Functional identification of Arabidopsis stress regulatory genes using the Controlled cDNA Overexpression System

Plants respond to environmental stresses by altering the expression of many genes via a complex signalling network. We created a Controlled cDNA Overexpression System (COS) to identify genes and regulatory factors which have important role in stress tolerance. An estradiol-inducible cDNA expression library was tested in three genetic screens by selecting for salt tolerance, ABA insensitive germination and activation of a stress responsive ADH1-LUC reporter gene construct. Several cDNAs conferring dominant, estradiol dependent stress tolerance phenotypes were identified. Screening for enhanced salt tolerance revealed that estradiol-controlled overexpression of 2-alkenal reductase (AER) cDNA confers considerably high level of salt insensitivity. Characterization of cDNA conferring insensitivity to 3μM ABA in germination assays has identified the full-length coding region of heat shock protein HSP17.6A, suggesting its implication in ABA signal transduction. Screening for transcriptional activation of ADH1-LUC reporter gene has identified the ERF/AP-type transcription factor RAP2.12, which sustained high level ADH1-LUC bioluminescence and enhanced ADH1 transcription in the presence of estradiol. Our data illustrate that application of inducible cDNA expression libraries such as the COS system provides an efficient tool for genetic identification and functional analysis of novel regulators of abiotic stress responses

Identification of Arabidopsis and Thellungiella genes involved in salt tolerance by novel genetic system

High salinity is a major constraint to plant growth and development. Plants respond to environmental stresses by altering gene expression pattern via a complex signaling network. We developed a novel genetic system based on conditional cDNA overexpression to isolate genes involved in plant salt tolerance. Transformation-ready Arabidopsis and Thellungiella cDNA libraries cloned in a plant expression vector under control of an inducible promoter were used to transfer into Arabidopsis, where activation of the inserted cDNA can lead to conditional phenotypes. Transgenic lines were tested in different screens (germination assay, growth-survival test). Our genetic system was suitable to identify not only well-known genes coding for proteins involved in stress tolerance, but several novel regulatory genes were discovered. Line N33 shows estradiol-dependent salt tolerant germination. It has a single T-DNA insertion; the full length cDNA encodes an unknown protein. This gene was designated as Novel Salt Tolerance (NSTO). The Thellungiella library allowed large scale random interspecific gene transfer and subsequent identification of novel regulatory genes which control stress tolerance in halophyte species. Our data illustrate that application of inducible cDNA expression libraries provides an efficient tool for genetic identification and functional analysis of novel positive or negative regulators of plant salt tolerance

Plk4 Is a Novel Substrate of Protein Phosphatase 5

The conserved Ser/Thr protein phosphatase 5 (PP5) is involved in the regulation of key cellular processes, including DNA damage repair and cell division in eukaryotes. As a co-chaperone of Hsp90, PP5 has been shown to modulate the maturation and activity of numerous oncogenic kinases. Here, we identify a novel substrate of PP5, the Polo-like kinase 4 (Plk4), which is the master regulator of centriole duplication in animal cells. We show that PP5 specifically interacts with Plk4, and is able to dephosphorylate the kinase in vitro and in vivo, which affects the interaction of Plk4 with its partner proteins. In addition, we provide evidence that PP5 and Plk4 co-localize to the centrosomes in Drosophila embryos and cultured cells. We demonstrate that PP5 is not essential; the null mutant flies are viable without a severe mitotic phenotype; however, its loss significantly reduces the fertility of the animals. Our results suggest that PP5 is a novel regulator of the Plk4 kinase in Drosophila

Protein Phosphatase 4 Is Required for Centrobin Function in DNA Damage Repair

Genome stability in human cells relies on the efficient repair of double-stranded DNA breaks, which is mainly achieved by homologous recombination (HR). Among the regulators of various cellular functions, Protein phosphatase 4 (PP4) plays a pivotal role in coordinating cellular response to DNA damage. Meanwhile, Centrobin (CNTRB), initially recognized for its association with centrosomal function and microtubule dynamics, has sparked interest due to its potential contribution to DNA repair processes. In this study, we investigate the involvement of PP4 and its interaction with CNTRB in HR-mediated DNA repair in human cells. Employing a range of experimental strategies, we investigate the physical interaction between PP4 and CNTRB and shed light on the importance of two specific motifs in CNTRB, the PP4-binding FRVP and the ATR kinase recognition SQ sequences, in the DNA repair process. Moreover, we examine cells depleted of PP4 or CNTRB and cells harboring FRVP and SQ mutations in CNTRB, which result in similar abnormal chromosome morphologies. This phenomenon likely results from the impaired resolution of Holliday junctions, which serve as crucial intermediates in HR. Taken together, our results provide new insights into the intricate mechanisms of PP4 and CNTRB-regulated HR repair and their interrelation

Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant

Host compatible rhizobia induce the formation of legume root nodules, symbiotic organs within which intracellular bacteria are present in plant-derived membrane compartments termed symbiosomes. In Medicago truncatula nodules, the Sinorhizobium microsymbionts undergo an irreversible differentiation process leading to the development of elongated polyploid noncultivable nitrogen fixing bacteroids that convert atmospheric dinitrogen into ammonia. This terminal differentiation is directed by the host plant and involves hundreds of nodule specific cysteine-rich peptides (NCRs). Except for certain in vitro activities of cationic peptides, the functional roles of individual NCR peptides in planta are not known. In this study, we demonstrate that the inability of M. truncatula dnf7 mutants to fix nitrogen is due to inactivation of a single NCR peptide, NCR169. In the absence of NCR169, bacterial differentiation was impaired and was associated with early senescence of the symbiotic cells. Introduction of the NCR169 gene into the dnf7-2/NCR169 deletion mutant restored symbiotic nitrogen fixation. Replacement of any of the cysteine residues in the NCR169 peptide with serine rendered it incapable of complementation, demonstrating an absolute requirement for all cysteines in planta. NCR169 was induced in the cell layers in which bacteroid elongation was most pronounced, and high expression persisted throughout the nitrogen-fixing nodule zone. Our results provide evidence for an essential role of NCR169 in the differentiation and persistence of nitrogen fixing bacteroids in M. truncatula

Az ozmotikus stresszválasz szabályozása magasabbrendű növényekben. = Regulation of osmotic stress responses in higher plants

Pályázatunkban egy új genetikai rendszert dolgoztunk ki, amely alkalmas a stressz jelátvitelben szerepet játszó növényi gének azonosítására. RT-PCR módszerrel jellemeztük több stressz indukált Arabidopsis gén aktivitását különböző stressz és homonkezelés után. A gének 5? promoter régióját megklónoztuk, és promoter nélküli luciferáz (LUC) illetve zöld fluorescens protein (GFP) riporter génekhez kapcsoltuk. A riporter gének aktivitását transzgenikus Arabidopsis növényekben tanulmányoztuk. Az új szabályozó faktorok azonosítása érdekében egy Arabidopsis cDNS könyvtárat hoztunk létre a pER8-GW expressziós vektorban, ami ösztradiol által indukálható expressziós kazettát hordoz. A cDNS könyvtár segítségével egy transzgenikus Arabidopsis növény populációt hoztunk létre. A transzgenikus növényeket só rezisztenciára, ABA érzékenységre illetve a már korábban beépített riporter gén aktivitásának megváltozására teszteltük. Több olyan Arabidopsis vonalat sikerült azonosítani, amelyekben az ösztradiol adása megnövekedett só vagy ABA toleranciával, illetve a riporter gén aktivitásával járt együtt. A C38-33 vonalban megemelkedett só toleranciát kaptunk a beépült cDNS transzkripciójának aktiválásával. A cDNS egy új, S1 domén-t tartalmazó fehérjét kódol. Az ADH-121 vonalban egy AP típusú transzkripció faktort azonosítottunk, ami képes volt az ADH-LUC riporter gén kontrukció aktiválására a külső környezeti tényezőktől függetlenül. | We have developed a genetic system to identify new regulatory factors, controlling stress responses in higher plants, namely in Arabidopsis. Using quantitative RT-PCR, we have characterized the expression of several stress-responsive genes in different conditions and hormonal treatments. The 5? promoter sequences of 5 stress-induced genes have been cloned and fused to promoterless reporter genes, such as the firefly luciferase (LUC) or the green fluorescence protein (GFP). Activity of the reporter gene constructs was characterized in transgenic Arabidopsis plants, using non-destructive assays. In order to identify new regulatory factors, a transformation-competent cDNA library was created in the plant expression vector pER8-GW, carrying an estradiol-responsive expression cassette. Large-scale Arabidopsis transformation generated a collection of transgenic plants, each carrying a cDNA clone. Transgenic plants were screened for salt tolerance, ABA insensitivity or activation of reporter gene constructs. Several salt tolerant or ABA insensitive lines were obtained and characterized. In some lines reporter genes were activated upon the induction of transgene expression, in the absence of stress. In the line C38-33 increased salt tolerance was obtained by the activation of a full length cDNA, coding for a previously unknown protein with S1 domain. In the line ADH-121, activation of an AP transcription factor lead to the increased expression of the ADH-LUC reporter construct

Differential regulation of the two metallothionein genes in common carp

The expression of two metallothionein (MT) genes was followed in carp (Cyprinus carpio) in vivo during exposure to As and Cu. Changes in the levels of MT-1 and MT-2 mRNA in the liver and kidney were detected by semi-quantitative RT-PCR. The inducibility of the two MT isoforms was tissue- and metal-specific. Regardless of whether As or Cu was applied, the liver was more responsive than the kidney. Copper influenced the expression of both isoforms: the MT-1 and MT-2 mRNA levels increased in both the liver and the kidney in a time- and dose-dependent manner. Arsenic affected mostly the MT-2 expression, while the level of the MT-1 transcript did not change significantly in either organ

Comparative analysis of new peptide conjugates of antitubercular drug candidates – model membrane and in vitro studies

Novel peptide conjugates of two antitubercular drug candidates were synthesised and characterised using new tuftsin peptide derivatives (OT14) as carrier moiety. As antitubercular drug candidates two pyridopyrimidine derivatives, TB803 (2-allylamino-4-oxopyrido[1,2-a]pyrimidine-3-carbaldehyde) and TB820 (4-oxo-2-(pyrrolidin-1-yl)-pyrido[1,2-a]pyrimidin-3-carbaldehyde) inhibiting vital enzyme of Mycobacterium tuberculosis were applied. Membrane affinity of the compounds TB803 and TB820 and their peptide conjugates was evaluated using experimental lipid mono- and bilayer models. Penetration ability was assessed tensiometrically from Langmuir monolayer study and applying quartz crystal microbalance for the supported lipid bilayer (SLB) system. Minimal inhibitory concentration (MIC) values remained in a similar micromolar range for both of the conjugates while their cellular uptake rate was improved significantly compared to the drug candidates. A correlation was found between membrane affinity properties and results of in vitro biological investigations. Analysis of physical/structural properties of SLB in contact with bioactive components and visualization of the structural change by atomic-force microscopy (AFM) provided information on the type and route of molecular interaction of drug construction with lipid layers. The possible role of electrostatic interactions between lipid layer and drug candidates was tested in Langmuir-balance experiments using negatively charged lipid mixture (DPPC+DPPG). Especially the peptide conjugates presented increased membrane affinity due to cationic character of the peptide sequence selected for the conjugate formation. That is supposed to be one reason for the enhanced cellular uptake observed in vitro for MonoMac6 cell line. The conjugation of antitubercular agents to a peptidic carrier is a promising approach to enhance membrane affinity, cellular uptake rate and in vitro selectivity

STABILON, a Novel Sequence Motif That Enhances the Expression and Accumulation of Intracellular and Secreted Proteins

The dynamic balance of transcriptional and translational regulation together with degron-controlled proteolysis shapes the ever-changing cellular proteome. While a large variety of degradation signals has been characterized, our knowledge of cis-acting protein motifs that can in vivo stabilize otherwise short-lived proteins is very limited. We have identified and characterized a conserved 13-mer protein segment derived from the p54/Rpn10 ubiquitin receptor subunit of the Drosophila 26S proteasome, which fulfills all the characteristics of a protein stabilization motif (STABILON). Attachment of STABILON to various intracellular as well as medically relevant secreted model proteins resulted in a significant increase in their cellular or extracellular concentration in mammalian cells. We demonstrate that STABILON acts as a universal and dual function motif that, on the one hand, increases the concentration of the corresponding mRNAs and, on the other hand, prevents the degradation of short-lived fusion proteins. Therefore, STABILON may lead to a breakthrough in biomedical recombinant protein production