Bacterial endophytes journey from the seed to the seedling: an analysis of community structural shifts and bacterial localization during colonization

Bacterial endophytes equip plants with the ability to navigate biotic and abiotic challenges. Introducing a consortium of these beneficial microbes into our crop plants would address the many challenges of sustainable agriculture, particularly if these endophytes were transmitted generationally. However, our knowledge of seedling colonization by seed endophytes is limited and has remained a data-bottleneck critical to resolving mechanisms that drive successful placement of seed endophytes within the plant body. In this work, we aimed to visualize these early stages of colonization and tie bacterial endophyte genera to key events in migration from the seedcoat to the seedling leaf. The requisite system for this study was an orchid, since their microscopic embryos and transparent seedcoat facilitated whole-seeding imaging during germination. Microbiome analysis revealed the dominant core genera as Sediminibacterium, Mesorhizobium, Bradyrhizobium, Ralstonia and Kocuria and demonstrated that as the germinating orchid protocorm formed a first leaf, and later, a root, the microbiome community structure shifted. All flagellated seed endophytes were visualized using LSCM with Alexa Fluor 488 during germination. The endophytes aggregated into bacterial mats, located to the shoot meristem and moved into the first leaf, where endophytes dispersed into smaller aggregates and colonized the stomata for access to the leaf interior. Many of the core genera were abundant during meristem/first leaf formation, while Pelomonas, Sphingomonas, Brevibacillus, and Rothia became prevalent during stomatal targeting. Immunolocalization studies with Arabidopsis during germination revealed a similar pattern of colonization. The whole-seeding approach in this study unveiled a detailed story of the dynamic changes that seed endophytes undergo during germination. Bacterial endophytes exhibited spatiotemporal patterns of colonization that suggest coordinated interspecies interactions as they migrate to the plant interior. Moreover, activities associated with the journey may explain the shifts in community structure as shoots and roots emerge during germination. Identification of genera and species associated with key colonization events provide a starting point for future co-culture experiments to understand the essential microbial interactions necessary to populate the seedling, and later in development, progeny seed.