A new set of differentially expressed signaling genes is early expressed in coffee leaf rust race II incompatible interaction

New races of coffee rust are overcoming resistance genes available in germplasm and cultivated cultivars and bringing recently some coffee-producing countries in severe economic challenge. The objective of this study was to identify the genes that are linked to host resistance to the major coffee rust race II. In our study, we have identified and studied a segregating population that has a single monogenic resistant gene to coffee rust. Coffee leaves of parents, resistant, and susceptible genotypes of the F₂ generation plants were inoculated with pathogen spores. A differential analysis was performed by combined cDNA-AFLP and bulk segregant analysis (BSA) in pooled samples collected 48 and 72 h postinoculation, increasing the selectiveness for differential gene expression. Of 108 differential expressed genes, between 33,000 gene fragments analyzed, 108 differential expressed genes were identified in resistant plants. About 20 and 22 % of these resistant-correlated genes are related to signaling and defense genes, respectively. Between signaling genes, the major subclass corresponds to receptor and resistant homolog genes, like nucleotide-binding site leucine-rich repeat (NBS-LRR), Pto-like, RLKs, Bger, and RGH1A, all not previously described in coffee rust responses. The second major subclass included kinases, where two mitogen-activated kinases (MAPK) are identified. Further gene expression analysis was performed for 21 selected genes by real-time PCR gene expression analysis at 0, 12, 24, 48, and 72 h postinoculation. The expression of genes involved in signaling and defense was higher at 24 and 72 h after inoculation, respectively. The NBS-LRR was the more differentially expressed gene between the signaling genes (four times more expressed in the resistant genotype), and thraumatin (PR5) was the more expressed between all genes (six times more expressed). Multivariate analysis reinforces the significance of the temporal separation of identified signaling and defense genes: early expression of signaling genes support the hypothesis that higher expression of the signaling components up regulates the defense genes identified. Additionally the increased gene expression of these two gene sets is associated with a single monogenic resistance trait to to leaf coffee rust in the interaction characterized here.     

Light control of the respiration of exogenous glycerol in the red macroalga Grateloupia doryphora

Heterotrophic activity in macroalgae has been little studied, but the red macroalga Grateloupia doryphora is known to grow in light at a higher rate in a glycerol-containing medium than in seawater. The effects of 0·1 M exogenous glycerol in seawater (SW90-gly) on the respiration rate of G. doryphora and the role played by light were investigated. The algae pretreated for 2 h in the light and in SW90-gly evolved oxygen and fixed carbon dioxide (H¹⁴CO₃ ^?), but also evolved radioactive ¹⁴CO₂ from [¹⁴C]glycerol. The rate of oxygen evolution was lower than that of samples in seawater, due to a high respiration rate and/or a partial inhibition of photosynthesis induced by glycerol. In contrast, the rate of inorganic carbon fixation was higher in SW90-gly than in control samples in seawater, suggesting that non-photosynthetic patterns were operating. In darkness, after pretreatment in the light in SW90-gly, samples showed a high oxygen uptake rate just after the light was turned off. Twenty minutes of darkness were enough to decrease this high respiration rate to that of samples in seawater. The oxygen uptake observed in all experiments with glycerol was mitochondrial as it was inhibited by potassium cyanide and salicylhydroxamic acid (SHAM). Pretreatment of samples in the light in SW90-gly with the photosynthetic inhibitor DCMU did not inhibit ensuing dark respiration, thus providing evidence for a non-photosynthetic effect of the light. The highest dark respiration rate was observed after the samples were pretreated in monochromatic blue light in glycerol-containing media.     

An Efficient Strategy for Small-Scale Screening and Production of Archaeal Membrane Transport Proteins in Escherichia coli

Background

Membrane proteins play a key role in many fundamental cellular processes such as transport of nutrients, sensing of environmental signals and energy transduction, and account for over 50% of all known drug targets. Despite their importance, structural and functional characterisation of membrane proteins still remains a challenge, partially due to the difficulties in recombinant expression and purification. Therefore the need for development of efficient methods for heterologous production is essential.

Methodology/Principal Findings

Fifteen integral membrane transport proteins from Archaea were selected as test targets, chosen to represent two superfamilies widespread in all organisms known as the Major Facilitator Superfamily (MFS) and the 5-Helix Inverted Repeat Transporter superfamily (5HIRT). These proteins typically have eleven to twelve predicted transmembrane helices and are putative transporters for sugar, metabolite, nucleobase, vitamin or neurotransmitter. They include a wide range of examples from the following families: Metabolite-H⁺-symporter; Sugar Porter; Nucleobase-Cation-Symporter-1; Nucleobase-Cation-Symporter-2; and neurotransmitter-sodium-symporter. Overproduction of transporters was evaluated with three vectors (pTTQ18, pET52b, pWarf) and two Escherichia coli strains (BL21 Star and C43 (DE3)). Thirteen transporter genes were successfully expressed; only two did not express in any of the tested vector-strain combinations. Initial trials showed that seven transporters could be purified and six of these yielded quantities of ≥ 0.4 mg per litre suitable for functional and structural studies. Size-exclusion chromatography confirmed that two purified transporters were almost homogeneous while four others were shown to be non-aggregating, indicating that they are ready for up-scale production and crystallisation trials.

Conclusions/Significance

Here, we describe an efficient strategy for heterologous production of membrane transport proteins in E. coli. Small-volume cultures (10 mL) produced sufficient amount of proteins to assess their purity and aggregation state. The methods described in this work are simple to implement and can be easily applied to many more membrane proteins.     

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 February 2013–31 March 2013

This article documents the addition of 142 microsatellite marker loci to the Molecular Ecology Resources database. Loci were developed for the following species: Agriophyllum squarrosum, Amazilia cyanocephala, Batillaria attramentaria, Fungal strain CTeY1 (Ascomycota), Gadopsis marmoratus, Juniperus phoenicea subsp. turbinata, Liriomyza sativae, Lupinus polyphyllus, Metschnikowia reukaufii, Puccinia striiformis and Xylocopa grisescens. These loci were cross‐tested on the following species: Amazilia beryllina, Amazilia candida, Amazilia rutila, Amazilia tzacatl, Amazilia violiceps, Amazilia yucatanensis, Campylopterus curvipennis, Cynanthus sordidus, Hylocharis leucotis, Juniperus brevifolia, Juniperus cedrus, Juniperus osteosperma, Juniperus oxycedrus, Juniperus thurifera, Liriomyza bryoniae, Liriomyza chinensis, Liriomyza huidobrensis and Liriomyza trifolii.     

Assessing the Effects of Aedes aegypti kdr Mutations on Pyrethroid Resistance and Its Fitness Cost

Pyrethroids are the most used insecticide class worldwide. They target the voltage gated sodium channel (Na_V), inducing the knockdown effect. In Aedes aegypti, the main dengue vector, the AaNa_V substitutions Val1016Ile and Phe1534Cys are the most important knockdown resistance (kdr) mutations. We evaluated the fitness cost of these kdr mutations related to distinct aspects of development and reproduction, in the absence of any other major resistance mechanism. To accomplish this, we initially set up 68 crosses with mosquitoes from a natural population. Allele-specific PCR revealed that one couple, the one originating the CIT-32 strain, had both parents homozygous for both kdr mutations. However, this pyrethroid resistant strain also presented high levels of detoxifying enzymes, which synergistically account for resistance, as revealed by biological and biochemical assays. Therefore, we carried out backcrosses between CIT-32 and Rockefeller (an insecticide susceptible strain) for eight generations in order to bring the kdr mutation into a susceptible genetic background. This new strain, named Rock-kdr, was highly resistant to pyrethroid and presented reduced alteration of detoxifying activity. Fitness of the Rock-kdr was then evaluated in comparison with Rockefeller. In this strain, larval development took longer, adults had an increased locomotor activity, fewer females laid eggs, and produced a lower number of eggs. Under an inter-strain competition scenario, the Rock-kdr larvae developed even slower. Moreover, when Rockefeller and Rock-kdr were reared together in population cage experiments during 15 generations in absence of insecticide, the mutant allele decreased in frequency. These results strongly suggest that the Ae. aegypti kdr mutations have a high fitness cost. Therefore, enhanced surveillance for resistance should be priority in localities where the kdr mutation is found before new adaptive alleles can be selected for diminishing the kdr deleterious effects.     

Gene Expression Profiling Specifies Chemokine,Mitochondrial and Lipid Metabolism Signatures in Leprosy

Herein, we performed microarray experiments in Schwann cells infected with live M. leprae and identified novel differentially expressed genes (DEG) in M. leprae infected cells. Also, we selected candidate genes associated or implicated with leprosy in genetic studies and biological experiments. Forty-seven genes were selected for validation in two independent types of samples by multiplex qPCR. First, an in vitro model using THP-1 cells was infected with live Mycobacterium leprae and M. bovis bacillus Calmette-Guérin (BCG). In a second situation, mRNA obtained from nerve biopsies from patients with leprosy or other peripheral neuropathies was tested. We detected DEGs that discriminate M. bovis BCG from M. leprae infection. Specific signatures of susceptible responses after M. leprae infection when compared to BCG lead to repression of genes, including CCL2, CCL3, IL8 and SOD2. The same 47-gene set was screened in nerve biopsies, which corroborated the down-regulation of CCL2 and CCL3 in leprosy, but also evidenced the down-regulation of genes involved in mitochondrial metabolism, and the up-regulation of genes involved in lipid metabolism and ubiquitination. Finally, a gene expression signature from DEG was identified in patients confirmed of having leprosy. A classification tree was able to ascertain 80% of the cases as leprosy or non-leprous peripheral neuropathy based on the expression of only LDLR and CCL4. A general immune and mitochondrial hypo-responsive state occurs in response to M. leprae infection. Also, the most important genes and pathways have been highlighted providing new tools for early diagnosis and treatment of leprosy.     

Prognostic and Predictive Significance of MYC and KRAS Alterations in Breast Cancer from Women Treated with Neoadjuvant Chemotherapy

Breast cancer is a complex disease, with heterogeneous clinical evolution. Several analyses have been performed to identify the risk factors for breast cancer progression and the patients who respond best to a specific treatment. We aimed to evaluate whether the hormone receptor expression, HER2 and MYC genes and their protein status, and KRAS codon 12 mutations may be prognostic or predictive biomarkers of breast cancer. Protein, gene and mutation status were concomitantly evaluated in 116 breast tumors from women who underwent neoadjuvant chemotherapy with doxorubicin plus cyclophosphamide. We observed that MYC expression was associated with luminal B and HER2 overexpression phenotypes compared to luminal A (p<0.05). The presence of MYC duplication or polysomy 8, as well as KRAS mutation, were also associated with the HER2 overexpression subtype (p<0.05). MYC expression and MYC gain were more frequently observed in early-onset compared to late-onset tumors (p<0.05). KRAS mutation was a risk factor of grade 3 tumors (p<0.05). A multivariate logistic regression demonstrated that MYC amplification defined as MYC/nucleus ratio of ≥2.5 was a protective factor for chemotherapy resistance. On the other hand, age and grade 2 tumors were a risk factor. Additionally, luminal B, HER2 overexpression, and triple-negative tumors presented increased odds of being resistant to chemotherapy relative to luminal A tumors. Thus, breast tumors with KRAS codon 12 mutations seem to present a worse prognosis. Additionally, MYC amplification may help in the identification of tumors that are sensitive to doxorubicin plus cyclophosphamide treatment. If confirmed in a large set of samples, these markers may be useful for clinical stratification and prognosis.     

Optimum bacteriocin production by Lactobacillus plantarum 17.2b requires absence of NaCl and apparently follows a mixed metabolite kinetics

Bacteriocins from lactic acid bacteria are ribosomally synthesized anti-microbial compounds that may find applications from food preservation to healthcare. Food preservation, using in situ bacteriocin production is the most obvious and simple. Frequently, the best conditions for bacteriocin production are those prevailing during food fermentation but a better understanding of the relationship between growth and bacteriocin production is required. In this work, we evaluate the effects of some environmental factors on bacteriocin production by Lactobacillus plantarum 17.2b. A first screening design showed that NaCl, temperature, pH and the type and concentration of carbon and nitrogen sources were most influents. A moderate stimulatory effect of ethanol and oleuropein was also registered. Two consecutive central composite designs were used to examine the effect of the selected variables and to compute its optimum. The evolution of changes produced by the alterations in environmental factors was further examined trough perturbation plots. Bacteriocin production by L. plantarum 17.2b was very sensitive to environmental conditions and uncoupled from growth. Maximum production required suboptimal growth temperatures, pH values above growth's optimum and no NaCl. A preliminary approach to kinetics showed that bacteriocin production by this strain apparently follows mixed metabolite kinetics.