Sequence and genome organization of papaya meleira virus infecting papaya in Brazil.

Papaya sticky disease ('meleira') was first observed in Brazil at the beginning of the 1980s. The disease is characterized by intense latex exudation from the fruit surface that becomes dark as it oxidizes, which makes it difficult to sell. The causal agent, which has been called papaya meleira virus (PMeV), has been identified as an isometric virus particle, approximately 50 nm in diameter, with a double-stranded RNA genome. Here, we report the first complete sequence and organization of the 8.7-kb viral dsRNA genome. Two ORFs coding for a putative coat protein and RNA-dependent RNA polymerase (RdRp) were predicted. In silico analysis revealed that the translated ORF2 contains the conserved domains characteristic of an RdRp protein (pfam02123:RdRP 4), which is a family that includes RdRps from members of the genera Luteovirus, Totivirus and Rotavirus. Evolutionary analysis with amino acid sequences with the RdRps from members of the family Totiviridae and some dsRNA viruses showed that PMeV RdRp did not root itself in any genus.

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.

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.

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.