A highly specific pathogen-responsive promoter element from the immediate-early activated CMPG1 gene in Petroselinum crispum.

Within the complex signalling network from pathogen-derived elicitor perception to defense-related gene activation, some immediate-early responding genes may have pivotal roles in downstream transcriptional regulation. We have identified the parsley (Petroselinum crispum) ELI17 gene as a particularly fast-responding gene possessing a new type of W box-containing, elicitor-responsive promoter element, E17. Highly selective E17-mediated reporter gene expression at pathogen infection sites in transgenic Arabidopsis thaliana plants demonstrated the potential of this promoter element for designing new strategies in resistance breeding as well as for further analysis of the early components of defense-related gene activation mechanisms. The protein encoded by the ELI17 gene exhibits various structural characteristics of established transcription factors and is designated as a CMPG protein according to the first four strictly conserved amino acids defining a newly emerging class of plant-specific proteins.

Accumulation of soluble and wall-bound indolic metabolites in Arabidopsis thaliana leaves infected with virulent or avirulent Pseudomonas syringae pathovar tomato strains.

The chemical structures and accumulation kinetics of several major soluble as well as wall-bound, alkali-hydrolyzable compounds induced upon infection of Arabidopsis thaliana leaves with Pseudomonas syringae pathovar tomato were established. All identified accumulating products were structurally related to tryptophan. Most prominent among the soluble substances were tryptophan, beta-d-glucopyranosyl indole-3-carboxylic acid, 6-hydroxyindole-3-carboxylic acid 6-O-beta-d-glucopyranoside, and the indolic phytoalexin camalexin. The single major accumulating wall component detectable under these conditions was indole-3-carboxylic acid. All of these compounds increased more rapidly, and camalexin as well as indole-3-carboxylic acid reached much higher levels, in the incompatible than in the compatible P. syringae/A. thaliana interaction. The only three prominent phenylpropanoid derivatives present in the soluble extract behaved differently. Two kaempferol glycosides remained largely unaffected, and sinapoyl malate decreased strongly upon bacterial infection with a time course inversely correlated with that of the accumulating tryptophan-related products. The accumulation patterns of both soluble and wall-bound compounds, as well as the disease resistance phenotypes, were essentially the same for infected wild-type and tt4 (no kaempferol glycosides) or fah1 (no sinapoyl malate) mutant plants. Largely different product combinations accumulated in wounded or senescing A. thaliana leaves. It seems unlikely that any one of the infection-induced compounds identified so far has a decisive role in the resistance response to P. syringae.

Two differentially regulated class II chitinases from parsley.

Two distinct cDNA clones, PcCHI1 and PcCHI2, with high sequence similarity to plant chitinases were isolated from parsley (Petroselinum crispum), expressed in Escherichia coli, and the encoded proteins functionally identified as endochitinases. Different expression patterns of the corresponding mRNAs and proteins in infected and uninfected parsley plants indicated distinct roles of the two isoforms in both pathogen defense and plant development. Infection of parsley leaf buds with Phytophthora sojae resulted in the rapid, transient and highly localized accumulation of PcCHI1 mRNA and protein around infection sites, whereas PcCHI2 mRNA and protein were systemically induced at later infection stages. Similar differences in the timing of induction were observed in elicitor-treated, suspension-cultured parsley cells. In uninfected plants, PcCHI1 mRNA was particularly abundant in the transmitting tract of healthy flowers, suggesting a role in the constitutive protection of susceptible transmitting tissue of the style against pathogen ingress and/or in the fertilization process, possibly by affecting pollen tube growth. Localization of PcCHI2 mRNA and protein in the parenchymatic collenchyme of young pedicels may indicate a function in the constitutive protection of this tissue. In addition to such distinct roles of PcCHI1 and PcCHI2 in preformed and induced pathogen defense, both chitinases may have endogenous regulatory functions in plant development.

UV light selectively coinduces supply pathways from primary metabolism and flavonoid secondary product formation in parsley.

The UV light-induced synthesis of UV-protective flavonoids diverts substantial amounts of substrates from primary metabolism into secondary product formation and thus causes major perturbations of the cellular homeostasis. Results from this study show that the mRNAs encoding representative enzymes from various supply pathways are coinduced in UV-irradiated parsley cells (Petroselinum crispum) with two mRNAs of flavonoid glycoside biosynthesis, encoding phenylalanine ammonia-lyase and chalcone synthase. Strong induction was observed for mRNAs encoding glucose 6-phosphate dehydrogenase (carbohydrate metabolism, providing substrates for the shikimate pathway), 3-deoxyarabinoheptulosonate 7-phosphate synthase (shikimate pathway, yielding phenylalanine), and acyl-CoA oxidase (fatty acid degradation, yielding acetyl-CoA), and moderate induction for an mRNA encoding S-adenosyl-homocysteine hydrolase (activated methyl cycle, yielding S-adenosyl-methionine for B-ring methylation). Ten arbitrarily selected mRNAs representing various unrelated metabolic activities remained unaffected. Comparative analysis of acyl-CoA oxidase and chalcone synthase with respect to mRNA expression modes and gene promoter structure and function revealed close similarities. These results indicate a fine-tuned regulatory network integrating those functionally related pathways of primary and secondary metabolism that are specifically required for protective adaptation to UV irradiation. Although the response of parsley cells to UV light is considerably broader than previously assumed, it contrasts greatly with the extensive metabolic reprogramming observed previously in elicitor-treated or fungus-infected cells.

Mutational analysis of 4-coumarate:CoA ligase identifies functionally important amino acids and verifies its close relationship to other adenylate-forming enzymes.

4-Coumarate:coenzyme A ligase (4CL) is a key enzyme of general phenylpropanoid metabolism which provides the precursors for a large variety of important plant secondary products, such as lignin, flavonoids, or phytoalexins. To identify amino acids important for 4CL activity, eight mutations were introduced into Arabidopsis thaliana At4CL2. Determination of specific activities and K(m) values for ATP and caffeate of the heterologously expressed and purified proteins identified four distinct classes of mutants: enzymes with little or no catalytic activity; enzymes with greatly reduced activity but wild-type K(m) values; enzymes with drastically altered K(m) values; and enzymes with almost wild-type properties. The latter class includes replacement of a cysteine residue which is strictly conserved in 4CLs and had previously been assumed to be directly involved in catalysis. These results substantiate the close relationship between 4CL and other adenylate-forming enzymes such as luciferases, peptide synthetases, and fatty acyl-CoA synthetases.

A novel regulatory element involved in rapid activation of parsley ELI7 gene family members by fungal elicitor or pathogen infection.

Abstract In parsley (Petroselinum crispum), members of the ELI7 gene family were rapidly transcriptionally activated following treatment with an elicitor derived from the phytopathogen Phytophthora sojae. Several cDNA and genomic ELI7 clones were isolated. The deduced amino acid sequences revealed close similarity to fatty acid desaturases/hydroxylases, however, the precise functions are still unknown. Analysis of the promoters of two strongly elicitor-induced family members, ELI7.1 and ELI7.2, allowed us to functionally pinpoint a novel, independently acting regulatory region (S box), the only major sequence similarity between the two gene promoters. In situ RNA/RNA hybridization using an ELI7.1 gene-specific probe demonstrated that expression of this gene is rapidly and locally induced around infection sites in planta as well.

Early nuclear events in plant defence signalling: rapid gene activation by WRKY transcription factors.

Parsley WRKY proteins comprise a family of plant-specific zinc-finger-type factors implicated in the regulation of genes associated with pathogen defence. In vitro, these proteins bind specifically to functionally defined TGAC-containing W box promoter elements within the Pathogenesis-Related Class10 (PR-10) genes. Here we present in vivo data demonstrating that WRKY1 is a transcriptional activator mediating fungal elicitor-induced gene expression by binding to W box elements. In situ RNA hybridization revealed that the WRKY1 gene is rapidly and locally activated in parsley leaf tissue around fungal infection sites. Transient expression studies in parsley protoplasts showed that a specific arrangement of W box elements in the WRKY1 promoter itself is necessary and sufficient for early activation and that WRKY1 binds to such elements. Our results demonstrate that WRKY transcription factors play an important role in the regulation of early defence-response genes including regulation of WRKY1.

Regulation and functional expression of cinnamate 4-hydroxylase from parsley.

A previously isolated parsley (Petroselinum crispum) cDNA with high sequence similarity to cinnamate 4-hydroxylase (C4H) cDNAs from several plant sources was expressed in yeast (Saccharomyces cerevisiae) containing a plant NADPH:cytochrome P450 oxidoreductase and verified as encoding a functional C4H (CYP73A10). Low genomic complexity and the occurrence of a single type of cDNA suggest the existence of only one C4H gene in parsley. The encoded mRNA and protein, in contrast to those of a functionally related NADPH:cytochrome P450 oxidoreductase, were strictly coregulated with phenylalanine ammonia-lyase mRNA and protein, respectively, as demonstrated by coinduction under various conditions and colocalization in situ in cross-sections from several different parsley tissues. These results support the hypothesis that the genes encoding the core reactions of phenylpropanoid metabolism form a tight regulatory unit.

Extensive reprogramming of primary and secondary metabolism by fungal elicitor or infection in parsley cells.

The transcription rates of numerous plant genes have previously been shown to be strongly affected by pathogen infection or elicitor treatment. Here we estimate the extent and complexity of this response by analyzing the patterns of mRNA induction in fungal elicitor-treated parsley cells (Petroselinum crispum) for several representatives from various primary and secondary metabolic pathways, cytosolic as well as plastidic. As a reference, we use the biphasic accumulation curve for the coordinately induced mRNAs encoding the three core enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase, cinnamate 4-hydroxylase and 4-coumarate:CoA ligase. Coincidence with this curve was observed for the mRNA induction kinetics of several, but not all, phenylpropanoid branch pathway-related reactions, whereas seven selected mRNAs from the pentose phosphate, glycolytic and shikimate pathways, including various cytosolic and plastidic isoforms, were induced with great differences in timing. Likewise unique and dissimilar from the reference curve were the induction patterns for various mRNAs encoding enzymes or proteins that are either more distantly or not at all related to phenylpropanoid metabolism. None of over 40 mRNAs tested so far remained unaffected. Using one strongly elicitor-responsive mRNA from carbohydrate metabolism, encoding a cytosolic glucose 6-phosphate dehydrogenase, for in situ RNA/RNA hybridization in fungus-infected parsley leaf tissue, we observed again the previously reported, close simulation of metabolic changes in true plant/fungus interactions by elicitor treatment of cultured cells. In addition to demonstrating extensive, highly complex functional, temporal and spatial patterns of changes in gene expression in infected plant cells, these results provide valuable information for the identification of pathogen-responsive promoters suitable for gene technology-assisted resistance breeding.

Knock-out mutants from an En-1 mutagenized Arabidopsis thaliana population generate phenylpropanoid biosynthesis phenotypes.

A collection of 8,000 Arabidopsis thaliana plants carrying 48,000 insertions of the maize transposable element En-1 has been generated. This population was used for reverse genetic analyses to identify insertions in individual gene loci. By using a PCR-based screening protocol, insertions were found in 55 genes. En-1 showed no preference for transcribed or untranscribed regions nor for a particular orientation relative to the gene of interest. In several cases, En-1 was inserted within a few kilobases upstream or downstream of the gene. En-1 was mobilized from such positions into the respective gene to cause gene disruption. Knock-out alleles of genes involved in flavonoid biosynthesis were generated. One mutant line contained an En-1 insertion in the flavonol synthase gene (FLS) and showed drastically reduced levels of kaempferol. Allelism tests with other lines containing En-1 insertions in the flavanone 3-hydroxylase gene (F3H) demonstrated that TRANSPARENT TESTA 6 (TT6) encodes flavanone 3-hydroxylase. The f3h and fls null mutants complete the set of A. thaliana lines defective in early steps of the flavonoid pathway. These experiments demonstrate the efficiency of the screening method and gene disruption strategy used for assigning functions to genes defined only by sequence.

Isolation of putative plant transcriptional coactivators using a modified two-hybrid system incorporating a GFP reporter gene.

Dual hybrid interacting screening in yeast led to the identification of two proteins from Arabidopsis both exhibiting sequence similarity to a family of transcriptional coactivators from a diverse range of organisms. Their discovery constitutes the first description of such plant proteins. A modified yeast two-hybrid approach utilising the green fluorescent protein (GFP) of Aequora victoria was developed and used to clone one of the putative plant transcriptional coactivators from an Arabidopsis cDNA library. The two proteins, designated KIWI and KELP, can associate both hetero- and homomerically and their genes were cloned and mapped on the Arabidopsis genome. Both proteins are believed to play a role in gene activation during pathogen defence and plant development. The involvement of these proteins in general plant transcription as well as the advantages of using GFP as a reporter gene for detecting protein-protein interactions are discussed.

Local mechanical stimulation induces components of the pathogen defense response in parsley.

Cell suspension cultures of parsley (Petroselinum crispum) have previously been used as a suitable system for studies of the nonhost resistance response to Phytophthora sojae. In this study, we replaced the penetrating fungus by local mechanical stimulation by using a needle of the same diameter as a fungal hypha, by local application of a structurally defined fungus-derived elicitor, or by a combination of the two stimuli. Similar to the fungal infection hypha, the local mechanical stimulus alone induced the translocation of cytoplasm and nucleus to the site of stimulation, the generation of intracellular reactive oxygen intermediates (ROI), and the expression of some, but not all, elicitor-responsive genes. When the elicitor was applied locally to the cell surface without mechanical stimulation, intracellular ROI also accumulated rapidly, but morphological changes were not detected. A combination of the mechanical stimulus with simultaneous application of low doses of elicitor closely simulated early reactions to fungal infection, including cytoplasmic aggregation, nuclear migration, and ROI accumulation. By contrast, cytoplasmic rearrangements were impaired at high elicitor concentrations. Neither papilla formation nor hypersensitive cell death occurred under the conditions tested. These results suggest that mechanical stimulation by the invading fungus is responsible for the observed intracellular rearrangements and may trigger some of the previously demonstrated changes in the activity of elicitor-responsive genes, whereas chemical stimulation is required for additional biochemical processes. As yet unidentified signals may be involved in papilla formation and hypersensitive cell death.

Rapid and transient induction of a parsley microsomal delta 12 fatty acid desaturase mRNA by fungal elicitor.

Treatment of cultured parsley (Petroselinum crispum L.) cells with a structurally defined peptide elicitor (Pep25) of fungal origin has previously been shown to cause rapid and large changes in the levels of various desaturated fatty acids. We isolated two distinct parsley cDNAs sharing high sequence similarity with microsomal omega-6 fatty acid desaturases (FADs). One of them was functionally identified as a delta 12 FAD by expression in the yeast Saccharomyces cerevisiae. Two dienoic fatty acids, hexadecadienoic and linoleic, which were not detectable in control cells, together constituted up to 12% of the total fatty acids in the transformed yeast cells. delta 12 FAD mRNA accumulated rapidly and transiently in elicitor-treated parsley cells, protoplasts, and leaves. These and previous results indicate that fatty acid desaturation is an important early component of the complex defense response of parsley to attempted fungal infection.

A novel type of pathogen defense-related cinnamyl alcohol dehydrogenase.

We describe an aromatic alcohol dehydrogenase with properties indicating a novel type of function in the defense response of plants to pathogens. To obtain the enzyme free of contamination with possible isoforms, a parsley (Petroselinum crispum) cDNA comprising the entire coding region of the elicitor-responsive gene, ELI3, was expressed in Escherichia coli. In accord with large amino acid sequence similarities with established cinnamyl and benzyl alcohol dehydrogenases from other plants, the enzyme efficiently reduced various cinnamyl and benzyl aldehydes using NADPH as a co-substrate. Highest substrate affinities were observed for cinnamaldehyde, 4-coumaraldehyde and coniferaldehyde, whereas sinapaldehyde, one of the most efficient substrates of several previously analyzed cinnamyl alcohol dehydrogenases and a characteristic precursor molecule of angiosperm lignin, was not converted. A single form of ELI3 mRNA was strongly and rapidly induced in fungal elicitor-treated parsley cells. These results, together with earlier findings that the ELI3 gene is strongly activated both in elicitor-treated parsley cells and at fungal infection sites in parsley leaves, but not in lignifying tissue, suggest a specific role of this enzyme in pathogen defense-related phenylpropanoid metabolism.

PcMYB1, a novel plant protein containing a DNA-binding domain with one MYB repeat, interacts in vivo with a light-regulatory promoter unit.

Light regulatory unit 1 (LRU1) is necessary for and sufficient to mediate light-dependent activation of the chalcone synthase (CHS) minimal promoter in Petroselinum crispum. This composite promoter unit consists of at least two distinct cis-acting elements, designated ACECHS and MRECHS, both of which are required for light induction. The ACGT-containing element ACECHS interacts with common plant regulatory factors (CPRFs) which belong to the basic region/leucine zipper (bZIP) class of transcription factors. Here, we demonstrate that MRECHS, originally identified as an in vivo DNA footprint, is a MYB recognition element. This element possesses a functional core that is essential for light responsiveness and is specifically recognized by two distantly related MYB-like proteins: MYB305 and the novel factor MYB1 from P. crispum. PcMYB1 was identified by both its specific binding to MRECHS in vitro and recognition of MRECHS in vivo. The deduced amino acid sequence revealed that PcMYB1 contains only one MYB-like repeat. This portion of the protein constitutes the DNA-binding domain. Mutational analysis of PcMYB1 in combination with sequence comparison suggests the presence of a helix-turn-helix structure containing a recognition helix that is sufficient for sequence-specific binding. The structure of this distinct MYB-like DNA-binding domain appears to be conserved in proteins from all three eukaryotic phyla.

Elicitor-stimulated ion fluxes and O2- from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley.

Fungal elicitor stimulates a multicomponent defense response in cultured parsley cells (Petroselinum crispum). Early elements of this receptor-mediated response are ion fluxes across the plasma membrane and the production of reactive oxygen species (ROS), sequentially followed by defense gene activation and phytoalexin accumulation. Omission of Ca2+ from the culture medium or inhibition of elicitor-stimulated ion fluxes by ion channel blockers prevented the latter three reactions, all of which were triggered in the absence of elicitor by amphotericin B-induced ion fluxes. Inhibition of elicitor-stimulated ROS production using diphenylene iodonium blocked defense gene activation and phytoalexin accumulation. O2- but not H2O2 stimulated phytoalexin accumulation, without inducing proton fluxes. These results demonstrate a causal relationship between early and late reactions of parsley cells to the elicitor and indicate a sequence of signaling events from receptor-mediated activation of ion channels via ROS production and defense gene activation to phytoalexin synthesis. Within this sequence, O2- rather than H2O2 appears to trigger the subsequent reactions.

Rapid, transient, and highly localized induction of plastidial omega-3 fatty acid desaturase mRNA at fungal infection sites in Petroselinum crispum.

Parsley (Petroselinum crispum) plants and suspension-cultured cells have been used extensively for studies of non-host-resistance mechanisms in plant/pathogen interactions. We now show that treatment of cultured parsley cells with a defined peptide elicitor of fungal origin causes rapid and large changes in the levels of various unsaturated fatty acids. While linoleic acid decreased and linolenic acid increased steadily for several hours, comparatively sharp increases in oleic acid followed a biphasic time course. In contrast, the overall level of stearic acid remained unaffected. Using a PCR-based approach, a parsley cDNA was isolated sharing high sequence similarity with omega-3 fatty acid desaturases. Subsequent isolation and characterization of a full-length cDNA enabled its functional identification as a plastid-localized omega-3 fatty acid desaturase by complementation of the Arabidopsis thaliana fad7/8 double mutant which is low in trienoic fatty acids. omega-3 Fatty acid desaturase mRNA accumulated rapidly and transiently in elicitor-treated cultured parsley cells, protoplasts, and leaves, as well as highly localized around fungal infection sites in parsley leaf buds. These results indicate that unsaturated fatty acid metabolism is yet another component of the highly complex, transcriptionally regulated pathogen defense response in plants.

Signal perception and intracellular signal transduction in plant pathogen defense.

Disease resistance in plant/pathogen interactions requires sensitive and specific recognition mechanisms for pathogen-derived signals in plants. Cultured parsley (Petroselinum crispum) cells respond to treatment with a crude cell wall preparation derived from the phytopathogenic fungus Phytophthora sojae with transcriptional activation of the same set of defense-related genes as are activated in parsley leaves upon infection with fungal spores. A 13 amino acid core sequence (Pep-13) of a 42 kDa fungal cell wall glycoprotein was identified, which stimulates the same responses as the crude cell wall elicitor, namely macroscopic Ca2+ and H(+)-influxes, effluxes of K(+)- and Cl- ions, production of active oxygen species (oxidative burst), defense-related gene activation, and formation of antifungal phytoalexins. Using [125I]Tyr-Pep-13 as ligand in binding assays, a single-class high-affinity binding site in parsley microsomal membranes and protoplasts could be detected. Binding was specific, saturable, and reversible. By chemical crosslinking, a 91 kDa parsley plasma membrane protein was identified to be the receptor of the peptide elicitor. Isolation of this receptor protein involved in pathogen defense in plants is under way.

Differentially regulated NADPH:cytochrome P450 oxidoreductases in parsley.

Two NADPH:cytochrome P450 oxidoreductases (CPRs) from parsley (Petroselinum crispum) were cloned, and the complete proteins were expressed and functionally identified in yeast. The two enzymes, designated CPR1 and CPR2, are 80% identical in amino acid sequence with one another and about 75% identical with CPRs from several other plant species. The mRNA accumulation patterns for CPR1 and CPR2 in fungal elicitor-treated or UV-irradiated cultured parsley cells and in developing or infected parsley plants were compared with those for cinnamate 4-hydroxylase (C4H), one of the most abundant CPR-dependent P450 enzymes in plants. All treatments strongly induced the mRNAs for C4H and CPR1 but not for CPR2, suggesting distinct metabolic roles of CPR1 and CPR2 and a functional relationship between CPR1 and C4H.

Arabidopsis thaliana defense-related protein ELI3 is an aromatic alcohol:NADP+ oxidoreductase.

We expressed a cDNA encoding the Arabidopsis thaliana defense-related protein ELI3-2 in Escherichia coli to determine its biochemical function. Based on a protein database search, this protein was recently predicted to be a mannitol dehydrogenase [Williamson, J. D., Stoop, J. M. H., Massel, M. O., Conkling, M. A. & Pharr, D. M. (1995) Proc. Natl. Acad. Sci. USA 92, 7148-7152]. Studies on the substrate specificity now revealed that ELI3-2 is an aromatic alcohol: NADP+ oxidoreductase (benzyl alcohol dehydrogenase). The enzyme showed a strong preference for various aromatic aldehydes as opposed to the corresponding alcohols. Highest substrate affinities were observed for 2-methoxybenzaldehyde, 3-methoxybenzaldehyde, salicylaldehyde, and benzaldehyde, in this order, whereas mannitol dehydrogenase activity could not be detected. These and previous results support the notion that ELI3-2 has an important role in resistance-related aromatic acid-derived metabolism.