Necessity of RNA degradosome assembly in Sinorhizobium meliloti for effective symbiosis - Project Summary / Abstract The proposed project will elucidate how subcellular compartmentalization of the RNase E endonuclease into bacterial ribonucleoprotein granules (BR-bodies) in Sinorhizobium meliloti contributes to successful host colonization. S. meliloti serves as a model for investigating infection by alpha-proteobacteria, which include human pathogens such as Brucella, Bartonella, and Rickettsia. Understanding the molecular and cellular mechanisms that underpin effective host colonization can provide critical insights for preventing and treating infectious diseases, particularly as bacterial resistance to antibiotics continues to emerge. S. meliloti establishes mutualistic symbiosis with compatible legumes, including the genetically tractable reference species Medicago truncatula, by inducing development of root nodules, colonizing them, and fixing nitrogen in exchange for nutrients from host plants. We found that removing the C-terminal intrinsically disordered region of RNase E in S. meliloti led to ineffective symbiosis, eliciting nodule development but producing etiolated plants that grew poorly compared to plants inoculated with wild-type bacteria. RNase E is an essential ribonuclease involved in RNA processing and decay, and its C-terminal domain, while dispensable for viability, was shown to be necessary and sufficient for the formation of liquid-liquid phase-separated biomolecular condensates, named BR-bodies, in alpha-proteobacteria. Sequestration of RNase E into such BR-bodies facilitates mRNA decay and modulates the cellular RNA pool. The goal of this project is to determine how phase separation of RNase E influences RNA processing in S. meliloti and enables effective symbiosis by accomplishing the following three specific aims. (1) We will assess the symbiotic defects of the RNase E C-terminal deletion mutant in detail by genetic analysis. (2) We will use microscopy to characterize the role of BR-bodies during host colonization. (3) We will examine how transcriptomes differ between wild- type and mutant S. meliloti during symbiosis, as well as compare gene expression profiles of host plants colonized by wild-type versus mutant bacteria. While RNase E has been linked to pathogenesis of various bacteria, including Brucella, relatively little is known about the specific microbe-host interactions involved. Results from the proposed investigation will help identify and provide better understanding of genetic and cellular factors that allow infecting bacteria to bypass host defenses. In addition to accomplishing the scientific objectives described above, this proposal will allow students, particularly those from underrepresented backgrounds, to gain research training and preparation for biomedical careers.
