In host-microbe interaction studies, genomic technologies have significantly advanced our understanding of host responses and structure of host-associated microbial communities (microbiota). However, spatial and physiological heterogeneity in host-microbe interactions has been more challenging to study. Plants are comprised of various cell types, each of which may interact with a distinct set of microbes and, at the same time, accomplish other tasks such as growth and nutrient acquisition/utilization. To date, interactions between individual plant cell types and the microbiota are poorly understood due to our lack of knowledge about cell type-specific responses of plants and the composition of associated microbes at the cellular level. Here, I employ high-throughput single cell trasncriptomics to analyze plant cell type-specific responses to the microbiota under nutrient (phosphate) replete and starvation conditions. I will mainly investigate immune response and phosphate starvation response and crosstalks between them in individual cell types. I will also develop a method for in situ microbiota community profiling by imaging-based sequencing of bacterial 16S rRNA, which provides a spatial map of the plant microbiota at the strain-level of resolution. By linking plant gene expression and microbiota distribution at the single cell resolution, I will investigate the roles of plant genes and microbiota members that show plant cell-type specificity. This project attempts to reveal plant cell type-specific responses to biotic and abiotic environmental signals and microbiota assembly that are significant and biologically meaningful.

A number of transformative molecular tools for biomedical research and therapeutics have been developed. However, a lack of safe, efficient delivery methods has precluded the widespread therapeutic application of these tools. One promising route to overcoming this limitation is to investigate the diverse biomolecule exchange systems found in nature and develop delivery tools based on these systems. Recently, a novel membrane vesicle (MV)-mediated horizontal gene transfer system was found in archaea. In this system, a relatively large 50 kbp plasmid is enclosed in a MV and delivered to other archaea. These plasmid vesicles (PVs) use a viral-like plasmid, pR1SE, that encodes the proteins required to assemble PVs and transfer the plasmid to other cells. As intercellular communication by MV is a universal process among the three domains of life, these systems may be exploited to deliver tools in human cells or the human microbiome. However, the mechanisms of archaeal MV and PV formation are largely unknown. I propose to identify the key proteins in the PV system. I will narrow down the candidates by remote homology detection among pR1SE and related viruses, and validate them by biochemical experiments. Furthermore, I will use genome-wide CRISPR screening to identify key proteins of host archaeal cells. Ultimately, I aim to reconstruct the PV system towards programmable synthetic vesicles production. This proposal will elucidate basic archaeal biology and provide a hint to the origin of viruses with a long-term objective of harnessing archaeal vesicle systems for delivery tools. Finally, this approach will serve as a paradigm for engineering archaea for molecular technologies.

Protein degradation systems maintain protein homeostasis. A failure of these systems causes various diseases, such as neurodegenerative diseases and cancers. In eukaryotic cells, ubiquitin is a general marker for selective degradation and determines protein lifetime in vivo. In selective degradation, E3 ubiquitin ligases determine target proteins. Although there are ~800 E3 ligases in human, only a handful of them have already shown to recognize specific short peptide motifs called “degrons”. Moreover, the feature(s) for unstable or misfolded structures recognized by E3 ligases remain unclear. In part, this is because we lack a comprehensive approach to investigate the global relationship between protein structural stability, ubiquitination status, and lifetime of the protein. To reveal the effect of protein folding stability (and other features) on ubiquitination and biological lifetime in vivo, I propose to measure these parameters for thousands of designed mini-proteins, whose folding stability has been previously characterized in detail. First, I will measure biological lifetime for these mini-proteins by flow cytometry, and monitor their ubiquitination status by using top-down proteomics approach. Then, I will analyze these data by using in silico analysis and decipher what factor(s) determine ubiquitination states and biological lifetime. This highly innovative and comprehensive approach using thousands of designed proteins will allow me to uncover the fundamental principle for protein lifetime in vivo and provide a mechanistic basis for designing better tools to manipulate protein lifetime.