While studying the nitrogen-fixing endophytes within poplar trees, the profound discovery was made that some of the isolates were yeast, a eukaryotic organism. Since it is believed that only prokaryotic organisms can utilize nitrogen from the air, this was an exciting discovery. In this small grant for exploratory research, the PIs will identify the eukaryotic genes from these nitrogen-fixing yeasts that allow growth on nitrogen-free medium, and attempt to isolate mutants that no longer grow in nitrogen-free medium. The nif gene cluster will be analyzed and compared with known diazotrophic bacteria and they will determine whether or not the genes have eukaryotic transcription signals. Identification of the known genes required for nitrogen fixation within the genome of the yeast strain will provide compelling evidence for nitrogen fixation by this eukaryotic organism. Verification that the genes known to be involved in nitrogen fixation are being expressed in the yeast strains will also provide further support for eukaryotic nitrogen fixation. If nif mutants can be isolated that no longer fix nitrogen, and if adding back the nitrogenase genes restores growth on nitrogen-free medium, it would provide unequivocal evidence of eukaryotic nitrogen fixation.
The broader impacts resulting from the proposed activity include an emphasis on training of women in science. Dr. Doty will continue to lead a graduate level seminar course, Plant-Microbe Interactions in which she shares her results about the nitrogen fixing endophytes of poplar. The goal is to publish the results in a first-rate journal, and give seminars to disseminate the news widely. If successful, this research would cause a paradigm shift in current understanding of nitrogen fixation since it would be the first example of a nitrogen-fixing eukaryotic organism.
