Distinct identities of leaf phloem cells revealed by single cell transcriptomics.

The leaf vasculature plays a key role in solute translocation. Veins consist of at least seven distinct cell types, with specific roles in transport, metabolism, and signaling. Little is known about leaf vascular cells, in particular the phloem parenchyma (PP). PP effluxes sucrose into the apoplasm as a basis for phloem loading, yet PP has been characterized only microscopically. Here, we enriched vascular cells from Arabidopsis leaves to generate a single-cell transcriptome atlas of leaf vasculature. We identified at least 19 cell clusters, encompassing epidermis, guard cells, hydathodes, mesophyll, and all vascular cell types, and used metabolic pathway analysis to define their roles. Clusters comprising PP cells were enriched for transporters, including SWEET11 and SWEET12 sucrose and UmamiT amino acid efflux carriers. We provide evidence that PP development occurs independently from ALTERED PHLOEM DEVELOPMENT, a transcription factor required for phloem differentiation. PP cells have a unique pattern of amino acid metabolism activity distinct from companion cells (CCs), explaining differential distribution/metabolism of amino acids in veins. The kinship relation of the vascular clusters is strikingly similar to the vein morphology, except for a clear separation of CC from the other vascular cells including PP. In summary, our single-cell RNA-sequencing analysis provides a wide range of information into the leaf vasculature and the role and relationship of the leaf cell types.

Development of a toolbox to dissect host-endosymbiont interactions and protein trafficking in the trypanosomatid Angomonas deanei.

BACKGROUND: Bacterial endosymbionts are found across the eukaryotic kingdom and profoundly impacted eukaryote evolution. In many endosymbiotic associations with vertically inherited symbionts, highly complementary metabolic functions encoded by host and endosymbiont genomes indicate integration of metabolic processes between the partner organisms. While endosymbionts were initially expected to exchange only metabolites with their hosts, recent evidence has demonstrated that also host-encoded proteins can be targeted to the bacterial symbionts in various endosymbiotic systems. These proteins seem to participate in regulating symbiont growth and physiology. However, mechanisms required for protein targeting and the specific endosymbiont targets of these trafficked proteins are currently unexplored owing to a lack of molecular tools that enable functional studies of endosymbiotic systems. RESULTS: Here we show that the trypanosomatid Angomonas deanei, which harbors a ß-proteobacterial endosymbiont, is readily amenable to genetic manipulation. Its rapid growth, availability of full genome and transcriptome sequences, ease of transfection, and high frequency of homologous recombination have allowed us to stably integrate transgenes into the A. deanei nuclear genome, efficiently generate null mutants, and elucidate protein localization by heterologous expression of a fluorescent protein fused to various putative targeting signals. Combining these novel tools with proteomic analysis was key for demonstrating the routing of a host-encoded protein to the endosymbiont, suggesting the existence of a specific endosymbiont-sorting machinery in A. deanei. CONCLUSIONS: After previous reports from plants, insects, and a cercozoan amoeba we found here that also in A. deanei, i.e. a member of a fourth eukaryotic supergroup, host-encoded proteins can be routed to the bacterial endosymbiont. This finding adds further evidence to our view that the targeting of host proteins is a general strategy of eukaryotes to gain control over and interact with a bacterial endosymbiont. The molecular resources reported here establish A. deanei as a time and cost efficient reference system that allows for a rigorous dissection of host-symbiont interactions that have been, and are still being shaped over evolutionary time. We expect this system to greatly enhance our understanding of the biology of endosymbiosis.