An E. coli biosensor for screening of cDNA libraries for isochorismate pyruvate lyase-encoding cDNAs.

Salicylic acid (SA) is an essential hormone for development and induced defense against biotrophic pathogens in plants. The formation of SA mainly derives from chorismate via demonstrated isochorismate synthase (ICS) and presumed isochorismate pyruvate lyase (IPL)-mediated steps in Arabidopsis thaliana, but so far no plant enzyme displaying IPL activity has been identified. Here, we developed an E. coli SA biosensor to screen for IPL activity based on the SalR regulator/salA promoter combination from Acinetobacter sp ADP1, to control the expression of the reporter luxCDABE. The biosensor was responsive to micromolar concentrations of exogenous SA, and to endogenous SA produced after transformation with a plasmid permitting IPTG-inducible expression of bacterial IPL in this biosensor strain. After screening a cDNA library constructed from turnip crinkle virus (TCV)-infected Arabidopsis ecotype Di-17, we identified an enzyme, PRXR1, as a putative IPL that converts isochorismate into SA. Our results provide a new experimental approach to identify IPL and new insights into the SA biosynthesis pathway in Arabidopsis.

Overexpression of AtWRKY50 is correlated with enhanced production of sinapic derivatives in Arabidopsis.

INTRODUCTION: WRKY proteins belong to a plant-specific class of transcription factors. Seventy-four WKRY genes have been identified in Arabidopsis and many WRKY proteins are known to be involved in responses to stress, especially to biotic stress. They may act either as transcriptional activators or as repressors of genes that play roles in the stress response. A number of studies have proposed the connection of Arabidopsis WRKY transcription factors in induced pathogenesis-related (PR) gene expression, although no direct evidence has been presented for specific WRKY-PR promoter interactions. OBJECTIVE: We previously identified AtWRKY50 as a transcriptional activator of SAR gene PR1. Although PR1 accumulates to high levels in plants after attack by pathogens, its function is still elusive. Here we investigated the effects of overexpression of several WRKY proteins, including AtWRKY50, on the metabolome of Arabidopsis thaliana. METHODS: The influence of overexpression of WRKY proteins on the metabolites of Arabidopsis was investigated by using an NMR spectroscopy-based metabolomic approach. The 1H NMR data was analysed using the multivariate data analysis methods, such as principal component analysis, hierarchical cluster analysis and partial least square-discriminant analysis. RESULTS: The results showed that the metabolome of transgenic Arabidopsis seedlings overexpressing AtWRKY50 was different from wild type Arabidopsis and transgenic Arabidopsis overexpressing other WRKY genes. Amongst other metabolites, sinapic acid and 1-O-sinapoyl-ß-D-glucose especially appeared to be the most prominent discriminating metabolites, accumulating to levels 2 to 3 times higher in the AtWRKY50 overexpressor lines. CONCLUSION: Our results indicate a possible involvement of AtWRKY50 in secondary metabolite production in Arabidopsis, in particular of hydroxycinnamates such as sinapic acid and 1-O-sinapoyl-ß-D-glucose.

Prospecting for genes involved in transcriptional regulation of plant defenses, a bioinformatics approach.

BACKGROUND: In order to comprehend the mechanisms of induced plant defense, knowledge of the biosynthesis and signaling pathways mediated by salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) is essential. Potentially, many transcription factors could be involved in the regulation of these pathways, although finding them is a difficult endeavor. Here we report the use of publicly available Arabidopsis microarray datasets to generate gene co-expression networks. RESULTS: Using 372 publicly available microarray data sets, a network was constructed in which Arabidopsis genes for known components of SA, JA and ET pathways together with the genes of over 1400 transcription factors were assayed for co-expression. After determining the Pearson Correlation Coefficient cutoff to obtain the most probable biologically relevant co-expressed genes, the resulting network confirmed the presence of many genes previously reported in literature to be relevant for stress responses and connections that fit current models of stress gene regulation, indicating the potential of our approach. In addition, the derived network suggested new candidate genes and associations that are potentially interesting for future research to further unravel their involvement in responses to stress. CONCLUSIONS: In this study large sets of stress related microarrays were used to reveal co-expression networks of transcription factors and signaling pathway components. These networks will benefit further characterization of the signal transduction pathways involved in plant defense.

WRKY transcription factors involved in activation of SA biosynthesis genes.

BACKGROUND: Increased defense against a variety of pathogens in plants is achieved through activation of a mechanism known as systemic acquired resistance (SAR). The broad-spectrum resistance brought about by SAR is mediated through salicylic acid (SA). An important step in SA biosynthesis in Arabidopsis is the conversion of chorismate to isochorismate through the action of isochorismate synthase, encoded by the ICS1 gene. Also AVRPPHB SUSCEPTIBLE 3 (PBS3) plays an important role in SA metabolism, as pbs3 mutants accumulate drastically reduced levels of SA-glucoside, a putative storage form of SA. Bioinformatics analysis previously performed by us identified WRKY28 and WRKY46 as possible regulators of ICS1 and PBS3. RESULTS: Expression studies with ICS1 promoter::ß-glucuronidase (GUS) genes in Arabidopsis thaliana protoplasts cotransfected with 35S::WRKY28 showed that over expression of WRKY28 resulted in a strong increase in GUS expression. Moreover, qRT-PCR analyses indicated that the endogenous ICS1 and PBS3 genes were highly expressed in protoplasts overexpressing WRKY28 or WRKY46, respectively. Electrophoretic mobility shift assays indentified potential WRKY28 binding sites in the ICS1 promoter, positioned -445 and -460 base pairs upstream of the transcription start site. Mutation of these sites in protoplast transactivation assays showed that these binding sites are functionally important for activation of the ICS1 promoter. Chromatin immunoprecipitation assays with haemagglutinin-epitope-tagged WRKY28 showed that the region of the ICS1 promoter containing the binding sites at -445 and -460 was highly enriched in the immunoprecipitated DNA. CONCLUSIONS: The results obtained here confirm results from our multiple microarray co-expression analyses indicating that WRKY28 and WRKY46 are transcriptional activators of ICS1 and PBS3, respectively, and support this in silico screening as a powerful tool for identifying new components of stress signaling pathways.

Effect of benzothiadiazole on the metabolome of Arabidopsis thaliana.

Benzothiadiazole (BTH) is a functional analog of the plant endogenous hormone-like compound, salicylic acid (SA), which is required for the induction of plant defense genes leading to systemic acquired resistance (SAR). Previous molecular and genetic studies have suggested that BTH itself might potentiate SAR resulting in the induction of several pathogenesis-related (PR) genes. However, the changes in the metabolome, which occur as a result of BTH-treatment, remain unclear. In this study, metabolic alterations in BTH-treated Arabidopsis thaliana were investigated using nuclear magnetic resonance (NMR) spectroscopy followed by multivariate data analyses such as principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA). Both PCA and PLS-DA show that increase of glucose, glutamine, inositol, malic acid, sucrose, and threonine as well as BTH and its degraded metabolites contribute to the clear discrimination of the metabolome of BTH-treated Arabidopsis from control plants. However, the levels of phenolic metabolites, which have generally been observed to be induced by other signaling molecules were significantly reduced in BTH-treated Arabidopsis. In addition to these changes due to BTH-treatment, it was also found that the EtOH used as a solvent in this treatment may per se act as an inducer of the accumulation of a flavonoid.

Ethylene as a modulator of disease resistance in plants.

The role of ethylene in the hormonal regulation of plant development has been well established. In addition, it has been implicated in biotic stress, both as a virulence factor of fungal and bacterial pathogens and as a signaling compound in disease resistance. This apparent discrepancy has stimulated research on the effects of various types of pathogens on mutant and transgenic plants that are impaired in ethylene production or perception. It has become clear that ethylene differentially affects resistance against pathogens with different lifestyles and plays an important role in mediating different types of induced resistance.

Arabidopsis WRKY50 and TGA Transcription Factors Synergistically Activate Expression of PR1.

Arabidopsis PR1 is a salicylic acid (SA) inducible marker gene for systemic acquired resistance (SAR). However, the regulation of PR1 in plants is poorly understood. In this study, we showed that AtWRKY50 transcription factor binds to two promoter elements of PR1 via its DNA binding domain. Interestingly, the DNA-binding sites for AtWRKY50 deviate significantly from the consensus WRKY binding W-box. The binding sites are located in close proximity to the binding sites for TGA transcription factors. Transactivation experiments in Arabidopsis protoplasts derived from wild type, npr1-1 and tga256 mutant plants indicated that AtWRKY50 alone was able to induce expression of a PR1::ß-glucuronidase (GUS) reporter gene, independent of TGAs or NPR1. However, co-expression of TGA2 or TGA5 with AtWRKY50 synergistically enhanced expression to high levels. Yeast-2-hybrid assays and bimolecular fluorescence complementation (BiFC) experiments revealed that AtWRKY50 could interact with TGA2 and TGA5. Using electrophoretic mobility shift assays (EMSA) it was established that AtWRKY50 and TGA2 or TGA5 simultaneously bind to the PR1 promoter. Taken together, these results support a role of AtWRKY50 in SA-induced expression of PR1. Highlights: AtWRKY50 specifically binds to LS10 region of PR1 promoter and interacts with TGAs to synergistically activate PR1 expression.

Vitamin K1 accumulation in tobacco plants overexpressing bacterial genes involved in the biosynthesis of salicylic acid.

Phylloquinone (Vitamin K(1)) is an essential component of the photosynthetic electron transfer. As isochorismate is required for the biosynthesis of Vitamin K(1), isochorismate synthase (ICS) activity is expected to be present in all green plants. In bacteria salicylic acid (SA) is synthesized via a two step pathway involving ICS and isochorismate pyruvate lyase (IPL). The effect of the introduction in tobacco plants of the bacterial ICS and IPL genes on the endogenous isochorismate pathway was investigated. Transgenic tobacco plants in which IPL was targeted to the chloroplast suffered severe growth retardation and had low Vitamin K(1) content. Probably because isochorismate was channeled towards SA production, the plants were no longer able to produce normal levels of Vitamin K(1). Transgenic tobacco plants in which the bacterial ICS was present in the chloroplast showed higher Vitamin K(1) contents than wild type plants.

Metabolomic analysis of methyl jasmonate treated Brassica rapa leaves by 2-dimensional NMR spectroscopy.

The metabolomic analysis of Brassica rapa leaves treated with methyl jasmonate was performed using 2-dimensional J-resolved NMR spectroscopy combined with multivariate data analysis. The principal component analysis of the J-resolved NMR spectra showed discrimination between control and methyl jasmonate treated plants by principal components 1 and 2. While the level of glucose, sucrose and amino acids showed a decrease after methyl jasmonate treatment, hydroxycinnamates and glucosinolate were highly increased. Methyl jasmonate treatment resulted in a long-term accumulation of indole glucosinolate and indole-3-acetic acid, lasting up to 14 days after treatment. Malate conjugated hydroxycinnamates also exhibited an increase until 14 days after methyl jasmonate treatment, these compounds might play an important role in plant defence responses mediated by methyl jasmonate.

Cloning and over-expression of a cDNA encoding a polyketide synthase from Cannabis sativa.

A polyketide synthase has been suggested to play an important role in cannabinoid biosynthesis in Cannabis sativa L. This enzyme catalyzes the biosynthesis of olivetolic acid, one of the precursors for cannabinoid biosynthesis. Using a reverse transcriptase-polymerase chain reaction (RT-PCR) based on the DNA homology of chalcone synthase (EC 2.3.1.156) and valerophenone synthase (EC 2.3.1.156) of hop (Humulus lupulus), a cDNA encoding a polyketide synthase in C. sativa was identified. The coding region of the gene is 1170 bp long encoding a 389 amino acid protein of a predicted 42.7 kDa molecular mass and with a pI of 6.04. The gene shares a high homology with a chalcone synthase gene of H. lupulus, 85% and 94% homology on the level of DNA and protein, respectively. Over-expression of the construct in Escherichia coli M15 resulted in a 45 kDa protein. The protein has chalcone synthase activity as well as valerophenone synthase activity, a chalcone synthase-like activity. Using n-hexanoyl-CoA and malonyl-CoA as substrates did not give olivetol or olivetolic acid as a product.

Tobacco Transcription Factor NtWRKY12 Interacts with TGA2.2 in vitro and in vivo.

The promoter of the salicylic acid-inducible PR-1a gene of Nicotiana tabacum contains binding sites for transcription factor NtWRKY12 (WK-box at position -564) and TGA factors (as-1-like element at position -592). Transactivation experiments in Arabidopsis protoplasts derived from wild type, npr1-1, tga256, and tga2356 mutant plants revealed that NtWRKY12 alone was able to induce a PR-1a::ß-glucuronidase (GUS) reporter gene to high levels, independent of co-expressed tobacco NtNPR1, TGA2.1, TGA2.2, or endogenous Arabidopsis NPR1, TGA2/3/5/6. By in vitro pull-down assays with GST and Strep fusion proteins and by Fluorescence Resonance Energy Transfer assays with protein-CFP and protein-YFP fusions in transfected protoplasts, it was shown that NtWRKY12 and TGA2.2 could interact in vitro and in vivo. Interaction of NtWRKY12 with TGA1a or TGA2.1 was not detectable by these techniques. A possible mechanism for the role of NtWRKY12 and TGA2.2 in PR-1a gene expression is discussed.

In silicio expression analysis of PKS genes isolated from Cannabis sativa L.

Cannabinoids, flavonoids, and stilbenoids have been identified in the annual dioecious plant Cannabis sativa L. Of these, the cannabinoids are the best known group of this plant's natural products. Polyketide synthases (PKSs) are responsible for the biosynthesis of diverse secondary metabolites, including flavonoids and stilbenoids. Biosynthetically, the cannabinoids are polyketide substituted with terpenoid moiety. Using an RT-PCR homology search, PKS cDNAs were isolated from cannabis plants. The deduced amino acid sequences showed 51%-73% identity to other CHS/STS type sequences of the PKS family. Further, phylogenetic analysis revealed that these PKS cDNAs grouped with other non-chalcone-producing PKSs. Homology modeling analysis of these cannabis PKSs predicts a 3D overall fold, similar to alfalfa CHS2, with small steric differences on the residues that shape the active site of the cannabis PKSs.

Metabolic changes of Brassica rapa transformed with a bacterial isochorismate synthase gene.

Metabolome analysis by 1-dimensional proton nuclear magnetic resonance (¹H NMR) coupled with multivariate data analysis was carried out in Brassica rapa plants transformed with a gene encoding bacterial isochorismate synthase (ICS). Partial least square-discrimination analysis (PLS-DA) on selected signals suggested that the resonances that were dominant in the transgenic plants corresponded to a glucosinolate (neoglucobrassicin), phenylpropanoids (sinapoyl malate, feruloyl malate, caffeoyl malate), organic acids (succinic acid and fumaric acid) and sugars (α- and ß-glucose). In contrast, amino acids alanine threonine, valine, leucine were dominant in the untransformed controls. In addition, HPLC data showed that the transgenic plant accumulated salicylic acid (SA) at significantly higher levels than the control plants, whereas the phylloquinone levels were not affected. The results suggest that the expression of the bacterial isochorismate synthase gene in B. rapa does not affect fluxes into pathways to other groups of secondary metabolites through competition for the same precursor. On the contrary, the biosynthesis of isochorismate-derived products (SA) seems to induce the competitive pathways via phenylalanine (phenylpropanoids) and tryptophan (IAA and indole glucosinolates).

A Novel WRKY transcription factor is required for induction of PR-1a gene expression by salicylic acid and bacterial elicitors.

PR-1a is a salicylic acid-inducible defense gene of tobacco (Nicotiana tabacum). One-hybrid screens identified a novel tobacco WRKY transcription factor (NtWRKY12) with specific binding sites in the PR-1a promoter at positions -564 (box WK(1)) and -859 (box WK(2)). NtWRKY12 belongs to the class of transcription factors in which the WRKY sequence is followed by a GKK rather than a GQK sequence. The binding sequence of NtWRKY12 (WK box TTTTCCAC) deviated significantly from the consensus sequence (W box TTGAC[C/T]) shown to be recognized by WRKY factors with the GQK sequence. Mutation of the GKK sequence in NtWRKY12 into GQK or GEK abolished binding to the WK box. The WK(1) box is in close proximity to binding sites in the PR-1a promoter for transcription factors TGA1a (as-1 box) and Myb1 (MBSII box). Expression studies with PR-1a promoterbeta-glucuronidase (GUS) genes in stably and transiently transformed tobacco indicated that NtWRKY12 and TGA1a act synergistically in PR-1a expression induced by salicylic acid and bacterial elicitors. Cotransfection of Arabidopsis thaliana protoplasts with 35SNtWRKY12 and PR-1aGUS promoter fusions showed that overexpression of NtWRKY12 resulted in a strong increase in GUS expression, which required functional WK boxes in the PR-1a promoter.

NMR metabolomics to revisit the tobacco mosaic virus infection in Nicotiana tabacum leaves.

Tobacco mosaic virus (TMV) infection of tobacco is a well-known and extensively studied model system for which a number of genes and proteins involved in the systemic acquired resistance (SAR) have been characterized. Little is known about the metabolic changes connected with the infection and SAR. Here we describe the use of NMR spectroscopy in combination with multivariate data analysis to study the metabolic changes. Particularly 2-D NMR methods, such as 2-D J-resolved spectra and their projected spectra, are shown to be powerful tools in the metabolomic studies. The macroscopic view of the metabolomes obtained by NMR spectroscopy of crude extracts enabled the identification of a series of totally different metabolites that seem connected with resistance, such as the clearly increased 5-caffeoylquinic acid, alpha-linolenic acid analogues, and sesqui- and diterpenoids in the infected plant parts.

In silicio expression analysis of PKS genes isolated from Cannabis sativa L.

Cannabinoids, flavonoids, and stilbenoids have been identified in the annual dioecious plant Cannabis sativa L. Of these, the cannabinoids are the best known group of this plant's natural products. Polyketide synthases (PKSs) are responsible for the biosynthesis of diverse secondary metabolites, including flavonoids and stilbenoids. Biosynthetically, the cannabinoids are polyketide substituted with terpenoid moiety. Using an RT-PCR homology search, PKS cDNAs were isolated from cannabis plants. The deduced amino acid sequences showed 51 percent-73 percent identity to other CHS/STS type sequences of the PKS family. Further, phylogenetic analysis revealed that these PKS cDNAs grouped with other non-chalcone-producing PKSs. Homology modeling analysis of these cannabis PKSs predicts a 3D overall fold, similar to alfalfa CHS2, with small steric differences on the residues that shape the active site of the cannabis PKSs.

Efficient translation of alfamovirus RNAs requires the binding of coat protein dimers to the 3' termini of the viral RNAs.

The coat protein (CP) of Alfalfa mosaic virus (AMV) is required to initiate infection by the viral tripartite RNA genome whereas infection by the tripartite Brome mosaic virus (BMV) genome is independent of CP. AMV CP stimulates translation of AMV RNA in vivo 50- to 100-fold. The 3' untranslated region (UTR) of the AMV subgenomic CP messenger RNA 4 contains at least two CP binding sites. A CP binding site in the 3'-terminal 112 nucleotides of RNA 4 was found to be required for efficient translation of the RNA whereas an upstream binding site was not. Binding of CP to the AMV 3' UTR induces a conformational change of the RNA but this change alone was not sufficient to stimulate translation. CP mutant R17A is unable to bind to the 3' UTR and translation in vivo of RNA 4 encoding this mutant occurs at undetectable levels. Replacement of the 3' UTR of this mutant RNA 4 by the 3' UTR of BMV RNA 4 restored translation of R17A-CP to wild-type levels. Apparently, the BMV 3' UTR stimulates translation independently of CP. AMV CP mutant N199 is defective in the formation of CP dimers and did not stimulate translation of RNA 4 in vivo although the mutant CP did bind to the 3' UTR. The finding that N199-CP does not promote AMV infection corroborates the notion that the requirement of CP in the inoculum reflects its role in translation of the viral RNAs.

Signaling of systemic acquired resistance in tobacco depends on ethylene perception.

The hypersensitive interaction between Tobacco mosaic virus (TMV) and tobacco results in accumulation of salicylic acid (SA), defense gene expression, and development of systemic acquired resistance (SAR) in uninfected leaves. The plant hormones SA and ethylene have been implicated in SAR. From a study with ethylene-insensitive (Tetr) tobacco, we concluded that ethylene perception is required to generate the systemic signal molecules in TMV-infected leaves that trigger SA accumulation, defense gene expression, and SAR development in uninfected leaves. Ethylene perception was not required for the responses of the plant to the systemic signal that leads to SAR development.

Method for the extraction of the volatile compound salicylic acid from tobacco leaf material.

Salicylic acid (SA) is a signalling compound in plants which is able to induce systemic acquired resistance. In the analysis of SA in plant tissues, the extraction recovery is often very low and variable. This is mainly caused by sublimation of SA, especially during evaporation of organic solvents. Techniques have been designed in order to overcome this problem. In the first part of the extraction procedure, sublimation of SA was prevented by addition of 0.2 M sodium hydroxide. At a later stage of the extraction procedure, sublimation of SA during solvent evaporation was controlled by the addition of a small amount of HPLC eluent. In this way, recoveries in the range of 71-91% for free SA and 65-79% for acid-hydrolysed SA were obtained. Recoveries could be further optimised by the use of an internal standard to correct for volume changes after the addition of the HPLC eluent.

Silencing of a gene encoding a protein component of the oxygen-evolving complex of photosystem II enhances virus replication in plants.

It has been suggested that, in addition to viral proteins, host proteins are involved in RNA virus replication. In this study the RNA helicase domain of the Tobacco mosaic virus (TMV) replicase proteins was used as bait in the yeast two-hybrid system to identify tobacco proteins with a putative role in TMV replication. Two host proteins were characterized. One protein (designated #3) belongs to a protein family of ATPases associated with various activities (AAA), while the second host protein (designated #13) is the 33K subunit of the oxygen-evolving complex of photosystem II. Using Tobacco rattle virus vectors, genes #3 and #13 were silenced in Nicotiana benthamiana, after which the plants were challenged by TMV infection. Silencing of gene #13 resulted in a 10-fold increase of TMV accumulation, whereas silencing of gene #3 caused a twofold reduction of TMV accumulation. Additionally, silencing of genes #3 and #13 decreased and increased, respectively, the accumulation of two other viruses. Similar to silencing of gene #13, inhibition of photosystem II by application of an herbicide increased TMV accumulation several fold. Infection of N. benthamiana with TMV resulted in a decrease of #13 mRNA levels. Silencing of gene #13 may reflect a novel strategy of TMV to suppress basal host defense mechanisms. The two-hybrid screenings did not identify tobacco proteins involved in helicase domain-induced N-mediated resistance.