Tuning the Sensitivity of the PDR5 Promoter-Based Detection of Diclofenac in Yeast Biosensors.

The commonly used drug diclofenac is an important environmental anthropogenic pollutant. Currently, detection of diclofenac is mainly based on chemical and physical methods. Here we describe a yeast biosensor that drives the diclofenac-dependent expression of a recombinant fluorescent protein from the authentic promoter of the PDR5 gene. This key component of the pleiotropic drug response encodes a multidrug transporter that is involved in cellular detoxification. We analyse the effects on diclofenac sensitivity of artificial PDR5 promoter derivatives in wild-type and various yeast mutant strains. This approach enabled us to generate sensor strains with elevated drug sensitivity.

Laser microdissection coupled to transcriptional profiling of Arabidopsis roots inoculated by Plasmodiophora brassicae indicates a role for brassinosteroids in clubroot formation.

The clubroot disease caused by the obligate biotrophic protist Plasmodiophora brassicae on host plants of the Brassicaceae family is characterized by enhanced cell division and cell expansion. Since a typical root section of an infected plant always includes different stages of the pathogen as well as uninfected cells, we were interested in investigating specific developmental stages of the pathogen and their effect on host transcriptional changes. We extended previous microarray studies on whole roots by using laser microdissection and pressure catapulting (LMPC) to isolate individual cells harboring defined developmental stages of the pathogen. In addition, we compared the central cylinder of infected plants with that of control plants. We were especially interested in elucidating the stage-specific hormonal network. The up-regulation of genes involved in auxin and cytokinin metabolism and signaling was confirmed. In addition, we found evidence that brassinosteroid (BR) synthesis and signal perception genes were in many cases up-regulated in enlarged cells and the central cylinder. This was confirmed by quantitative PCR. Treatment of wild-type plants with the BR biosynthesis inhibitor propiconazole reduced gall formation, and the analysis of the BR receptor mutant bri1-6 revealed less severe gall formation than in the respective wild type. Our results identify novel hormone pathways involved in clubroot development. Using LMPC to generate pools of homogeneous cell type populations combined with transcriptome analysis has been very useful to elucidate the regulation of gall growth by this obligate biotropic pathogen in a cell- and stage-specific manner.

Overexpression of ctr1Δ300, a high-affinity copper transporter with deletion of the cytosolic C-terminus in Saccharomyces cerevisiae under excess copper, leads to disruption of transition metal homeostasis and transcriptional remodelling of cellular processes.

In an approach to generating Saccharomyces cerevisiae strains with increased intracellular copper amounts for technical applications, we overexpressed the copper transporter CTR1 and a variant of CTR1 with a truncation in the C-terminus after the 300th amino acid (ctr1Δ300). We determined the copper sensitivity of the generated strains and used inductively coupled plasma spectrometry analysis (ICP-OES and ICP-MS) to investigate the effects of overexpression of both constructs under excess copper on the cellular content of different elements in S. cerevisiae. In addition, we performed DNA microarray analysis to obtain the gene expression profile under the changed element contents. Overexpression of CTR1 increased the copper content in the cells to 160% and 78 genes were differentially regulated. Overexpression of the truncated ctr1Δ300 resulted in an increased copper, iron and zinc content of > 200% and 980 genes showed differential expression. We found that transition metal ion homeostasis was disrupted in ctr1Δ300-overexpressing strains under excess copper and that this was combined with a transcriptional remodelling of cellular processes.

Transcriptome analysis of Arabidopsis clubroots indicate a key role for cytokinins in disease development.

The clubroot disease of the family Brassicaceae is caused by the obligate biotrophic protist Plasmodiophora brassicae. Infected roots undergo a developmental switch that results in the formation of aberrant roots (clubs). To investigate host gene expression during the development of the disease, we have used the Arabidopsis ATH1 genome array. Two timepoints were chosen, an early timepoint at which the pathogen has colonized the root but has induced only very limited change of host cell and root morphology and a later timepoint at which more than 60% of the host root cells were colonized and root morphology was drastically altered. At both timepoints, more than 1,000 genes were differentially expressed in infected versus control roots. These included genes associated with growth and cell cycle, sugar phosphate metabolism, and defense. The involvement of plant hormones in club development was further supported; genes involved in auxin homeostasis, such as nitrilases and members of the GH3 family, were upregulated, whereas genes involved in cytokinin homeostasis (cytokinin synthases and cytokinin oxidases/dehydrogenases) were already strongly downregulated at the early timepoint. Cytokinin oxidase/dehydrogenase overexpressing lines were disease resistant, clearly indicating the importance of cytokinin as a key factor in clubroot disease development.

Induction of trehalase in Arabidopsis plants infected with the trehalose-producing pathogen Plasmodiophora brassicae.

Various microorganisms produce the disaccharide trehalose during their symbiotic and pathogenic interactions with plants. Trehalose has strong effects on plant metabolism and growth; therefore, we became interested to study its possible role in the interaction of Arabidopsis thaliana with Plasmodiophora brassicae, the causal agent of clubroot disease. We found that trehalose accumulated strongly in the infected organs (i.e., the roots and hypocotyls) and, to a lesser extent, in the leaves and stems of infected plants. This accumulation pattern of trehalose correlated with the expression of a putative trehalose-6-phosphate synthase (EC 2.4.1.15) gene from P. brassicae, PbTPS1. Clubroot formation also resulted in an induction of the Arabidopsis trehalase gene, ATTRE1, and in a concomitant increase in trehalase (EC 3.2.1.28) activity in the roots and hypocotyls, but not in the leaves and stems of infected plants. Thus, induction of ATTRE1 expression was probably responsible for the increased trehalase activity. Trehalase activity increased before trehalose accumulated; therefore, it is unlikely that trehalase was induced by its substrate. The induction of trehalase may be part of the plant's defense response and may prevent excess accumulation of trehalose in the plant cells, where it could interfere with the regulation of carbon metabolism.