Public perception of folate-biofortified genetically modified lettuce varieties in Brazil.

Lettuce is one of the most widely consumed vegetables in the world, commonly eaten fresh in salads, sandwiches, wraps, and as a garnish in various dishes. Consequently, it is a very promising vehicle to deliver vitamins, such as folate (vitamin B9), to a specific population using biofortified varieties generated by conventional or molecular breeding. A new genetically modified lettuce was generated with increased folate content. However, some issues related to public perception regarding this technology should still be evaluated. The aim of this study was to analyze whether consumers are willing to accept a folate-biofortified GM lettuce that could become available to the Brazilian market. A questionnaire involving several issues regarding lettuce consumption was answered by 2,391 people from almost all Brazilian states. When informed that the folic acid biofortified lettuce is a transgenic plant, 46.1% of respondents stated that they would eat it and 30.5% stated that it would be a possibility. This study demonstrated that if there is any explanation regarding the advantage in relation to the use of biotechnology, like enrichment with folic acid, the number of people who accept it increases.

Use of health services by quilombola elderly people / Utilização de serviços de saúde por idosos quilombolas

Objective: To analyze the use of health services by quilombola elderly people. Methods: This is a cross-sectional and household-based study conducted in 11 quilombola communities, with 236 elderly people ≥60 years old. Statistical differences were found in the estimates of indicators of the use of health services according to gender and age group. Pearson's Chi-square or Fisher's Exact tests were performed. Differences were considered statistically significant when p<0.05. Results: Most of the elderly people did not have a health insurance plan, seeking mainly public hospital/outpatient unit. The last medical consultation for 80.3% of the participants was performed in the 12 months prior to the interviews, with fewer consultations for men (p= 0.027). There was a low hospitalization rate in the last year and a low demand for health services in the last two weeks. Conclusion: Quilombola women and long-lived elderly people use health services more and, in general, the elderly citizens depend on SUS to exercise their right to health. The public hospital/outpatient unit was the most used service, and PHCU was little sought (AU).

Objetivo: Analisar a utilização de serviços de saúde por idosos quilombolas. Métodos: Trata-se de um estudo transversal, de base domiciliar, realizado em 11 comunidades quilombolas, com 236 idosos ≥60 anos. Verificou-se diferenças estatísticas nas estimativas dos indicadores de uso de serviços de saúde segundo sexo e faixa etária. Realizaram-se testes de Qui-quadrado de Pearson ou Exato de Fisher. As diferenças foram consideradas estatisticamente significantes quando p<0,05. Resultados: A maioria dos idosos não possuía plano de saúde, buscando, principalmente, hospital/ambulatório público. A última consulta médica de 80,3% dos participantes foi realizada nos 12 meses anteriores às entrevistas, com número menor de consulta para os homens (p= 0,027). Houve baixa internação hospitalar no último ano e procura de algum serviço de saúde nas duas últimas semanas. Conclusão: As mulheres quilombolas e os idosos mais velhos utilizam mais os serviços de saúde e, no geral, os idosos dependem do SUS para exercer o seu direito à saúde. O hospital/ambulatório público foi o serviço mais utilizado, e a UBS pouco procurada (AU).

A Capsid Protein Fragment of a Fusagra-like Virus Found in Carica papaya Latex Interacts with the 50S Ribosomal Protein L17.

Papaya sticky disease is caused by the association of a fusagra-like and an umbra-like virus, named papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2), respectively. Both viral genomes are encapsidated in particles formed by the PMeV ORF1 product, which has the potential to encode a protein with 1563 amino acids (aa). However, the structural components of the viral capsid are unknown. To characterize the structural proteins of PMeV and PMeV2, virions were purified from Carica papaya latex. SDS-PAGE analysis of purified virus revealed two major proteins of ~40 kDa and ~55 kDa. Amino-terminal sequencing of the ~55 kDa protein and LC-MS/MS of purified virions indicated that this protein starts at aa 263 of the deduced ORF1 product as a result of either degradation or proteolytic processing. A yeast two-hybrid assay was used to identify Arabidopsis proteins interacting with two PMeV ORF1 product fragments (aa 321-670 and 961-1200). The 50S ribosomal protein L17 (AtRPL17) was identified as potentially associated with modulated translation-related proteins. In plant cells, AtRPL17 co-localized and interacted with the PMeV ORF1 fragments. These findings support the hypothesis that the interaction between PMeV/PMeV2 structural proteins and RPL17 is important for virus-host interactions.

Whitefly-tolerant transgenic common bean (Phaseolus vulgaris) line.

Common bean (Phaseolus vulgaris L.) is a staple food in Brazil with both nutritional and socioeconomic importance. As an orphan crop, it has not received as much research attention as the commodity crops. Crop losses are strongly related to virus diseases transmitted by the whitefly Bemisia tabaci, one of the most important agricultural pests in the world. The main method of managing whitefly-transmitted viruses has been the application of insecticides to reduce vector populations. Compared to chemical vector control, a more sustainable strategy for managing insect-borne viruses is the development of resistant/tolerant cultivars. RNA interference has been applied to develop plant lines resistant to the whitefly in other species, such as tomato, lettuce and tobacco. Still, no whitefly-resistant plant has been made commercially available to date. Common bean is a recalcitrant species to in vitro regeneration; therefore, stable genetic transformation of this plant has been achieved only at low frequencies (<1%) using particle bombardment. In the present work, two transgenic common bean lines were obtained with an intron-hairpin construct to induce post-transcriptional gene silencing against the B. tabaci vATPase (Bt-vATPase) gene, with stable expression of siRNA. Northern blot analysis revealed the presence of bands of expected size for siRNA in leaf samples of the line Bt-22.5, while in the other line (11.5), the amount of siRNA produced was significantly smaller. Bioassays were conducted with both lines, but only the line Bt-22.5 was associated with significant mortality of adult insects (97% when insects were fed on detached leaves and 59% on the whole plant). The expression of the Bt-vATPase gene was 50% lower (p < 0.05) in insects that fed on the transgenic line Bt-22.5, when compared to non-transgenic controls. The transgenic line did not affect the virus transmission ability of the insects. Moreover, no effect was observed on the reproduction of non-target organisms, such as the black aphid Aphis craccivora, the leafminer Liriomyza sp. and the whitefly parasitoid Encarsia formosa. The results presented here serve as a basis for the development of whitefly-tolerant transgenic elite common bean cultivars, with potential to contribute to the management of the whitefly and virus diseases.

Antinutritional factors, nutritional improvement, and future food use of common beans: A perspective.

Common bean seeds are an excellent source of protein as well as of carbohydrates, minerals, vitamins, and bioactive compounds reducing, when in the diet, the risks of diseases. The presence of bioactive compounds with antinutritional properties (e.g., phytic acid, lectins, raffinosaccharides, protease inhibitors) limits, however, the bean's nutritional value and its wider use in food preparations. In the last decades, concerted efforts have been, therefore, made to develop new common bean genotypes with reduced antinutritional compounds by exploiting the natural genetic variability of common bean and also applying induced mutagenesis. However, possible negative, or positive, pleiotropic effects due to these modifications, in terms of plant performance in response to stresses or in the resulting technological properties of the developed mutant genotypes, have yet not been thoroughly investigated. The purpose of the perspective paper is to first highlight the current advances, which have been already made in mutant bean characterization. A view will be further provided on future research directions to specifically explore further advantages and disadvantages of these bean mutants, their potential use in innovative foods and representing a valuable genetic reservoir of combinations to assess the true functional role of specific seed bioactive components directly in the food matrix.

Herbicide tolerance and gene silencing stability over generations in the ricin bio-detoxicated castor bean.

Castor bean (Ricinus communis L.) is an important cultivated oilseed. Seeds contain ricinoleic acid, a valuable product for a variety of industries. Castor cake is a residue of ricinoleic manufacture and could be used as animal feed due to its high amount of protein. However, castor cake contains ricin and RCA120, both highly toxic and allergenic proteins. In 2017, we reported the development of a transgenic event (named TB14S-5D) with an undetectable amount of ricin/RCA120. In the present work, we evaluate TB14S-5D for tolerance to the herbicide imazapyr, as it contains the selectable marker gene, ahas, which was previously isolated from Arabidopsis thaliana and contains a mutation at position 653 bp. In addition, we demonstrated that the ricin coding genes are stably silenced over three generations.

Two efficient CRISPR/Cas9 systems for gene editing in soybean.

Genome editing using CRISPR/Cas9 has been highlighted as a powerful tool for crop improvement. Nevertheless, its efficiency can be improved, especially for crops with a complex genome, such as soybean. In this work, using the CRISPR/Cas9 technology we evaluated two CRISPR systems, a one-component vs. a two-component strategy. In a simplified system, the single transcriptional unit (STU), SpCas9 and sgRNA are driven by only one promoter, and in the conventional system, the two-component transcriptional unit (TCTU), SpCas9, is under the control of a pol II promoter and the sgRNAs are under the control of a pol III promoter. A multiplex system with three targets was designed targeting two different genes, GmIPK1 and GmIPK2, coding for enzymes from the phytic acid synthesis pathway. Both systems were tested using the hairy root soybean methodology. Results showed gene-specific edition. For the GmIPK1 gene, edition was observed in both configurations, with a deletion of 1 to 749 base pairs; however, the TCTU showed higher indel frequencies. For GmIPK2 major exclusions were observed in both systems, but the editing efficiency was low for STU. Both systems (STU or TCTU) have been shown to be capable of promoting effective gene editing in soybean. The TCTU configuration proved to be preferable, since it was more efficient. The STU system was less efficient, but the size of the CRISPR/Cas cassette was smaller.

Generation of Trichoderma harzianum with pyr4 auxotrophic marker by using the CRISPR/Cas9 system.

Trichoderma harzianum is a filamentous fungus used as a biological control agent for agricultural pests. Genes of this microorganism have been studied, and their applications are patented for use in biofungicides and plant breeding strategies. Gene editing technologies would be of great importance for genetic characterization of this species, but have not yet been reported. This work describes mutants obtained with an auxotrophic marker in this species using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/ Cas (CRISPR-associated) system. For this, sequences for a guide RNA and Cas9 overexpression were inserted via biolistics, and the sequencing approach confirmed deletions and insertions at the pyr4 gene. Phenotypic characterization demonstrated a reduction in the growth of mutants in the absence of uridine, as well as resistance to 5-fluorotic acid. In addition, the gene disruption did not reduce mycoparasitc activity against phytopathogens. Thus, target disruption of the pyr4 gene in T. harzianum using the CRISPR/Cas9 system was demonstrated, and it was also shown that endogenous expression of the system did not interfere with the biological control activity of pathogens. This work is the first report of CRISPR Cas9-based editing in this biocontrol species, and the mutants expressing Cas9 have potential for the generation of useful technologies in agricultural biotechnology.

Histomorphometric evaluation of human extraction sockets treated with autologous fibrin, sticky bone or biphasic calcium phosphate.

It is essential to maintain the alveolar bone ridge to ensure the success of implant therapy. Platelet-rich fibrin (PRF) may benefit bone repair, but its quantitative microscopic results are still inconclusive. The aim of this study was to histomorphometrically analyze human dental alveoli after extraction treated with autologous fibrin, biphasic calcium phosphate or sticky bone. The sample consisted of healthy adult volunteer patients, with clinical and tomographic indication for single post-extraction graft of upper premolars for maintenance of the alveolar ridge and subsequent implantation. The 10 remaining patients in the study were divided into three groups according to the type of filling used in the dental socket: autologous PRF plug covered by a PRF membrane (G1), PRF associated with an alloplastic graft based on hydroxyapatite with beta tricalcium phosphate covered by a collagen membrane (G2) or alloplastic graft based on beta tricalcium phosphate covered by collagen membrane (control). After 8 months, bone biopsies were performed at the grafted sites and the patients underwent implant-prosthetic rehabilitation. Paraffin-embedded tissue blocks were routinely processed and sectionsfrom different depths were mounted in 3 slides and stained with HE. The histomorphometric evaluation analyzed 30 photomicrographs per block, quantifying the percentage presence of newly formed bone, connective tissue and remaining biomaterial using the ImageJ software. Parametric data enabled intergroup comparisons using ANOVA and Tukey's post-hoc test for multiple comparison with statistical significance of 5% (p<0.05), with normality of the 3 groups by the Jarque-Bera test (p>0.05). There was a higher mean of newly formed bone in G1 (68.83%) compared to G2 (35.69%) and control (16.28%). There was greater presence of connective tissue in the control (61.56%). Remaining biomaterial was higher in G2 (15.75%), but did not differ statistically from the control. Bone regeneration obtained with PRF alone or sticky bone suggests the efficacy of these therapies, encouraging the clinical use of this blood concentrate in dental procedures.

A manutenção do rebordo ósseo alveolar é prerrogativa para o sucesso da terapia com implantes. A fibrina rica em plaquetas (PRF) poderia beneficiar o reparo ósseo, mas seus resultados microscópicos quantitativos são ainda inconclusivos. O objetivo deste trabalho foi analisar histomorfometricamente alvéolos dentários humanos pós-extração tratados com fibrina autóloga, fosfato de cálcio bifásico ou sua associação. A amostra consistiu de pacientes adultos voluntários saudáveis, com indicação clínica e tomográfica de enxerto unitário pós-exodontia de pré-molares superiores para manutenção de rebordo alveolar e posterior implante. Os 10 pacientes remanescentes no estudo foram divididos em 3 grupos de acordo com o tipo de preenchimento usado no alvéolo dentário: plug de PRF autóloga recoberto por membrana de PRF (G1), PRF associada a enxerto aloplástico de hidroxiapatita com beta fosfato tricálcio recoberto por membrana de colágeno (G2) ou enxerto aloplástico de beta fosfato tricálcio recoberto por membrana de colágeno (controle). Após 8 meses, foram realizadas biópsias ósseas nos locais enxertados e os pacientes seguiram a reabilitação implanto-protético. Blocos histológicos incluídos em parafina foram microtomizados para gerar 3 lâminas de secções em diferentes profundidades, que foram coradas em HE. A avaliação histomorfométrica analisou 30 fotomicrografias por bloco, quantificando a presença percentual de osso neoformado, tecido conjuntivo e biomaterial remanescente pelo programa ImageJ. Os dados paramétricos permitiram comparações intergrupos usando ANOVA e pós-teste de comparações múltiplas de Tukey com significância estatística de 5% (p<0,05), havendo normalidade dos 3 grupos pelo teste Jarque-Bera (p>0,05). Houve maior média de osso neoformado em G1 (68,83%) em comparação a G2 (35,69%) e controle (16,28%). Houve maior presença de tecido conjuntivo no controle (61,56 %). Biomaterial remanescente foi maior em G2 (15,75%), mas não diferiu estatisticamente para o controle. A regeneração óssea obtida com PRF isolada ou em associação sugere a eficácia destas terapias, encorajando o uso clínico deste concentrado sanguíneo em procedimentos odontológicos.

Near-fatal poisoning after ricin injection.

OBJECTIVE: To report a near-fatal poisoning after intentional injection of ricin from a castor bean (Ricinus communis) extract. CASE REPORT: A 21 year-old man self-injected ∼3 mL of a castor bean extract intramuscularly and subcutaneously in the left antecubital fossa. Upon admission to our ED (1 h post-exposure; day 1, D1) he was awake and alert, but complained of mild local pain and showed slight local edema and erythema. He evolved to refractory shock (∼24 h post-exposure) that required the administration of a large volume of fluids and high doses of norepinephrine and vasopressin, mainly from D2 to D4. During this period, he developed clinical and laboratory features compatible with systemic inflammatory response syndrome, multiple organ dysfunction, capillary leak syndrome, rhabdomyolysis, necrotizing fasciitis and possible compartment syndrome. The patient underwent forearm fasciotomy on D4 and there was progressive improvement of the hemodynamic status from D7 onwards. Wound management involved several debridements, broad-spectrum antibiotics and two skin grafts. Major laboratory findings within 12 days post-exposure revealed hypoalbuminemia, proteinuria, thrombocytopenia, leukocytosis and increases in cytokines (IL-6, IL-10 and TNF-α), troponin and creatine kinase. Ricin A-chain (ELISA) was detected in serum up to D3 (peak at 24 h post-exposure), with ∼79% being excreted in the urine within 64 h post-exposure. Ricinine was detected in serum and urine by LC-MS up to D5. A ricin A-chain concentration of 246 µg/mL was found in the seed extract, corresponding to the injection of ∼738 µg of ricin A-chain (∼10.5 µg/kg). The patient was discharged on D71, with limited range of motion and function of the left forearm and hand. CONCLUSION: Ricin injection resulted in a near-fatal poisoning that evolved with septic shock-like syndrome, multiple organ dysfunction and necrotizing fasciitis, all of which were successfully treated with supportive care.

Histomorphometric evaluation of human extraction sockets treated with autologous fibrin, sticky bone or biphasic calcium phosphate / Avaliação histomorfométrica de alvéolos dentários humanos pós-extração tratados com fibrina autóloga, aglutinado ósseo ou fosfato de cálcio bifásico

ABSTRACT It is essential to maintain the alveolar bone ridge to ensure the success of implant therapy. Platelet-rich fibrin (PRF) may benefit bone repair, but its quantitative microscopic results are still inconclusive. The aim of this study was to histomorphometrically analyze human dental alveoli after extraction treated with autologous fibrin, biphasic calcium phosphate or sticky bone. The sample consisted of healthy adult volunteer patients, with clinical and tomographic indication for single post-extraction graft of upper premolars for maintenance of the alveolar ridge and subsequent implantation. The 10 remaining patients in the study were divided into three groups according to the type of filling used in the dental socket: autologous PRF plug covered by a PRF membrane (G1), PRF associated with an alloplastic graft based on hydroxyapatite with beta tricalcium phosphate covered by a collagen membrane (G2) or alloplastic graft based on beta tricalcium phosphate covered by collagen membrane (control). After 8 months, bone biopsies were performed at the grafted sites and the patients underwent implant-prosthetic rehabilitation. Paraffin-embedded tissue blocks were routinely processed and sectionsfrom different depths were mounted in 3 slides and stained with HE. The histomorphometric evaluation analyzed 30 photomicrographs per block, quantifying the percentage presence of newly formed bone, connective tissue and remaining biomaterial using the ImageJ software. Parametric data enabled intergroup comparisons using ANOVA and Tukey's post-hoc test for multiple comparison with statistical significance of 5% (p<0.05), with normality of the 3 groups by the Jarque-Bera test (p>0.05). There was a higher mean of newly formed bone in G1 (68.83%) compared to G2 (35.69%) and control (16.28%). There was greater presence of connective tissue in the control (61.56%). Remaining biomaterial was higher in G2 (15.75%), but did not differ statistically from the control. Bone regeneration obtained with PRF alone or sticky bone suggests the efficacy of these therapies, encouraging the clinical use of this blood concentrate in dental procedures.

RESUMO A manutenção do rebordo ósseo alveolar é prerrogativa para o sucesso da terapia com implantes. A fibrina rica em plaquetas (PRF) poderia beneficiar o reparo ósseo, mas seus resultados microscópicos quantitativos são ainda inconclusivos. O objetivo deste trabalho foi analisar histomorfometricamente alvéolos dentários humanos pós-extração tratados com fibrina autóloga, fosfato de cálcio bifásico ou sua associação. A amostra consistiu de pacientes adultos voluntários saudáveis, com indicação clínica e tomográfica de enxerto unitário pós-exodontia de pré-molares superiores para manutenção de rebordo alveolar e posterior implante. Os 10 pacientes remanescentes no estudo foram divididos em 3 grupos de acordo com o tipo de preenchimento usado no alvéolo dentário: plug de PRF autóloga recoberto por membrana de PRF (G1), PRF associada a enxerto aloplástico de hidroxiapatita com beta fosfato tricálcio recoberto por membrana de colágeno (G2) ou enxerto aloplástico de beta fosfato tricálcio recoberto por membrana de colágeno (controle). Após 8 meses, foram realizadas biópsias ósseas nos locais enxertados e os pacientes seguiram a reabilitação implanto-protético. Blocos histológicos incluídos em parafina foram microtomizados para gerar 3 lâminas de secções em diferentes profundidades, que foram coradas em HE. A avaliação histomorfométrica analisou 30 fotomicrografias por bloco, quantificando a presença percentual de osso neoformado, tecido conjuntivo e biomaterial remanescente pelo programa ImageJ. Os dados paramétricos permitiram comparações intergrupos usando ANOVA e pós-teste de comparações múltiplas de Tukey com significância estatística de 5% (p<0,05), havendo normalidade dos 3 grupos pelo teste Jarque-Bera (p>0,05). Houve maior média de osso neoformado em G1 (68,83%) em comparação a G2 (35,69%) e controle (16,28%). Houve maior presença de tecido conjuntivo no controle (61,56 %). Biomaterial remanescente foi maior em G2 (15,75%), mas não diferiu estatisticamente para o controle. A regeneração óssea obtida com PRF isolada ou em associação sugere a eficácia destas terapias, encorajando o uso clínico deste concentrado sanguíneo em procedimentos odontológicos.

Evaluation of lettuce chloroplast and soybean cotyledon as platforms for production of functional bone morphogenetic protein 2.

The bone morphogenetic protein BMP2 plays a crucial role in the formation and regeneration of bone and cartilage, which is critical for maintaining skeletal integrity and bone fracture repair. Because of its important role in osteogenic properties it has been commercially produced for clinical use. Here we report attempts to express human BMP2 using two plant systems (lettuce chloroplast and soybean seeds). The rhBMP2 gene (coding for the 13 kDa active polypeptide) was introduced in two regions of the lettuce chloroplast genome. Two homoplasmic events were achieved and RT-PCR demonstrated that the BMP2 gene was transcribed. However, it was not possible to detect accumulation of rhBMP2 in leaves. Two soybean events were achieved to express a full-length hBMP2 gene (coding for the 45 kDa pro-BMP2) fused with the α-coixin signal peptide, under control of the ß-conglycinin promoter. Pro-BMP2 was expressed in the transgenic seeds at levels of up to 9.28% of the total soluble seed protein as determined by ELISA. It was demonstrated that this recombinant form was biologically active upon administration to C2C12 cell cultures, because it was able to induce an osteogenic cascade, as observed by the enhanced expression of SP7 (osterix) and ALPI (alkaline phosphatase) genes. Collectively, these results corroborated our previous observation that soybean seeds provide an effective strategy for achieving stable accumulation of functional therapeutic proteins, such as BMP2.

Bio-detoxification of ricin in castor bean (Ricinus communis L.) seeds.

Ricin is a highly toxic ribosome-inactivating lectin occurring in the seeds of castor bean (Ricinus communis L.). Castor bean grows throughout tropical and sub-tropical regions and is a very important crop due to its high seed content of ricinoleic acid, an unusual fatty acid, which has several industrial applications. However, due to the presence of the toxin, castor bean can cause death after the exposure of animals to low doses of ricin through skin contact, injection, inhalation or oral routes. Aiming to generate a detoxified genotype, we explored the RNAi concept in order to silence the ricin coding genes in the endosperm of castor bean seeds. Results indicated that ricin genes were effectively silenced in genetically modified (GM) plants, and ricin proteins were not detected by ELISA. Hemagglutination activity was not observed with proteins isolated from GM seeds. In addition, we demonstrated that seed proteins from GM plants were not toxic to rat intestine epithelial cells or to Swiss Webster mice. After oil extraction, bio-detoxified castor bean cake, which is very rich in valuable proteins, can be used for animal feeding. Gene silencing would make castor bean cultivation safer for farmers, industrial workers and society.

Overexpression of an aquaglyceroporin gene from Trichoderma harzianum improves water-use efficiency and drought tolerance in Nicotiana tabacum.

Aquaporins (AQPs) and aquaglyceroporins (AQGPs) are integral membrane proteins that mediate the transport of water and solutes, such as glycerol and urea, across membranes. AQP and AQGP genes represent a valuable tool for biotechnological improvement of plant tolerance to environmental stresses. We previously isolated a gene encoding for an aquaglyceroporin (ThAQGP), which was up-regulated in Trichoderma harzianum during interaction with the plant pathogen Fusarium solani. This gene was introduced into Nicotiana tabacum and plants were physiologically characterized. Under favorable growth conditions, transgenic progenies did not had differences in both germination and growth rates when compared to wild type. However, physiological responses under drought stress revealed that transgenic plants presented significantly higher transpiration rate, stomatal conductance, photosynthetic efficiency and faster turgor recovery than wild type. Quantitative RT-PCR analysis demonstrated the presence of ThAQGP transcripts in transgenic lines, showing the cause-effect relationship between the observed phenotype and the expression of the transgene. Our results underscore the high potential of T. harzianum as a source of genes with promising applications in transgenic plants tolerant to drought stress.

RNAi-mediated resistance to whitefly (Bemisia tabaci) in genetically engineered lettuce (Lactuca sativa).

RNA interference (RNAi)-based transgenic technologies have evolved as potent biochemical tools for silencing specific genes of plant pathogens and pests. The approach has been demonstrated to be useful in silencing genes in insect species. Here, we report on the successful construction of RNAi-based plasmid containing an interfering cassette designed to generate dsRNAs that target a novel v-ATPase transcript in whitefly (Bemisia tabaci), an important agricultural pest in tropical and sub-tropical regions. The presence of the transgene was confirmed in T0 and T1 generations of transgenic lettuce lines, segregating in a Mendelian fashion. Seven lines were infested with whiteflies and monitored over a period of 32 days. Analysis of mortality showed that within five days of feeding, insects on transgenic plants showed a mortality rate of 83.8-98.1%. In addition, a reduced number of eggs (95 fold less) was observed in flies feeding on transgenic lettuce plants than insects on control lines. Quantitative reverse transcription PCR showed decreased expression level of endogenous v-ATPase gene in whiteflies feeding on transgenic plants. This technology is a foundation for the production of whitefly-resistant commercial crops, improving agricultural sustainability and food security, reducing the use of more environmentally aggressive methods of pest control.

Construction of an agroinfectious clone of bean rugose mosaic virus using Gibson Assembly.

Construction of agroinfectious viral clones usually requires many steps of cloning and sub-cloning and also a binary vector, which makes the process laborious, time-consuming, and frequently susceptible to some degree of plasmid instability. Nowadays, novel methods have been applied to the assembly of infectious viral clones, and here we have applied isothermal, single-step Gibson Assembly (GA) to construct an agroinfectious clone of Bean rugose mosaic virus (BRMV) using a small binary vector. The procedure has drastically reduced the cloning steps, and BRMV could be recovered from agroinfiltrated common bean twenty days after inoculation, indicating that the infectious clone could spread in the plant tissues and efficiently generate a systemic infection. The virus was also recovered from leaves of common bean and soybean cultivars mechanically inoculated with infectious clone two weeks after inoculation, confirming the efficiency of GA cloning procedure to produce the first BRMV agroinfectious clone to bean and soybean.

Use of transgenic Aedes aegypti in Brazil: risk perception and assessment.

The OX513A strain of Aedes aegypti, which was developed by the British company Oxitec, expresses a self-limiting transgene that prevents larvae from developing to adulthood. In April 2014, the Brazilian National Technical Commission on Biosafety completed a risk assessment of OX513A and concluded that the strain did not present new biological risks to humans or the environment and could be released in Brazil. At that point, Brazil became the first country to approve the unconstrained release of a genetically modified mosquito. During the assessment, the commission produced a comprehensive list of - and systematically analysed - the perceived hazards. Such hazards included the potential survival to adulthood of immature stages carrying the transgene - should the transgene fail to be expressed or be turned off by exposure to sufficient environmental tetracycline. Other perceived hazards included the potential allergenicity and/or toxicity of the proteins expressed by the gene, the potential for gene flow or increased transmission of human pathogens and the occupation of vacant breeding sites by other vector species. The Zika epidemic both elevated the perceived importance of Ae. aegypti as a vector - among policy-makers and regulators as well as the general public - and increased concerns over the release of males of the OX513A strain. We have therefore reassessed the potential hazards. We found that release of the transgenic mosquitoes would still be both safe and of great potential value in the control of diseases spread by Ae. aegypti, such as chikungunya, dengue and Zika.

La souche OX513A d'Aedes aegypti, qui a été créée par la société britannique Oxitec, exprime un transgène autolimitant qui empêche les larves de se développer et de devenir adultes. En avril 2014, la Commission technique nationale de biosécurité du Brésil a procédé à une évaluation des risques liés à la souche OX513A et conclu qu'elle ne présentait pas de nouveaux risques biologiques pour les êtres humains ou l'environnement et pouvait être lâchée au Brésil. Le Brésil est donc devenu le premier pays à approuver le lâcher non contraint d'un moustique génétiquement modifié. Au cours de l'évaluation, la commission a établi une liste exhaustive des risques perçus, qu'elle a par ailleurs systématiquement analysés. Ces risques incluaient la survie potentielle à l'âge adulte des larves immatures porteuses du transgène ­ si le transgène ne s'exprime pas ou est désactivé par une exposition à la tétracycline suffisante dans l'environnement. Les autres risques perçus incluaient les potentielles propriétés allergisantes et/ou la toxicité des protéines exprimées par le gène, l'éventualité d'un flux de gènes ou d'une transmission accrue d'agents pathogènes pour l'homme et l'occupation de sites de reproduction vacants par d'autres espèces vectrices. L'épidémie d'infections à virus Zika a accentué l'importance accordée par les responsables politiques, les organismes de réglementation ainsi que le grand public à Ae. aegypti en tant que moustique vecteur, et a accru l'inquiétude relative au lâcher de mâles de la souche OX513A. Nous avons donc réévalué les risques potentiels. Nous estimons que le lâcher de moustiques transgéniques serait à la fois sans danger et extrêmement utile pour lutter contre les maladies transmises par Ae. aegypti, telles que le chikungunya, la dengue et le virus Zika.

La cepa OX513A de Aedes aegypti, que desarrolló la empresa británica Oxitec, expresa un transgén autolimitado que impide que las larvas se desarrollen hasta la edad adulta. En abril de 2014, la Comisión Nacional Técnica de Bioseguridad de Brasil realizó una evaluación de riesgos de OX513A y concluyó que la cepa no presentaba nuevos riesgos biológicos para los humanos o el medioambiente y que podría liberarse en Brasil. En ese momento, Brasil se convirtió en el primer país en aprobar la liberación ilimitada de un mosquito modificado genéticamente. A lo largo de la evaluación, la comisión redactó una lista completa, y analizada sistemáticamente, de las posibles contingencias. Entre dichos peligros se encontraba la posible supervivencia hasta la edad adulta de etapas inmaduras que portan el transgén, en caso de que éste no consiga expresarse o se inutilice debido a la exposición a la suficiente tetraciclina medioambiental. Otras posibles contingencias eran la alergia y/o toxicidad de las proteínas expresadas por el gen, la posibilidad de un flujo genético o el aumento de la transmisión de patógenos humanos y la ocupación de lugares de cría desocupados por parte de otras especies vectores. La epidemia por el virus de Zika aumentó la importancia de Ae. aegypti como vector, entre los responsables y reguladores políticos, así como entre el público general, y aumentó las preocupaciones acerca de la liberación de machos de la cepa OX513A. Por lo tanto, se han vuelto a evaluar los posibles riesgos. Se ha descubierto que la liberación de mosquitos transgénicos sería segura y tendría un gran valor potencial en el control de la propagación de enfermedades por Ae. aegypti, como la fiebre chikungunya, el dengue y la enfermedad por el virus de Zika.

The Stress-Induced Soybean NAC Transcription Factor GmNAC81 Plays a Positive Role in Developmentally Programmed Leaf Senescence.

The onset of leaf senescence is a highly regulated developmental change that is controlled by both genetics and the environment. Senescence is triggered by massive transcriptional reprogramming, but functional information about its underlying regulatory mechanisms is limited. In the current investigation, we performed a functional analysis of the soybean (Glycine max) osmotic stress- and endoplasmic reticulum (ER) stress-induced NAC transcription factor GmNAC81 during natural leaf senescence using overexpression studies and reverse genetics. GmNAC81-overexpressing lines displayed accelerated flowering and leaf senescence but otherwise developed normally. The precocious leaf senescence of GmNAC81-overexpressing lines was associated with greater Chl loss, faster photosynthetic decay and higher expression of hydrolytic enzyme-encoding GmNAC81 target genes, including the vacuolar processing enzyme (VPE), an executioner of vacuole-triggered programmed cell death (PCD). Conversely, virus-induced gene silencing-mediated silencing of GmNAC81 delayed leaf senescence and was associated with reductions in Chl loss, lipid peroxidation and the expression of GmNAC81 direct targets. Promoter-reporter studies revealed that the expression pattern of GmNAC81 was associated with senescence in soybean leaves. Our data indicate that GmNAC81 is a positive regulator of age-dependent senescence and may integrate osmotic stress- and ER stress-induced PCD responses with natural leaf senescence through the GmNAC81/VPE regulatory circuit.

Metabolic engineering of folate and its precursors in Mexican common bean (Phaseolus vulgaris L.).

Folate (vitamin B9) deficiency causes several health problems globally. However, folate biofortification of major staple crops is one alternative that can be used to improve vitamin intakes in populations at risk. We increased the folate levels in common bean by engineering the pteridine branch required for their biosynthesis. GTP cyclohydrolase I from Arabidopsis (AtGchI) was stably introduced into three common bean Pinto cultivars by particle bombardment. Seed-specific overexpression of AtGCHI caused significant increases of up to 150-fold in biosynthetic pteridines in the transformed lines. The pteridine boost enhanced folate levels in raw desiccated seeds by up to threefold (325 µg in a 100 g portion), which would represent 81% of the adult recommended daily allowance. Unexpectedly, the engineering also triggered a general increase in PABA levels, the other folate precursor. This was not observed in previous engineering studies and was probably caused by a feedforward mechanism that remains to be elucidated. Results from this work also show that common bean grains accumulate considerable amounts of oxidized pteridines that might represent products of folate degradation in desiccating seeds. Our study uncovers a probable different regulation of folate homoeostasis in these legume grains than that observed in other engineering works. Legumes are good sources of folates, and this work shows that they can be engineered to accumulate even greater amounts of folate that, when consumed, can improve folate status. Biofortification of common bean with folates and other micronutrients represents a promising strategy to improve the nutritional status of populations around the world.