WRKY Transcription Factors Involved in Activation of SA Biosynthesis Genes

<p>Abstract</p> <p>Background</p> <p>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 <it>ICS1 </it>gene. Also <it>AVR</it>_{<it>PPHB </it>}<it>SUSCEPTIBLE 3 </it>(<it>PBS3</it>) plays an important role in SA metabolism, as <it>pbs3 </it>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 <it>ICS1 </it>and <it>PBS3</it>.</p> <p>Results</p> <p>Expression studies with <it>ICS1 promoter::β-glucuronidase </it>(<it>GUS</it>) genes in <it>Arabidopsis thaliana </it>protoplasts cotransfected with <it>35S::WRKY28 </it>showed that over expression of WRKY28 resulted in a strong increase in GUS expression. Moreover, qRT-PCR analyses indicated that the endogenous <it>ICS1 </it>and <it>PBS3 </it>genes were highly expressed in protoplasts overexpressing WRKY28 or WRKY46, respectively. Electrophoretic mobility shift assays indentified potential WRKY28 binding sites in the <it>ICS1 </it>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 <it>ICS1 </it>promoter. Chromatin immunoprecipitation assays with haemagglutinin-epitope-tagged WRKY28 showed that the region of the <it>ICS1 </it>promoter containing the binding sites at -445 and -460 was highly enriched in the immunoprecipitated DNA.</p> <p>Conclusions</p> <p>The results obtained here confirm results from our multiple microarray co-expression analyses indicating that WRKY28 and WRKY46 are transcriptional activators of <it>ICS1 </it>and <it>PBS3</it>, respectively, and support this <it>in silico </it>screening as a powerful tool for identifying new components of stress signaling pathways.</p

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

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

Tobacco mosaic virus helicase domain induces necrosis in N gene-carrying tobacco in the absence of virus replication

Tobacco mosaic virus (TMV) elicits a hypersensitive response (HR) in tobacco plants that carry the N gene. To identify the elicitor of this HR, Agrobacteriumn tumefaciens was used as a vector for the transient expression of TMV replicase proteins, movement protein, and coat protein in NN and nn tobacco. Transient expression of the 126K protein and fragments thereof containing the helicase motifs induced necrosis and systemic expression of the pathogenesis-related PR-1a gene in NN plants but not in nn plants. The results confirm previous evidence that the TMV helicase sequence is the elicitor of the HR (H. S. Padgett, Y. Watanabe, and R. N. Beachy, Mol. Plant-Microbe Interact. 10:709-715, 1997) and demonstrate that this helicase sequence acts as an elicitor of HR in the absence of other viral proteins or RNA replication

A Novel WRKY Transcription Factor Is Required for Induction of PR-1a Gene Expression by Salicylic Acid and Bacterial Elicitors[C][W]

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 WK1) and −859 (box WK2). 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 WK1 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 promoter∷β-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 35S∷NtWRKY12 and PR-1a∷GUS 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