A genome-reduced Corynebacterium glutamicum derivative discloses a hidden pathway relevant for 1,2-propanediol production.

BACKGROUND: 1,2-propanediol (1,2-PDO) is widely used in the cosmetic, food, and drug industries with a worldwide consumption of over 1.5 million metric tons per year. Although efforts have been made to engineer microbial hosts such as Corynebacterium glutamicum to produce 1,2-PDO from renewable resources, the performance of such strains is still improvable to be competitive with existing petrochemical production routes. RESULTS: In this study, we enabled 1,2-PDO production in the genome-reduced strain C. glutamicum PC2 by introducing previously described modifications. The resulting strain showed reduced product formation but secreted 50 ± 1 mM D-lactate as byproduct. C. glutamicum PC2 lacks the D-lactate dehydrogenase which pointed to a yet unknown pathway relevant for 1,2-PDO production. Further analysis indicated that in C. glutamicum methylglyoxal, the precursor for 1,2-PDO synthesis, is detoxified with the antioxidant native mycothiol (MSH) by a glyoxalase-like system to lactoylmycothiol and converted to D-lactate which is rerouted into the central carbon metabolism at the level of pyruvate. Metabolomics of cell extracts of the empty vector-carrying wildtype, a 1,2-PDO producer and its derivative with inactive D-lactate dehydrogenase identified major mass peaks characteristic for lactoylmycothiol and its precursors MSH and glucosaminyl-myo-inositol, whereas the respective mass peaks were absent in a production strain with inactivated MSH synthesis. Deletion of mshA, encoding MSH synthase, in the 1,2-PDO producing strain C. glutamicum ΔhdpAΔldh(pEKEx3-mgsA-yqhD-gldA) improved the product yield by 56% to 0.53 ± 0.01 mM1,2-PDO mMglucose-1 which is the highest value for C. glutamicum reported so far. CONCLUSIONS: Genome reduced-strains are a useful basis to unravel metabolic constraints for strain engineering and disclosed in this study the pathway to detoxify methylglyoxal which represents a precursor for 1,2-PDO production. Subsequent inactivation of the competing pathway significantly improved the 1,2-PDO yield.

The Formation of a Camalexin Biosynthetic Metabolon.

Arabidopsis (Arabidopsis thaliana) efficiently synthesizes the antifungal phytoalexin camalexin without the apparent release of bioactive intermediates, such as indole-3-acetaldoxime, suggesting that the biosynthetic pathway of this compound is channeled by the formation of an enzyme complex. To identify such protein interactions, we used two independent untargeted coimmunoprecipitation (co-IP) approaches with the biosynthetic enzymes CYP71B15 and CYP71A13 as baits and determined that the camalexin biosynthetic P450 enzymes copurified with these enzymes. These interactions were confirmed by targeted co-IP and Förster resonance energy transfer measurements based on fluorescence lifetime microscopy (FRET-FLIM). Furthermore, the interaction of CYP71A13 and Arabidopsis P450 Reductase1 was observed. We detected increased substrate affinity of CYP79B2 in the presence of CYP71A13, indicating an allosteric interaction. Camalexin biosynthesis involves glutathionylation of the intermediary indole-3-cyanohydrin, which is synthesized by CYP71A12 and especially CYP71A13. FRET-FLIM and co-IP demonstrated that the glutathione transferase GSTU4, which is coexpressed with Trp- and camalexin-specific enzymes, is physically recruited to the complex. Surprisingly, camalexin concentrations were elevated in knockout and reduced in GSTU4-overexpressing plants. This shows that GSTU4 is not directly involved in camalexin biosynthesis but rather plays a role in a competing mechanism.

PROTEIN PHOSPHATASE 2A-B'γ Controls Botrytis cinerea Resistance and Developmental Leaf Senescence.

Plants optimize their growth and survival through highly integrated regulatory networks that coordinate defensive measures and developmental transitions in response to environmental cues. Protein phosphatase 2A (PP2A) is a key signaling component that controls stress reactions and growth at different stages of plant development, and the PP2A regulatory subunit PP2A-B'γ is required for negative regulation of pathogenesis responses and for maintenance of cell homeostasis in short-day conditions. Here, we report molecular mechanisms by which PP2A-B'γ regulates Botrytis cinerea resistance and leaf senescence in Arabidopsis (Arabidopsis thaliana). We extend the molecular functionality of PP2A-B'γ to a protein kinase-phosphatase interaction with the defense-associated calcium-dependent protein kinase CPK1 and present indications this interaction may function to control CPK1 activity. In presenescent leaf tissues, PP2A-B'γ is also required to negatively control the expression of salicylic acid-related defense genes, which have recently proven vital in plant resistance to necrotrophic fungal pathogens. In addition, we find the premature leaf yellowing of pp2a-b'γ depends on salicylic acid biosynthesis via SALICYLIC ACID INDUCTION DEFICIENT2 and bears the hallmarks of developmental leaf senescence. We propose PP2A-B'γ age-dependently controls salicylic acid-related signaling in plant immunity and developmental leaf senescence.

The role of CYP71A12 monooxygenase in pathogen-triggered tryptophan metabolism and Arabidopsis immunity.

Effective defense of Arabidopsis against filamentous pathogens requires two mechanisms, both of which involve biosynthesis of tryptophan (Trp)-derived metabolites. Extracellular resistance involves products of PEN2-dependent metabolism of indole glucosinolates (IGs). Restriction of further fungal growth requires PAD3-dependent camalexin and other, as yet uncharacterized, indolics. This study focuses on the function of CYP71A12 monooxygenase in pathogen-triggered Trp metabolism, including the biosynthesis of indole-3-carboxylic acid (ICA). Moreover, to investigate the contribution of CYP71A12 and its products to Arabidopsis immunity, we analyzed infection phenotypes of multiple mutant lines combining pen2 with pad3, cyp71A12, cyp71A13 or cyp82C2. Metabolite profiling of cyp71A12 lines revealed a reduction in ICA accumulation. Additionally, analysis of mutant plants showed that low amounts of ICA can form during an immune response by CYP71B6/AAO1-dependent metabolism of indole acetonitrile, but not via IG hydrolysis. Infection assays with Plectosphaerella cucumerina and Colletotrichum tropicale, two pathogens with different lifestyles, revealed cyp71A12-, cyp71A13- and cyp82C2-associated defects associated with Arabidopsis immunity. Our results indicate that CYP71A12, but not CYP71A13, is the major enzyme responsible for the accumulation of ICA in Arabidopsis in response to pathogen ingression. We also show that both enzymes are key players in the resistance of Arabidopsis against selected filamentous pathogens after they invade.

Short-Term Exposure to Nitrogen Dioxide Provides Basal Pathogen Resistance.

Nitrogen dioxide (NO2) forms in plants under stress conditions, but little is known about its physiological functions. Here, we explored the physiological functions of NO2 in plant cells using short-term fumigation of Arabidopsis (Arabidopsis thaliana) for 1 h with 10 µL L-1 NO2. Although leaf symptoms were absent, the expression of genes related to pathogen resistance was induced. Fumigated plants developed basal disease resistance, or pattern-triggered immunity, against the necrotrophic fungus Botrytis cinerea and the hemibiotrophic bacterium Pseudomonas syringae Functional salicylic acid and jasmonic acid (JA) signaling pathways were both required for the full expression of NO2-induced resistance against B. cinerea An early peak of salicylic acid accumulation immediately after NO2 exposure was followed by a transient accumulation of oxophytodienoic acid. The simultaneous NO2-induced expression of genes involved in jasmonate biosynthesis and jasmonate catabolism resulted in the complete suppression of JA and JA-isoleucine (JA-Ile) accumulation, which was accompanied by a rise in the levels of their catabolic intermediates 12-OH-JA, 12-OH-JA-Ile, and 12-COOH-JA-Ile. NO2-treated plants emitted the volatile monoterpene α-pinene and the sesquiterpene longifolene (syn. junipene), which could function in signaling or direct defense against pathogens. NO2-triggered B. cinerea resistance was dependent on enhanced early callose deposition and CYTOCHROME P450 79B2 (CYP79B2), CYP79B3, and PHYTOALEXIN DEFICIENT3 gene functions but independent of camalexin, CYP81F2, and 4-OH-indol-3-ylmethylglucosinolate derivatives. In sum, exogenous NO2 triggers basal pathogen resistance, pointing to a possible role for endogenous NO2 in defense signaling. Additionally, this study revealed the involvement of jasmonate catabolism and volatiles in pathogen immunity.

PP2A-B'γ modulates foliar trans-methylation capacity and the formation of 4-methoxy-indol-3-yl-methyl glucosinolate in Arabidopsis leaves.

Glucosinolates (GSL) of cruciferous plants comprise a major group of structurally diverse secondary compounds which act as deterrents against aphids and microbial pathogens and have large commercial and ecological impacts. While the transcriptional regulation governing the biosynthesis and modification of GSL is now relatively well understood, post-translational regulatory components that specifically determine the structural variation of indole glucosinolates have not been reported. We show that the cytoplasmic protein phosphatase 2A regulatory subunit B'γ (PP2A-B'γ) physically interacts with indole glucosinolate methyltransferases and controls the methoxylation of indole glucosinolates and the formation of 4-methoxy-indol-3-yl-methyl glucosinolate in Arabidopsis leaves. By taking advantage of proteomic approaches and metabolic analysis we further demonstrate that PP2A-B'γ is required to control the abundance of oligomeric protein complexes functionally linked with the activated methyl cycle and the trans-methylation capacity of leaf cells. These findings highlight the key regulatory role of PP2A-B'γ in methionine metabolism and provide a previously unrecognized perspective for metabolic engineering of glucosinolate metabolism in cruciferous plants.

TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE-Mediated Generation and Metabolic Analysis of Camalexin-Deficient cyp71a12 cyp71a13 Double Knockout Lines.

In Arabidopsis (Arabidopsis thaliana), a number of defense-related metabolites are synthesized via indole-3-acetonitrile (IAN), including camalexin and indole-3-carboxylic acid (ICOOH) derivatives. Cytochrome P450 71A13 (CYP71A13) is a key enzyme for camalexin biosynthesis and catalyzes the conversion of indole-3-acetaldoxime (IAOx) to IAN. The CYP71A13 gene is located in tandem with its close homolog CYP71A12, also encoding an IAOx dehydratase. However, for CYP71A12, indole-3-carbaldehyde and cyanide were identified as major reaction products. To clarify CYP71A12 function in vivo and to better understand IAN metabolism, we generated two cyp71a12 cyp71a13 double knockout mutant lines. CYP71A12-specific transcription activator-like effector nucleases were introduced into the cyp71a13 background, and very efficient somatic mutagenesis was achieved. We observed stable transmission of the cyp71a12 mutation to the following generations, which is a major challenge for targeted mutagenesis in Arabidopsis. In contrast to cyp71a13 plants, in which camalexin accumulation is partially reduced, double mutants synthesized only traces of camalexin, demonstrating that CYP71A12 contributes to camalexin biosynthesis in leaf tissue. A major role of CYP71A12 was identified for the inducible biosynthesis of ICOOH. Specifically, the ICOOH methyl ester was reduced to 12% of the wild-type level in AgNO3-challenged cyp71a12 leaves. In contrast, indole-3-carbaldehyde derivatives apparently are synthesized via alternative pathways, such as the degradation of indole glucosinolates. Based on these results, we present a model for this surprisingly complex metabolic network with multiple IAN sources and channeling of IAOx-derived IAN into camalexin biosynthesis. In conclusion, transcription activator-like effector nuclease-mediated mutation is a powerful tool for functional analysis of tandem genes in secondary metabolism.

Substantial reprogramming of the Eutrema salsugineum (Thellungiella salsuginea) transcriptome in response to UV and silver nitrate challenge.

BACKGROUND: Cruciferous plants synthesize a large variety of tryptophan-derived phytoalexins in response to pathogen infection, UV irradiation, or high dosages of heavy metals. The major phytoalexins of Eutrema salsugineum (Thellungiella salsuginea), which has recently been established as an extremophile model plant, are probably derivatives of indole glucosinolates, in contrast to Arabidopsis, which synthesizes characteristic camalexin from the glucosinolate precursor indole-3-acetaldoxime. RESULTS: The transcriptional response of E. salsugineum to UV irradiation and AgNO3 was monitored by RNAseq and microarray analysis. Most transcripts (respectively 70% and 78%) were significantly differentially regulated and a large overlap between the two treatments was observed (54% of total). While core genes of the biosynthesis of aliphatic glucosinolates were repressed, tryptophan and indole glucosinolate biosynthetic genes, as well as defence-related WRKY transcription factors, were consistently upregulated. The putative Eutrema WRKY33 ortholog was functionally tested and shown to complement camalexin deficiency in Atwrky33 mutant. CONCLUSIONS: In E. salsugineum, UV irradiation or heavy metal application resulted in substantial transcriptional reprogramming. Consistently induced genes of indole glucosinolate biosynthesis and modification will serve as candidate genes for the biosynthesis of Eutrema-specific phytoalexins.

Sustained exposure to abscisic acid enhances the colonization potential of the mutualist fungus Piriformospora indica on Arabidopsis thaliana roots.

Root colonization by the beneficial fungus Piriformospora indica is controlled by plant innate immunity, but factors that channel this interaction into a mutualistic relationship are not known. We have explored the impact of abscisic acid (ABA) and osmotic stress on the P. indica interaction with Arabidopsis thaliana. The activation of plant innate immunity in roots was determined by measuring the concentration of the phytoalexin camalexin and expression of transcription factors regulating the biosynthesis of tryptophan-related defence metabolites. Furthermore, the impact of the fungus on the content of ABA, salicylic acid, jasmonic acid (JA) and JA-related metabolites was examined. We demonstrated that treatment with exogenous ABA or the ABA analogue pyrabactin increased fungal colonization efficiency without impairment of plant fitness. Concomitantly, ABA-deficient mutants of A. thaliana (aba1-6 and aba2-1) were less colonized, while plants exposed to moderate stress were more colonized than corresponding controls. Sustained exposure to ABA attenuated expression of transcription factors MYB51, MYB122 and WRKY33 in roots upon P. indica challenge or chitin treatment, and prevented an increase in camalexin content. The results indicate that ABA can strengthen the interaction with P. indica as a consequence of its impact on plant innate immunity. Consequently, ABA will be relevant for the establishment and outcome of the symbiosis under stress conditions.

The Biosynthetic Pathway of Indole-3-Carbaldehyde and Indole-3-Carboxylic Acid Derivatives in Arabidopsis.

Indolic secondary metabolites play an important role in pathogen defense in cruciferous plants. In Arabidopsis (Arabidopsis thaliana), in addition to the characteristic phytoalexin camalexin, derivatives of indole-3-carbaldehyde (ICHO) and indole-3-carboxylic acid (ICOOH) are synthesized from tryptophan via the intermediates indole-3-acetaldoxime and indole-3-acetonitrile. Based on feeding experiments combined with nontargeted metabolite profiling, their composition in nontreated and silver nitrate (AgNO3)-treated leaf tissue was comprehensively analyzed. As major derivatives, glucose conjugates of 5-hydroxyindole-3-carbaldehyde, ICOOH, and 6-hydroxyindole-3-carboxylic acid were identified. Quantification of ICHO and ICOOH derivative pools after glucosidase treatment revealed that, in response to AgNO3 treatment, their total accumulation level was similar to that of camalexin. ARABIDOPSIS ALDEHYDE OXIDASE1 (AAO1), initially discussed to be involved in the biosynthesis of indole-3-acetic acid, and Cytochrome P450 (CYP) 71B6 were found to be transcriptionally coexpressed with camalexin biosynthetic genes. CYP71B6 was expressed in Saccharomyces cerevisiae and shown to efficiently convert indole-3-acetonitrile into ICHO and ICOOH, thereby releasing cyanide. To evaluate the role of both enzymes in the biosynthesis of ICHO and ICOOH derivatives, knockout and overexpression lines for CYP71B6 and AAO1 were established and analyzed for indolic metabolites. The observed metabolic phenotypes suggest that AAO1 functions in the oxidation of ICHO to ICOOH in both nontreated and AgNO3-treated leaves, whereas CYP71B6 is relevant for ICOOH derivative biosynthesis specifically after induction. In summary, a model for the biosynthesis of ICHO and ICOOH derivatives is presented.

Indole-3-acetaldoxime-derived compounds restrict root colonization in the beneficial interaction between Arabidopsis roots and the endophyte Piriformospora indica.

The growth-promoting and root-colonizing endophyte Piriformospora indica induces camalexin and the expression of CYP79B2, CYP79B3, CYP71A13, PAD3, and WRKY33 required for the synthesis of indole-3-acetaldoxime (IAOx)-derived compounds in the roots of Arabidopsis seedlings. Upregulation of the mRNA levels by P. indica requires cytoplasmic calcium elevation and mitogen-activated protein kinase 3 but not root-hair-deficient 2, radical oxygen production, or the 3-phosphoinositide-dependent kinase 1/oxidative signal-inducible 1 pathway. Because P. indica-mediated growth promotion is impaired in cyp79B2 cyp79B3 seedlings, while pad3 seedlings-which do not accumulate camalexin-still respond to the fungus, IAOx-derived compounds other than camalexin (e.g., indole glucosinolates) are required during early phases of the beneficial interaction. The roots of cyp79B2 cyp79B3 seedlings are more colonized than wild-type roots, and upregulation of the defense genes pathogenesis-related (PR)-1, PR-3, PDF1.2, phenylalanine ammonia lyase, and germin indicates that the mutant responds to the lack of IAOx-derived compounds by activating other defense processes. After 6 weeks on soil, defense genes are no longer upregulated in wild-type, cyp79B2 cyp79B3, and pad3 roots. This results in uncontrolled fungal growth in the mutant roots and reduced performance of the mutants. We propose that a long-term harmony between the two symbionts requires restriction of root colonization by IAOx-derived compounds.

The phytoalexins from cultivated and wild crucifers: chemistry and biology.

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 chemical synthesis, biosynthesis, metabolism by plant fungal pathogens and biological activities. A summary table containing all phytoalexins, their cultivated and wild cruciferous sources, their synthetic starting materials, biotransformation products and biological activities is provided.

Inducible expression of a Nep1-like protein serves as a model trigger system of camalexin biosynthesis.

Camalexin, the major Arabidopsis phytoalexin, is synthesized in response to a great variety of pathogens. Specific pathogen-associated molecular patterns, such as Nep1-like proteins from oomycetes act as signals triggering the transcriptional activation of the camalexin biosynthetic genes. PaNie, a Nep1-like protein from Pythiumaphanidermatum was expressed in Arabidopsis under the control of an ethanol-inducible promoter. This system was developed as a tool to study the regulation of camalexin biosynthesis. It allowed induction of camalexin preceded by strong transcriptional activation of the tryptophan and camalexin biosynthetic genes. In flowers and green siliques PaNie expression elicited only minor camalexin formation, indicating low capability for phytoalexin synthesis in reproductive organs in contrast to leaf and stem tissue.

Dissection of the network of indolic defence compounds in Arabidopsis thaliana by multiple mutant analysis.

Characteristic for cruciferous plants is the synthesis of a complex array of defence-related indolic compounds. In Arabidopsis, these include indol-3-ylmethyl glucosinolates (IMGs), as well as stress-inducible indole-3-carbaldehyde (ICHO)/indole-3-carboxylic acid (ICOOH) derivatives and camalexin. Key enzymes in the biosynthesis of the inducible metabolites are the cytochrome P450 enzymes CYP71A12, CYP71A13 and CYP71B6 and Arabidopsis Aldehyde Oxidase 1 (AAO1). Multiple mutants in the corresponding genes were generated and their metabolic phenotypes were comprehensively analysed in untreated, UV exposed and silver nitrate-treated leaves. Most strikingly, ICOOH and ICHO derivatives synthesized in response to UV exposure were not metabolically related. While ICHO concentrations correlated with IMGs, ICOOH derivatives were anti-correlated with IMGs and partially dependent on CYP71B6. The AAO1 genotype was shown to not only be important for ICHO metabolism but also for the accumulation of 4-pyridoxic acid, suggesting a dual role of AAO1 in vitamin B6 metabolism and IMG degradation in Arabidopsis.

Characterisation of the tryptophan synthase alpha subunit in maize.

BACKGROUND: In bacteria, such as Salmonella typhimurium, tryptophan is synthesized from indole-3-glycerole phosphate (IGP) by a tryptophan synthase alphabetabetaalpha heterotetramer. Plants have evolved multiple alpha (TSA) and beta (TSB) homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS) complex in Arabidopsis. On the other hand maize (Zea mays) expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB. RESULTS: In order to clarify, how tryptophan is synthesized in maize, two TSA homologs, hitherto uncharacterized ZmTSA and ZmTSAlike, were functionally analyzed. ZmTSA is localized in plastids, the major site of tryptophan biosynthesis in plants. It catalyzes the tryptophan synthase alpha-reaction (cleavage of IGP), and forms a tryptophan synthase complex with ZmTSB1 in vitro. The catalytic efficiency of the alpha-reaction is strongly enhanced upon complex formation. A 160 kD tryptophan synthase complex was partially purified from maize leaves and ZmTSA was identified as native alpha-subunit of this complex by mass spectrometry. ZmTSAlike, for which no in vitro activity was detected, is localized in the cytosol. ZmTSAlike, BX1, and IGL were not detectable in the native tryptophan synthase complex in leaves. CONCLUSION: It was demonstrated in vivo and in vitro that maize forms a tryptophan synthase complex and ZmTSA functions as alpha-subunit in this complex.

Biosynthetic origin of BE-10988 in Streptomyces sp. BA10988.

The biosynthetic origin of the tumor-inhibitory derivative, BE-10988, was studied in Streptomyces sp . BA10988 by retrobiosynthetic NMR analysis using [U-(13)C6]glucose as a precursor. The isotopologue compositions of the indole moieties of BE-10988 and tryptophan were virtually identical. This indicates that tryptophan or a closely related metabolite served as a biosynthetic precursor of BE-10988 in analogy to the biosynthetic pathway of camalexin, a structurally related phytoalexin in Arabidopsis thaliana. Labeling experiments with [U-(13)C8(15)N]indole, L-[ring-(2)H5]tryptophan, or L-[U-(13)C3(15)N]cysteine confirmed this hypothesis. However, transfer of the label from [ring-(2)H5]indole pyruvic acid, but not from the known camalexin precursor, [ring-(2)H5]indole-3-acetaldoxime, showed that plants and bacteria have evolved independent mechanisms of tryptophan modification in the biosynthesis of thiazolylindole derivatives.

Maize nitrilases have a dual role in auxin homeostasis and beta-cyanoalanine hydrolysis.

The auxin indole-3-acetic acid (IAA), which is essential for plant growth and development, is suggested to be synthesized via several redundant pathways. In maize (Zea mays), the nitrilase ZmNIT2 is expressed in auxin-synthesizing tissues and efficiently hydrolyses indole-3-acetonitrile to IAA. Zmnit2 transposon insertion mutants were compromised in root growth in young seedlings and sensitivity to indole-3-acetonitrile, and accumulated lower quantities of IAA conjugates in kernels and root tips, suggesting a substantial contribution of ZmNIT2 to total IAA biosynthesis in maize. An additional enzymatic function, turnover of beta-cyanoalanine, is acquired when ZmNIT2 forms heteromers with the homologue ZmNIT1. In plants carrying an insertion mutation in either nitrilase gene this activity was strongly reduced. A dual role for ZmNIT2 in auxin biosynthesis and in cyanide detoxification as a heteromer with ZmNIT1 is therefore proposed.

Camalexin.

Camalexin (3-thiazol-2'-yl-indole) is the characteristic phytoalexin of Arabidopsis thaliana, which is induced by a great variety of plant pathogens. While particular pathogens, as well as a human tumour cell line, were growth inhibited by camalexin, some fungi show resistance due to active degradation. Camalexin originates from tryptophan and its biosynthesis involves the cytochrome P450 enzymes CYP79B2 and CYP71B15 (PAD3). Camalexin induction is a complex process, for which triggering by reactive oxygen species (ROS), salicylic acid signalling, and the glutathione status are important. Targets of the signalling cascade are the tryptophan and camalexin biosynthetic genes, which are strongly transcriptionally upregulated at the sites of pathogen infection. The important knowledge on camalexin, which is reviewed in this paper, will help to establish camalexin as a model for the investigation of the significance of phytoalexins in response pathogen challenge.

Regulatory variability of camalexin biosynthesis.

The anthranilate synthase ASA1, CYP79B2 and CYP71B15 (PAD3) are biosynthetic genes of the Arabidopsis phytoalexin camalexin, which are induced after pathogen infection and abiotic treatments like silver nitrate spraying. The natural variation of camalexin biosynthesis in response to Pseudomonas syringae infection was determined in several ecotypes, and differential CYP71B15 regulation as a potential basis for this variation was investigated. The expression of camalexin biosynthetic genes was restricted to the tissue undergoing cell death. After droplet infection with Alternaria alternata, a potent camalexin inducer in the Col-0 ecotype, camalexin formation and the induction of ASA1, CYP79B2 and CYP71B15 were strictly co-localized with the infection site.

Expression of antimicrobial peptides under control of a camalexin-biosynthetic promoter confers enhanced resistance against Pseudomonas syringae.

In Arabidopsis thaliana phytoalexin biosynthesis is tightly regulated. The camalexin biosynthetic gene CYP71B15/PAD3 is highly expressed in response to pathogens and specific abiotic triggers, while constitutive expression is very low. Based on this property we expressed artificial antimicrobial peptides under control of the CYP71B15 promoter avoiding potential toxic effects to the plant related to constitutive expression. Significant and substantial growth inhibition of Pseudomonas syringae was observed, demonstrating that expression of these peptides under control of a phytoalexin promoter is an effective approach for enhancement of resistance against bacterial pathogens.