Josias C. Faria

RNAi-Mediated Resistance to Bean golden mosaic virus in Genetically Engineered Common Bean (Phaseolus vulgaris)

Bean golden mosaic virus (BGMV) is transmitted by the whitefly Bemisia tabaci in a persistent, circulative manner, causing the golden mosaic of common bean (Phaseolus vulgaris L.). The characteristic symptoms are yellow-green mosaic of leaves, stunted growth, or distorted pods. The disease is the largest constraint to bean production in Latin America and causes severe yield losses (40 to 100%). Here, we explored the concept of using an RNA interference construct to silence the sequence region of the AC1 viral gene and generate highly resistant transgenic common bean plants. Eighteen transgenic common bean lines were obtained with an intron-hairpin construction to induce post-transcriptional gene silencing against the AC1 gene. One line (named 5.1) presented high resistance (approximately 93% of the plants were free of symptoms) upon inoculation at high pressure (more than 300 viruliferous whiteflies per plant during the whole plant life cycle) and at a very early stage of plant development. Transgene-specific small interfering RNAs were detected in both inoculated and non-inoculated transgenic plants. A semiquantitative polymerase chain reaction analysis revealed the presence of viral DNA in transgenic plants exposed to viruliferous whiteflies for a period of 6 days. However, when insects were removed, no virus DNA could be detected after an additional period of 6 days.

First transgenic geminivirus-resistant plant in the field

volume 27 number 12 december 2009 nature biotechnology unsatisfactory, and commercial cultivars are susceptible to early, moderate or severe infection2,4. After the first demonstration of pathogenderived resistance (PDR) in the pioneering work from Roger Beachy ́s lab describing coat protein–mediated resistance to tobacco mosaic virus5, several strategies have been used to genetically engineer tolerance or immunity to viruses in transgenic plants. These strategies are based on two broad classes: protein-mediated resistance and RNA silencing–mediated resistance. Now that we better understand the mechanisms of RNA interference (RNAi) and its biological functions, it is possible to look back on initial experiments from a new perspective. It is now known that plants naturally process viral RNAs to generate small sequences of a pathogen’s genetic material that can be specifically used against that pathogen through the RNA-induced silencing complex. An RNA-silencing (posttranscriptional gene silencing) mechanism was recognized as being responsible for resistance to RNA viruses. This mechanism depends on the formation of doublestranded RNA (dsRNA) whose antisense strand is complementary to the transcript of a targeted gene. These discoveries led to the introduction of constructs to produce intracellular generation of small interfering RNA (siRNA)-like species in transgenic plants, inducing targeted gene silencing and virus resistance. This is an important tool in generating plants resistant to a broad range of viruses6. However, not all viral genes used in transgenic constructs render plants resistant to infection. The use of inverted repeat constructs, resulting in dsRNA transcripts, is the most efficient means of generating transformed lines showing effective gene knockdown or virus resistance7. The most likely reason for this is that dsRNAs are fed directly into the silencing pathway at the level of the RNaseIII-like enzyme Dicer, and therefore they are not reliant on the action of plant-encoded RNA-dependent RNA polymerase proteins. Nevertheless, most examples of RNAi-mediated virus resistance pertain to RNA plant viruses. Indeed, attempts to obtain robust PDR to geminiviruses have not been as successful as those against RNA viruses, and development of geminivirus-resistant plants is considered a major challenge8. Because of the social and economic importance of common bean as a source of protein in the diet of over a billion people worldwide, we have been attempting since should be done remains undone.” If it is not clearly stated with meaning that weeds are a major constraint on the quality of life of most women in the developing world, then what should be done remains undone, and gender issues have not been adequately addressed with biotechnology.

Transgenic beans (Phaseolus vulgaris L.) engineered to express viral antisense RNAs show delayed and attenuated symptoms to bean golden mosaic geminivirus

The genes Rep-TrAP-REn and BC1 from the Brazilian isolate bean golden mosaic geminivirus (BGMV-BR) were cloned in antisense orientation under the transcriptional control of the CaMV 35S promoter. This construct was used to transform common bean (Phaseolus vulgaris L.) using the biolistic method. Transgenic plants from the R3 and R4 generations were challenged by inoculation with a BGMV-BR viruliferous whitefly population. Of the four transgenic lines tested, two had both delayed and attenuated viral symptoms. Un-transformed plants or plants transformed with a construct containing only the gus-neo gene developed typical BGMV-BR symptoms 10–15 days after inoculation.

Molecular characterization of the first commercial transgenic common bean immune to the Bean golden mosaic virus.

Golden mosaic of common bean is caused by the Bean golden mosaic virus (BGMV). The disease is one of the greatest constraints on bean production in Latin America and causes significant yield losses. The RNAi concept was explored to silence the rep (AC1) viral gene and a transgenic bean line immune to BGMV upon inoculation at high pressure was previously generated. Identification of the transgene insert confirmed the presence of a single locus corresponding to two intact copies of the RNAi cassette in opposite orientation and three intact copies of the AtAhas gene. It is flanked by Phaseolus genomic sequences and interspersed by one nuclear and three chloroplastic genomic sequences. Southern analyses showed that the transgenes were structurally stable for eight self-pollinated generations and after backcrosses with a non transgenic commercial variety. Transgene expression analyses revealed similar levels of siRNA in leaves of transgenic plants cultivated under field conditions in three distinct regions. siRNA were also analyzed during seed development in common bean transgenic plants. siRNA signals were also detected in seeds, albeit at significantly lower levels than those observed in leaves, and could not be detected in seeds cooked during 10 min. This information is relevant to demonstrate that GM beans are free of siRNA signals after cooking and therefore suitable for human consumption. Additionally, characterization of the locus where the transgene was integrated in the common bean genome provides a valuable tool to trace this GM bean material in the field and in the market.

Three distinct begomoviruses associated with soybean in central Brazil.

We report the complete nucleotide sequences of geminiviruses of the genus Begomovirus infecting soybean (Glycine max) in central Brazil. Samples obtained from soybean plants collected at Santo Antonio de Goiás, Goiás State, showing typical symptoms of viral infection, were analyzed. Infection was confirmed by PCR-based amplification of a DNA-A fragment with universal begomovirus primers. Total DNA from infected plants was then subjected to rolling-circle amplification (RCA), and 2.6-kb molecules were cloned into plasmid vectors. Sequencing of the three DNA-A and two DNA-B clones thus obtained confirmed infection by three distinct begomoviruses: bean golden mosaic virus, Sida micrantha mosaic virus and okra mottle virus, the last of which was reported recently to be a novel virus infecting okra plants in Brazil. Begomovirus infection of soybean plants has been reported sporadically in Brazil and has generally not been considered to be of economic relevance.

Distinction between a transgenic and a conventional common bean genotype by 1H HR-MAS NMR.

In this work, it is proposed a methodology which allows to differentiate a conventional and a specific transgenic common beans, grown in greenhouse or under field conditions, based on modifications in chemical composition using (1)H HR-MAS NMR. It is demonstrated that the influence of typical variables from field planting conditions had no significant influence on the ability to set apart transgenic from conventional. This methodology was corroborated by multivariate data analysis of the (1)H NMR and IR spectra. This study also points out the simplicity of using the HR-MAS NMR technique for food analyses. The measurement is highly simplified because it does not require any pretreatment of the sample apart from the addition of a small amount of D2O necessary to produce homogeneous dough and a field frequency lock. Moreover, due to the high concentration of the sample, measurement time in HR-MAS NMR is very short.

Molecular and biological characterization of a new Brazilian begomovirus, euphorbia yellow mosaic virus (EuYMV), infecting Euphorbia heterophylla plants

To date, no begomovirus has been fully characterized from Euphorbia heterophylla, a widely distributed weed, in Brazil. Here, we show the occurrence of a new begomovirus on E. heterophylla plants showing bright yellow mosaic. The bipartite viral genome was cloned from 10 samples, and all clones are almost identical to each other (95.6-98.8% nucleotide sequence identity). The DNA-A sequences shared a maximum nucleotide sequence identity of 87.3% with euphorbia mosaic Peru virus (EuMPV) and thus were classified as belonging to a novel begomovirus species, tentatively named Euphorbia yellow mosaic virus (EuYMV). The EuYMV DNA-B sequences share a maximum nucleotide sequence identity of 56.2% with a euphorbia mosaic virus (EuMV) isolate from Mexico. Phylogenetic analysis demonstrated that this new virus belongs to a different lineage than EuMV isolates from Central America.

Primers and probes development for specific PCR detection of genetically modified common bean (Phaseolus vulgaris) Embrapa 5.1.

The genetically modified common bean Embrapa 5.1, developed by Brazilian Agricultural Research Corporation (Embrapa), is the first commercial GM plant produced in Latin America. It presents high resistance to the Bean golden mosaic virus. In this work, primers and probes targeting a taxon-specific reference DNA sequence for the common bean (Phaseolus vulgaris L.) and a construct-specific DNA sequence of Embrapa 5.1 GM common bean were successfully developed. The primers and probes showed high specificity for the target detection. Both methods showed suitable efficiency and performance to be used as an endogenous target for detection of common bean DNA and for construct-specific detection of GM common bean Embrapa 5.1, respectively. Both real-time PCR assays proved to be valuable for future assessment of interlaboratory studies.

Comparative analysis of nutritional compositions of transgenic RNAi-mediated virus-resistant bean (event EMB-PV051-1) with its non-transgenic counterpart

Golden mosaic is among the most economically important diseases that severely reduce bean production in Latin America. In 2011, a transgenic bean event named Embrapa 5.1 (EMB-PV051-1), resistant to bean golden mosaic virus, was approved for commercial release in Brazil. The aim of this study was to measure and evaluate the nutritional components of the beans, as well as the anti-nutrient levels in the primary transgenic line and its derived near-isogenic lines after crosses and backcrosses with two commercial cultivars. Nutritional assessment of transgenic crops used for human consumption is an important aspect of safety evaluations. Results demonstrated that the transgenic bean event, cultivated under field conditions, was substantially equivalent to that of the non-transgenic bean plants. In addition, the amounts of the nutritional components are within the range of values observed for several bean commercial varieties grown across a range of environments and seasons.

Development of Plasmid DNA Reference Material for the Quantification of Genetically Modified Common Bean Embrapa 5.1

The genetically modified (GM) common bean Embrapa 5.1 was recently approved for commercialization. The reliable detection and quantification of GM organisms is strongly dependent on validated methods as well as calibration systems. This work presents the development of a calibrant plasmid for Embrapa 5.1 common bean detection. The reaction parameters were determined and compared for both the plasmid DNA (pDNA) and the genomic DNA (gDNA). PCR efficiencies for pDNA were 81% for the construction-specific assays and 76% for the taxon-specific assay, whereas for gDNA efficiencies were 94 and 93%, respectively. The limits of detection (LOD) in both qPCR assays were 10(2) and 10(3) copies of gDNA and pDNA per PCR reaction, respectively. This is sufficient to detect 0.067 and 0.67% of GM common bean in 100 ng of DNA, respectively, which is in agreement with detecting the 1% GM content required by the Brazilian legislation.