Unravelling the mechanisms of biocontrol in the tomato-fusarium oxysporum f. sp. lycopersici-Brevibacillus brevis interaction

Thesis is devoted to the investigation of lipid signaling processes as a universal mechanism mediating cellular responses to phytohormones and elicitors thus playing a key role in cell metabolism remodeling during plant adaptation to environmental changes. Phospholipase D (PLD) and its product phosphatidic acid (PA) were found to be involved to the SA-induced signaling cascades in Arabidopsis thaliana guard cells. Using radioactive labeling of phospholipids we found an activation of PLD and production of PA in leaves of 4-week old plants after salicylic acid (SA) treatment. Using histochemical assay, inhibitor assay and transgenic lines knock-out by different isoforms of NADPH-oxidases, we showed the involvement of PLD and NADPH-oxidase RbohD to PA-mediated superoxide formation in Arabidopsis tissues infiltrated by SA and SA-induced stomatal closure. SA- crosstalk with abscisic acid (ABA) in transcriptome remodeling induced by these hormones was investigated in suspension cell culture. Both SA and ABA inhibited basal activity of phosphatidylinositol dependent phospholipase C (PI-PLC) in vivo, while SA (but not ABA) also induced the phosphorylation of phosphatidylinositols. Total transcriptomes of suspension cells after SA or ABA treatment were compared to those obtained from suspension cells treated with U73122 (PI-PLC inhibitor) or wortmannin (inhibitor of phosphatidylinositol-4-kinases (PI4K) that provide the substrate for PI-PLC catalyzed reactions). We found a specific gene clusters, for those the effect of ABA and inhibitors was similar; SA-dependent genes, regulated via the balance of phosphoinositides, and SA-dependent genes, regulated via PLD-mediated pathway. Based on the bioinformatic analysis of the promoters of all selected gene sets, we claim a phosphoinositides level regulation to be an important factor mediating basal cell transcriptome and expression changes induced by SA and ABA.The effect of bacterial peptide flg22 on phospholipid turnover was detected in both suspension cells and seedlings. Flg22 induced accumulation of PA by the activation of PI-PLC coupled with diacylglycerolkinase (DGK) and a corresponding parallel increase of phosphatidylinositol-4,5-biphosphate content, that is a substrate of PI-PLC. Inhibitor analysis revealed the involvement of Ca2+ ions in lipid signaling enzymes reaction to flagellin treatment. We showed the role of DGK and PI-PLC in production of reactive oxygen species (ROS) induced by flg22. PA-production was placed in signaling cascade downstream of flagellin recognition by FLS2-BAK1 receptor complex receptor, but upstream or ROS formation by NADPH-oxidase RbohD. DGK5 was found to be the main source of the detected PA. The role of DGK5 was characterized in basal transcriptome regulation and its flagellin-induced remodeling; in flg22-induced callose accumulation in apoplast and resistance to biotrophic pathogen Pseudomonas syringae pv tomato DC3000. We proposed a new model of flagellin perception that includes PI-PLC and DGK5. Role of phosphoinositides in auxin and cytokinin signaling cascades was revealed studying root morphogenesis in Arabidopsis mutant pi4kb1b2 deficient for two PI4K genes, and pi4kb1b2sid2 that had additional mutation it key enzyme of SA biosynthesis, thus allowing us to separate SA-dependent and independent effects of the PI4K deficiency. pi4kb1b2 mutant plants exhibit the dwarf phenotype both in leaf and root parts, while pi4kb1b2sid2 show the normal rosette growth compared to WT, but still shorter roots. We analyzed root meristem anatomy, cortical cells elongation, gravitropic response, responses to exogenic hormones and firstly showed the connection of PI4K activity with auxin and cytokinin effects during root morphogenesis and gravitropism. Our results broaden the knowledge about the nature of plant phytohormonal signaling and can be used as a basis for increasing the resistance of agriculturally important crop plants to environmental stresses

Deciphering the role of lipid signalling in plant response to environmental stresses and developmental cues

This thesis presents an investigation into the transcriptional regulation of TaxolTM biosynthsis in Taxus cuspidata cell suspension cultures. The potent anticancer drug TaxolTM has been shown to be successful in the treatment of breast, lung and ovarian cancer and the acquired immunodeficiency syndrome (AIDS) related Kaposi’s sarcoma. Produced by all species of yew, TaxolTM belongs to the class of taxane diterpenoids and is of huge pharmaceutical importance. The plant material utilised in this thesis is a cell suspension culture initiated from isolated procambium cells of T. cuspidata. The latter is a meristematic tissue giving rise to the conductive tissue of plants. This un-differentiated cell suspension culture exhibits an increased and stable production of TaxolTM in response to the plant hormone elicitor methyljasmonate, limited cell aggregation and fast growth when compared to a cell suspension culture initiated from differentiated cells (somatic) of T. cuspidata. In order to assess the stem cell characteristics of the employed procambium cell suspension culture, the transcriptome of T. cuspidata was sequenced utilising Roche/ 454 and Illumina/ Solexa NlaIII tag sequencing technolo-

Transcriptional regulation of taxol™ biosynthesis in Taxus cuspidate procambium cells

Zusammenfassung 7 1 ABSTRACT/ZUSAMMENFASSUNG

Analysis of differential protein and gene expression in Arabidopsis thaliana depending on iron supply and the abundance of the central iron uptake regulator FIT (Fer-like Iron Deficiency-induced Transcription Factor) and investigations on possible post-translational modifications of FIT

To study the regulatory mechanism of PHA biosynthesis and possibly e nhance PHA production when grown on carbon monoxide and carbon dioxide mineral salt medium (RRNCO), which is simulating synthesis gas medium, R. rubrum strains were cultured in nitrogen-limiting RRNCO medium and were examined for PHA producti on. Six PHA biosynthetic genes ( phaC1AB, phaC2, phaC3, phaJ ) were over-expressed individually or in combination in R. rubrum and nine over-expressing strains and one control strain harboring empty vector were generated. PHA content analyses revealed tha t over-expressing phaC1 and phaC2 alone significantly enhanced PHA production, whereas over-expressi ng phaC3 and phaJ alone did not have significant effect on PHA production. PHA content anal yses and western blot experiments showed that over-expressing each gene of op r n phaC1AB alone significantly increased PHA production and over-expressing phaB resulted in the increasing of the PHA content the most. Furthermore, the data showed that the ex pression level of phaB

Genetic, biochemical and physiological studies of acetyl-CoA metabolism via acyl-condensation

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome They were synthesized by PCR amplification from genomic DNA Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines These versatile GST resources provide novel and powerful tools for functional genomics

Versatile gene-specific sequence tags for arabidopsis functional genomics : transcript profiling and reserve genetics applications

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Versatile gene-specific sequence tags for arabidopsis functional genomics : transcript profiling and reserve genetics applications