EDGE FGT: Essential New Molecular Genetic Tools for Defining Phenotype in the Global, Harmful Algal Bloom-producing Diatom, Pseudo-nitzchia spp.

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Harmful algal blooms (HABs) threatening ecosystems, fisheries and human health worldwide, are driven by one diatom genus, Pseudo-nitzschia, along with several dinoflagellate genera and cyanobacteria. In the last decade, various species of Pseudo-nitzschia have produced devasting blooms of the neurotoxin, domoic acid (DA), in the coastal waters of Australia, Brazil, California, Chile, France, the Gulf of Maine, Indonesia, Italy and Tunisia. Currently there is great uncertainty regarding how ocean acidification, increased concentrations of atmospheric CO2, precipitation and nutrient stress will shape the productivity, and global range of Pseudo-nitzschia species' HABs. Indeed, long term trends have emerged that link toxic Pseudo-nitzschia spp. blooms to more frequent DA contamination of shellfish fisheries in the warmer ocean temperatures that prevail during warmer years. Our collective experience with diatom molecular genetics, current diatom model organisms, marine biochemistry and microscopy and our recent discovery and characterization of the hitherto unknown pathway for DA biosynthesis places us in ideal position to establish, Pseudo-nitzschia sp., as new model diatom. In so doing, molecular biological methods will be developed to enable genome-to-phenome investigations of the regulation and biosynthesis of DA. Specifically, a method to transform Pseudo-nitzschia sp driven by bacterial conjugation will be developed and disseminated to marine labs worldwide. Our research will establish a new model diatom, Pseudo-nitzschia sp., and produce and disseminate state-of-the-art molecular biological methods to characterize the genome-phenome linkages that drive domoic acid (DA), harmful algal blooms worldwide. Taking a two-track approach, we propose to significantly upgrade the functional genomic and molecular biological tools currently used by the diatom community. Track I, We will construct a bacteria-diatom conjugation protocol and a concomitant, maintainable episome for transgenic gene delivery in Pseudo-nitzschia australis and multistriata, two globally dispersed, highly-toxigenic diatom species. Developing and disseminating new methods for diatom transformation will increase the number, and variety of species of diatoms available for functional genomics studies. Track II, With robust genetic manipulation tools for Pseudo-nitzschia spp. in place, we will develop a new diatom toolkit for molecular and biochemical applications. The first three tools to be developed are a) nanobody-fluorescent proteins (Nb-FPs) that can either repress or activate the target protein's function in vivo; b) inducible fluorescent biosensors (UnaG) that can detect DA in laboratory or seawater cultures; c) proximity-labeling enzymes: TurboID and miniTurbo, that provide insights into protein-protein interactions in vivo. As the development of the conjugative transformation protocol for Pseudo-nitzschia spp proceeds, we will express and troubleshoot each of the proteomic tools in the model organism, Phaeodactylum tricornutum.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.