-heterocyclizations in cabbage phytoalexin biosynthesis. Nat. Chem. Biol. 2015, 11, 837, DOI: 10.1038/nchembio.191418Two cytochromes P450 catalyze S-heterocyclizations in cabbage phytoalexin biosynthesisKlein, Andrew P.; Sattely, Elizabeth S.Nature Chemical Biology (2015), 11 (11), 837-839CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Phytoalexins are abundant in edible crucifers and have important biol. activities, yet no dedicated gene for their biosynthesis is known. Here, we report two new cytochromes P 450 from Brassica rapa (Chinese cabbage) that catalyze unprecedented S-heterocyclizations in cyclobrassinin and spirobrassinin biosynthesis. Our results provide genetic and biochem. insights into the biosynthesis of a prominent pair of dietary metabolites and have implications for pathway discovery across >20 recently sequenced crucifers.
- 19Runguphan, W.; Qu, X.; O’Connor, S. E. Integrating carbon–halogen bond formation into medicinal plant metabolism. Nature 2010, 468, 461, DOI: 10.1038/nature0952419Integrating carbon-halogen bond formation into medicinal plant metabolismRunguphan, Weerawat; Qu, Xu-Dong; O'Connor, Sarah E.Nature (London, United Kingdom) (2010), 468 (7322), 461-464CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Halogenation, which was once considered a rare occurrence in nature, has now been obsd. in many natural product biosynthetic pathways. However, only a small fraction of halogenated compds. have been isolated from terrestrial plants. Given the impact that halogenation can have on the biol. activity of natural products, we reasoned that the introduction of halides into medicinal plant metab. would provide the opportunity to rationally bioengineer a broad variety of novel plant products with altered, and perhaps improved, pharmacol. properties. Here we report that chlorination biosynthetic machinery from soil bacteria can be successfully introduced into the medicinal plant Catharanthus roseus (Madagascar periwinkle). These prokaryotic halogenases function within the context of the plant cell to generate chlorinated tryptophan, which is then shuttled into monoterpene indole alkaloid metab. to yield chlorinated alkaloids. A new functional group-a halide-is thereby introduced into the complex metab. of C. roseus, and is incorporated in a predictable and regioselective manner onto the plant alkaloid products. Medicinal plants, despite their genetic and developmental complexity, therefore seem to be a viable platform for synthetic biol. efforts.
- 20Menon, B. R. K. RadH: A Versatile Halogenase for Integration into Synthetic Pathways. Angew. Chem., Int. Ed. 2017, 56, 11841– 11845, DOI: 10.1002/anie.20170634220RadH: A Versatile Halogenase for Integration into Synthetic PathwaysMenon, Binuraj R. K.; Brandenburger, Eileen; Sharif, Humera H.; Klemstein, Ulrike; Shepherd, Sarah A.; Greaney, Michael F.; Micklefield, JasonAngewandte Chemie, International Edition (2017), 56 (39), 11841-11845CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)Flavin-dependent halogenases are useful enzymes for providing halogenated mols. with improved biol. activity, or intermediates for synthetic derivatization. We demonstrate how the fungal halogenase RadH can be used to regioselectively halogenate a range of bioactive arom. scaffolds. Site-directed mutagenesis of RadH was used to identify catalytic residues and provide insight into the mechanism of fungal halogenases. A high-throughput fluorescence screen was also developed, which enabled a RadH mutant to be evolved with improved properties. Finally we demonstrate how biosynthetic genes from fungi, bacteria, and plants can be combined to encode a new pathway to generate a novel chlorinated coumarin "non-natural" product in E. coli.
- 21Dean, R. The Top 10 fungal pathogens in molecular plant pathology. Mol. Plant Pathol. 2012, 13, 414– 430, DOI: 10.1111/j.1364-3703.2011.00783.x21The Top 10 fungal pathogens in molecular plant pathologyDean Ralph; Van Kan Jan A L; Pretorius Zacharias A; Hammond-Kosack Kim E; Di Pietro Antonio; Spanu Pietro D; Rudd Jason J; Dickman Marty; Kahmann Regine; Ellis Jeff; Foster Gary DMolecular plant pathology (2012), 13 (4), 414-30 ISSN:.The aim of this review was to survey all fungal pathologists with an association with the journal Molecular Plant Pathology and ask them to nominate which fungal pathogens they would place in a 'Top 10' based on scientific/economic importance. The survey generated 495 votes from the international community, and resulted in the generation of a Top 10 fungal plant pathogen list for Molecular Plant Pathology. The Top 10 list includes, in rank order, (1) Magnaporthe oryzae; (2) Botrytis cinerea; (3) Puccinia spp.; (4) Fusarium graminearum; (5) Fusarium oxysporum; (6) Blumeria graminis; (7) Mycosphaerella graminicola; (8) Colletotrichum spp.; (9) Ustilago maydis; (10) Melampsora lini, with honourable mentions for fungi just missing out on the Top 10, including Phakopsora pachyrhizi and Rhizoctonia solani. This article presents a short resume of each fungus in the Top 10 list and its importance, with the intent of initiating discussion and debate amongst the plant mycology community, as well as laying down a bench-mark. It will be interesting to see in future years how perceptions change and what fungi will comprise any future Top 10.
- 22Fritz, R.; Lanen, C.; Colas, V.; Leroux, P. Inhibition of Methionine Biosynthesis in Botrytis cinerea by the Anilinopyrimidine Fungicide Pyrimethanil. Pestic. Sci. 1997, 49, 40– 46, DOI: 10.1002/(SICI)1096-9063(199701)49:1<40::AID-PS470>3.0.CO;2-Y22Inhibition of methionine biosynthesis in Botrytis cinerea by the anilinopyrimidine fungicide pyrimethanilFritz, Rene; Lanen, Catherine; Colas, Virginie; Leroux, PierrePesticide Science (1997), 49 (1), 40-46CODEN: PSSCBG; ISSN:0031-613X. (Wiley)When mycelium of B. cinerea was treated with low concns. of pyrimethanil, the total amt. of free amino acids increased. Qual. variations were also induced: alanine, glutamine, lysine, glycine, histidine, asparagine, arginine, threonine, α-aminobutyrate and β-alanine were accumulated; cyst(e)ine, valine, leucine and citrulline were reduced. When mycelium of B. cinerea was incubated with Na2[35S]O4, pyurimethanil, at 1·5 μM, induced a decrease of [35S]methionine and simultaneously an increase of [35S]cystathionine. Thus, pyrimethanil inhibits the biosynthesis of methionine and suggest that the primary target could be the cystathionine β-lyase.
- 23Milling, R. J.; Richardson, C. J. Mode of action of the anilino-pyrimidine fungicide Pyrimethanil. 2. Effects on enzyme secretion in Botrytis cinerea. Pestic. Sci. 1995, 45, 43– 48, DOI: 10.1002/ps.278045010723Mode of action of the anilino-pyrimidine fungicide pyrimethanil. 2. Effects on enzyme secretion in Botrytis cinereaMilling, Richard J.; Richardson, Caroline J.Pesticide Science (1995), 45 (1), 43-8CODEN: PSSCBG; ISSN:0031-613X. (Wiley)The effect of pyrimethanil on the levels of cell wall degrading enzymes secreted by Botrytis cinerea Pers. was investigated in diseased plant tissues and in liq. B. cinerea cultures. Total proteinase activity isolated from infected carrot slices which were treated with 5·0 μM pyrimethanil was decreased by 76%, 3 d after inoculation. Polygalacturonase, cellulase, proteinase and laccase activities were all decreased in the medium of three day-old cultures grown in the presence of pyrimethanil. The pyrimethanil concns. resulting in 50% redn. in total enzyme activities (IC50) were approx. 0·25 μM for polygalacturonase, cellulase and proteinase, and approx. 1·0 μM for laccase. No significant growth inhibition was obsd. at these pyrimethanil concns. Pyrimethanil did not inhibit the enzymes directly, nor did it inhibit the synthesis of cytosolic proteins. Therefore, it was proposed that the fungicide inhibits protein secretion at a post-translational stage in the secretory pathway. Large differences were found in the effects of pyrimethanil on the growth of B. cinerea in liq. cultures and on agar plates, depending on the compn. of the medium. In liq. media contg. cellulose and protein as carbon and nitrogen sources, growth inhibition occurred at 5·0 μM pyrimethanil, while no growth inhibition was obsd. with 50 μM pyrimethanil in malt ext. Similarly, growth occurred on potato/dextrose agar (PDA) at 0·5 μM pyrimethanil, but no growth was seen at this concn. on agars contg. cellulose and protein. Thus it appears that pyrimethanil is most active in media where the fungus has to utilize extracellular enzymes to mobilize the nutrients it requires for growth.
- 24Li, X.; Fernández-Ortuño, D.; Grabke, A.; Schnabel, G. Resistance to Fludioxonil in Botrytis cinerea Isolates from Blackberry and Strawberry. Phytopathology 2014, 104, 724– 732, DOI: 10.1094/PHYTO-11-13-0308-R24Resistance to fludioxonil in Botrytis cinerea isolates from blackberry and strawberryLi, Xingpeng; Fernandez-Ortuno, Dolores; Grabke, Anja; Schnabel, GuidoPhytopathology (2014), 104 (7), 724-732CODEN: PHYTAJ; ISSN:0031-949X. (American Phytopathological Society)Site-specific fungicides, including the phenylpyrrole fludioxonil, are frequently used for gray mold control but are at risk for the development of resistance. In this study, field isolates that were low-resistant (LR) and moderately resistant (MR) to fludioxonil from blackberry and strawberry fields of North Carolina, South Carolina, and Virginia were characterized. Genes involved in osmoregulation, including bcsak1, BcOS4, bos5, and BRRG-1, were cloned and sequenced to detect potential target gene alterations; however, none were found. A previously described mutation (R632I) in transcription factor Mrr1, which is known to increase the expression of ATP-binding cassette transporter AtrB, was found in MR but not in sensitive (S) or LR isolates. Expression of atrB in MR isolates was ≈200-fold increased compared with an S isolate; however, 30- to 100-fold overexpression was also detected in LR isolates. Both MR isolates exhibited increased sensitivity to salt stress in the form of mycelial growth inhibition at 4% NaCl, indicating a disruption of osmoregulatory processes in those strains. However, the glycerol content was indistinguishable between S, LR, and MR isolates with and without exposure to fludioxonil, suggesting that the glycerol synthesis pathway may not be a part of the resistance mechanism in LR or MR strains. An investigation into the origin of LR and MR isolates from blackberry revealed two insertions in the mrr1 gene consistent with those found in the Botrytis clade group S. The emergence of strains overexpressing atrB in European and now in North American strawberry fields underscores the importance of this resistance mechanism for development of resistance to fludioxonil in Botrytis cinerea.
- 25Pimentel, D.; Burgess, M. In Integrated Pest Management; Peshin, R., Pimentel, D., Eds.; Springer: Dordrecht, 2014.There is no corresponding record for this reference.
- 26van der Werf, H. M. G. Assessing the impact of pesticides on the environment. Agric., Ecosyst. Environ. 1996, 60, 81– 96, DOI: 10.1016/S0167-8809(96)01096-126Assessing the impact of pesticides on the environmentvan der Werf, Hayo M. G.Agriculture, Ecosystems & Environment (1996), 60 (2,3), 81-96CODEN: AEENDO; ISSN:0167-8809. (Elsevier)A review with many refs. on factors which should be taken into consideration to assess pesticide environmental impact, and how can impact be quantified. Six recent approaches to assess the impact of pesticides on the environment are compared regarding choice, transformation and aggregation of input parameters. The use of simulation models to assess environmental impact is discussed.
- 27Dong, O. X.; Ronald, P. C. Genetic Engineering for Disease Resistance in Plants: Recent Progress and Future Perspectives. Plant Physiol. 2019, 180, 26, DOI: 10.1104/pp.18.0122427Genetic engineering for disease resistance in plants: recent progress and future perspectivesDong, Oliver Xiaoou; Ronald, Pamela C.Plant Physiology (2019), 180 (1), 26-38CODEN: PLPHAY; ISSN:1532-2548. (American Society of Plant Biologists)A review. This article discusses about the application of genetic engineering as powerful tools that allows new genes into vegetatively propagated crops including Musa, Manihot esculenta, and Solanum tuberosum for enhancing disease resistance against plant pathogens. First, it enables the introduction, removal, modification, or fine-tuning of specific genes of interest with minimal undesired changes to the rest of the crop genome. Second, genetic engineering allows for interchange of genetic material across species. Thus, the raw genetic materials that can be exploited for this process is not restricted to the genes available within the species. Third, plant transformation during genetic engineeringallows the introduction of new genes into vegetatively propagated crops such as banana (Musa sp.), cassava (Manihot esculenta), and potato (Solanum tuberosum). As a result, crops exhibiting desired agronomic traits can be obtained in fewer generations compared with conventional breeding.
- 28Griffioen, G. Indole amide derivatives and related compounds for use in the treatment of neurodegenerative diseases. US Patent US9434722B2, 2013.There is no corresponding record for this reference.
- 29Sainsbury, F.; Thuenemann, E. C.; Lomonossoff, G. P. pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol. J. 2009, 7, 682– 693, DOI: 10.1111/j.1467-7652.2009.00434.x29pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plantsSainsbury, Frank; Thuenemann, Eva C.; Lomonossoff, George P.Plant Biotechnology Journal (2009), 7 (7), 682-693CODEN: PBJLAE; ISSN:1467-7644. (Wiley-Blackwell)Agro-infiltration of leaf tissue with binary vectors harbouring a sequence of interest is a rapid method of expressing proteins in plants. It has recently been shown that flanking the sequence to be expressed with a modified 5'-untranslated region (UTR) and the 3'-UTR from Cowpea mosaic virus (CPMV) RNA-2 (CPMV-HT) within the binary vector pBINPLUS greatly enhances the level of expression that can be achieved. To exploit this finding, a series of small binary vectors tailored for transient expression (termed the pEAQ vectors) has been created. In these, more than 7 kb of nonessential sequence was removed from the pBINPLUS backbone and T-DNA region, and unique restriction sites were introduced to allow for accommodation of multiple expression cassettes, including that for a suppressor of silencing, on the same plasmid. These vectors allow the high-level simultaneous expression of multiple polypeptides from a single plasmid within a few days. Furthermore, vectors have been developed which allow the direct cloning of genes into the binary plasmid by both restriction enzyme-based cloning and GATEWAY recombination. In both cases, N- or C-terminal histidine tags may be fused to the target sequence as required. These vectors provide an easy and quick tool for the prodn. of milligram quantities of recombinant proteins from plants with std. plant research techniques at a bench-top scale.
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