A highly efficient sulfadiazine selection system for the generation of transgenic plants and algae

Abstract

The genetic transformation of plant cells is critically dependent on the availability of efficient selectable marker gene. Sulfonamides are herbicides that, by inhibiting the folic acid biosynthetic pathway, suppress the growth of untransformed cells. Sulfonamide resistance genes that were previously developed as selectable markers for plant transformation were based on the assumption that, in plants, the folic acid biosynthetic pathway resides in the chloroplast compartment. Consequently, the Sul resistance protein, a herbicide-insensitive dihydropteroate synthase, was targeted to the chloroplast. Although these vectors produce transgenic plants, the transformation efficiencies are low compared to other markers. Here, we show that this inefficiency is due to the erroneous assumption that the folic acid pathway is located in chloroplasts. When the RbcS transit peptide was replaced by a transit peptide for protein import into mitochondria, the compartment where folic acid biosynthesis takes place in yeast, much higher resistance to sulfonamide and much higher transformation efficiencies are obtained, suggesting that current sul vectors are likely to function due to low-level mistargeting of the resistance protein to mitochondria. We constructed a series of optimized transformation vectors and demonstrate that they produce transgenic events at very high frequency in both the seed plant tobacco and the green alga Chlamydomonas reinhardtii. Co-transformation experiments in tobacco revealed that sul is even superior to nptII, the currently most efficient selectable marker gene, and thus provides an attractive marker for the high-throughput genetic transformation of plants and algae.

Targeting specificity of nuclear-encoded organelle proteins with a self-assembling split-fluorescent protein toolkit

“…Similarly, the transit peptide of RbcS is able to translocate the GFP11 tag into mitochondria, but this property remained undetected with the fluorescent protein tagging approach. Remarkably, supporting the above observation, such dual targeting of the RbcS transit peptide was also witnessed in a recent study employing sulfadiazine-resistant plants (Tabatabaei et al, 2019). Conversely, the dual targeting of the yeast CoxIV transport signal had never been reported previously.…”

Determining targeting specificity of nuclear-encoded organelle proteins with the self-assembling split-fluorescent protein toolkit

“…Similarly, as shown here, the transit peptide of RbcS is able to translocate the GFP11 tag into mitochondria, but this property remained undetected with the FP-tagging approach. Remarkably, such mitochondria targeting properties of the RbcS transit peptide were also found in a recent study employing sulfadiazine-resistant plants (Tabatabaei et al, 2018). In contrast, dual targeting of the transport signal of yeast CoxIV had never been reported earlier.…”

The Synthetic Biology Toolkit for Photosynthetic Microorganisms

“…Several antibiotic resistance genes are available in Chlamydomonas, including the newly developed sulfadiazine resistance gene (Tabatabaei et al, 2019) and the well-established selectable marker genes conferring resistance to paromomycin, hygromycin, spectinomycin, kanamycin, and zeocin. Most of these genes are functional in numerous other algal species (Doron et al, 2016;Poliner et al, 2018).…”