The role of microRNAs in NBS-LRR gene expression and its implications for plant immunity and crop development.

Plants evolved, over millions of years, complex defense systems against pathogens. Once infected, the interaction between pathogen effector molecules and host receptors triggers plant immune responses, which include apoptosis, systemic immune response, among others. An important protein family responsible for pathogen effector recognition is the nucleotide binding site-leucine repeat rich (NBS-LRR) proteins. The NBS-LRR gene family is the largest disease resistance gene class in plants. These proteins are widely distributed in vascular plants and have a complex multigenic cluster distribution in plant genomes. To counteract the genetic load of such a large gene family on fitness cost, plants evolved a mechanism using post transcriptional gene silencing induced by small RNAs, particularly microRNAs. For the NBS-LRR gene family, the small RNAs involved in this silencing mechanism are mainly the microRNA482/2118 superfamily. This suppression mechanism is relieved upon pathogen infection, thus allowing increased NBS-LRR expression and triggering plant immunity. In this review, we will discuss the biogenesis of microRNAs and secondary RNAs involved in this silencing mechanism, biochemical and structural features of NBS-LRR proteins in response to pathogen effectors and the evolution of microRNA-based silencing mechanism with a focus on the miR482/2118 family. Furthermore, the biotechnological manipulation of microRNA expression, using both transgenic or genome editing approaches to improve cultivated plants will be discussed, with a focus on the miR482/2118 family in soybean.

Soybean seed protein storage vacuoles for expression of recombinant molecules.

Soybean is one of the most important protein sources for human consumption and livestock feed. Soy production also allows the biosynthesis of edible oils, biodiesel, and biofertilizers. With the advent of modern agricultural biotechnology, soybean plants have also converted into bioreactors of therapeutic proteins and industrial enzymes. Soybean's characteristics, such as protein storage vacuoles (PSVs) and other unique organelles, allow the plant to be exploited as an accumulator of heterologous proteins under high stability and scalability conditions, and that maintains its basic functions. This review reports the main aspects of heterologous protein accumulation in soybean PSVs.

Biosseguridade para sistemas de produção de peixes em tanque-rede em função da colmatação agravada por Limnoperna fortunei / Biosecurity for fishfarming cage systems due to the highly clogged screen aggravated by Limnoperna fortunei

Este trabalho avaliou a colmatação por Limnoperna fortunei em diferentes materiais para confecção de telas de arame, a dinâmica da colmatação pelo molusco e a relação custo-benefício dos materiais usados em tanques-rede. Utilizaram-se amostras de telas de simples torção de malha 19mm, de cinco tipos diferentes de revestimentos, colocadas na barragem de Salto Caxias, no Rio Iguaçu, estado do Paraná, entre julho de 2012 e julho de 2014. A cada seis meses, foi verificado o ganho de peso das telas em razão da colmatação, além da contagem do número de mexilhões aderidos. Após as coletas dos dados, foi realizado o teste estatístico de Kruskal-Wallis para se avaliar o ganho de peso das telas devido à colmatação. Realizou-se também uma pesquisa com 21 empresas para se obter o preço comercializado de telas para confecção de tanques-rede e se avaliar a expectativa de durabilidade dos materiais. Verificou-se que os diferentes materiais avaliados para as telas influenciam na colmatação e aderência do mexilhão-dourado; além disso, constatou-se que a tela mais eficiente quanto ao custo/ano é a de arame galvanizado plastificado e, para baixa colmatação, a de arame Bezinal.(AU)

This research evaluated degree of clogging by Limnoperna fortunei in different materials for wire mesh fabrication, the dynamics of mollusk sealing and the cost-benefit ratio of these materials used in tank-nets. Samples of single-twist screens of 19 mm mesh were used, from five different types of coatings were placed in the Salto Caxias dam on the Iguaçu River in the Paraná state, between July 2012 and July 2014. Every six months, it was verified the weight gain of the screens due to clogging and counting of the number of mussels adhered. After the data collection was performed Kruskal-Wallis statistical test to evaluate the weight gain of the screens due to clogging. In addition, a survey was carried out with 21 companies to obtain the commercialized price of screens for the production of net tanks and to evaluate the expected durability of the materials. The different materials evaluated for the screens influenced the sealing and adhesion of the golden mussel. In addition, the most cost-per-year screen is that of plastic-coated galvanized wire and for low clogging it is the Bezinal wire.(AU)

The efficiency of wire nets in enhancing the biosecurity of poultry in Brazil.

The aim of this study was to evaluate the efficiency of the use of wire nets of various mesh sizes to enhance biosecurity in the poultry industry in Brazil by preventing other bird species from entering chicken houses. The Brazilian poultry industry is technologically advanced and employs updated technology. The current Brazilian guidelines recommend the use of 25.40 mm mesh. However, scientific evidence of the efficiency of the nets recommended by these guidelines is lacking. In this study, a bird biometric methodology was developed to evaluate bird species. The methodology was based on the body dimensions of the animal, and it employed a new statistical design to analyse the data. Three groups of bird species were designated according to their importance. The value of this criterion (the importance of the species) was estimated by assessing the ability of birds to pass through the net. The paradigm was used to study 23 wild avian species that are naturally present in Brazil. The best results were observed for nets with a mesh size < or = 19.11 mm. This mesh size was able to efficiently restrain all of the species studied. However, in the same test, the net with 25.40 mm mesh could not restrain 11 bird species, one of which was Passer domesticus, which is found worldwide. On the basis of these results, the use of 19.11 mm mesh should be strongly recommended in order to achieve biosecurity of poultry houses.

Soybeans as bioreactors for biopharmaceuticals and industrial proteins.

Plants present various advantages for the production of biomolecules, including low risk of contamination with prions, viruses and other pathogens, scalability, low production costs, and available agronomical systems. Plants are also versatile vehicles for the production of recombinant molecules because they allow protein expression in various organs, such as tubers and seeds, which naturally accumulate large amounts of protein. Among crop plants, soybean is an excellent protein producer. Soybean plants are also a good source of abundant and cheap biomass and can be cultivated under controlled greenhouse conditions. Under containment, the plant cycle can be manipulated and the final seed yield can be maximized for large-scale protein production within a small and controlled area. Exploitation of specific regulatory sequences capable of directing and accumulating recombinant proteins in protein storage vacuoles in soybean seeds, associated with recently developed biological research tools and purification systems, has great potential to accelerate preliminary characterization of plant-derived biopharmaceuticals and industrial macromolecules. This is an important step in the development of genetically engineered products that are inexpensive and safe for medicinal, food and other uses.

A minimal DNA cassette as a vector for genetic transformation of soybean (Glycine max).

Currently, the market demands products committed to protecting human health and the environment, known as clean products. We developed a protocol using DNA fragments containing only the gene sequence of interest, to replace the circular vectors containing genes for antibiotic resistance and other undesirable sequences, for obtaining transgenic soybeans for microparticle bombardment. Vector pAC321 was digested with the restriction enzyme PvuII to produce the 6159 bp ahas fragment, which contains the mutated ahas gene from Arabidopsis thaliana (Brassicaceae), under the control of its own promoter and terminator. This gene confers resistance against imazapyr, a herbicidal molecule of the imidazolinone class, capable of systemically translocating and concentrating in the apical meristematic region of the plant, the same region used for the introduction of the transgenes. This fragment was used to generate 10 putative transgenic soybean lines.

Correct targeting of proinsulin in protein storage vacuoles of transgenic soybean seeds.

Soybean plants are promising bioreactors for the expression of biochemically complex proteins that cannot be produced in a safe and/or economically viable way in microorganisms, eukaryotic culture cells or secreted by transgenic animal glands. Soybeans present many desirable agronomic characteristics for high scale protein production, such as high productivity, short reproductive cycle, photoperiod sensitivity, and natural organs destined for protein accumulation in the seeds. The significant similarities between plant and human cells in terms of protein synthesis processes, folding, assembly, and post-translational processing are important for efficient accumulation of recombinant proteins. We obtained two transgenic lines using biolystics, incorporating the human proinsulin gene under control of the monocot tissue-specific promoter from sorghum gamma-kafirin seed storage protein gene and the alpha-coixin cotyledonary vacuolar signal peptide from Coix lacryma-jobi (Poaceae). Transgenic plants expressed the proinsulin gene and accumulated the polypeptide in mature seeds. Protein targeting to cotyledonary protein storage vacuoles was successfully achieved and confirmed with immunocytochemistry assays. The combination of different regulatory sequences was apparently responsible for high stability in protein accumulation, since human proinsulin was detected after seven years under room temperature storage conditions.

Gene flow from transgenic to nontransgenic soybean plants in the Cerrado region of Brazil.

Evaluation of transgenic crops under field conditions is a fundamental step for the production of genetically engineered varieties. In order to determine if there is pollen dispersal from transgenic to nontransgenic soybean plants, a field release experiment was conducted in the Cerrado region of Brazil. Nontransgenic plants were cultivated in plots surrounding Roundup Ready transgenic plants carrying the cp4 epsps gene, which confers herbicide tolerance against glyphosate herbicide, and pollen dispersal was evaluated by checking for the dominant gene. The percentage of cross-pollination was calculated as a fraction of herbicide-tolerant and -nontolerant plants. The greatest amount of transgenic pollen dispersion was observed in the first row, located at one meter from the central (transgenic) plot, with a 0.52% average frequency. The frequency of pollen dispersion decreased to 0.12% in row 2, reaching 0% when the plants were up to 10 m distance from the central plot. Under these conditions pollen flow was higher for a short distance. This fact suggests that the management necessary to avoid cross-pollination from transgenic to nontransgenic plants in the seed production fields should be similar to the procedures currently utilized to produce commercial seeds.

Gene flow from transgenic to nontransgenic soybean plants in the Cerrado region of Brazil

Evaluation of transgenic crops under field conditions is a fundamental step for the production of genetically engineered varieties. In order to determine if there is pollen dispersal from transgenic to nontransgenic soybean plants, a field release experiment was conducted in the Cerrado region of Brazil. Nontransgenic plants were cultivated in plots surrounding Roundup Ready transgenic plants carrying the cp4 epsps gene, which confers herbicide tolerance against glyphosate herbicide, and pollen dispersal was evaluated by checking for the dominant gene. The percentage of cross-pollination was calculated as a fraction of herbicide-tolerant and -nontolerant plants. The greatest amount of transgenic pollen dispersion was observed in the first row, located at one meter from the central (transgenic) plot, with a 0.52% average frequency. The frequency of pollen dispersion decreased to 0.12% in row 2, reaching 0% when the plants were up to 10 m distance from the central plot. Under these conditions pollen flow was higher for a short distance. This fact suggests that the management necessary to avoid cross-pollination from transgenic to nontransgenic plants in the seed production fields should be similar to the procedures currently utilized to produce commercial seeds.

Effectiveness of liposomes to transfect livestock fibroblasts

The development of an efficient transfection system in livestock cells is an important step towards investigating gene transfer and the functioning and production of transgenic animals. Important factors involved in cationic liposome mediated gene transfer were evaluated through in vitro transfection of bovine, caprine and ovine fibroblast cells. Transfection of plasmid DNA complexes of different commercially available liposomes (Lipofectamine, Lipofectin, Cellfectin and DMRIE-C; Gibco-BRL, USA) was evaluated utilizing the following parameters: DNA/liposome ratio, cell density, DNA conformation, and the effect of transfection time on the efficiency of bovine fibroblasts to express a reporter gene. The effects and concentrations of liposomes were also evaluated in caprine and ovine fibroblasts. Lipofectamine alone and Lipofectamine with Plus reagent induced high-frequency expression of beta-galactosidase and neo genes in all cells evaluated (47 and 88.3%, respectively). Regarding phenotype, chromosomal stability was similar in transfected and non-transfected cells. The parameters set in this study will establish a foundation for utilizing transfected fibroblast cells to generate transgenic animals through nuclear transfer technology and gene function studies.

Development of bovine embryos reconstructed by nuclear transfer of transfected and non-transfected adult fibroblast cells

An association of two techniques, nuclear transfer (NT), and transfection of somatic animal cells, has numerous potential applications and considerable impact, mainly in agriculture, medicine, pharmacy, and fundamental biology. In addition, somatic cell nuclear transfer is the most efficient alternative to produce large transgenic animals. We compared in vitro and in vivo developmental capacities of NT using fibroblast cells isolated from a 14-month-old cloned Simmental heifer (FCE) vs the same line transfected with a plasmid containing neomycin-resistant genes (TFCE). There were no significant differences (P > 0.5) in either fusion (116/149 = 78% vs 216/301 = 72%), cleavage (78/116 = 67% vs 141/216 = 65%) and blastocyst (35/116 = 30% vs 52/216 = 24%) rates or in pregnancy rate at 30 to 35 days after embryo transfer (2/17 vs 3/17) between NT using FCE and TFCE, respectively. Transfection and long-term in vitro culture of transfected cells did not affect developmental capacity of NT embryos up to 40 days of gestation

Inheritance of a recessive transgene-associated character controlling albinism in transgenic bean (Phaseolus vulgaris L.).

We identified a transgenic line exhibiting albinism during our work to introduce genes through genetic engineering in dry bean (Phaseolus vulgaris). The transgenic mother plant (R0) presented a normal phenotype and generated albino and normal green plants in the first generation (R1). The segregation ratio of the albino character in the R1 and R2 generations fitted the expected ratio for a character controlled by a single recessive gene linked to a foreign gus gene, suggesting that albinism could be a consequence of insertional mutation caused by introduction of the exogenous gene. Analysis by electron microscope revealed that the albino cells possessed no chloroplasts and a greater number of mitochondria when compared to normal green plants. This transgenic bean line may be used in understanding the genetic control of chloroplast genesis, for acquiring additional knowledge of genomic structure or in physiological studies. This is the first described transgene-associated mutant bean plant.