Seed-expressed fluorescent proteins as versatile tools for easy (co)transformation and high-throughput functional genomics in Arabidopsis.

We demonstrate that fluorescent proteins can be used as visual selection markers for the transformation of Arabidopsis thaliana by the floral dip method. Seed-specific expression of green fluorescent protein (GFP) variants, as well as DsRed, permits the identification of mature transformed seeds in a large background of untransformed seeds by fluorescence microscopy. In planta visualization of transformed seeds in siliques shows that susceptibility to floral dip transformation is limited to a small, defined window in flower development. In the competent stage, the random transformation of up to 25% of the seeds within a single silique may occur. The use of fluorescent proteins with different spectral characteristics allows a rapid identification and genetic analysis of seeds that have received multiple genes-of-interest in co-transformation experiments. The data reveal that co-transformation does not occur at random, since the co-transformed genes are integrated at a single genetic locus in approximately 70% of the cases. This genetic linkage of the co-transformed genes greatly simplifies metabolic pathway engineering by reverse genetics in Arabidopsis. Additional advantages of using visual selection instead of antibiotic resistance include a rapid identification of the effect of the T-DNA insertion or the transgene on seed development and/or germination. This technology, of tagging and identifying transformed seeds by fluorescence provides a novel high-throughput screening system with many potential applications in plant biotechnology.

Overexpression of the tomato Asc-1 gene mediates high insensitivity to AAL toxins and fumonisin B1 in tomato hairy roots and confers resistance to Alternaria alternata f. sp. lycopersici in Nicotiana umbratica plants.

The sphinganine-analog mycotoxins (SAMs) fumonisin B1 and AAL toxins are inhibitors of eukaryotic sphinganine N-acyltransferase in vitro. Treatment of eukaryotes with SAMs generally results in an accumulation of sphingoid base precursors and a depletion of complex sphingolipids. The asc,asc genotypes of tomato (Lycopersicon esculentum) and Nicotiana umbratica are sensitive to SAMs and host of the AAL toxin-producing fungus Alternaria alternata f. sp. lycopersici. Codominant insensitivity to SAMs in tomato is mediated by the Asc-1 gene, and sensitivity is associated with a frame-shift mutation present in asc-1. We investigated the function of Asc-1 in mediating insensitivity to SAMs and resistance to the fungus by overexpression of asc-1 and Asc-1. In this study, it is shown that overexpression of these genes did not lead to visual symptoms in tomato hairy roots and N. umbratica plants. Overexpression of asc-1 did not influence the (in)sensitivity to SAMs. Overexpression of Asc-1 in SAM-sensitive hairy roots and N. umbratica plants, however, mediated a high insensitivity to SAMs and resistance to plant infection by Alternaria alternata f. sp. lycopersici.