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The plant-specific phi class of glutathione transferases (GSTFs) are often highly stress-inducible and expressed in a tissue-specific manner, suggestive of them having important protective roles. To date, these functions remain largely unknown, although activities associated with the binding and transport of reactive metabolites have been proposed. Using a sensitive and selective binding screen, we have probed the Arabidopsis thaliana GSTFs for natural product ligands from bacteria and plants. Uniquely, when overexpressed in bacteria, family members GSTF2 and GSTF3 bound a series of heterocyclic compounds, including lumichrome, harmane, norharmane and indole-3-aldehyde. When screened against total metabolite extracts from A. thaliana, GSTF2 also selectively bound the indole-derived phytoalexin camalexin, as well as the flavonol quercetin-3-O-rhamnoside. In each case, isothermal titration calorimetry revealed high-affinity binding (typically Kd

Plants respond to both abiotic and biotic stresses with alterations in the expression of genes required to produce protective metabolites. Sometimes plants can be challenged with different stresses simultaneously and as they cannot evade from this situation, priorities have to be set to deal with the most urgent threat. The abiotic stress ultraviolet-B (UV-B) light induces the production of UV-protective flavonols in Arabidopsis Col-0 cell suspension cultures and this accumulation is attenuated by concurrent application of the bacterial elicitor flg22 (simulating biotic stress). This inhibition correlates with strong suppression of the flavonol biosynthesis genes. In parallel, flg22 induces the production of defence-related compounds, such as the phytoalexins, camalexin and scopoletin, as well as lignin, a structural barrier thought to restrict pathogen spread. This correlated positively with flg22-mediated expression of enzymes for lignin, scopoletin and camalexin production. As flavonols, lignin and scopoletin are all derived from phenylalanine, it appears that the plant focuses the metabolism on production of scopoletin and lignin at the expense of flavonol production. Furthermore, it appears that this crosstalk involves antagonistic regulation of two opposing MYB transcription factors, the positive regulator of the flavonol pathway MYB12 (UV-B-induced and flg22-suppressed) and the negative regulator MYB4 (UV-B- and flg22-induced).

flg22 treatment increases levels of miR393, a microRNA that targets auxin receptors. Over-expression of miR393 renders plants more resistant to biotroph pathogens and more susceptible to necrotroph pathogens. In contrast, over-expression of AFB1, an auxin receptor whose mRNA is partially resistant to miR393 degradation, renders the plant more susceptible to biotroph pathogens. Here we investigate the mechanism by which auxin signalling and miR393 influence plant defence. We show that auxin signalling represses SA levels and signalling. We also show that miR393 represses auxin signalling, preventing it from antagonizing SA signalling. In addition, over-expression of miR393 increases glucosinolate levels and decreases the levels of camalexin. Further studies on pathogen interactions in auxin signalling mutants revealed that ARF1 and ARF9 negatively regulate glucosinolate accumulation, and that ARF9 positively regulates camalexin accumulation. We propose that the action of miR393 on auxin signalling triggers two complementary responses. First, it prevents suppression of SA levels by auxin. Second, it stabilizes ARF1 and ARF9 in inactive complexes. As a result, the plant is able to mount a full SA response and to re-direct metabolic flow toward the most effective anti-microbial compounds for biotroph resistance. We propose that miR393 levels can fine-tune plant defences and prioritize resources.

The defense-related plant metabolites known as glucosinolates play important roles in agriculture, ecology, and human health. Despite an advanced biochemical understanding of the glucosinolate pathway, the source of the reduced sulfur atom in the core glucosinolate structure remains unknown. Recent evidence has pointed toward GSH, which would require further involvement of a GSH conjugate processing enzyme. In this article, we show that an Arabidopsis thaliana mutant impaired in the production of the γ-glutamyl peptidases GGP1 and GGP3 has altered glucosinolate levels and accumulates up to 10 related GSH conjugates. We also show that the double mutant is impaired in the production of camalexin and accumulates high amounts of the camalexin intermediate GS-IAN upon induction. In addition, we demonstrate that the cellular and subcellular localization of GGP1 and GGP3 matches that of known glucosinolate and camalexin enzymes. Finally, we show that the purified recombinant GGPs can metabolize at least nine of the 10 glucosinolate-related GSH conjugates as well as GS-IAN. Our results demonstrate that GSH is the sulfur donor in the biosynthesis of glucosinolates and establish an in vivo function for the only known cytosolic plant γ-glutamyl peptidases, namely, the processing of GSH conjugates in the glucosinolate and camalexin pathways.

Trichoderma spp. are common soil fungi used as biocontrol agents due to their capacity to produce antibiotics, induce systemic resistance in plants and parasitize phytopathogenic fungi of major agricultural importance. The present study investigated whether colonization of Arabidopsis thaliana seedlings by Trichoderma atroviride affected plant growth and development. Here it is shown that T. atroviride promotes growth in Arabidopsis. Moreover, T. atroviride produced indole compounds in liquid cultures. These results suggest that indoleacetic acid-related indoles (IAA-related indoles) produced by T. atroviride may have a stimulatory effect on plant growth. In addition, whether colonization of Arabidopsis roots by T. atroviride can induce systemic protection against foliar pathogens was tested. Arabidopsis roots inoculation with T. atroviride provided systemic protection to the leaves inoculated with bacterial and fungal pathogens. To investigate the possible pathway involved in the systemic resistance induced by T. atroviride, the expression profile of salicylic acid, jasmonic acid/ethylene, oxidative burst and camalexin related genes was assessed in Arabidopsis. T. atroviride induced an overlapped expression of defence-related genes of SA and JA/ET pathways, and of the gene involved in the synthesis of the antimicrobial phytoalexin, camalexin, both locally and systemically. This is the first report where colonization of Arabidopsis roots by T. atroviride induces the expression of SA and JA/ET pathways simultaneously to confer resistance against hemibiotrophic and necrotrophic phytopathogens. The beneficial effects induced by the inoculation of Arabidopsis roots with T. atroviride and the induction of the plant defence system suggest a molecular dialogue between these organisms.

In plants, autophagy has been assigned 'pro-death' and 'pro-survival' roles in controlling programmed cell death associated with microbial effector-triggered immunity. The role of autophagy in basal immunity to virulent pathogens has not been addressed systematically, however. Using several autophagy-deficient (atg) genotypes, we determined the function of autophagy in basal plant immunity. Arabidopsis mutants lacking ATG5, ATG10 and ATG18a develop spreading necrosis upon infection with the necrotrophic fungal pathogen, Alternaria brassicicola, which is accompanied by the production of reactive oxygen intermediates and by enhanced hyphal growth. Likewise, treatment with the fungal toxin fumonisin B1 causes spreading lesion formation in atg mutant genotypes. We suggest that autophagy constitutes a 'pro-survival' mechanism that controls the containment of host tissue-destructive microbial infections. In contrast, atg plants do not show spreading necrosis, but exhibit marked resistance against the virulent biotrophic phytopathogen, Pseudomonas syringae pv. tomato. Inducible defenses associated with basal plant immunity, such as callose production or mitogen-activated protein kinase activation, were unaltered in atg genotypes. However, phytohormone analysis revealed that salicylic acid (SA) levels in non-infected and bacteria-infected atg plants were slightly higher than those in Col-0 plants, and were accompanied by elevated SA-dependent gene expression and camalexin production. This suggests that previously undetected moderate infection-induced rises in SA result in measurably enhanced bacterial resistance, and that autophagy negatively controls SA-dependent defenses and basal immunity to bacterial infection. We infer that the way in which autophagy contributes to plant immunity to different pathogens is mechanistically diverse, and thus resembles the complex role of this process in animal innate immunity.

Indole-3-acetaldoxime (IAOx) is a branch point compound of tryptophan (Trp) metabolism in glucosinolate-producing species such as Arabidopsis, serving as a precursor to indole-glucosinolates (IGs), the defense compound camalexin, indole-3-acetonitrile (IAN) and indole-3-acetic acid (IAA). We synthesized [(2)H(5)] and [(13)C(10)(15)N(2)]IAOx and [(13)C(6)], [(2)H(5)] and [2',2'-(2)H(2)]IAN in order to quantify endogenous IAOx and IAN in Arabidopsis and tobacco, a non-IG producing species. We found that side chain-labeled [2',2'-(2)H(2)]IAN overestimated the amount of IAN by 2-fold compared to when [(2)H(5)]IAN was used as internal standard, presumably due to protium-deuterium exchange within the internal standard during extraction of plant tissue. We also determined that [(13)C(1)]IAN underestimated the amount of IAN when the ratio of [(13)C(1)]IAN standard to endogenous IAN was greater than five to one, whereas either [(2)H(5)]IAN or [(13)C(6)]IAN showed a linear relationship with endogenous IAN over a broader range of concentrations. Transgenic tobacco vector control lines did not have detectable levels of IAOx or IAN (limit of detection∼100 pg/gfr.wt), while lines expressing either the IAOx-producing CYP79B2 or CYP79B3 genes from Arabidopsis under CaMV 35S promoter control accumulated IAOx in the range of 1-9 μg/gfr.wt. IAN levels in these lines ranged from 0.6 to 6.7 μg/gfr.wt, and IAA levels were ∼9-14-fold above levels in control lines. An Arabidopsis line expressing the same CYP79B2 overexpression construct accumulated IAOx in two of three lines measured (∼200 and 400 ng/gfr.wt) and accumulated IAN in all three lines. IAN is proposed to be a metabolite of IAOx or an enzymatic breakdown product of IGs induced upon tissue damage. Since tobacco does not produce detectable IGs, the tobacco data are consistent with IAN being a metabolite of IAOx. IAOx and IAN were also examined in the Arabidopsis activation tagged yucca mutant, and no accumulation of IAOx was found above the limits of detection but accumulation of IAN (3-fold above wt) occurred. The latter was surprising in light of recent reports that rule out IAOx and IAN as intermediates in YUCCA-mediated IAA synthesis.

Camalexin, a major phytoalexin in Arabidopsis thaliana, consists of an indole ring and a thiazole ring. The indole ring is produced from Trp, which is converted to indole-3-acetonitrile (IAN) by CYP79B2/CYP79B3 and CYP71A13. Conversion of Cys(IAN) to dihydrocamalexic acid and subsequently to camalexin is catalyzed by CYP71B15. Recent studies proposed that Cys derivative, not Cys itself, is the precursor of the thiazole ring that conjugates with IAN. The nature of the Cys derivative and how it conjugates to IAN and subsequently forms Cys(IAN) remain obscure. We found that protein accumulation of multiple glutathione S-transferases (GSTs), elevation of GST activity, and consumption of glutathione (GSH) coincided with camalexin production. GSTF6 overexpression increased and GSTF6-knockout reduced camalexin production. Arabidopsis GSTF6 expressed in yeast cells catalyzed GSH(IAN) formation. GSH(IAN), (IAN)CysGly, and γGluCys(IAN) were determined to be intermediates within the camalexin biosynthetic pathway. Inhibitor treatments and mutant analyses revealed the involvement of γ-glutamyl transpeptidases (GGTs) and phytochelatin synthase (PCS) in the catabolism of GSH(IAN). The expression of GSTF6, GGT1, GGT2, and PCS1 was coordinately upregulated during camalexin biosynthesis. These results suggest that GSH is the Cys derivative used during camalexin biosynthesis, that the conjugation of GSH with IAN is catalyzed by GSTF6, and that GGTs and PCS are involved in camalexin biosynthesis.

When plants sense the attack of microbial organisms, they initiate a series of defense responses. One of the most important defense components is the production of phytoalexins that are newly synthesized as anti-microbial secondary metabolites; however, knowledge about the signaling components regulating phytoalexin biosynthesis is limited. Mitogen-activated protein kinase (MAPK) cascades are key components in the defense signaling evoked by recognition of microbe-associated molecular patterns (MAMPs) that regulate several defense responses including phytoalexin biosynthesis. In Arabidopsis, biosynthesis of an indole-derived phytoalexin, camalexin, is regulated by MAPK cascades including AtMPK3, AtMPK4 and AtMPK6. Recently, we characterized a novel MAPK cascade in rice (OsMKK4-OsMPK3/OsMPK6) that induces production of diterpenoid phytoalexins by regulating the expression of their biosynthetic genes. Downstream signals of MAPK cascades are thought to be mediated by several transcription factors. To date, AtWRKY33 and OsTGAP1 have been identified as transcriptional activators of phytoalexin biosynthesis in Arabidopsis and rice. Here, we discuss and compare the regulatory mechanisms for phytoalexin biosynthesis through MAPK cascades and transcription factors in Arabidopsis and rice.

*† SUMMARY The two closely related Arabidopsis transcription factors, WRKY18 and WRKY40, play a major and partly redundant role in PAMP-triggered basal defense. We monitored the transcriptional reprogramming induced by the powdery mildew fungus, Golovinomyces orontii, during early stages of infection with respect to the role of WRKY18/40. Expression of >1300 Arabidopsis genes was differentially altered already 8 hours post infection (hpi), indicating rapid pre-penetration signaling between the pathogen and the host. We found that WRKY18/ 40 negatively affects pre-invasion host defenses and deduced a subset of genes that appear to be under WRKY18/40 control. A mutant lacking the WRKY18/40 repressors executes pathogen-dependent but exaggerated expression of some defense genes leading, for example, to strongly elevated levels of camalexin. This implies that WRKY18/40 act in a feedback repression system controlling basal defense. Moreover, using chromatin immunoprecipitation (ChIP), direct in vivo interactions of WRKY40 to promoter regions containing W box elements of the regulatory gene EDS1, the AP2-type transcription factor gene RRTF1 and to JAZ8 ,a member of the JA-signaling repressor gene family were demonstrated. Our data support a model in which WRKY18/40 negatively modulate the expression of positive regulators of defense such as CYP71A13, EDS1 and PAD4, but positively modulate the expression of some key JA-signaling genes by partly suppressing the expression of JAZ repressors.

Camalexin, the characteristic phytoalexin of Arabidopsis thaliana, inhibits growth of the fungal necrotroph Alternaria brassicicola. This plant metabolite probably exerts its antifungal toxicity by causing cell membrane damage. Here we observed that activation of a cellular response to this damage requires cell wall integrity (CWI) and the high osmolarity glycerol (HOG) pathways. Camalexin was found to activate both AbHog1 and AbSlt2 MAP kinases, and activation of the latter was abrogated in a AbHog1 deficient strain. Mutant strains lacking functional MAP kinases showed hypersensitivity to camalexin and brassinin, a structurally related phytoalexin produced by several cultivated Brassica species. Enhanced susceptibility to the membrane permeabilization activity of camalexin was observed for MAP kinase deficient mutants. These results suggest that the two signalling pathways have a pivotal role in regulating a cellular compensatory response to preserve cell integrity during exposure to camalexin. AbHog1 and AbSlt2 deficient mutants had reduced virulence on host plants that may, at least for the latter mutants, partially result from their inability to cope with defence metabolites such as indolic phytoalexins. This constitutes the first evidence that a phytoalexin activates fungal MAP kinases and that outputs of activated cascades contribute to protecting the fungus against antimicrobial plant metabolites.

While it is well established that reactive oxygen species can induce cell death, intracellularly generated oxidative stress does not induce lesions in the Arabidopsis (Arabidopsis thaliana) photorespiratory mutant cat2 when plants are grown in short days (SD). One interpretation of this observation is that a function necessary to couple peroxisomal hydrogen peroxide (H2O2)-triggered oxidative stress to cell death is only operative in long days (LD). Like lesion formation, pathogenesis-related genes and camalexin were only induced in cat2 in LD, despite less severe intracellular redox perturbation compared with SD. Lesion formation triggered by peroxisomal H2O2 was modified by introducing secondary mutations into the cat2 background and was completely absent in cat2 sid2 double mutants, in which ISOCHORISMATE SYNTHASE1 (ICS1) activity is defective. In addition to H2O2-induced salicylic acid (SA) accumulation, the sid2 mutation in ICS1 abolished a range of LD-dependent pathogen responses in cat2, while supplementation of cat2 with SA in SD activated these responses. Nontargeted transcript and metabolite profiling identified clusters of genes and small molecules associated with the daylength-dependent ICS1-mediated relay of H2O2 signaling. The effect of oxidative stress in cat2 on resistance to biotic challenge was dependent on both growth daylength and ICS1. We conclude that (1) lesions induced by intracellular oxidative stress originating in the peroxisomes can be genetically reverted; (2) the isochorismate pathway of SA synthesis couples intracellular oxidative stress to cell death and associated disease resistance responses; and (3) camalexin accumulation was strictly dependent on the simultaneous presence of both H2O2 and SA signals.

Plants adapt to challenging environmental factors by modulating morphogenetic processes. Although it has been speculated that activation of defense responses against pathogens leads to plant growth adjustment, little is known about developmental and architectural responses to defense stimulators. In this report we evaluated the activity of oligogalacturonides (OGs), a class of molecules directly involved in plant immunity, to modulate root system architecture in Arabidopsis thaliana. We show that OGs induce PAD3 expression and camalexin synthesis, two well-known markers of defense responses. These effects were related to primary root growth inhibition and increased lateral root and root hair formation, which are reminiscent of altered auxin responses. Cellular analysis showed that the effect of these compounds on primary root growth was due to changes in cell elongation and increased flavonoid accumulation at the root elongation region. Moreover, the observations that similar changes in primary root growth were induced by naphthylphthalamic acid supply and that auxin- or flavonoid-related mutants tir1, doc1, pgp1, pgp4, pgp19, and tt4-1 show differential responses to primary root growth inhibition by OGs suggest that auxin homeostasis plays a role in the oligogalacturonide-induced alteration of root cell patterning. Our results suggest that OGs might play a dual function in adaptation of plants to pathogen challenge by inducing defense responses and plant architecture adjustment.

Jasmonic acid (JA) is a fatty acid-derived signaling molecule that regulates a broad range of plant defense responses against herbivores and some microbial pathogens. Molecular genetic studies have established that JA also performs a critical role in several aspects of plant development. Here, we describe the characterization of the Arabidopsis mutant jasmonic acid-hypersensitive1-1 (jah1-1), which is defective in several aspects of JA responses. Although the mutant exhibits increased sensitivity to JA in root growth inhibition, it shows decreased expression of JA-inducible defense genes and reduced resistance to the necrotrophic fungus Botrytis cinerea . Gene cloning studies indicate that these defects are caused by a mutation in the cytochrome P450 protein CYP82C2. We provide evidence showing that the compromised resistance of the jah1-1 mutant to B . cinerea is accompanied by decreased expression of JA-induced defense genes and reduced accumulation of JA-induced indole glucosinolates (IGs). Conversely, the enhanced resistance to B. cinerea in CYP82C2-overexpressing plants is accompanied by increased expression of JA-induced defense genes and elevated levels of JA-induced IGs. We demonstrate that CYP82C2 affects JA-induced accumulation of the IG biosynthetic precursor tryptophan (Trp), but not the JA-induced IAA or pathogen-induced camalexin. Together, our results support a hypothesis that CYP82C2 may act in the metabolism of Trp-derived secondary metabolites under conditions in which JA levels are elevated. The jah1-1 mutant should thus be important in future studies toward understanding the mechanisms underlying the complexity of JA-mediated differential responses, which are important for plants to adapt their growth to the ever-changing environments.

*SUMMARY We have analysed the role of tryptophan-derived secondary metabolites in disease resistance of Arabidopsis to the oomycete pathogen Phytophthora brassicae. Transcript analysis revealed that genes encoding enzymes involved in tryptophan, camalexin and indole glucosinolate (iGS) biosynthesis are coordinately induced in response to P. brassicae. However, a deficiency in either camalexin or iGS accumulation has only a minor effect on the disease resistance of Arabidopsis mutants. In contrast, the double mutant cyp79B2 cyp79B3, which has a blockage in the production of indole-3-aldoxime (IAOx), the common precursor of tryptophan-derived metabolites including camalexin and iGS, is highly susceptible to P. brassicae. Because cyp79B2 cyp79B3 shows no deficiencies in other tested disease resistance responses, we concluded that the lack of IAOx-derived compounds renders Arabidopsis susceptible despite wild-type-like pathogen-induced hypersensitive cell death, stress hormone signaling and callose deposition. The susceptibility of the double mutant pen2-1 pad3-1, which has a combined defect in camalexin synthesis and PEN2-catalysed hydrolysis of iGS compounds, demonstrates that both camalexin and products of iGS hydrolysis are important for disease resistance to P. brassicae. Products of iGS hydrolysis play an early defensive role, as indicated by enhanced epidermal penetration rates of Arabidopsis mutants affected in iGS synthesis or degradation. Our results show that disease resistance of Arabidopsis to P. brassicae is established by the sequential activity of the phytoanticipin iGS and the phytoalexin camalexin.

Despite the fact that roots are the organs most subject to microbial interactions, very little is known about the response of roots to microbe-associated molecular patterns (MAMPs). By monitoring transcriptional activation of β-glucuronidase reporters and MAMP-elicited callose deposition, we show that three MAMPs, the flagellar peptide Flg22, peptidoglycan, and chitin, trigger a strong tissue-specific response in Arabidopsis thaliana roots, either at the elongation zone for Flg22 and peptidoglycan or in the mature parts of the roots for chitin. Ethylene signaling, the 4-methoxy-indole-3-ylmethylglucosinolate biosynthetic pathway, and the PEN2 myrosinase, but not salicylic acid or jasmonic acid signaling, play major roles in this MAMP response. We also show that Flg22 induces the cytochrome P450 CYP71A12-dependent exudation of the phytoalexin camalexin by Arabidopsis roots. The phytotoxin coronatine, an Ile-jasmonic acid mimic produced by Pseudomonas syringae pathovars, suppresses MAMP-activated responses in the roots. This suppression requires the E3 ubiquitin ligase COI1 as well as the transcription factor JIN1/MYC2 but does not rely on salicylic acid–jasmonic acid antagonism. These experiments demonstrate the presence of highly orchestrated and tissue-specific MAMP responses in roots and potential pathogen-encoded mechanisms to block these MAMP-elicited signaling pathways.

Mitogen-activated protein (MAP) kinase phosphatases are important negative regulators of the levels and kinetics of MAP kinase activation that modulate cellular responses. The dual-specificity phosphatase MAP KINASE PHOSPHATASE1 (MKP1) was previously shown to regulate MAP KINASE6 (MPK6) activation levels and abiotic stress responses in Arabidopsis thaliana. Here, we report that the mkp1 null mutation in the Columbia (Col) accession results in growth defects and constitutive biotic defense responses, including elevated levels of salicylic acid, camalexin, PR gene expression, and resistance to the bacterial pathogen Pseudomonas syringae. PROTEIN TYROSINE PHOSPHATASE1 (PTP1) also interacts with MPK6, but the ptp1 null mutant shows no aberrant growth phenotype. However, the pronounced constitutive defense response of the mkp1 ptp1 double mutant reveals that MKP1 and PTP1 repress defense responses in a coordinated fashion. Moreover, mutations in MPK3 and MPK6 distinctly suppress mkp1 and mkp1 ptp1 phenotypes, indicating that MKP1 and PTP1 act as repressors of inappropriate MPK3/MPK6-dependent stress signaling. Finally, we provide evidence that the natural modifier of mkp1 in Col is largely the disease resistance gene homolog SUPPRESSOR OF npr1-1, CONSTITUTIVE 1 (SNC1) that is absent in the Wassilewskija accession. Our data thus indicate a major role of MKP1 and PTP1 in repressing salicylic acid biosynthesis in the autoimmune-like response caused by SNC1.

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, γ-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.

Arabidopsis thaliana is known to produce the phytoalexin camalexin in response to abiotic and biotic stress. Here we studied the mechanisms of tolerance to camalexin in the fungus Botrytis cinerea, a necrotrophic pathogen of A. thaliana. Exposure of B. cinerea to camalexin induces expression of BcatrB, an ABC transporter that functions in the efflux of fungitoxic compounds. B. cinerea inoculated on wild-type A. thaliana plants yields smaller lesions than on camalexin-deficient A. thaliana mutants. A B. cinerea strain lacking functional BcatrB is more sensitive to camalexin in vitro and less virulent on wild-type plants, but is still fully virulent on camalexin-deficient mutants. Pre-treatment of A. thaliana with UV-C leads to increased camalexin accumulation and substantial resistance to B. cinerea. UV-C-induced resistance was not seen in the camalexin-deficient mutants cyp79B2/B3, cyp71A13, pad3 or pad2, and was strongly reduced in ups1. Here we demonstrate that an ABC transporter is a virulence factor that increases tolerance of the pathogen towards a phytoalexin, and the complete restoration of virulence on host plants lacking this phytoalexin.

In plants the amino acid tryptophan (Trp) is used to synthesize proteins and a wide variety of compounds that control development and defense. Recent studies in the reference plant Arabidopsis thaliana have elucidated a number of tryptophan secondary metabolism pathways derived from the Trp metabolite, indole-3-acetaldoxime (IAOx), particularly the pathway for synthesis of indolic glucosinolate (IG) herbivory defense compounds. Analyses of mutants, natural variants, and transgenic strains with perturbations in the IG pathway have revealed that regulation of this pathway is networked at many levels, including interfaces with Trp synthesis, other Trp secondary metabolism pathways, other glucosinolate synthesis pathways, and sulfur metabolism. Transcriptional regulatory mechanisms have been particularly well characterized, but additional mechanisms such as metabolic channeling may also contribute to the homeostasis of Trp secondary metabolism. The IG pathway thus serves as a paradigm for regulatory cross-talk between primary and secondary metabolism and among inter-related secondary metabolic processes.

*Summary Physical injury inflicted on living tissue makes it vulnerable to invasion by pathogens. Wounding of Arabidopsis thaliana leaves, however, does not conform to this concept and leads to immunity to Botrytis cinerea, the causal agent of grey mould. In wounded leaves, hyphal growth was strongly inhibited compared to unwounded controls. Wound-induced resistance was not associated with salicylic acid-, jasmonic acid- or ethylene-dependent defence responses. The phytoalexin camalexin was found to be involved in this defence response as camalexin-deficient mutants were not protected after wounding and the B. cinerea strains used here were sensitive to this compound. Wounding alone did not lead to camalexin production but primed its accumulation after inoculation with B. cinerea, further supporting the role of camalexin in wound-induced resistance. In parallel with increased camalexin production, genes involved in the biosynthesis of camalexin were induced faster in wounded and infected plants in comparison with unwounded and infected plants. Glutathione was also found to be required for resistance, as mutants deficient in c-glutamylcysteine synthetase showed susceptibility to B. cinerea after wounding, indicating that wild-type basal levels of glutathione are required for the wound-induced resistance. Furthermore, expression of the gene encoding glutathione-S-transferase 1 was primed by wounding in leaves inoculated with B. cinerea. In addition, the priming of MAP kinase activity was observed after inoculation of wounded leaves with B. cinerea compared to unwounded inoculated controls. Our results demonstrate how abiotic stress can induce immunity to virulent strains of B. cinerea, a process that involves camalexin and glutathione.

Plant and animal perception of microbes through pathogen surveillance proteins leads to MAP kinase signalling and the expression of defence genes. However, little is known about how plant MAP kinases regulate specific gene expression. We report that, in the absence of pathogens, Arabidopsis MAP kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further supported by the suppression of PAD3 expression in mpk4-wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation.

Plant recognition of pathogens leads to rapid activation of MPK3 and MPK6, two Arabidopsis mitogen-activated protein kinases (MAPKs), and their orthologs in other species. Here, we report that synthesis of camalexin, the major phytoalexin in Arabidopsis , is regulated by the MPK3/MPK6 cascade. Activation of MPK3/MPK6 by expression of active upstream MAPK kinase (MAPKK) or MAPKK kinase (MAPKKK) was sufficient to induce camalexin synthesis in the absence of pathogen attack. Induction of camalexin by Botrytis cinerea was preceded by MPK3/MPK6 activation, and compromised in mpk3 and mpk6 mutants. Genetic analysis placed the MPK3/MPK6 cascade upstream of PHYTOALEXIN DEFICIENT 2 (PAD2) and PAD3, but independent or downstream of PAD1 and PAD4. Camalexin induction after MPK3/MPK6 activation was preceded by rapid and coordinated up-regulation of multiple genes encoding enzymes in the tryptophan (Trp) biosynthetic pathway, in the conversion of Trp to indole-3-acetaldoxime (IAOx, a branch point between primary and secondary metabolism), and in the camalexin biosynthetic pathway downstream of IAOx. These results indicate that the MPK3/MPK6 cascade regulates camalexin synthesis through transcriptional regulation of the biosynthetic genes after pathogen infection.

Root galls of Brassicaceae caused by Plasmodiophora brassicae are dependent on increased auxin and cytokinin formation. In this study we investigated whether indole glucosinolates are involved in indole-3-acetic acid (IAA) biosynthesis in root galls, by using a genetic approach. The cytochrome P450 enzymes, CYP79B2 and CYP79B3, convert tryptophan to indole-3-acetaldoxime (IAOx), which is a precursor for indole glucosinolates and the phytoalexin camalexin in Arabidopsis thaliana. Root galls of the Arabidopsis ecotypes Wassilewskija (WS) and Columbia (Col) accumulated camalexin, WS at levels up to 320 μg/g dry weight. By contrast, camalexin was absent in root galls of cyp79b2/b3 double mutants. Infection rate and disease index as a measure of club development in mutant and wild-type plants of the two ecotypes were investigated and no differences were found in gall formation. This demonstrates that camalexin is an ineffective inhibitor of P. brassicae and indole glucosinolates are not the source of elevated levels of IAA in galls, because free IAA levels in mutant galls were comparable with those in wild type.

Investigation of phytoalexin production using abiotic elicitation showed that the phytoalexin rapalexin A was produced by both Thellungiella halophila and Arabidopsis thaliana, but while A. thaliana produced camalexin, T. halophila produced wasalexins A and B and methoxybrassenin B. Considering that the genome of T. halophila is being sequenced currently and that the wasalexin pathway present in T. halophila is expected to involve a number of genes also present in Brassica species, our discovery should facilitate the isolation of genes involved in biosynthetic pathways of phytoalexins of the most economically important crucifer species.

Lipopolysaccharides (LPSs), as MAMP molecules, trigger the activation of signal transduction pathways involved in defence. Currently, plant metabolomics is providing new dimensions into understanding the intracellular adaptive responses to external stimuli. The effect of LPS on the metabolomes of Arabidopsis thaliana cells and leaf tissue was investigated over a 24 h period. Cellular metabolites and those secreted into the medium were extracted with methanol and liquid chromatography coupled to mass spectrometry was used for quantitative and qualitative analyses. Multivariate statistical data analyses were used to extract interpretable information from the generated multidimensional LC-MS data. The results show that LPS perception triggered differential changes in the metabolomes of cells and leaves, leading to variation in the biosynthesis of specialised secondary metabolites. Time-dependent changes in metabolite profiles were observed and biomarkers associated with the LPS-induced response were tentatively identified. These include the phytohormones salicylic acid and jasmonic acid, and also the associated methyl esters and sugar conjugates. The induced defensive state resulted in increases in indole—and other glucosinolates, indole derivatives, camalexin as well as cinnamic acid derivatives and other phenylpropanoids. These annotated metabolites indicate dynamic reprogramming of metabolic pathways that are functionally related towards creating an enhanced defensive capacity. The results reveal new insights into the mode of action of LPS as an activator of plant innate immunity, broadens knowledge about the defence metabolite pathways involved in Arabidopsis responses to LPS, and identifies specialised metabolites of functional importance that can be employed to enhance immunity against pathogen infection.

SUMMARY Plant immunity against the majority of microbial pathogens is conveyed by a phenomenon termed non-host resistance (NHR). This multifactorial trait provides durable protection against a given pathogen species. We investigated the molecular basis of NHR in Arabidopsis against the wheat leaf rust pathogen, Puccinia triticina (Ptr). Urediospores germinated with high efficiency and grew randomly over the Arabidopsis leaf surface. However, only 12% of urediospores produced a germ tube that successfully located a stoma and just 0.2% of urediospores went on to produce a haustorium within a penetrated mesophyll cell. Attempted Ptr infection induced the production of reactive oxygen intermediates (ROIs), nitric oxide (NO), salicylic acid (SA) and camalexin. The expression of SA, jasmonic acid (JA) and ROI-dependent genes was also detected. A series of well-characterized defence-related mutants were challenged with Ptr, but none of these lines exhibited significantly increased susceptibility to this fungus. Our findings also suggest that attempted Ptr infection triggers transient stomatal closure in Arabidopsis. We assessed the response of a collection of 79 geographically diverse Arabidopsis accessions to Ptr. Wa-1 plants supported a striking increase in Ptr substomatal vesicle frequency relative to all other tested accessions. Furthermore, SA and camalexin levels became elevated in Wa-1 plants relative to the Col reference line, in response to attempted Ptr infection. Additionally, the kinetics of SA-dependent gene expression was expedited in this accession relative to Col plants. To uncover the genetic architecture of NHR against Ptr, we assayed the response of the Arabidopsis Landsberg erecta (Ler) x Columbia (Col) recombinant inbred population to this fungus. Multiple small-to-medium effect quantitative trait loci were identified that govern the expression of NHR in Arabidopsis against Ptr.

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.

Systemic acquired resistance (SAR) is usually described as a phenomenon whereby localized inoculation with a necrotizing pathogen renders a plant more resistant to subsequent pathogen infection. Here we show that Pseudomonas syringae strains for which Arabidopsis thaliana represents a non-host plant systemically elevate resistance although the underlying interactions neither trigger a hypersensitive response nor cause necrotic disease symptoms. A similar enhancement of systemic resistance was observed when elicitor-active preparations of two typical bacterial pathogen-associated molecular patterns (PAMPs), flagellin and lipopolysaccharides (LPS), were applied in a localized manner. Several lines of evidence indicate that the observed systemic resistance responses are identical to SAR. Localized applications of non-adapted bacteria, flagellin or LPS elevate levels of the SAR regulatory metabolite salicylic acid (SA) and pathogenesis-related (PR) gene expression not only in treated but also in distant leaves. All treatments also systemically increase expression of the SAR marker gene FLAVIN-DEPENDENT MONOOXYGENASE 1. Further, a whole set of SAR-deficient Arabidopsis lines, including mutants in SA biosynthesis and signalling, are impaired in establishing the systemic resistance response triggered by non-host bacteria or PAMPs. We also show that the magnitude of defence reactions such as SA accumulation, PR gene expression or camalexin accumulation induced at sites of virulent or avirulent P. syringae inoculation but not the extent of tissue necrosis during these interactions determines the extent of SAR in distant leaves. Our data indicate that PAMPs significantly contribute to SAR initiation in Arabidopsis and that tissue necroses at inoculation sites are dispensable for SAR activation.

Abscisic acid (ABA) is a defense hormone with influence on callose-dependent and -independent resistance against Leptosphaeria maculans acting in the RLM1Col pathway. ABA-deficient and -insensitive mutants in Ler-0 background (aba1-3 and abi1-1) displayed susceptibility to L. maculans, along with a significantly decreased level of callose depositions, whereas abi2-1 and abi3-1 remained resistant, together with the abi5-1 mutant of Ws-0 background. Suppressor mutants of abi1-1 confirmed that the L. maculans-susceptible response was due to the dominant negative nature of the abi1-1 mutant. Highly induced camalexin levels made ABA mutants in Col-0 background (aba2-1, aba3-1, and abi4-1) appear resistant, but displayed enhanced susceptibility as double mutants with pad3-1, impaired in camalexin biosynthesis. β-Aminobutyric acid (BABA) pretreatment of Ler-0 contributed to an elevated level of endogenous ABA after L. maculans inoculation. Comparisons between (RLM1Col)pad3 and rlm1Lerpad3 showed that ABA and BABA enhancement of callose deposition requires induction from RLM1Col·ABI1, but not ABI2, was found to be involved in a feedback mechanism that modulates RLM1Col expression. Genetic analysis showed further that this feedback occurs upstream of ABI4 and that components downstream of ABI4 modulate ABI1 activity. ABA and BABA treatments of the L. maculans-susceptible callose synthase mutant pmr4 showed that ABA also induces a callose-independent resistance. Similar treatments enhanced callose depositions and induced resistance to L. maculans in oilseed rape, and BABA-induced resistance was found to be independent of salicylic acid.

SUMMARY Susceptibility was evaluated of host and non-host plants to three pathogenic Botrytis species: the generalist B. cinerea and the specialists B. elliptica (lily) and B. tulipae (tulip). B. tulipae was, unexpectedly, able to infect plant species other than tulip, and to a similar extent as B. cinerea. To study host and non-host interactions in more detail, the three Botrytis species were inoculated on Arabidopsis wild-types and 23 mutant genotypes. Disease development was monitored macroscopically by quantifying the lesion area and microscopically by bright-field and fluorescence microscopy following histochemical staining. B. cinerea and B. tulipae were very similar in their ability to infect the tested Arabidopsis genotypes, whereas B. elliptica caused disease only on a few Arabidopsis mutant genotypes. Arabidopsis mutants with a delayed or reduced cell death response were generally more resistant to Botrytis infection, whereas mutants in which cell death was accelerated were more susceptible. Differences in susceptibility between genotypes were generally gradual. Only the camalexin-deficient mutant pad3 was fully susceptible to all three Botrytis species. Cellular changes were monitored during compatible and incompatible interactions. The formation of papillae, the presence of lysosome-like vesicles and the intracellular accumulation of H(2)O(2) and nitric oxide were visualized in the infection zones using fluorescent probes. Based on histology and responses of Arabidopsis mutants, a model is proposed in which resistance against Botrytis, besides the production of camalexin, depends on the balance between cell death and survival.

Summary 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.

Root-knot nematodes (RKNs; Meloidogyne spp.) are plant parasites with a broad host range causing great losses worldwide. To parasitize their hosts, RKNs establish feeding sites in roots known as giant cells. The majority of work studying plant-RKN interactions in susceptible hosts addresses establishment of the giant cells and there is limited information on the early defense responses. Here we characterized early defense or pattern-triggered immunity (PTI) against RKNs in Arabidopsis thaliana. To address PTI, we evaluated known canonical PTI signaling mutants with RKNs and investigated the expression of PTI marker genes after RKN infection using both quantitative PCR and β-glucuronidase reporter transgenic lines. We showed that PTI-compromised plants have enhanced susceptibility to RKNs, including the bak1-5 mutant. BAK1 is a common partner of distinct receptors of microbe- and damage-associated molecular patterns. Furthermore, our data indicated that nematode recognition leading to PTI responses involves camalexin and glucosinolate biosynthesis. While the RKN-induced glucosinolate biosynthetic pathway was BAK1-dependent, the camalexin biosynthetic pathway was only partially dependent on BAK1. Combined, our results indicate the presence of BAK1-dependent and -independent PTI against RKNs in A. thaliana, suggesting the existence of diverse nematode recognition mechanisms.

Antimicrobial compounds have critical roles in plant immunity; for example, Arabidopsis thaliana and other crucifers deploy phytoalexins and glucosinolate derivatives in defense against pathogens. The pathogen-responsive MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3) and MPK6 have essential functions in the induction of camalexin, the major phytoalexin in Arabidopsis. In search of cyanide, a coproduct of ethylene and camalexin biosynthesis, we found that MPK3 and MPK6 also affect the accumulation of extracellular thiocyanate ion derived from the indole glucosinolate (IGS) pathway. Botrytis cinerea infection activates MPK3/MPK6, which promote indole-3-yl-methylglucosinolate (I3G) biosynthesis and its conversion to 4-methoxyindole-3-yl-methylglucosinolate (4MI3G). Gain- and loss-of-function analyses demonstrated that MPK3/MPK6 regulate the expression of MYB51 and MYB122, two key regulators of IGS biosynthesis, as well as CYP81F2 and IGMT1/IGMT2, which encode enzymes in the conversion of I3G to 4MI3G, through ETHYLENE RESPONSE FACTOR6 (ERF6), a substrate of MPK3/MPK6. Under the action of PENETRATION2 (PEN2), an atypical myrosinase, and PEN3, an ATP binding cassette transporter, 4MI3G is converted to extracellular unstable antimicrobial compounds, possibly isothiocyanates that can react with nucleophiles and release the stable thiocyanate ion. Recent studies demonstrated the importance of PEN2/PEN3-dependent IGS derivatives in plant immunity. Here, we report that MPK3/MPK6 and their substrate ERF6 promote the biosynthesis of IGSs and the conversion of I3G to 4MI3G, a target of PEN2/PEN3-dependent chemical defenses in plant immunity.

Trichoderma species are present in many ecosystems and some strains have the ability to reduce the severity of plant diseases by activating various defense pathways via specific biologically active signaling molecules. Hence we investigated the effects of low molecular weight volatile compounds of Trichoderma asperellum IsmT5 on Arabidopsis thaliana. During co-cultivation of T. asperellum IsmT5 without physical contact to A. thaliana we observed smaller but vital and robust plants. The exposed plants exhibit increased trichome numbers, accumulation of defense-related compounds such as H2O2, anthocyanin, camalexin, and increased expression of defense-related genes. We conclude that A. thaliana perceives the Trichoderma volatiles as stress compounds and subsequently initiates multilayered adaptations including activation of signaling cascades to withstand this environmental influence. The prominent headspace volatile of T. asperellum IsmT5 was identified to be 6-pentyl-α-pyrone (6PP), which was solely applied to A. thaliana to verify the growth and defense reactions. Most noticeable is that A. thaliana preexposed to 6PP showed significantly reduced symptoms when challenged with Botrytis cinerea and Alternaria brassicicola, indicating that defense-activated plants subsequently became more resistant to pathogen attack. Together, these results support that products that are based on Trichoderma volatiles have the potential being a useful biocontrol agent in agriculture.

The phytoalexin camalexin and indolic glucosinolates share not only a common evolutionary origin and a tightly interconnected biosynthetic pathway, but regulatory proteins controlling the shared enzymatic steps are also modulated by the same R2R3-MYB transcription factors. The indolic phytoalexin camalexin is a crucial defense metabolite in the model plant Arabidopsis. Indolic phytoalexins and glucosinolates appear to have a common evolutionary origin and are interconnected on the biosynthetic level: a key intermediate in the biosynthesis of camalexin, indole-3-acetaldoxime (IAOx), is also required for the biosynthesis of indolic glucosinolates and is under tight control by the transcription factors MYB34, MYB51, and MYB122. The abundance of camalexin was strongly reduced in myb34/51 and myb51/122 double and in triple myb mutant, suggesting that these transcription factors are important in camalexin biosynthesis. Furthermore, expression of MYB51 and MYB122 was significantly increased by biotic and abiotic camalexin-inducing agents. Feeding of the triple myb34/51/122 mutant with IAOx or indole-3-acetonitrile largely restored camalexin biosynthesis. Conversely, tryptophan could not complement the low camalexin phenotype of this mutant, which supports a role for the three MYB factors in camalexin biosynthesis upstream of IAOx. Consistently expression of the camalexin biosynthesis genes CYP71B15/PAD3 and CYP71A13 was not negatively affected in the triple myb mutant and the MYBs could not activate pCYP71B15::uidA expression in trans-activation assays with cultured Arabidopsis cells. In conclusion, this study reveals the importance of MYB factors regulating the generation of IAOx as precursor of camalexin.

The Arabidopsis mutant wrky33 is highly susceptible to Botrytis cinerea. We identified >1680 Botrytis-induced WRKY33 binding sites associated with 1576 Arabidopsis genes. Transcriptional profiling defined 318 functional direct target genes at 14 hr post inoculation. Comparative analyses revealed that WRKY33 possesses dual functionality acting either as a repressor or as an activator in a promoter-context dependent manner. We confirmed known WRKY33 targets involved in hormone signaling and phytoalexin biosynthesis, but also uncovered a novel negative role of abscisic acid (ABA) in resistance towards B. cinerea 2100. The ABA biosynthesis genes NCED3 and NCED5 were identified as direct targets required for WRKY33-mediated resistance. Loss-of-WRKY33 function resulted in elevated ABA levels and genetic studies confirmed that WRKY33 acts upstream of NCED3/NCED5 to negatively regulate ABA biosynthesis. This study provides the first detailed view of the genome-wide contribution of a specific plant transcription factor in modulating the transcriptional network associated with plant immunity. DOI: http://dx.doi.org/10.7554/eLife.07295.001, eLife digest Crop yields can be badly affected by diseases caused by some fungi and other microbes. One fungus called Botrytis cinerea is able to infect many different species of crop plants—including tomatoes and grapes—and can cause severe damage both before and after harvest. This fungus belongs to a group of microbes that kill the plant cells they invade and then extract the nutrients from the dead cells. Some plants are able to resist infection by B. cinerea and researchers have identified several proteins that are involved in this resistance. One such protein is called WRKY33, which is able to bind to DNA to regulate the activity of particular genes. However, it was not clear exactly which genes were involved in the response to B. cinerea. Arabidopsis thaliana is a small flowering plant that is often used in research. Mutant A. thaliana plants lacking WRKY33 are very susceptible to infection with B. cinerea. Here, Liu et al. use several genetic techniques to find out which genes WRKY33 regulates when A. thaliana plants are exposed to the fungus. The experiments indicate that WRKY33 can alter the activity of over 300 genes. Some of these genes had previously been shown to be targets of WRKY33 and are involved in cell responses to plant hormones and the production of an antimicrobial molecule called camalexin. Liu et al. also show that two genes called NCED3 and NCED5—which are required for the production of a plant hormone called abscisic acid—are repressed by WRKY33. Mutant plants lacking WRKY33 had increased levels of abscisic acid and further experiments suggested that the repression of NCED3 and NCED5 by WRKY33 is important to resistance against the fungus. Liu et al.'s findings provide the first detailed view of which genes in A. thaliana are regulated by WRKY33 when the plant is exposed to B. cinerea. A future challenge is to understand how blocking the production of abscisic acid protects plants against B. cinerea and other similar fungi. DOI: http://dx.doi.org/10.7554/eLife.07295.002

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.

Summary Verticillium dahliae is a destructive, soil-borne fungal pathogen that causes vascular wilt disease in many economically important crops worldwide. A polyamine oxidase (PAO) gene was identified and cloned by screening suppression subtractive hybridisation and cDNA libraries of cotton genotypes tolerant to Verticillium wilt and was induced early and strongly by inoculation with V. dahliae and application of plant hormone. Recombinant cotton polyamine oxidase (GhPAO) was found to catalyse the conversion of spermine (Spm) to spermidine (Spd) in vitro. Constitutive expression of GhPAO in Arabidopsis thaliana produced improved resistance to V. dahliae and maintained putrescine, Spd and Spm at high levels. Hydrogen peroxide (H2O2), salicylic acid and camalexin (a phytoalexin) levels were distinctly increased in GhPAO-overexpressing Arabidopsis plants during V. dahliae infection when compared with wild-type plants, and Spm and camalexin efficiently inhibited growth of V. dahliae in vitro. Spermine promoted the accumulation of camalexin by inducing the expression of mitogen-activated protein kinases and cytochrome P450 proteins in Arabidopsis and cotton plants. The three polyamines all showed higher accumulation in tolerant cotton cultivars than in susceptible cotton cultivars after inoculation with V. dahliae. GhPAO silencing in cotton significantly reduced the Spd level and increased the Spm level, leading to enhanced susceptibility to infection by V. dahliae, and the levels of H2O2 and camalexin were distinctly lower in GhPAO-silenced cotton plants after V. dahliae infection. Together, these results suggest that GhPAO contributes to resistance of the plant against V. dahliae through the mediation of Spm and camalexin signalling.

*† ‡ Summary The Arabidopsis pad2-1 mutant belongs to a series of non-allelic camalexin-deficient mutants. It was originally described as showing enhanced susceptibility to virulent strains of Pseudomonas syringae and was later shown to be hyper-susceptible to the oomycete pathogen Phytophthora brassicae (formerly P. porri). Surprisingly, in both pathosystems, the disease susceptibility of pad2-1 was not caused by the camalexin deficiency, suggesting additional roles of PAD2 in disease resistance. The susceptibility of pad2-1 to P. brassicae was used to map the mutation to the gene At4g23100, which encodes c-glutamylcysteine synthetase (c-ECS, GSH1). GSH1 catalyzes the first committed step of glutathione (GSH) biosynthesis. The pad2-1 mutation caused an S to N transition at amino acid position 298 close to the active center. The conclusion that PAD2 encodes GSH1 is supported by several lines of evidence: (i) pad2-1 mutants contained only about 22% of wild-type amounts of GSH, (ii) genetic complementation of pad2-1 with wild-type GSH1 cDNA restored GSH production, accumulation of camalexin in response to P. syringae and resistance to P. brassicae and P. syringae, (iii) another GSH1 mutant, cad2-1, showed pad2-like phenotypes, and (iv) feeding of GSH to excised leaves of pad2-1 restored camalexin production and resistance to P. brassicae. Inoculation of Col-0 with P. brassicae caused a coordinated increase in the transcript abundance of GSH1 and GSH2, the gene encoding the second enzyme in GSH biosynthesis, and resulted in enhanced foliar GSH accumulation. The pad2-1 mutant showed enhanced susceptibility to additional pathogens, suggesting an important general role of GSH in disease resistance of Arabidopsis.

Anthranilate synthase (AS) is a key enzyme in tryptophan (Trp) biosynthesis. Metabolic changes in transgenic Arabidopsis plants expressing the feedback-resistant anthranilate synthase α subunit gene OASA1D were investigated with respect to Trp synthesis and effects on secondary metabolism. The Trp content varied depending on the transgenic line, with some lines showing an approximately 200-fold increase. The levels of AS activity in crude extracts from the transgenic lines were comparable to those in the wild type. On the other hand, the enzyme prepared from the lines accumulating high levels of Trp showed a relaxed feedback sensitivity. The AS activity, determined in the presence of 50 μM l -Trp, correlated well with the amount of free Trp in the transgenic lines, indicating the important role of feedback inhibition in control of Trp pool size. In Arabidopsis, Trp is a precursor of multiple secondary metabolites, including indole glucosinolates and camalexin. The amount of indol-3-ylmethyl glucosinolate (I3M) in rosette leaves of the high-Trp accumulating lines was 1.5- to 2.1-fold greater than that in wild type. The treatment of the leaves with jasmonic acid resulted in a more pronounced accumulation of I3M in the high-Trp accumulating lines than in wild type. The induction of camalexin formation after the inoculation of Alternaria brassicicola was not affected by the accumulation of a large amount of Trp. The accumulation of constitutive phenylpropanoids and flavonoids was suppressed in high-Trp accumulating lines, while the amounts of Phe and Tyr increased, thereby indicating an interaction between the Trp branch and the Phe and Tyr branch in the shikimate pathway.

Here we have examined genetically well-defined interactions in leaves of Arabidopsis ecotype Col-5 during compatible and incompatible interactions with isogenic pairs of Pseudomonas syringae pathovars tomato (Pst) DC3000 and phaseolicola (Pph) 1448A strains differing only in the presence or absences of cloned avr genes that determine the compatibility and incompatibility of the reaction. Strain Pst DC3000 was virulent causing formation of a spreading lesion surrounded by a chlorotic halo. The bean pathogen Pph 1448A (as a non-host pathogen) caused no visible symptoms apart from very patchy discolouration after 3 days. The presence of the avirulence genes avrPpiA and avrPphB in Pph 1448A led to a rapid or slow HR owing to interaction with the resistance genes RPM1 and RPS5 respectively. The hrpL mutant of Pph failed to produce symptoms. Microscopical and biochemical studies were used for in situ localization of plant molecules involved in plant resistance such as autofluorescent phenolics, lignin and camalexin. A strong correlation was observed between the timing and extent of cell death and high levels of phenolic and lignin accumulation in cell-walls and cytoplasm of cells undergoing the HR, whereas no staining of lignin-like material was observed during the compatible interaction. Accumulation of phytoalexin, camalexin, was associated with tissue necrosis whether during the HR or a susceptible response. Although high levels of camalexin were found within tissues inoculated with the virulent strain DC3000, the earliest detection of camalexin occurred during HR following inoculation with avirulent strain. The results indicate that accumulation of phenolics but not camalexin may be involved in resistance against bacterial pathogens used in this study.

Numerous studies have suggested that plant/pathogen interactions are partially mediated via plant secondary metabolite production and corresponding pathogen tolerance. However, there are inconsistent reports on the ability of particular compounds to provide resistance to a pathogen. Most of these studies have focused on individual isolates of a given pathogen, suggesting that pathogens vary in their sensitivity to plant-produced toxins. We tested variability in virulence among pathogen isolates, and the impact on this by plant production of, and pathogen tolerance to, secondary metabolites. Botrytis cinerea isolates showed differing sensitivity to purified camalexin, and camalexin-sensitive isolates produced larger lesions on camalexin-deficient Arabidopsis genotypes than on the wild type. In contrast, the camalexin-insensitive isolate produced lesions of similar size on wild-type and camalexin-deficient Arabidopsis. Additional analysis with Arabidopsis secondary metabolite biosynthetic mutants suggests that Botrytis also has variable sensitivity to phenylpropanoids and glucosinolates. Furthermore, Botrytis infection generates a gradient of secondary metabolite responses emanating from the developing lesion, with the Botrytis isolate used determining the accumulation pattern. Collectively, our results indicate that Arabidopsis/Botrytis interactions are influenced at the metabolic level by variations in toxin production in the host and sensitivity in the pathogen.

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

We report the characterization of an Arabidopsis thaliana mutant, ups1, isolated on the basis of reduced expression of phosphoribosylanthranilate transferase, a tryptophan biosynthetic enzyme. ups1 also exhibits defects in a wide range of defence responses. After infection with Pseudomonas syringae or Botrytis cinerea, the expression of genes regulated by both the salicylic acid and jasmonic acid/ethylene pathways is reduced in ups1 compared with wild type. Camalexin accumulation in ups1 is greatly reduced after infection with these two pathogens, as well as after amino acid starvation or oxidative stress. Reactive oxygen species (ROS)-mediated gene expression is also compromised in ups1 indicating that this mutant is defective in signalling pathways activated in response to both biotic and abiotic stress. The fact that all three major defence signalling pathways are disrupted in ups1, together with the oxidative stress phenotype, leads us to suggest that UPS1 is involved in ROS signal transduction.

Despite the identification of several key mechanisms underlying disease resistance, comparatively little is known about the way developmental factors affect the capability of plants to defend themselves against microbial pathogens. Here, the influence of leaf age on inducible local and systemic defence responses in the incompatible interaction between Arabidopsis thaliana and Pseudomonas syringae pv. maculicola (avrRPM1) is investigated. Comparison of the local defence behaviour of rosette leaves differing in age revealed that younger leaves generally had to invest in more pronounced inducible defences than older (non-senescent) leaves to achieve a similar degree of resistance. For instance, younger leaves exhibited a more distinctive oxidative burst and a stronger hypersensitive cell death response than older ones. The pathogen-induced expression of the defence related genes phenylalanine ammonia lyase (PAL) and glutathione-S-transferase (GST) proved to be higher and faster in younger leaves, respectively, whereas gene induction of PR-1 (pathogenesis-related protein 1) was largely independent of leaf age. Similarly, upon bacterial inoculation, the defence signal salicylic acid (SA) and the phytoalexin camalexin accumulated to higher amounts in younger than in older leaves. Despite these differences in inducible defences, bacterial growth as a measure of disease resistance proved to be similar in inoculated younger and older leaves. In tissue distant from the inoculation site, systemic acquired resistance (SAR) developed most effectively in younger leaves, and this response was associated with strong accumulation of SA and PR-1 transcripts. However, older leaves still exhibited SAR to a certain degree, and this resistance developed essentially without systemic SA or PR-1 accumulation. These results suggest that mechanisms independent of SA signalling in systemic leaves contribute to realise SAR.