DEFINING THE TAXONOMY, FUNCTION, AND PLANT HEALTH IMPACT OF PROTIST-ASSOCIATED BACTERIAL COMMUNITIES IN THE RHIZOSPHERE

Predation is a leading contributor to bacterial mortality in the environment, and bacterivore protists have an important role in shaping rhizosphere microbiome composition and plant health. Protists may also enhance the survival, dominance, and behavior of bacteria that resist predation, including plant beneficial or endophytic bacteria, while some bacteria form symbiotic relationships with protists. However, there is still a limited understanding of the role of protists in positive selection of bacteria in the context of the rhizosphere. We hypothesize that rhizosphere protists help promote the survival of some plant endophytic bacterial clades, and that these interactions may depend on the presence of the plant. The goal of this project is to understand taxonomic patterns governing protist enhancement of bacteria in the rhizosphere, the genetic features enriched and expressed in protist-associated rhizosphere bacteria, and the impact of these associations on root colonization and plant growth. The project will result in an understanding of how protist-bacterial associations affect the rhizosphere success of specific bacteria and protists, and the resulting implications for plant health.The project has three major objectives:Objective 1: Characterize protist-associated bacterial communities. In this objective, we will characterize the bulk soil and rhizosphere bacterial communities selected by protist isolates after repeated passaging in vitro, and for a smaller selection of protists, after passaging in soil or maize seedling mesocosms. Passaged cultures and samples will be profiled by amplicon sequencing to identify bacterial taxa selected by specific or diverse predators, and to determine whether the communities selected show patterns across protist taxon or trophic style. Selected protist cultures will be profiled after cell sorting and antibiotic passaging to identify candidate protist-attached, encysted, or obligate bacterial taxa. This objective will result in the identification of novel bacterial taxa enriched by different soil protists in the rhizosphere, including candidate physical protist-bacterial associations, and the determination of potential taxonomic and soil compartment patterns governing this selection.Objective 2: Genetic and functional analysis of protist-bacterial associations in the rhizosphere. Bacteria selected by some protist cultures will be identified and characterized for plant beneficial traits, predation resistance, effects on protist growth, and gene content (2.1). Protists and their bacterial communities will also be metagenomically characterized to identify rare and uncultured bacteria associated with the protists, and to identify candidate antipredation and symbiosis strategies (2.2). Transcriptome analysis (2.3) followed by targeted LC-MS analysis of selected metabolites (2.4) will be performed to identify metabolic pathways that are expressed during the protist-bacterial interaction, with a special focus on identification of traits with potential relevance to plant-microbe interactions such as production of phytohormones, antibiotics, and signals. This objective will identify candidate mechanisms of protist-bacterial associations, will determine how they may affect important rhizosphere functions, and will generate a resource of genetic information for plant protistology research.Objective 3: Determine how protist-bacterial associations affect rhizosphere colonization and plant health. We hypothesize that protists increase colonization of their associated bacteria on the root, although the benefits may be less pronounced in complex bacterial or protist mixtures. In this objective, duplex droplet digital PCR (ddPCR) assays will be developed to simultaneously detect the absolute abundance of protist and bacterial pairs, and validated through testing on target and non-target organisms (3.1). Protist and bacterial pairs will be inoculated singly or in combination on maize seedlings, and ddPCR will be performed on root samples to quantify protist and bacterial abundance (3.2). The experiments will be performed with and without the addition of defined protist or bacterial communities, to determine the effects of inter-protist and inter-bacterial competition on protist-enhanced colonization. Plant health will be assessed using a panel of metrics including root and shoot biomass, height, root morphology, and photosynthetic parameters. This objective will determine whether bacterial colonization and plant health is impacted by associated predators, and will determine the role of competition on the phenomenon.