Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format

BACKGROUND: The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. Protein-protein interactions occur in cells in two ways, constitutive and regulative. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis. RESULTS: A split luciferase complementation assay was applied to detect the regulated interactions in rice. A transformation method of rice protoplasts in a 96-well plate was first established for a large-scale analysis. In addition, an antibody that specifically recognizes a carboxyl-terminal fragment of Renilla luciferase was newly developed. A pair of antibodies that recognize amino- and carboxyl- terminal fragments of Renilla luciferase, respectively, was then used to monitor quality and quantity of interacting recombinant-proteins accumulated in the cells. For a proof-of-concept, the method was applied to detect the gibberellin-dependent interaction between GIBBERELLIN INSENSITIVE DWARF1 and SLENDER RICE 1. CONCLUSIONS: A method to detect regulated protein-protein interactions was developed towards establishment of the rice interactome

Pseudo-Response Regulator (PRR) Homologues of the Moss Physcomitrella patens: Insights into the Evolution of the PRR Family in Land Plants

The pseudo-response regulators (PRRs) are the circadian clock component proteins in the model dicot Arabidopsis thaliana. They contain a receiver-like domain (RLD) similar to the receiver domains of the RRs in the His–Asp phosphorelay system, but the RLDs lack the phosphoacceptor aspartic acid residue invariably conserved in the receiver domains. To study the evolution of PRR genes in plants, here we characterize their homologue genes, PpPRR1, PpPRR2, PpPRR3 and PpPRR4, from the moss Physcomitrella patens. In the phylogenetic analysis, PpPRRs cluster together, sister to an angiosperm PRR gene subfamily, illustrating their close relationships with the angiosperm PRRs. However, distinct from the angiosperm sequences, the RLDs of PpPRR2/3/4 exhibit a potential phosphoacceptor aspartic acid–aspartic acid–lysine (DDK) motif. Consistently, the PpPRR2 RLD had phosphotransfer ability in vitro, suggesting that PpPRR2 functions as an RR. The PpPRR1 RLD, on the other hand, shows a partially diverged DDK motif, and it did not show phosphotransfer ability. All PpPRRs were expressed in a circadian and light-dependent manner, with differential regulation between PpPRR2/4 and PpPRR1/3. Altogether, our results illustrate that PRRs originated from an RR(s) and that there are intraspecific divergences among PpPRRs. Finally, we offer scenarios for the evolution of the PRR family in land plants

Rice early flowering1, a CKI, phosphorylates DELLA protein SLR1 to negatively regulate gibberellin signalling

The plant hormone gibberellin (GA) is crucial for multiple aspects of plant growth and development. To study the relevant regulatory mechanisms, we isolated a rice mutant earlier flowering1, el1, which is deficient in a casein kinase I that has critical roles in both plants and animals. el1 had an enhanced GA response, consistent with the suppression of EL1 expression by exogenous GA3. Biochemical characterization showed that EL1 specifically phosphorylates the rice DELLA protein SLR1, proving a direct evidence for SLR1 phosphorylation. Overexpression of SLR1 in wild-type plants caused a severe dwarf phenotype, which was significantly suppressed by EL1 deficiency, indicating the negative effect of SLR1 on GA signalling requires the EL1 function. Further studies showed that the phosphorylation of SLR1 is important for maintaining its activity and stability, and mutation of the candidate phosphorylation site of SLR1 results in the altered GA signalling. This study shows EL1 a novel and key regulator of the GA response and provided important clues on casein kinase I activities in GA signalling and plant development

A Genome-Wide Compilation of the Two-Component Systems in Lotus japonicus

The two-component systems (TCS), or histidine-to-aspartate phosphorelays, are evolutionarily conserved common signal transduction mechanisms that are implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes including plants. Among higher plants, legumes including Lotus japonicus have a unique ability to engage in beneficial symbiosis with nitrogen-fixing bacteria. We previously presented a genome-wide compiled list of TCS-associated components of Mesorhizobium loti, which is a symbiont specific to L. japonicus (Hagiwara et al. 2004, DNA Res., 11, 57–65). To gain both general and specific insights into TCS of this currently attractive model legume, here we compiled TCS-associated components as many as possible from a genome-wide viewpoint by taking advantage that the efforts of whole genome sequencing of L. japonicus are almost at final stage. In the current database (http://www.kazusa.or.jp/lotus/index.html), it was found that L. japonicus has, at least, 14 genes each encoding a histidine kinase, 7 histidine-containing phosphotransmitter-related genes, 7 type-A response regulator (RR)-related genes, 11 type-B RR-related genes, and also 5 circadian clock-associated pseudo-RR genes. These results suggested that most of the L. japonicus TCS-associated genes have already been uncovered in this genome-wide analysis, if not all. Here, characteristics of these TCS-associated components of L. japonicus were inspected, one by one, in comparison with those of Arabidopsis thaliana. In addition, some critical experiments were also done to gain further insights into the functions of L. japonicus TCS-associated genes with special reference to cytokinin-mediated signal transduction and circadian clock

Inter- and intra-molecular interactions of Arabidopsis thaliana DELLA protein RGL1

The phytohormone gibberellin and the DELLA proteins act together to control key aspects of plant development. Gibberellin induces degradation of DELLA proteins by recruitment of an F-box protein using a molecular switch: a gibberellin-bound nuclear receptor interacts with the N-terminal domain of DELLA proteins, and this event primes the DELLA C-terminal domain for interaction with the F-box protein. However, the mechanism of signalling between the N- and C-terminal domains of DELLA proteins is unresolved. In the present study, we used in vivo and in vitro approaches to characterize di- and tri-partite interactions of the DELLA protein RGL1 (REPRESSOR OF GA1-3-LIKE 1) of Arabidopsis thaliana with the gibberellin receptor GID1A (GIBBERELLIC ACID-INSENSITIVE DWARF-1A) and the F-box protein SLY1 (SLEEPY1). Deuterium-exchange MS unequivocally showed that the entire N-terminal domain of RGL1 is disordered prior to interaction with the GID1A; furthermore, association/dissociation kinetics, determined by surface plasmon resonance, predicts a two-state conformational change of the RGL1 N-terminal domain upon interaction with GID1A. Additionally, competition assays with monoclonal antibodies revealed that contacts mediated by the short helix Asp-Glu-Leu-Leu of the hallmark DELLA motif are not essential for the GID1A–RGL1 N-terminal domain interaction. Finally, yeast two- and three-hybrid experiments determined that unabated communication between N- and C-terminal domains of RGL1 is required for recruitment of the F-box protein SLY1

Evaluation of Microbubbles as Contrast Agents for Ultrasonography and Magnetic Resonance Imaging

Background: Microbubbles (MBs) can serve as an ultrasound contrast agent, and has the potential for magnetic resonance imaging (MRI). Due to the relatively low effect of MBs on MRI, it is necessary to develop new MBs that are more suitable for MRI. In this study, we evaluate the properties of SonoVueH and custom-made Fe 3O 4-nanoparticle-embedded microbubbles (Fe3O4-MBs) in terms of contrast agents for ultrsonography (US) and MRI. Methodology/Principal Findings: A total of 20 HepG2 subcutaneous-tumor-bearing nude mice were randomly assigned to 2 groups (i.e., n = 10 mice each group), one for US test and the other for MRI test. Within each group, two tests were performed for each mouse. The contrast agent for the first test is SonoVueH, and the second is Fe 3O 4-MBs. US was performed using a Technos MPX US system (Esaote, Italy) with a contrast-tuned imaging (CnTI TM) mode. MRI was performed using a 7.0T Micro-MRI (PharmaScan, Bruker Biospin GmbH, Germany) with an EPI-T2 * sequence. The data of signal-to-noise ratio (SNR) from the region-of-interest of each US and MR image was calculated by ImageJ (National Institute of Health, USA). In group 1, enhancement of SonoVueH was significantly higher than Fe 3O 4-MBs on US (P,0.001). In group 2, negative enhancement of Fe3O4-MBs was significantly higher than SonoVueH on MRI (P,0.001). The time to peak showed no significant differences between US and MRI, both of which used the same MBs (P.0.05). The SNR analysis of the enhancement process reveals a strong negative correlation in both cases (i.e., SonoVueH r=20.733, Fe 3O 4-MBs r = 20.903

Phosphorylation Alters the Interaction of the Arabidopsis Phosphotransfer Protein AHP1 with Its Sensor Kinase ETR1

The ethylene receptor ethylene response 1 (ETR1) and the Arabidopsis histidine-containing phosphotransfer protein 1 (AHP1) form a tight complex in vitro. According to our current model ETR1 and AHP1 together with a response regulator form a phosphorelay system controlling the gene expression response to the plant hormone ethylene, similar to the two-component signaling in bacteria. The model implies that ETR1 functions as a sensor kinase and is autophosphorylated in the absence of ethylene. The phosphoryl group is then transferred onto a histidine at the canonical phosphorylation site in AHP1. For phosphoryl group transfer both binding partners need to form a tight complex. After ethylene binding the receptor is switched to the non-phosphorylated state. This switch is accompanied by a conformational change that decreases the affinity to the phosphorylated AHP1. To test this model we used fluorescence polarization and examined how the phosphorylation status of the proteins affects formation of the suggested ETR1−AHP1 signaling complex. We have employed various mutants of ETR1 and AHP1 mimicking permanent phosphorylation or preventing phosphorylation, respectively. Our results show that phosphorylation plays an important role in complex formation as affinity is dramatically reduced when the signaling partners are either both in their non-phosphorylated form or both in their phosphorylated form. On the other hand, affinity is greatly enhanced when either protein is in the phosphorylated state and the corresponding partner in its non-phosphorylated form. Our results indicate that interaction of ETR1 and AHP1 requires that ETR1 is a dimer, as in its functional state as receptor in planta

Genome-Wide Haplotype Changes Produced by Artificial Selection during Modern Rice Breeding in Japan

During the last 90 years, the breeding of rice has delivered cultivars with improved agronomic and economic characteristics. Crossing of different lines and successive artificial selection of progeny based on their phenotypes have changed the chromosomal constitution of the ancestors of modern rice; however, the nature of these changes is unclear. The recent accumulation of data for genome-wide single-nucleotide polymorphisms (SNPs) in rice has allowed us to investigate the change in haplotype structure and composition. To assess the impact of these changes during modern breeding, we studied 177 Japanese rice accessions, which were categorized into three groups: landraces, improved cultivars developed from 1931 to 1974 (the early breeding phase), and improved cultivars developed from 1975 to 2005 (the late breeding phase). Phylogenetic tree and structure analysis indicated genetic differentiation between non-irrigated (upland) and irrigated (lowland) rice groups as well as genetic structuring within the irrigated rice group that corresponded to the existence of three subgroups. Pedigree analysis revealed that a limited number of landraces and cultivars was used for breeding at the beginning of the period of systematic breeding and that 11 landraces accounted for 70% of the ancestors of the modern improved cultivars. The values for linkage disequilibrium estimated from SNP alleles and the haplotype diversity determined from consecutive alleles in five-SNP windows indicated that haplotype blocks became less diverse over time as a result of the breeding process. A decrease in haplotype diversity, caused by a reduced number of polymorphisms in the haplotype blocks, was observed in several chromosomal regions. However, our results also indicate that new haplotype polymorphisms have been generated across the genome during the breeding process. These findings will facilitate our understanding of the association between particular haplotypes and desirable phenotypes in modern Japanese rice cultivars

Digital chest radiography: an update on modern technology, dose containment and control of image quality

The introduction of digital radiography not only has revolutionized communication between radiologists and clinicians, but also has improved image quality and allowed for further reduction of patient exposure. However, digital radiography also poses risks, such as unnoticed increases in patient dose and suboptimum image processing that may lead to suppression of diagnostic information. Advanced processing techniques, such as temporal subtraction, dual-energy subtraction and computer-aided detection (CAD) will play an increasing role in the future and are all targeted to decrease the influence of distracting anatomic background structures and to ease the detection of focal and subtle lesions. This review summarizes the most recent technical developments with regard to new detector techniques, options for dose reduction and optimized image processing. It explains the meaning of the exposure indicator or the dose reference level as tools for the radiologist to control the dose. It also provides an overview over the multitude of studies conducted in recent years to evaluate the options of these new developments to realize the principle of ALARA. The focus of the review is hereby on adult applications, the relationship between dose and image quality and the differences between the various detector systems

Fate of the H-NS–Repressed bgl Operon in Evolution of Escherichia coli

In the enterobacterial species Escherichia coli and Salmonella enterica, expression of horizontally acquired genes with a higher than average AT content is repressed by the nucleoid-associated protein H-NS. A classical example of an H-NS–repressed locus is the bgl (aryl-β,D-glucoside) operon of E. coli. This locus is “cryptic,” as no laboratory growth conditions are known to relieve repression of bgl by H-NS in E. coli K12. However, repression can be relieved by spontaneous mutations. Here, we investigated the phylogeny of the bgl operon. Typing of bgl in a representative collection of E. coli demonstrated that it evolved clonally and that it is present in strains of the phylogenetic groups A, B1, and B2, while it is presumably replaced by a cluster of ORFans in the phylogenetic group D. Interestingly, the bgl operon is mutated in 20% of the strains of phylogenetic groups A and B1, suggesting erosion of bgl in these groups. However, bgl is functional in almost all B2 isolates and, in approximately 50% of them, it is weakly expressed at laboratory growth conditions. Homologs of bgl genes exist in Klebsiella, Enterobacter, and Erwinia species and also in low GC-content Gram-positive bacteria, while absent in E. albertii and Salmonella sp. This suggests horizontal transfer of bgl genes to an ancestral Enterobacterium. Conservation and weak expression of bgl in isolates of phylogenetic group B2 may indicate a functional role of bgl in extraintestinal pathogenic E. coli