Aladár Pettkó-Szandtner

Prediction of functional regions of the maize streak virus replication-associated proteins by protein-protein interaction analysis

The replication of the geminiviruses depends on the viral encoded early (complementary-sense) proteins and on host genome encoded factors. Additionally, some of the early proteins (the AL2 protein of subgroup III, and the RepA (formerly known as C1) or Rep (C1:C2) proteins of subgroup I geminiviruses) can function as transcriptional activators of virion- (V-)sense gene expression. The yeast two-hybrid system has allowed us to predict some of the functionally important regions of the maize streak virus (MSV) early proteins RepA and Rep. Defined domains of these proteins were shown to act as transactivators in yeast cells. We detected the association of the RepA and Rep proteins, and their subfragments, with the maize retinoblastoma protein (ZmRb1) which is likely to be one of the interacting host proteins. We showed the self-association capability of the MSV proteins and suggest that homo- or hetero-oligomerization may play an important role in virus replication. These results provide new insights into the role of different regions of the MSV proteins in relation to transcriptional activation and regulation of viral DNA replication.

The involvement of reactive oxygen species (ROS) in the cell cycle activation (G0-to-G1 transition) of plant cells

Reactive oxygen species (ROS) are involved in various cellular processes in plants. Among those, resistance to abiotic stress, defence mechanisms and cell expansion have been intensively studied during the last years. We recently demonstrated that ROS, in concert with auxin, have a role in cell cycle activation of differentiated leaf cells.1 In this addendum we provide further evidence to show that oxidative stress/ROS accelerate auxin-mediated cell cycle entry (G0-to-G1 Addendum to: Pasternak TP, Ötvös K, Domoki M, Fehér A. Linked activation of cell division and oxidative stress defense in alfalfa leaf protoplast-derived cells is dependent on exogenous auxin. Plant Growth Regul 2007; 51:109-17.

Transcriptional repression by MYB3R proteins regulates plant organ growth

In multicellular organisms, temporal and spatial regulation of cell proliferation is central for generating organs with defined sizes and morphologies. For establishing and maintaining the post‐mitotic quiescent state during cell differentiation, it is important to repress genes with mitotic functions. We found that three of the Arabidopsis MYB3R transcription factors synergistically maintain G2/M‐specific genes repressed in post‐mitotic cells and restrict the time window of mitotic gene expression in proliferating cells. The combined mutants of the three repressor‐type MYB3R genes displayed long roots, enlarged leaves, embryos, and seeds. Genome‐wide chromatin immunoprecipitation revealed that MYB3R3 binds to the promoters of G2/M‐specific genes and to E2F target genes. MYB3R3 associates with the repressor‐type E2F, E2FC, and the RETINOBLASTOMA RELATED proteins. In contrast, the activator MYB3R4 was in complex with E2FB in proliferating cells. With mass spectrometry and pairwise interaction assays, we identified some of the other conserved components of the multiprotein complexes, known as DREAM/dREAM in human and flies. In plants, these repressor complexes are important for periodic expression during cell cycle and to establish a post‐mitotic quiescent state determining organ size.

Mitosis-specific promoter of the alfalfa cyclin-dependent kinase gene (Medsa;CDKB2;1) is activated by wounding and ethylene in a non-cell division-dependent manner

Cyclin-dependent serine/threonine kinases (CDKs) have pivotal roles in regulating the eukaryotic cell cycle. Plants possess a unique class of CDKs (B-type CDKs) with preferential protein accumulation at G2/M-phases; however, their exact functions are still enigmatic. Here we describe the functional characterization of a 360-bp promoter region of the alfalfa (Medicago sativa) CDKB2;1 gene in transgenic plants and cell lines. It is shown that the activity of the analyzed promoter was characteristic for proliferating meristematic regions in planta and specific for cells in the G2/M-phases in synchronized cell cultures. Immunohistochemical analysis of transgenic root sections further confirmed the correlation of the expression of the CDKB2;1 promoter-linked reporter genes with the accumulation of the correspondent kinase. It was found that, in addition to auxin (2,4-dichlorophenoxyacetic acid) treatment, wounding could also induce both the reporter and endogenous genes in transgenic leaf explants. Furthermore, ethylene, known as a wound-response mediator, had a similar effect. The gene activation in response to wounding or ethephon was faster and occurred without the induction of cell cycle progression in contrast to the control auxin treatment. In silico analysis of this promoter indeed revealed the presence of a set of cis-elements, indicating not only cell cycle- but wound- and ethylene-dependent regulation of this CDK gene. Based on the presented data, we discuss the functional significance of the complex regulation of mitosis-specific CDK genes in plants.

Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control

The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX‐labelled DNA damage foci in an ATM‐ and ATR‐dependent manner. These γH2AX‐labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co‐localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta. Genetic interaction between the RBR‐silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity.

Isolation and characterization of a novel n-alkane-degrading strain, Acinetobacter haemolyticus AR-46

Abstract Strain AR-46, isolated and identified as Acinetobacter haemolyticus, evolutionally distant from the known hydrocarbon-degrading Acinetobacter spp., proved to have excellent longchain n-alkane-degrading ability. This is the first detailed report on an n-alkane-utilizing strain belonging to this species. The preferred substrate is n-hexadecane, with an optimal temperature of 37 °C under aerobic conditions. Five complete and two partial open reading frames were sequenced and correlated with the early steps of monoterminal oxidation-initiated n-alkane mineralization. The encoded protein sequences and the arrangement of these genes displayed high similarity to those found in Acinetobacter sp. M-1, but AR-46 seemed to have only one alkane hydroxylase gene, with a completely different induction profile. Unique behaviour was also observed in n-alkane bioavailability. Substrate uptake occurred through the hydrophobic surface of n-alkane droplet-adhered cells possessing long, thick fimbriae, which were presumed to play a major role in n-alkane solubilization. A majority of the cells was in detached form, with thick, but short fimbriae. These free cells were permanently hydrophilic, unlike the cells of other Acinetobacter strains.

The phosphomimetic mutation of syndecan-4 binds and inhibits Tiam1 modulating Rac1 activity in PDZ interaction–dependent manner

The small GTPases of the Rho family comprising RhoA, Rac1 and Cdc42 function as molecular switches controlling several essential biochemical pathways in eukaryotic cells. Their activity is cycling between an active GTP-bound and an inactive GDP-bound conformation. The exchange of GDP to GTP is catalyzed by guanine nucleotide exchange factors (GEFs). Here we report a novel regulatory mechanism of Rac1 activity, which is controlled by a phosphomimetic (Ser179Glu) mutant of syndecan-4 (SDC4). SDC4 is a ubiquitously expressed transmembrane, heparan sulfate proteoglycan. In this study we show that the Ser179Glu mutant binds strongly Tiam1, a Rac1-GEF reducing Rac1-GTP by 3-fold in MCF-7 breast adenocarcinoma cells. Mutational analysis unravels the PDZ interaction between SDC4 and Tiam1 is indispensable for the suppression of the Rac1 activity. Neither of the SDC4 interactions is effective alone to block the Rac1 activity, on the contrary, lack of either of interactions can increase the activity of Rac1, therefore the Rac1 activity is the resultant of the inhibitory and stimulatory effects. In addition, SDC4 can bind and tether RhoGDI1 (GDP-dissociation inhibitor 1) to the membrane. Expression of the phosphomimetic SDC4 results in the accumulation of the Rac1–RhoGDI1 complex. Co-immunoprecipitation assays (co-IP-s) reveal that SDC4 can form complexes with RhoGDI1. Together, the regulation of the basal activity of Rac1 is fine tuned and SDC4 is implicated in multiple ways.

Hsp70-associated chaperones have a critical role in buffering protein production costs

Proteins are necessary for cellular growth. Concurrently, however, protein production has high energetic demands associated with transcription and translation. Here, we propose that activity of molecular chaperones shape protein burden, that is the fitness costs associated with expression of unneeded proteins. To test this hypothesis, we performed a genome-wide genetic interaction screen in bakers yeast. Impairment of transcription, translation, and protein folding rendered cells hypersensitive to protein burden. Specifically, deletion of specific regulators of the Hsp70-associated chaperone network increased protein burden. In agreement with expectation, temperature stress, increased mistranslation and a chemical misfolding agent all substantially enhanced protein burden. Finally, unneeded protein perturbed interactions between key components of the Hsp70-Hsp90 network involved in folding of native proteins. We conclude that specific chaperones contribute to protein burden. Our work indicates that by minimizing the damaging impact of gratuitous protein overproduction, chaperones enable tolerance to massive changes in genomic expression.