Crystal structure of the di-haem cytochrome c peroxidase from Pseudomonas aeruginosa

AbstractBackground: Cytochrome c peroxidase from Pseudomonas aeruginosa (PsCCP) represents a new class of peroxidases which work without the need to create a semi-stable free radical for catalysis. The enzyme is located in the bacterial periplasm where its likely function is to provide protection against toxic peroxides. The soluble 323-residue single polypeptide chain contains two covalent c-type haems with very different properties: one of them is a low-potential (–330 mV) centre where hydrogen peroxide is reduced (the peroxidatic site); the other is a high-potential (+320 mV) centre which feeds electrons to the peroxidatic site from soluble electron-shuttle proteins such as cytochrome c and azurin.Results The crystal structure of the oxidized form of PsCCP has been determined to 2.4 å resolution by multiple isomorphous replacement, and refined to an R-factor of 19.2%. PsCCP is organized into two domains, both of them containing a covalent c-haem in a structure reminiscent of class 1 cytochromes c. The domains are related by a quasi-twofold axis. The domain interface holds a newly discovered calcium-binding site with an unusual set of ligands.Conclusion The likely function of the calcium site is to maintain the structural integrity of the enzyme and/or to modulate electron transfer between the two haem domains. The low-potential haem has two histidine axial ligands (His55 and His71) and the high-potential haem is ligated by His201 and Met275. There are no polar residues at the peroxidatic site in the inactive oxidized enzyme. The structure suggests that, in the half-reduced functional form of the enzyme, the low-potential haem has to shed His71 in order to make the enzyme catalytically competent. This process is likely to trigger a reorganization of the active site, and may introduce new residues into the haem pocket

Using clinical trial data and linked administrative health data to reduce the risk of adverse events associated with the uptake of newly released drugs by older Australians: a model process

BackgroundThe study was undertaken to evaluate the contribution of a process which uses clinical trial data plus linked de-identified administrative health data to forecast potential risk of adverse events associated with the use of newly released drugs by older Australian patients. MethodsThe study uses publicly available data from the clinical trials of a newly released drug to ascertain which patient age groups, gender, comorbidities and co-medications were excluded in the trials. It then uses linked de-identified hospital morbidity and medications dispensing data to investigate the comorbidities and co-medications of patients who suffer from the target morbidity of the new drug and who are the likely target population for the drug. The clinical trial information and the linked morbidity and medication data are compared to assess which patient groups could potentially be at risk of an adverse event associated with use of the new drug. ResultsApplying the model in a retrospective real-world scenario identified that the majority of the sample group of Australian patients aged 65 years and over with the target morbidity of the newly released COX-2-selective NSAID rofecoxib also suffered from a major morbidity excluded in the trials of that drug, indicating a substantial potential risk of adverse events amongst those patients. This risk was borne out in post-release morbidity and mortality associated with use of that drug. ConclusionsClinical trial data and linked administrative health data can together support a prospective assessment of patient groups who could be at risk of an adverse event if they are prescribed a newly released drug in the context of their age, gender, comorbidities and/or co-medications. Communication of this independent risk information to prescribers has the potential to reduce adverse events in the period after the release of the new drug, which is when the risk is greatest. Note: The terms \u27adverse drug reaction\u27 and \u27adverse drug event\u27 have come to be used interchangeably in the current literature. For consistency, the authors have chosen to use the wider term \u27adverse drug event\u27 (ADE). <br /

A change in temperature modulates defence to yellow (stripe) rust in wheat line UC1041 independently of resistance gene Yr36

Background Rust diseases are of major importance in wheat production worldwide. With the constant evolution of new rust strains and their adaptation to higher temperatures, consistent and durable disease resistance is a key challenge. Environmental conditions affect resistance gene performance, but the basis for this is poorly understood. Results Here we show that a change in day temperature affects wheat resistance to Puccinia striiformis f. sp tritici (Pst), the causal agent of yellow (or stripe) rust. Using adult plants of near-isogenic lines UC1041 +/- Yr36, there was no significant difference between Pst percentage uredia coverage in plants grown at day temperatures of 18°C or 25°C in adult UC1041 + Yr36 plants. However, when plants were transferred to the lower day temperature at the time of Pst inoculation, infection increased up to two fold. Interestingly, this response was independent of Yr36, which has previously been reported as a temperature-responsive resistance gene as Pst development in adult UC1041 -Yr36 plants was similarly affected by the plants experiencing a temperature reduction. In addition, UC1041 -Yr36 plants grown at the lower temperature then transferred to the higher temperature were effectively resistant and a temperature change in either direction was shown to affect Pst development up to 8 days prior to inoculation. Results for seedlings were similar, but more variable compared to adult plants. Enhanced resistance to Pst was observed in seedlings of UC1041 and the cultivar Shamrock when transferred to the higher temperature. Resistance was not affected in seedlings of cultivar Solstice by a temperature change in either direction. Conclusions Yr36 is effective at 18°C, refining the lower range of temperature at which resistance against Pst is conferred compared to previous studies. Results reveal previously uncharacterised defence temperature sensitivity in the UC1041 background which is caused by a change in temperature and independently of Yr36. This novel phenotype is present in some cultivars but absent in others, suggesting that Pst defence may be more stable in some cultivars than others when plants are exposed to varying temperatures

Necrosis and ethylene-inducing-like peptide patterns from crop pathogens induce differential responses within seven brassicaceous species

Translational research is required to advance fundamental knowledge on plant immunity towards application in crop improvement. Recognition of microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) triggers a first layer of immunity in plants. The broadly occurring family of necrosis- and ethylene-inducing peptide 1 (NEP1)-like proteins (NLPs) contains immunogenic peptide patterns that are recognized by a number of plant species. Arabidopsis can recognize NLPs by the pattern recognition receptor AtRLP23 and its co-receptors SOBIR1, BAK1, and BKK1, leading to induction of defence responses including the production of reactive oxygen species (ROS) and elevation of intracellular [Ca2+]. However, little is known about NLP perception in Brassica crop species. Within 12 diverse accessions for each of six Brassica crop species, we demonstrate variation in response to Botrytis cinerea NLP BcNEP2, with Brassica oleracea (CC genome) being nonresponsive and only two Brassica napus cultivars responding to BcNEP2. Peptides derived from four fungal pathogens of these crop species elicited responses similar to BcNEP2 in B. napus and Arabidopsis. Induction of ROS by NLP peptides was strongly reduced in Atrlp23, Atsobir1 and Atbak1-5 Atbkk1-1 mutants, confirming that recognition of Brassica pathogen NLPs occurs in a similar manner to that of HaNLP3 from Hyaloperonospora arabidopsidis in Arabidopsis. In silico analysis of the genomes of two B. napus accessions showed similar presence of homologues for AtBAK1, AtBKK1 and AtSOBIR1 but variation in the organization of AtRLP23 homologues. We could not detect a strong correlation between the ability to respond to NLP peptides and resistance to B. cinerea

Coevolution between a Family of Parasite Virulence Effectors and a Class of LINE-1 Retrotransposons

Parasites are able to evolve rapidly and overcome host defense mechanisms, but the molecular basis of this adaptation is poorly understood. Powdery mildew fungi (Erysiphales, Ascomycota) are obligate biotrophic parasites infecting nearly 10,000 plant genera. They obtain their nutrients from host plants through specialized feeding structures known as haustoria. We previously identified the AVRk1 powdery mildew-specific gene family encoding effectors that contribute to the successful establishment of haustoria. Here, we report the extensive proliferation of the AVRk1 gene family throughout the genome of B. graminis, with sequences diverging in formae speciales adapted to infect different hosts. Also, importantly, we have discovered that the effectors have coevolved with a particular family of LINE-1 retrotransposons, named TE1a. The coevolution of these two entities indicates a mutual benefit to the association, which could ultimately contribute to parasite adaptation and success. We propose that the association would benefit 1) the powdery mildew fungus, by providing a mechanism for amplifying and diversifying effectors and 2) the associated retrotransposons, by providing a basis for their maintenance through selection in the fungal genome

Positive Selection Differs between Protein Secondary Structure Elements in Drosophila

Different protein secondary structure elements have different physicochemical properties and roles in the protein, which may determine their evolutionary flexibility. However, it is not clear to what extent protein structure affects the way Darwinian selection acts at the amino acid level. Using phylogeny-based likelihood tests for positive selection, we have examined the relationship between protein secondary structure and selection across six species of Drosophila. We find that amino acids that form disordered regions, such as random coils, are far more likely to be under positive selection than expected from their proportion in the proteins, and residues in helices and β-structures are subject to less positive selection than predicted. In addition, it appears that sites undergoing positive selection are more likely than expected to occur close to one another in the protein sequence. Finally, on a genome-wide scale, we have determined that positively selected sites are found more frequently toward the gene ends. Our results demonstrate that protein structures with a greater degree of organization and strong hydrophobicity, represented here as helices and β-structures, are less tolerant to molecular adaptation than disordered, hydrophilic regions, across a diverse set of proteins

Silencing of Aphid Genes by dsRNA Feeding from Plants

RNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms, including hemipteran insects such as aphids. Previous work has shown that RNAi-mediated knockdown of pea aphid (Acyrthosiphon pisum) genes can be achieved through direct injection of double-stranded RNA (dsRNA) or small-interfering RNAs (siRNA) into the pea aphid hemolymph or by feeding these insects on artificial diets containing the small RNAs.In this study, we have developed the plant-mediated RNAi technology for aphids to allow for gene silencing in the aphid natural environment and minimize handling of these insects during experiments. The green peach aphid M. persicae was selected because it has a broad plant host range that includes the model plants Nicotiana benthamiana and Arabidopsis thaliana for which transgenic materials can relatively quickly be generated. We targeted M. persicae Rack1, which is predominantly expressed in the gut, and M. persicae C002 (MpC002), which is predominantly expressed in the salivary glands. The aphids were fed on N. benthamiana leaf disks transiently producing dsRNA corresponding to these genes and on A. thaliana plants stably producing the dsRNAs. MpC002 and Rack-1 expression were knocked down by up to 60% on transgenic N. benthamiana and A. thaliana. Moreover, silenced M. persicae produced less progeny consistent with these genes having essential functions.Similar levels of gene silencing were achieved in our plant-mediated RNAi approach and published silencing methods for aphids. Furthermore, the N. benthamiana leaf disk assay can be developed into a screen to assess which genes are essential for aphid survival on plants. Our results also demonstrate the feasibility of the plant-mediated RNAi approach for aphid control

Differential expression of genes possibly involved in incompatible interaction between barley and powdery mildew

Bu çalışma, 28 Eylül-01 Ekim 2011 tarihleri arasında İstanbul[Türkiye]’da düzenlenen European Biotechnology Congress’da bildiri olarak sunulmuştur.European Biotechnol Themat Network Asso

Differential expression of genes possibly involved in incompatible interaction between barley and powdery mildew

The various domains of a plant disease resistance protein from wheat were found to be interacting with yeast proteins when screened via yeast two hybrid analyses. These genes are considered to play roles in disease resistance response. Thus, the expression levels in Mla3 mediated Powdery Mildew (Blumeria graminis f.sp. hordei, Bgh) disease resistance in barley were determined. The barley homologs of ARD1, CPR7, CSE1, GCN2 and SRP72, were partially cloned and sequenced. Their differential expression was confirmed using qRT-RCR at 6 hpi, 12 hpi, 24 hpi and 72 hpi upon incompatible Bgh infection in a resistant barley line (Pallas-02). All of the genes except HvCPR7 showed maximum expression levels at 12 hpi and gradually dropped at 24 and 72 hpi. On the other hand, HvCPR7 showed highest induction at 6 hpi, indicating that HvCRP7 is an even earlier responding gene. Most of the human homologues of these genes have been widely studied and found to have roles in apoptosis. As apoptosis is very important in the plant disease resistance response, qRT-PCR was performed to check if the genes are induced in resistant barley after powdery mildew inoculation. All the five genes were found to be differentially expressed in incompatible interactions between barley and powdery mildew