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.

In vivo trans-specific gene silencing in fungal cells by in planta expression of a double-stranded RNA.

BACKGROUND: Self-complementary RNA transcripts form a double-stranded RNA (dsRNA) that triggers a sequence-specific mRNA degradation, in a process known as RNA interference (RNAi), leading to gene silencing. In vascular plants, RNAi molecules trafficking occur between cells and systemically throughout the plant. RNAi signals can spread systemically throughout a plant, even across graft junctions from transgenic to non-transgenic stocks. There is also a great interest in applying RNAi to pathogenic fungi. Specific inhibition of gene expression by RNAi has been shown to be suitable for a multitude of phytopathogenic filamentous fungi. However, double-stranded (ds)RNA/small interfering (si)RNA silencing effect has not been observed in vivo. RESULTS: This study demonstrates for the first time the in vivo interference phenomenon in the pathogenic fungus Fusarium verticillioides, in which expression of an individual fungal transgene was specifically abolished by inoculating mycelial cells in transgenic tobacco plants engineered to express siRNAs from a dsRNA corresponding to the particular transgene. CONCLUSION: The results provide a powerful tool for further studies on molecular plant-microbe and symbiotic interactions. From a biotechnological perspective, silencing of fungal genes by generating siRNAs in the host provides a novel strategy for the development of broad fungi-resistance strategies in plants and other organisms.