A protoplast generation and transformation method for soybean sudden death syndrome causal agents and .

BACKGROUND

Soybean production around the globe faces significant annual yield losses due to pests and diseases. One of the most significant causes of soybean yield loss annually in the U.S. is sudden death syndrome (SDS), caused by soil-borne fungi in the species complex. Two of these species, and , have been discovered in the U.S. The genetic mechanisms that these pathogens employ to induce root rot and SDS are largely unknown. Previous methods describing protoplast generation and transformation have been used to study gene function, but these methods lack important details and controls. In addition, no reports of protoplast generation and genetic transformation have been made for .

RESULTS

We developed a new protocol for developing fungal protoplasts in these species and test the protoplasts for the ability to take up foreign DNA. We show that wild-type strains of and are sensitive to the antibiotics hygromycin and nourseothricin, but strains transformed with resistance genes displayed resistance to these antibiotics. In addition, integration of fluorescent protein reporter genes demonstrates that the foreign DNA is expressed and results in a functional protein, providing fluorescence to both pathogens.

CONCLUSIONS

This protocol provides significant details for reproducibly producing protoplasts and transforming and . The protocol can be used to develop high quality protoplasts for further investigations into genetic mechanisms of growth and pathogenicity of and . Fluorescent strains developed in this study can be used to investigate temporal colonization and potential host preferences of these species.