Comparison of the Role of 5′ Terminal Sequences of Alfalfa Mosaic Virus RNAs 1, 2, and 3 in Viral RNA Replication

AbstractThe 5′ untranslated regions (UTRs) of the genomic RNAs 1, 2, and 3 of alfalfa mosaic virus (AMV) are 100, 54, and 345 nucleotides (nt) long, respectively, and lack extensive sequence similarity to each other. RNA 3 encodes the movement protein P3 and the coat protein and can be replicated in transgenic tobacco plants expressing the replicase proteins P1 and P2 (P12 plants). 5′Cis-acting sequences involved in RNA 3 replication have been shown to be confined to the 5′ UTR. When the 5′ UTR of RNA 3 was replaced by the 5′ UTRs of RNAs 1 or 2, the recombinant RNA was not infectious to P12 plants. Also, when the P3 gene in RNA 3 was put under the control of a subgenomic promoter and the 5′ UTR of this RNA was replaced by 5′ terminal RNA 1 sequences of 103 to 860 nt long or RNA 2 sequences of 57 to 612 nt long, no accumulation of the hybrid RNAs was observed. Deletion of the 5′ 22 nucleotides of RNA 3 resulted in the accumulation of a major progeny that lacked the 5′ 79 nt. However, when the 5′ 22 nucleotides of RNA 3 were replaced by the complete 5′ UTR of RNA 1 or 5′ sequences of RNAs 1, 2, or 3 with a length of 5 to 15 nt, accumulation of the full-length mutant RNAs was observed. The effect of mutations in the 5′ viral sequences of 5 to 15 nt was analyzed. It is concluded that although elements within nucleotides 80–345 of the 5′ UTR of RNA 3 are sufficient for replication, a specific sequence of 3 to 5 nt is required to target the replicase to an initiation site corresponding to the 5′ end of the RNA

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

Combining functional weed ecology and crop stable isotope ratios to identify cultivation intensity: a comparison of cereal production regimes in Haute Provence, France and Asturias, Spain

This investigation combines two independent methods of identifying crop growing conditions and husbandry practices—functional weed ecology and crop stable carbon and nitrogen isotope analysis—in order to assess their potential for inferring the intensity of past cereal production systems using archaeobotanical assemblages. Present-day organic cereal farming in Haute Provence, France features crop varieties adapted to low-nutrient soils managed through crop rotation, with little to no manuring. Weed quadrat survey of 60 crop field transects in this region revealed that floristic variation primarily reflects geographical differences. Functional ecological weed data clearly distinguish the Provence fields from those surveyed in a previous study of intensively managed spelt wheat in Asturias, north-western Spain: as expected, weed ecological data reflect higher soil fertility and disturbance in Asturias. Similarly, crop stable nitrogen isotope values distinguish between intensive manuring in Asturias and long-term cultivation with minimal manuring in Haute Provence. The new model of cereal cultivation intensity based on weed ecology and crop isotope values in Haute Provence and Asturias was tested through application to two other present-day regimes, successfully identifying a high-intensity regime in the Sighisoara region, Romania, and low-intensity production in Kastamonu, Turkey. Application of this new model to Neolithic archaeobotanical assemblages in central Europe suggests that early farming tended to be intensive, and likely incorporated manuring, but also exhibited considerable variation, providing a finer grained understanding of cultivation intensity than previously available