The Intracellular Scots Pine Shoot Symbiont Methylobacterium extorquens DSM13060 Aggregates around the Host Nucleus and Encodes Eukaryote-Like Proteins

Abstract

ABSTRACT Endophytes are microbes that inhabit plant tissues without any apparent signs of infection, often fundamentally altering plant phenotypes. While endophytes are typically studied in plant roots, where they colonize the apoplast or dead cells, Methylobacterium extorquens strain DSM13060 is a facultatively intracellular symbiont of the meristematic cells of Scots pine (Pinus sylvestris L.) shoot tips. The bacterium promotes host growth and development without the production of known plant growth-stimulating factors. Our objective was to examine intracellular colonization by M. extorquens DSM13060 of Scots pine and sequence its genome to identify novel molecular mechanisms potentially involved in intracellular colonization and plant growth promotion. Reporter construct analysis of known growth promotion genes demonstrated that these were only weakly active inside the plant or not expressed at all. We found that bacterial cells accumulate near the nucleus in intact, living pine cells, pointing to host nuclear processes as the target of the symbiont's activity. Genome analysis identified a set of eukaryote-like functions that are common as effectors in intracellular bacterial pathogens, supporting the notion of intracellular bacterial activity. These include ankyrin repeats, transcription factors, and host-defense silencing functions and may be secreted by a recently imported type IV secretion system. Potential factors involved in host growth include three copies of phospholipase A2, an enzyme that is rare in bacteria but implicated in a range of plant cellular processes, and proteins putatively involved in gibberellin biosynthesis. Our results describe a novel endophytic niche and create a foundation for postgenomic studies of a symbiosis with potential applications in forestry and agriculture. IMPORTANCE All multicellular eukaryotes host communities of essential microbes, but most of these interactions are still poorly understood. In plants, bacterial endophytes are found inside all tissues. M. extorquens DSM13060 occupies an unusual niche inside cells of the dividing shoot tissues of a pine and stimulates seedling growth without producing cytokinin, auxin, or other plant hormones commonly synthesized by plant-associated bacteria. Here, we tracked the bacteria using a fluorescent tag and confocal laser scanning microscopy and found that they localize near the nucleus of the plant cell. This prompted us to sequence the genome and identify proteins that may affect host growth by targeting processes in the host cytoplasm and nucleus. We found many novel genes whose products may modulate plant processes from within the plant cell. Our results open up new avenues to better understand how bacteria assist in plant growth, with broad implications for plant science, forestry, and agriculture.