In the Gramineae, the cyclic hydroxamic acids 2,4-dihydroxy-1, 4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7-methoxy-1, 4-benzoxazin-3-one (DIMBOA) form part of the defense against insects and microbial pathogens. Five genes, Bx1 through Bx5, are required for DIBOA biosynthesis in maize. The functions of these five genes, clustered on chromosome 4, were demonstrated in vitro. Bx1 encodes a tryptophan synthase alpha homolog that catalyzes the formation of indole for the production of secondary metabolites rather than tryptophan, thereby defining the branch point from primary to secondary metabolism. Bx2 through Bx5 encode cytochrome P450-dependent monooxygenases that catalyze four consecutive hydroxylations and one ring expansion to form the highly oxidized DIBOA.

Glucosinolates are natural plant products that function in the defense toward herbivores and pathogens. Plant defense is regulated by multiple signal transduction pathways in which salicylic acid (SA), jasmonic acid, and ethylene function as signaling molecules. Glucosinolate content was analyzed in Arabidopsis wild-type plants in response to single or combinatorial treatments with methyljasmonate (MeJA), 2,6-dichloro-isonicotinic acid, ethylene, and 2,4-dichloro-phenoxyacetic acid, or by wounding. In addition, several signal transduction mutants and the SA-depleted transgenic NahG line were analyzed. In parallel, expression of glucosinolate biosynthetic genes of the CYP79 gene family and the UDPG:thiohydroximate glucosyltransferase was monitored. After MeJA treatment, the amount of indole glucosinolates increased 3-to 4-fold, and the corresponding Trp-metabolizing genes CYP79B2 and CYP79B3 were both highly induced. Specifically, the indole glucosinolate N-methoxy-indol-3-ylmethylglucosinolate accumulated 10-fold in response to MeJA treatment, whereas 4-methoxy-indol-3-ylmethylglucosinolate accumulated 1.5-fold in response to 2,6-dichloro-isonicotinic acid. In general, few changes were seen for the levels of aliphatic glucosinolates, although increases in the levels of 8-methylthiooctyl glucosinolate and 8-methylsulfinyloctyl glucosinolate were observed, particularly after MeJA treatments. The findings were supported by the composition of glucosinolates in the coronatine-insensitive mutant coi1, the ctr1 mutant displaying constitutive triple response, and the SA-overproducing mpk4 and cpr1 mutants. The present data indicate that different indole glucosinolate methoxylating enzymes are induced by the jasmonate and the SA signal transduction pathways, whereas the aliphatic glucosinolates appear to be primarily genetically and not environmentally controlled. Thus, different defense pathways activate subsets of biosynthetic enzymes, leading to the accumulation of specific glucosinolates.Glucosinolates are amino acid-derived natural plant products that function in the defense against herbivores and microorganisms. Upon tissue disruption, e.g. caused by insect feeding, glucosinolates are hydrolyzed by specific thioglucosidases called myrosinases to produce an array of biologically active compounds, typically isothiocyanates, nitriles, and thiocyanates (for review, see Halkier, 1999;Rask et al., 2000). These compounds function as inhibitors of microbial growth (Mari et al., 1993;Manici et al., 1997), as attractants for specialist insects, and as deterrents of generalist herbivores. For humans, glucosinolates are important as flavor compounds, as cancer-preventive agents, and as biopesticides in agriculture.Glucosinolate biosynthesis is considered a threestep process: First, the amino acid may enter the chain elongation pathway, in which the condensing enzymes MAM1 and MAM-L have recently been identified (de Quiros et al., 2000;Kroymann et al., 2001). Second, the core glucosinolate structure is formed (see below); and t...

Camalexin (3-thiazol-2-yl-indole) is an indole alkaloid phytoalexin produced by Arabidopsis thaliana that is thought to be important for resistance to necrotrophic fungal pathogens, such as Alternaria brassicicola and Botrytis cinerea. It is produced from Trp, which is converted to indole acetaldoxime (IAOx) by the action of cytochrome P450 monooxygenases CYP79B2 and CYP79B3. The remaining biosynthetic steps are unknown except for the last step, which is conversion of dihydrocamalexic acid to camalexin by CYP71B15 (PAD3). This article reports characterization of CYP71A13. Plants carrying cyp71A13 mutations produce greatly reduced amounts of camalexin after infection by Pseudomonas syringae or A. brassicicola and are susceptible to A. brassicicola, as are pad3 and cyp79B2 cyp79B3 mutants. Expression levels of CYP71A13 and PAD3 are coregulated. CYP71A13 expressed in Escherichia coli converted IAOx to indole-3-acetonitrile (IAN). Expression of CYP79B2 and CYP71A13 in Nicotiana benthamiana resulted in conversion of Trp to IAN. Exogenously supplied IAN restored camalexin production in cyp71A13 mutant plants. Together, these results lead to the conclusion that CYP71A13 catalyzes the conversion of IAOx to IAN in camalexin synthesis and provide further support for the role of camalexin in resistance to A. brassicicola.

We show that oomycete-derived Nep1 (for necrosis and ethylene-inducing peptide1)-like proteins (NLPs) trigger a comprehensive immune response in Arabidopsis thaliana, comprising posttranslational activation of mitogen-activated protein kinase activity, deposition of callose, production of nitric oxide, reactive oxygen intermediates, ethylene, and the phytoalexin camalexin, as well as cell death. Transcript profiling experiments revealed that NLPs trigger extensive reprogramming of the Arabidopsis transcriptome closely resembling that evoked by bacteria-derived flagellin. NLP-induced cell death is an active, light-dependent process requiring HSP90 but not caspase activity, salicylic acid, jasmonic acid, ethylene, or functional SGT1a/SGT1b. Studies on animal, yeast, moss, and plant cells revealed that sensitivity to NLPs is not a general characteristic of phospholipid bilayer systems but appears to be restricted to dicot plants. NLP-induced cell death does not require an intact plant cell wall, and ectopic expression of NLP in dicot plants resulted in cell death only when the protein was delivered to the apoplast. Our findings strongly suggest that NLP-induced necrosis requires interaction with a target site that is unique to the extracytoplasmic side of dicot plant plasma membranes. We propose that NLPs play dual roles in plant pathogen interactions as toxin-like virulence factors and as triggers of plant innate immune responses.

Characteristic for cruciferous plants is their production of N-and S-containing indole phytoalexins with disease resistance and cancer-preventive properties, previously proposed to be synthesized from indole independently of tryptophan. We show that camalexin, the indole phytoalexin of Arabidopsis thaliana, is synthesized from tryptophan via indole-3-acetaldoxime (IAOx) in a reaction catalyzed by CYP79B2 and CYP79B3. Cyp79B2͞cyp79B3 double knockout mutant is devoid of camalexin, as it is also devoid of indole glucosinolates [Zhao, Y., Hull, A. K., Gupta, N. R., Goss, K. A., Alonso, J., Ecker, J. R., Normanly, J., Chory, J. & Celenza, J. L. (2002) Genes Dev. 16, 3100 -3112], and isotope-labeled IAOx is incorporated into camalexin. These results demonstrate that only CYP79B2 and CYP79B3 contribute significantly to the IAOx pool from which camalexin and indole glucosinolates are synthesized. Furthermore, production of camalexin in the sur1 mutant devoid of glucosinolates excludes the possibility that camalexin is derived from indole glucosinolates. CYP79B2 plays an important role in camalexin biosynthesis in that the transcript level of CYP79B2, but not CYP79B3, is increased upon induction of camalexin by silver nitrate as evidenced by microarray analysis and promoter-␤-glucuronidase data. The structural similarity between cruciferous indole phytoalexins suggests that these compounds are biogenetically related and synthesized from tryptophan via IAOx by CYP79B homologues. The data show that IAOx is a key branching point between several secondary metabolic pathways as well as primary metabolism, where IAOx has been shown to play a critical role in IAA homeostasis.C haracteristic for cruciferous plants is the synthesis of a wide range of species-specific phytoalexins that structurally are sulfur-containing indole alkaloids (1) and the synthesis of glucosinolates (reviewed in ref.2). Both groups of natural products are involved in plant defense and have cancer-preventive properties (3, 4). Very little is known about the biosynthetic pathway of the S-containing indole phytoalexins. Their similar structure with N-and S-containing side chains at C-3 of the indole ring suggests a biogenetic relationship (5). Camalexin (3-thiazol-2Ј-yl-indole) is produced in the model plant Arabidopsis thaliana (6). It is induced by a variety of microorganisms, e.g., Pseudomonas syringae (6) and Alternaria brassisicola (7), and by abiotic factors, such as AgNO 3 (8). These findings make camalexin a good model compound for studying biosynthesis and regulation of cruciferous indole phytoalexins. In vivo feeding experiments where radiolabeled anthranilate and tryptophan were applied on leaves treated with AgNO 3 led to the suggestion that tryptophan was not a precursor in camalexin biosynthesis because tryptophan was much less efficiently incorporated into camalexin compared with anthranilate (8, 9). The data were further supported by labeling studies performed in three tryptophan mutants (8), where reduced levels of camalexin accumulated in ...

(M.M., A.S.) Camalexin represents the main phytoalexin in Arabidopsis (Arabidopsis thaliana). The camalexin-deficient phytoalexin deficient 3 (pad3) mutant has been widely used to assess the biological role of camalexin, although the exact substrate of the cytochrome P450 enzyme 71B15 encoded by PAD3 remained elusive. 2-(Indol-3-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid (dihydrocamalexic acid) was identified as likely intermediate in camalexin biosynthesis downstream of indole-3-acetaldoxime, as it accumulated in leaves of silver nitrate-induced pad3 mutant plants and it complemented the camalexin-deficient phenotype of a cyp79b2/cyp79b3 double-knockout mutant. Recombinant CYP71B15 heterologously expressed in yeast catalyzed the conversion of dihydrocamalexic acid to camalexin with preference of the (S)-enantiomer. Arabidopsis microsomes isolated from leaves of CYP71B15-overexpressing and induced wild-type plants were capable of the same reaction but not microsomes from induced leaves of pad3 mutants. In conclusion, CYP71B15 catalyzes the final step in camalexin biosynthesis.

Accumulation of camalexin, the characteristic phytoalexin of Arabidopsis thaliana, is induced by a great variety of plant pathogens. It is derived from Trp, which is converted to indole-3-acetonitrile (IAN) by successive action of the cytochrome P450 enzymes CYP79B2/B3 and CYP71A13. Extracts from wild-type plants and camalexin biosynthetic mutants, treated with silver nitrate or inoculated with Phytophthora infestans, were comprehensively analyzed by ultra-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry. This metabolomics approach was combined with precursor feeding experiments to characterize the IAN metabolic network and to identify novel biosynthetic intermediates and metabolites of camalexin. Indole-3-carbaldehyde and indole-3-carboxylic acid derivatives were shown to originate from IAN. IAN conjugates with glutathione, g-glutamylcysteine, and cysteine [Cys(IAN)] accumulated in challenged phytoalexin deficient3 (pad3) mutants. Cys(IAN) rescued the camalexin-deficient phenotype of cyp79b2 cyp79b3 and was itself converted to dihydrocamalexic acid (DHCA), the known substrate of CYP71B15 (PAD3), by microsomes isolated from silver nitrate-treated Arabidopsis leaves. Surprisingly, yeast-expressed CYP71B15 also catalyzed thiazoline ring closure, DHCA formation, and cyanide release with Cys(IAN) as substrate. In conclusion, in the camalexin biosynthetic pathway, IAN is derivatized to the intermediate Cys (IAN), which serves as substrate of the multifunctional cytochrome P450 enzyme CYP71B15.

The innate immune system is a host defense mechanism that is evolutionarily conserved from insects to human and is mainly involved in the recognition and control of the early stage of infection in all animals (1). Over the last decade, it has become increasingly evident that also plants have acquired the ability to recognize "non self" via sensitive perception systems for components of microorganisms called pathogen-associated molecular patterns (PAMPs) 2 (2-4). As classically defined, PAMPs are highly characteristic of potentially infectious microbes, but are not present in the host. In addition, such patterns are often vital for microbial survival and are therefore not subject to mutational variation. PAMPs that trigger innate immune responses in various vertebrate and non-vertebrate organisms include lipopolysaccharides (LPS) from Gram-negative bacteria, eubacterial flagellin, viral, and bacterial nucleic acids, fungal cell wall-derived glucans, chitins, mannans, or proteins and peptidoglycans (PGN) from Gram-positive bacteria (5-8). Peptidoglycan (PGN) is an essential and unique component of the bacterial envelope that provides rigidity and structure to the bacterial cell. Virtually all bacteria contain a layer of PGN, but the amount, location, and specific composition vary. PGN is a polymer of alternating N-acetylglucosamine (GlcNAc) and N-acetyl-muramic acid (MurNAc) residues in ␤-1-4 linkage which are cross-linked by short peptides (9, 10). The glycan chains display little variation among different bacterial species while the peptide subunit and the interpeptide bridge reveal species specific differences. PGN from Staphylococcus aureus belongs to the L-lysine (Lys)-type, which is primarily found in Gram-positive bacteria whereas meso-diaminopimelate (Dap)-type PGN is typical for many Gram-negative bacteria.As PGNs are located on most bacterial surfaces they constitute excellent targets for recognition by the innate immune system. Indeed, PGN is known for a long time to promote an innate immune response in vertebrates and insects (11-13), and a breakdown product of PGN, muramyl dipeptide (MurNAc-L-Ala-D-Glu; MDP) was found to be the minimal chemical structure required for PAMP activity in mammals (14). PGN is perceived in animals via various pattern recognition receptors (PRRs), including scavenger receptors, nucleotide-binding oligomerization domain-containing proteins (NODs), a family of peptidoglycan recognition proteins (PGRPs), PGN-lytic enzymes and Toll-like receptor TLR2 (15-19).Remarkable similarities have been uncovered in the molecular mode of PAMP perception in animals and plants (2,20,21). Perception of flagellin in Arabidopsis was shown to be depend-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.