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.

Prevalência de hanseníase em São Luís–Maranhão entre os anos de2013 a 2015 / Prevalence of leprosy in São Luís-Maranhão from 2013 to 2015

Objetivo: analisar e representar, espacialmente, a evolução da distribuição geográfica dasocorrências de Hanseníase no Maranhão entre os anos de 2013 a 2015, utilizando técnicas degeoprocessamento. Método: realizou-se análise, utilizando mapas e tabelas temáticas, com base nosregistros do Sistema de Informação de Agravos de Notificação. Resultados: foram identificados 1.879casos, somados entre 219 bairros residenciais. Para o ano de 2013, o distrito mais afetado foi oTirirical; em 2014, aumentou a prevalência nos Distritos de Bequimão e Coroadinho e em 2015, houveuma redução de prevalências nos distritos de Tirirical Cohab, Coroadinho e Itaqui Bacanga.Conclusão: apesar de algumas localidades estudadas terem apresentado uma diminuição daprevalência entre 2013 e 2014, de 2014 a 2015 houve um aumento na quantidade de casos. O estudode prevalências possibilitou essa identificação de casos, podendo ser observada a imprescindibilidadede subsidiar estratégias de controle.

Objective: to analyze and spatially represent the evolution of the geographic distribution ofoccurrences of Leprosy in Maranhão between the years 2013 to 2015 using geoprocessing techniques.Method: the analysis was performed using maps and thematic tables, based on the records of theNotifiable Diseases Information System. Results: 1.879 cases were identified, among 219 residentialdistricts. For the year 2013, the district most affected was Tirirical; in 2014, increased prevalencein the Districts of Bequimão and Coroadinho and in 2015, there was a reduction of prevalences in thedistricts of Tirirical Cohab, Coroadinho and Itaqui Bacanga. Conclusion: although some localitiesstudied showed decrease in their prevalence between 2013 and 2014; there was increase in thenumber of cases from 2014 to 2015. The prevalence study made it possible to identify cases, and itis possible to observe the necessity of subsidizing control strategies.

New sources of resistance to leafminers (Liriomyza sativae) in melon (Cucumis melo L.) germplasm.

Leafminers (Liriomyza sativae) are the main melon (Cucumis melo L.) pests in Northeast Brazil, which is the main region for the production and export of the fruit in Brazil. Of the integrated management strategies available, genetic resistance is the best method of preventing damage by these insects. The aim of this study was to select sources of resistance to leafminers in melon germplasm. Seven experiments were conducted in the laboratory, field, and greenhouse, with and without choice, using 52 melon accessions and 4 commercial hybrids as controls. Genetic variability among the accessions made it possible to select four new sources of resistance: 'CNPH 11-1072' and 'CNPH 11-1077', because they exhibited lower levels of infestation by the insect (antixenosis); and 'CNPH 00-915(R)' and 'BAGMEL 56(R)', because the pest larvae died soon after beginning to feed on the leaf mesophyll (antibiosis).

Molecular marker-based genetic diversity analysis of scantly studied Brazilian accessions of a medicinal plant, Morinda citrifolia L. (noni).

Morinda citrifolia L., commonly known as noni, has been used for the treatment of various diseases for over two centuries. It was introduced and widely disseminated in Brazil because of its high market value and ease of adaptation to the soil and climatic conditions of the country. The aim of this study was to estimate the genetic variability of noni accessions from the collection of Embrapa Agroindústria Tropical in Brazil. We evaluated 36 plants of the 13 accessions of noni from the germplasm collection of M. citrifolia. Several methods of DNA extraction were tested. After definition of the method, the DNA of each sample was subjected to polymerase chain reactions using 20 random amplified polymorphic DNA primers. The band patterns on agarose gel were converted into a binary data matrix, which was used to estimate the genetic distances between the plants and to perform the cluster analyses. Of the total number of markers used in this study, 125 (81.1%) were polymorphic. The genetic distances between the genotypes ranged from 0.04 to 0.49. Regardless of the high number of polymorphic bands, the genetic variability of the noni plants evaluated was low since most of the genotypes belonged to the same cluster as shown by the dendrogram and Tocher's cluster analysis. The low genetic diversity among the studied noni individuals indicates that additional variability should be introduced in the germplasm collection of noni by gathering new individuals and/or by hybridizing contrasting individuals.

Should menopausal characteristics be considered during cardiorespiratory exercise prescription in postmenopausal women?

OBJECTIVE: Menopausal characteristics (i.e. the nature of menopause, hormone therapy, and time elapsed since menopause) are known to affect women's health-related quality of life. The purpose of this study was to determine whether menopausal characteristics affect the cardiorespiratory exercise response and which characteristics should be considered for exercise prescription. METHODS: Fifty-eight postmenopausal women (60.21 ± 4.49 years of age; 66.26 ± 8.99 kg body weight; 157.09 ± 4.92 cm in height; 29.70 ± 4.79 ml·kg(-1)·min(-1) maximal oxygen uptake) participated in this study. A graded 25-W/min(2) cycle ergometer exercise protocol was applied to assess aerobic power and ventilatory thresholds. Participants' heart rates and gas-exchange variables were measured continuously using a COSMED K4b(2) portable gas analyzer system. The first and the second ventilatory thresholds were determined by the time-course curves of ventilation and oxygen and carbon dioxide ventilatory equivalents. Using age as a covariate, an analysis of covariance was performed to assess the effect of menopause characteristics upon the data. RESULTS: Regardless of the nature of menopause, use of hormone therapy, time elapsed since menopause, and the interaction between these characteristics, the participants presented no differences in maximal oxygen uptake values, neither on submaximal variables often used in evaluations of exercise prescription, such as percent of maximal oxygen uptake, maximal heart rate, and heart rate reserve, nor in respiratory exchange ratio and gas exchange energy expenditure at aerobic and anaerobic ventilatory thresholds. CONCLUSIONS: These data suggest that a personalized cardiorespiratory target zone for this population should be set according to the published literature, and that consideration of the individual menopausal characteristics seems to be unnecessary.

Effects of a 12-month multi-component exercise program on the body composition of postmenopausal women.

OBJECTIVE: The aims of this study were to identify the effects of a 12-month exercise program on the body composition of postmenopausal women and to examine the interaction of menopause characteristics (nature and time since menopause, hormone therapy) with exercise. METHODS: A total of 158 postmenopausal Caucasian women were analyzed in this study (70 in the control and 88 in the exercise group). This subset is part of the 'Shape up during menopause' which is a program that aims to develop exercise and health promotion in postmenopausal women. Exercise and control groups were tested before and at the end of the program. Data related to menopause were obtained through medical history. Measurements of fat mass, visceral fat area, skeletal muscle mass, fat-free mass, soft lean mass, and basal metabolic rate were assessed by octopolar bioimpedance. RESULTS: Alongside basal metabolic rate, all the anthropometric and body composition variables were influenced by the exercise program. The major differences between groups were found in skeletal muscle mass, total soft lean mass, fat-free mass, and skeletal muscle mass index (effect sizes ranged from 0.89 to 6.64). There were no interactive effects found between exercise and menopause characteristics. CONCLUSIONS: There were positive changes in all measured variables and no significant interactive effects with menopause characteristics; therefore, our data suggest that exercise alone promoted improvements in the body composition of postmenopausal women.

Development of transgenic imazapyr-tolerant cowpea (Vigna unguiculata).

KEY MESSAGE: Here we present the development of cowpea lines tolerant to a herbicide from imidazoline class (imazapyr). Plants presented tolerance to fourfold the commercial recommended dose for weed control. Cowpea is one of the most important and widely cultivated legumes in many parts of the world. Its cultivation is drastically affected by weeds, causing damages during growth and development of plants, competing for light, nutrients and water. Consequently, weed control is critical, especially using no-tillage farming systems. In tropical regions, no-till farming is much easier with the use of herbicides to control weeds. This study was conducted to evaluate the possibility of obtaining transgenic cowpea plants resistant to imidazolinone, which would facilitate weed control during the summer season. The biolistic process was used to insert a mutated acetohydroxyacid synthase coding gene (Atahas) which confers tolerance to imazapyr. The transgene integration was confirmed by Southern blot analysis. Out of ten lines tested for tolerance to 100 g ha(-1) imazapyr, eight presented some tolerance. One line (named 59) revealed high herbicide tolerance and developmental growth comparable to non-transgenic plants. This line was further tested for tolerance to higher herbicide concentrations and presented tolerance to 400 g ha(-1) imazapyr (fourfold the commercial recommended dose) with no visible symptoms. Line 59 will be the foundation for generating imidazolinone-tolerant cowpea varieties, which will facilitate cultivation of this crop in large areas.

Genetic divergence among accessions of melon from traditional agriculture of the Brazilian Northeast.

The genetic divergence of 38 melon accessions from traditional agriculture of the Brazilian Northeast and three commercial hybrids were evaluated using fruit descriptors and microsatellite markers. The melon germplasm belongs to the botanic varieties cantalupensis (19), momordica (7), conomon (4), and inodorus (3), and to eight genotypes that were identified only at the species level. The fruit descriptors evaluated were: number of fruits per plant (NPF), fruit mass (FM; kg), fruit longitudinal diameter (LD; cm), fruit transversal diameter (TD; cm), shape index based on the LD/TD ratio, flesh pulp thickness, cavity thickness (CT; cm), firmness fruit pulp (N), and soluble solids (SS; °Brix). The results showed high variability for all descriptors, especially for NPF, LD, and FM. The grouping analysis based on fruit descriptors produced eight groups without taxonomic criteria. The LD (22.52%), NPF (19.70%), CT (16.13%), and SS (9.57%) characteristics were the descriptors that contributed the most to genotype dissimilarity. The 17 simple sequence repeat polymorphic markers amplified 41 alleles with an average of 2.41 alleles and three genotypes per locus. Some markers presented a high frequency for the main allele. The genetic diversity ranged from 0.07 to 0.60, the observed heterozygosity had very low values, and the mean polymorphism information content was 0.32. Molecular genetic similarity analyses clustered the accessions in 13 groups, also not following taxonomic ranks. There was no association between morphoagronomic and molecular groupings. In conclusion, there was great variability among the accessions and among and within botanic groups.

Diversity among isolates of cowpea severe mosaic virus infecting cowpeas in northeastern Brazil.

Eleven isolates of cowpea severe mosaic virus (CPSMV), a member of the genus Comovirus, were selected from 50 samples collected of nine cowpea fields in Northeastern Brazil (Piauí, Ceará, Rio Grande do Norte, Paraíba, Pernambuco, Alagoas, Sergipe, Bahia, and Distrito Federal) and partially sequenced. The RNA1 partial sequence, corresponding to the helicase, viral genome-linked protein, picornain 3C-like protease, and the RNA-directed RNA polymerase genes from CPSMV, had high identity among isolates, varying from 98 to 100%. No evidence was found for intermolecular or intramolecular recombination. Phylogenetic analysis confirmed that the Brazilian CPSMV isolates are substantially different from the CPSMV strain USA. Despite the low variability found among Brazilian CPSMV isolates, there were notable differences in the symptomatology of infected cowpea plants, ranging from mild to moderate. Previous reports have demonstrated an association between CPSMV symptom determinants and helicase. However, we found no correlation between the helicase mutations and symptoms caused by CPSMV. Nevertheless, all isolates with mutation R to K in the protease provoked severe symptoms. This type of information can provide a foundation for the development of strategies to produce durable resistant cowpea lines. It is crucial for strategies based on DNA sequence-dependent technologies, such as inhibition with RNAi.

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.

Isolation and characterization of the promoter sequence of a cassava gene coding for Pt2L4, a glutamic acid-rich protein differentially expressed in storage roots.

Cassava is one of the most important tropical food crops for more than 600 million people worldwide. Transgenic technologies can be useful for increasing its nutritional value and its resistance to viral diseases and insect pests. However, tissue-specific promoters that guarantee correct expression of transgenes would be necessary. We used inverse polymerase chain reaction to isolate a promoter sequence of the Mec1 gene coding for Pt2L4, a glutamic acid-rich protein differentially expressed in cassava storage roots. In silico analysis revealed putative cis-acting regulatory elements within this promoter sequence, including root-specific elements that may be required for its expression in vascular tissues. Transient expression experiments showed that the Mec1 promoter is functional, since this sequence was able to drive GUS expression in bean embryonic axes. Results from our computational analysis can serve as a guide for functional experiments to identify regions with tissue-specific Mec1 promoter activity. The DNA sequence that we identified is a new promoter that could be a candidate for genetic engineering of cassava roots.

Suppression of SERK gene expression affects fungus tolerance and somatic embryogenesis in transgenic lettuce.

The Somatic embryogenesis receptor-like kinase (SERK) gene plays an important role in plant somatic and zygotic embryogenesis induction. The gene encodes an LRR-containing receptor-like kinase protein. Studies have been carried out focusing on different aspects of its function, but definitive conclusions on its role are far from being reached. SERK expression is generally detected in cells in which somatic or zygotic embryogenesis has been triggered. Transgenic lettuce lines were produced to silence the endogenous SERK gene using antisense RNA. The average number of seeds per flower in the R(1) and R(2) generations was similar for both transgenic and non-transgenic lines. However, a reduction in the number of viable grained seeds was observed in four studied transgenic lines. Endogenous SERK expression analysis revealed the absence of detectable LsSERK gene transcripts in three transgenic lines, which presented a reduction in their ability to form in vitro somatic embryonic structures. In addition, transgenic lines showed enhanced susceptibility to the pathogenic fungus Sclerotinia sclerotiorum, when compared to control plants. The results support the idea that SERK genes might not only be involved in plant growth and development, but probably also in a general mechanism of biotic and abiotic stress perception.

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.

Transgene elimination in genetically modified dry bean and soybean lines

Transgene elimination is a poorly studied phenomenon in plants. We made genetic and molecular studies of a transgenic dry bean line immune to bean golden mosaic geminivirus and a soybean line. In both lines, the transgenes were stable during the vegetative phase but were eliminated during meiosis. Due to its potential biotechnological value, this transgenic line was micropropagated by grafting and the vegetative copies were studied for more than two years. More than 300 plants of progeny were obtained during this period, demonstrating that the phenomenon of elimination was consistently repeated and offering an opportunity for detailed study of transgene elimination, including the characterization of the integration sites. Cloning and sequencing of the transgenic loci, reciprocal crosses to untransformed plants, genomic DNA blots, and GUS assays were performed in the transgenic lines. Based on the molecular and genetic characterization, possible mechanisms involved in transgene elimination include intrachromosomal recombination, genetic instability resulting from the tissue culture manipulations, and co-elimination of transgenes, triggered by a process of genome defense.

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.

Expression of human bone morphogenetic protein (BMP-2 and BMP-4) genes in transgenic bovine fibroblasts

cDNAs dos genes bone morphogenetic protein-2 (BMP-2) e bone morphogenetic protein-4 (BMP-4) foram sintetizados a partir de RNA total extraído de tecidos ósseos de pacientes que apresentavam trauma facial (fraturas do maxilar entre o 7º e o 10º dia pós-trauma) e clonados num vetor para expressão em células mamíferas, sob controle do promotor de citomegalovírus (CMV). Os vetores contendo os genes BMP-2 e o BMP-4 foram utilizados para a transfecção de fibroblastos bovinos. mRNAs foram indiretamente detectados por RT-PCR nas células transfectadas. As proteínas BMP-2 e BMP-4 foram detectadas mediante análises de Western blot. Os resultados demonstram a possibilidade de produção desses fatores de crescimento celular em fibroblastos bovinos. Essas células poderão ser utilizadas como fontes doadoras de material genético para a técnica de transferência nuclear na geração de animais transgênicos.

Biolistic co-transformation of Metarhizium anisopliae var. acridum strain CG423 with green fluorescent protein and resistance to glufosinate ammonium.

Metarhizium anisopliae var. acridum (syn. M. flavoviride) is recognized as a highly specific and virulent mycopathogen of locusts and grasshoppers and is currently being developed as a biological control agent for this group of insects in Brazil. Intact conidia of M. anisopliae var. acridum strain CG423 were transformed using microparticle bombardment. Plasmids used were: (1) pBARKS1 carrying the bar gene of Streptomyces hygroscopicus fused to the Aspergillus nidulans trpC promoter, encoding resistance to glufosinate ammonium (or phosphinothricin) and modified by addition of the telomeric repeat (TTAGGG)(18) of Fusarium oxysporum and 2.pEGFP/gpd/tel carrying a red-shifted variant gene for Aequorea victoria green fluorescent protein (EGFP) which we have fused to the A. nidulans gpd promoter and trpC terminator. Highly fluorescent co-transformants were selected on solid minimal medium containing 100 microg ml(-1) glufosinate ammonium using an inverted microscope with 450-490 nm excitation/510 nm emission filter set. Southern blot analysis of co-transformants revealed varying multiple chromosomal integrations of both bar and egfp genes at both telomeric and non-telomeric loci. Transformants retained pathogenicity in bioassays against Rhammatocerus schistocercoides and showed unaltered lack of pathogenicity against larvae of the non-target insect Anticarsia gemmatalis. One co-transformant from four tested, however, showed a significant, but non-dose-dependent, elevation in virulence against Tenebrio molitor.