RNAi-Mediated Suppression of Laccase2 Impairs Cuticle Tanning and Molting in the Cotton Boll Weevil (Anthonomus grandis).

The cotton boll weevil, Anthonomus grandis, is the most economically important pest of cotton in Brazil. Pest management programs focused on A. grandis are based mostly on the use of chemical insecticides, which may cause serious ecological impacts. Furthermore, A. grandis has developed resistance to some insecticides after their long-term use. Therefore, alternative control approaches that are more sustainable and have reduced environmental impacts are highly desirable to protect cotton crops from this destructive pest. RNA interference (RNAi) is a valuable reverse genetics tool for the investigation of gene function and has been explored for the development of strategies to control agricultural insect pests. This study aimed to evaluate the biological role of the Laccase2 (AgraLac2) gene in A. grandis and its potential as an RNAi target for the control of this insect pest. We found that AgraLac2 is expressed throughout the development of A. grandis with significantly higher expression in pupal and adult developmental stages. In addition, the immunolocalization of the AgraLac2 protein in third-instar larvae using specific antibodies revealed that AgraLac2 is distributed throughout the epithelial tissue, the cuticle and the tracheal system. We also verified that the knockdown of AgraLac2 in A. grandis resulted in an altered cuticle tanning process, molting defects and arrested development. Remarkably, insects injected with dsAgraLac2 exhibited defects in cuticle hardening and pigmentation. As a consequence, the development of dsAgraLac2-treated insects was compromised, and in cases of severe phenotypic defects, the insects subsequently died. On the contrary, insects subjected to control treatments did not show any visible phenotypic defects in cuticle formation and successfully molted to the pupal and adult stages. Taken together, our data indicate that AgraLac2 is involved in the cuticle tanning process in A. grandis and may be a promising target for the development of RNAi-based technologies.

Dissecting the Subcellular Localization, Intracellular Trafficking, Interactions, Membrane Association, and Topology of Citrus Leprosis Virus C Proteins.

Citrus leprosis (CL) is a re-emergent viral disease affecting citrus crops in the Americas, and citrus leprosis virus C (CiLV-C), belonging to the genus Cilevirus, is the main pathogen responsible for the disease. Despite the economic importance of CL to the citrus industry, very little is known about the performance of viral proteins. Here, we present a robust in vivo study around functionality of p29, p15, p61, MP, and p24 CiLV-C proteins in the host cells. The intracellular sub-localization of all those viral proteins in plant cells are shown, and their co-localization with the endoplasmic reticulum (ER), Golgi complex (GC) (p15, MP, p61 and p24), actin filaments (p29, p15 and p24), nucleus (p15), and plasmodesmata (MP) are described. Several features are disclosed, including i) ER remodeling and redistribution of GC apparatus, ii) trafficking of the p29 and MP along the ER network system, iii) self-interaction of the p29, p15, and p24 and hetero-association between p29-p15, p29-MP, p29-p24, and p15-MP proteins in vivo. We also showed that all proteins are associated with biological membranes; whilst p15 is peripherally associated, p29, p24, and MP are integrally bound to cell membranes. Furthermore, while p24 exposes an N-cytoplasm-C-lumen topology, p29, and p15 are oriented toward the cytoplasmic face of the biological membrane. Based on our findings, we discuss the possible performance of each protein in the context of infection and a hypothetical model encompassing the virus spread and sites for replication and particle assembly is suggested.

Review: Potential biotechnological assets related to plant immunity modulation applicable in engineering disease-resistant crops.

This review emphasizes the biotechnological potential of molecules implicated in the different layers of plant immunity, including, pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), effector-triggered susceptibility (ETS), and effector-triggered immunity (ETI) that can be applied in the development of disease-resistant genetically modified (GM) plants. These biomolecules are produced by pathogens (viruses, bacteria, fungi, oomycetes) or plants during their mutual interactions. Biomolecules involved in the first layers of plant immunity, PTI and ETS, include inhibitors of pathogen cell-wall-degrading enzymes (CWDEs), plant pattern recognition receptors (PRRs) and susceptibility (S) proteins, while the ETI-related biomolecules include plant resistance (R) proteins. The biomolecules involved in plant defense PTI/ETI responses described herein also include antimicrobial peptides (AMPs), pathogenesis-related (PR) proteins and ribosome-inhibiting proteins (RIPs), as well as enzymes involved in plant defensive secondary metabolite biosynthesis (phytoanticipins and phytoalexins). Moreover, the regulation of immunity by RNA interference (RNAi) in GM disease-resistant plants is also considered. Therefore, the present review does not cover all the classes of biomolecules involved in plant innate immunity that may be applied in the development of disease-resistant GM crops but instead highlights the most common strategies in the literature, as well as their advantages and disadvantages.

Transgenic cotton expressing Cry10Aa toxin confers high resistance to the cotton boll weevil.

Genetically modified (GM) cotton plants that effectively control cotton boll weevil (CBW), which is the most destructive cotton insect pest in South America, are reported here for the first time. This work presents the successful development of a new GM cotton with high resistance to CBW conferred by Cry10Aa toxin, a protein encoded by entomopathogenic Bacillus thuringiensis (Bt) gene. The plant transformation vector harbouring cry10Aa gene driven by the cotton ubiquitination-related promoter uceA1.7 was introduced into a Brazilian cotton cultivar by biolistic transformation. Quantitative PCR (qPCR) assays revealed high transcription levels of cry10Aa in both T0 GM cotton leaf and flower bud tissues. Southern blot and qPCR-based 2-ΔΔCt analyses revealed that T0 GM plants had either one or two transgene copies. Quantitative and qualitative analyses of Cry10Aa protein expression showed variable protein expression levels in both flower buds and leaves tissues of T0 GM cotton plants, ranging from approximately 3.0 to 14.0 µg g-1 fresh tissue. CBW susceptibility bioassays, performed by feeding adults and larvae with T0 GM cotton leaves and flower buds, respectively, demonstrated a significant entomotoxic effect and a high level of CBW mortality (up to 100%). Molecular analysis revealed that transgene stability and entomotoxic effect to CBW were maintained in T1 generation as the Cry10Aa toxin expression levels remained high in both tissues, ranging from 4.05 to 19.57 µg g-1 fresh tissue, and the CBW mortality rate remained around 100%. In conclusion, these Cry10Aa GM cotton plants represent a great advance in the control of the devastating CBW insect pest and can substantially impact cotton agribusiness.

The recombinant pea defensin Drr230a is active against impacting soybean and cotton pathogenic fungi from the genera Fusarium, Colletotrichum and Phakopsora.

Plant defensins are antifungal peptides produced by the innate immune system plants developed to circumvent fungal infection. The defensin Drr230a, originally isolated from pea, has been previously shown to be active against various entomopathogenic and phytopathogenic fungi. In the present study, the activity of a yeast-expressed recombinant Drr230a protein (rDrr230a) was tested against impacting soybean and cotton fungi. First, the gene was subcloned into the yeast expression vector pPICZαA and expressed in Pichia pastoris. Resulting rDrr230a exhibited in vitro activity against fungal growth and spore germination of Fusarium tucumaniae, which causes soybean sudden death syndrome, and against Colletotrichum gossypii var. cephalosporioides, which causes cotton ramulosis. The rDrr230a IC50 corresponding to inhibition of fungal growth of F. tucumaniae and C. gossypii var. cephalosporioides was 7.67 and 0.84 µM, respectively, demonstrating moderate activity against F. tucumaniae and high potency against C. gossypii var. cephalosporioides. Additionally, rDrr230a at 25 ng/µl (3.83 µM) resulted in 100 % inhibition of spore germination of both fungi, demonstrating that rDrr230a affects fungal development since spore germination. Moreover, rDrr230a at 3 µg/µl (460.12 µM) inhibited 100 % of in vitro spore germination of the obligatory biotrophic fungus Phakopsora pachyrhizi, which causes Asian soybean rust. Interestingly, rDrr230a substantially decreased the severity of Asian rust, as demonstrated by in planta assay. To our knowledge, this is the first report of a plant defensin active against an obligatory biotrophic phytopathogenic fungus. Results revealed the potential of rDrr230a as a candidate to be used in plant genetic engineering to control relevant cotton and soybean fungal diseases.

The effect of polymerization mode on monomer conversion, free radical entrapment, and interaction with hydroxyapatite of commercial self-adhesive cements.

OBJECTIVES: This study evaluated the degree of conversion, the free radical entrapment, and the chemical interaction of self-adhesive resin cements mixed with pure hydroxyapatite, as a function of the polymerization activation mode among a variety of commercial self-adhesive cements. MATERIALS AND METHODS: Four cements (Embrace WetBond, MaxCem Elite, Bifix SE, and RelyX U200) were mixed, combined with hydroxyapatite, dispensed into molds, and distributed into three groups, according to polymerization protocols: IP (photoactivation for 40s); DP (delayed photoactivation, 10 min self-curing plus 40s light-activated); and CA (chemical activation, no light exposure). Infrared (IR) spectra were obtained and monomer conversion (%) was calculated by comparing the aliphatic-to-aromatic IR absorption peak ratio before and after polymerization (n=10). The free radical entrapment values of the resin cements were characterized using Electron Paramagnetic Resonance (EPR) and the concentration of spins (number of spins/mass) calculated (n=3). Values were compared using two-way ANOVA and Tukey's post-hoc test (α=5%). X-ray diffraction (XRD) characterized the crystallinity of hydroxyapatite as a function of the chemical interactions with the resin cements. RESULTS: The tested parameters varied as a function of resin cement and polymerization protocol. Embrace WetBond and RelyX U200 demonstrated dependence on photoactivation (immediate or delayed), whereas MaxCem Elite exhibited dependence on the chemical activation mode. Bifix SE presented the best balance based on the parameters analyzed, irrespective of the activation protocol. CONCLUSIONS: Choice of polymerization protocol affects the degree of conversion, free radical entrapment, and the chemical interaction between hydroxyapatite and self-adhesive resin cement mixtures.

Domestic violence against elderly with disability.

Abuse against elders with disabilities is a growing problem as the world population ages. Though they require mandatory reporting, these cases are most frequently not detected or not reported by health professionals for a variety of reasons, including the difficulty of making an accurate diagnosis. By performing a retrospective analysis of alleged domestic violence cases against elders with moderate or severe disability, presented to medical forensic examination at the North Branch of the National Institute of Legal Medicine and Forensic Sciences of Portugal, in Porto, between 2005 and 2013 (n = 70), we aimed to improve our knowledge of some demographic and forensic characteristics of these cases as well as improve their detection and prevention. The most frequently reported type of abuse was physical (86%), allegedly perpetrated by male abusers (63%) living with their victims (90%), who were most commonly their children (47%) or partners (49%; when victims are married). The victims were most frequently female (63%) who had motor disabilities (49%) and presented a history of previous episodes of abuse in 74% of cases; however, only 28% were previously reported. The physical consequences were most frequently minor injuries (95%) with permanent consequences (scars) in only 6.8% of the cases. The injuries were multiple in the majority of the cases (64%), and the preferred locations were the head and neck (75%). Elderly females with motor disabilities appear to have a greatest risk of domestic violence, which translates, most frequently, into multiple injuries that are mainly in the head and neck.

Compatibility between silorane adhesive and simplified methacrylate-based adhesive systems.

The purpose of this study was to evaluate the impact of replacing P90 primer with simplified adhesive systems (with a conventional, two-step adhesive or a self-etching, one-step adhesive) on bond strength to dentin in Class I restorations. The interfaces were also analyzed using confocal microscopy by adding a fluorophore to the adhesive components and SEM using silver nitrate for nanoleakage investigation. X-ray diffraction (XRD) characterized the chemical interaction of the adhesives. Failure mode and nanoleakage varied among groups. Characteristic micromorphology and higher nanoleakage were noted for silorane combinations. On the other hand, no signs of phase crystallization in the silorane adhesive combinations were noted. Replacing the silorane primer with simplified adhesives proved successful as the modified systems provided bonding to dentin comparable to that of the unmodified silorane systems. However, the dedicated adhesive exhibited signs of degradation immediately after application, which may impact the longevity of restorations in short periods.

Hydrolytic degradation of silorane- and methacrylate-based composite restorations: Evaluation of push-out strength and marginal adaptation.

OBJECTIVES: This study compared the hydrolytic degradation of composite restorations based on methacrylate and silorane systems regarding bond strength and marginal adaptation. Materials and methods. Sixty bovine incisors were ground flat to obtain a 2-mm thick slice in which conical preparations were made. The specimens were randomly distributed into four groups (n = 15) according to the restorative system (silorane-Filtek LS/P90 adhesive; methacrylate-Filtek P60/Adper Easy Bond) and the degradation protocol (control: immediate evaluation; hydrolytic degradation: 6 months storage in water at 37°C). Marginal adaptation was evaluated using a dye staining technique. Digital images of the stained gaps were obtained to calculate the marginal gap (%), the ratio between the stained margins and the total length of the margin. Push-out bond strength test was conducted (0.5 mm/min). Marginal adaptation data was submitted to Kruskal-Wallis test and the bond strength data to two-way ANOVA/Tukey's test (α = 0.05). Results. The marginal adaptation was neither affected by the restorative system nor by the degradation protocol, although the number of perfect sealed reduced after 6 months. No significance was observed among the groups. No significance was noted between the silorane- and the methacrylate-based restorations for immediate bond strength. After the hydrolytic degradation, the silorane system showed higher bond strength then the methacrylate restorations. Conclusion. The silorane and methacrylate restorative systems produce restorations with similar immediate interfacial quality and 6 months of water storage does not cause significant bonding degradation for both systems. The silorane restorations show an increase in the bond strength after 6 months.

Effects of curing protocols on fluid kinetics and hardness of resin cements.

The effects of polymerization protocols on water sorption/solubility, the diffusion coefficient (D), the flux (J), and the hardness (KH) of two resin cements were evaluated. The materials were manipulated and divided into three groups (n=6) according to the curing protocol: PA=photoactivation (40 s); DP=delayed photoactivation (10 min self-curing plus 40 s photoactivated); CA=chemical activation. After desiccation, the specimens were weighed, stored in water (37ºC), evaluated over 28-days, and hardness recorded. Chemical activation resulted in lower net water uptake, D, and J for RelyX ARC (RX). For Variolink II (VL), CA yielded equivalent D and lower J; however, photoactivation resulted in lower net water uptake. Hardness of VL was less affected by the water storage, irrespective of the polymerization protocol. Considering the water diffusion parameters, VL demonstrated immediate photoactivation dependence; for RX, a chemical activation. Different polymerization protocols affect the fluid kinetics and the hardness of the resin cements tested.

Caracterização molecular e variabilidade genética de acessos elite de mandioca para fins industriais / Molecular characterization and genetic variability of elite cassava accessions for industrial purpose

Marcadores moleculares são ferramentas úteis na caracterização molecular de acessos de mandioca, em razão de apresentarem elevada capacidade de detecção das informações contidas no genoma. O objetivo deste trabalho foi caracterizar, por meio de marcadores RAPD, 20 acessos de mandioca para fins industriais conservados no Banco Regional de Germoplasma de Mandioca do Cerrado (BGMC). Em laboratório, os acessos foram avaliados por meio de marcadores RAPD, sendo posteriormente estimada a matriz de similaridade genética entre os acessos, por meio do índice de Jaccard. A análise, feita através de 11 iniciadores, gerou um total de 120 marcadores RAPD, dos quais 74 (62 por cento) foram polimórficos, revelando a presença de elevada variabilidade genética no grupo de acessos avaliados. A análise de agrupamento revelou a formação de apenas um agrupamento forte, formado pelos acessos BGMC 1130, BGMC 788, BGMC 1270 e BGMC 1107, o que indica que, no melhoramento genético de mandioca, não devem ser priorizadas hibridações entre esses acessos, sob pena de efeitos de endogamia. Por sua vez, o acesso BGMC 436 foi o mais divergente em relação aos demais e, como expressa elevado potencial produtivo na região do Cerrado do Brasil Central, representa boa opção como genitor para o melhoramento de mandioca para essa região. O estudo comprovou que os marcadores RAPD são eficientes na determinação da variabilidade genética dos acessos avaliados e que neste grupo existe elevada variabilidade genética passível de ser utilizada no melhoramento genético.

Molecular markers are useful tools for the molecular characterization of cassava accessions since they present high capacity to detect information within the genome. The aim of this research was to characterize through RAPD molecular markers, 20 industrial cassava accessions conserved in the Cerrado Cassava Regional Germoplasm Bank ("Banco Regional de Germoplasma de Mandioca do Cerrado"-BGMC). Upon laboratory conditions, the accessions were evaluated though RAPD markers, being afterwards estimated by the matrix of genetic similarity among the accessions, through the Jaccard index. The analyses through 11 primers generated a total of 120 RAPD markers, among which 74 (62 percent) were polymorphic, revealing the presence of high genetic variability within the group of evaluated accessions. The clustering analyses revealed the formation of a single strong group, comprised of the accessions BGMC 1130, BGMC 788, BGMC 1270 and BGMC 1107, which indicates that the cassava genetic breeding hybridizations among these accessions should not be prioritized, upon risk of endogamy effects. The accession BGMC 436 was the most divergent as compared to the others and, as it expresses high productive potential within the Central Brazil Cerrado, it represents a good option as parent for the cassava breeding in this region. The study confirmed that the RAPD markers are efficient for the determination of genetic variability among evaluated accessions and that within the group there is high genetic variability useful for the plant breeding.

Susceptibility of Anthonomus grandis (cotton boll weevil) and Spodoptera frugiperda (fall armyworm) to a cry1ia-type toxin from a Brazilian Bacillus thuringiensis strain.

Different isolates of the soil bacterium Bacillus thuringiensis produce multiple crystal (Cry) proteins toxic to a variety of insects, nematodes and protozoans. These insecticidal Cry toxins are known to be active against specific insect orders, being harmless to mammals, birds, amphibians, and reptiles. Due to these characteristics, genes encoding several Cry toxins have been engineered in order to be expressed by a variety of crop plants to control insectpests. The cotton boll weevil, Anthonomus grandis, and the fall armyworm, Spodoptera frugiperda, are the major economically devastating pests of cotton crop in Brazil, causing severe losses, mainly due to their endophytic habit, which results in damages to the cotton boll and floral bud structures. A cry1Ia-type gene, designated cry1Ia12, was isolated and cloned from the Bt S811 strain. Nucleotide sequencing of the cry1Ia12 gene revealed an open reading frame of 2160 bp, encoding a protein of 719 amino acid residues in length, with a predicted molecular mass of 81 kDa. The amino acid sequence of Cry1Ia12 is 99% identical to the known Cry1Ia proteins and differs from them only in one or two amino acid residues positioned along the three domains involved in the insecticidal activity of the toxin. The recombinant Cry1Ia12 protein, corresponding to the cry1Ia12 gene expressed in Escherichia coli cells, showed moderate toxicity towards first instar larvae of both cotton boll weevil and fall armyworm. The highest concentration of the recombinant Cry1Ia12 tested to achieve the maximum toxicities against cotton boll weevil larvae and fall armyworm larvae were 230 microg/mL and 5 microg/mL, respectively. The herein demonstrated insecticidal activity of the recombinant Cry1Ia12 toxin against cotton boll weevil and fall armyworm larvae opens promising perspectives for the genetic engineering of cotton crop resistant to both these devastating pests in Brazil.

Signaling pathways in a Citrus EST database

Citrus spp. are economically important crops, which in Brazil are grown mainly in the State of São Paulo. Citrus cultures are attacked by several pathogens, causing severe yield losses. In order to better understand this culture, the Millenium Project (IAC Cordeirópolis) was launched in order to sequence Citrus ESTs (expressed sequence tags) from different tissues, including leaf, bark, fruit, root and flower. Plants were submitted to biotic and abiotic stresses and investigated under different development stages (adult vs. juvenile). Several cDNA libraries were constructed and the sequences obtained formed the Citrus ESTs database with almost 200,000 sequences. Searches were performed in the Citrus database to investigate the presence of different signaling pathway components. Several of the genes involved in the signaling of sugar, calcium, cytokinin, plant hormones, inositol phosphate, MAPKinase and COP9 were found in the citrus genome and are discussed in this paper. The results obtained may indicate that similar mechanisms described in other plants, such as Arabidopsis, occur in citrus. Further experimental studies must be conducted in order to understand the different signaling pathways present.

Genes associated with hypersensitive response (HR) in the citrus EST database (CitEST)

Plants are continuously exposed to pathogen attack, but successful infection is rare because they protect themselves against pathogens using a wide range of response mechanisms. One of them is the hypersensitive response (HR), which is a form of cell death often associated with plant resistance to pathogen infection to prevent the spreadsebpg@cnpq.br sebpg@cnpq.br of the potential pathogen from infected to uninfected tissues. Cell death is activated by recognition of pathogen-derived molecules by the resistance (R) gene products, and is associated with the massive accumulation of reactive oxygen species (ROS), salicylic acid (SA), and other pro-death signals such as nitric oxide (NO). The analysis of the citrus EST (CitEST) database revealed the presence of putative genes likely to be involved in HR through their products, like metacaspases, lipoxygenases, phospholipases, pathogenesis-related proteins, glutathione transferases/peroxidases, enzymes involved in the phenylpropanoid pathway and in the formation and detoxification of ROS, as well as those involved in the formation and regulation of ion channels, SA and NO. By analysis of the EST database of Citrus, it was possible to identify several putative genes that code for key enzymes involved in HR triggering and also in plant defense against biotic and abiotic stress.