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Structure function relationships of the phyllosphere microbiota

Periodic Reporting for period 3 - PhyMo (Structure function relationships of the phyllosphere microbiota)

Reporting period: 2018-09-01 to 2020-02-29

The phyllosphere comprises the aerial parts of the plants that carry out the bulk of terrestrial carbon dioxide fixation. As such, the phyllosphere represents a major biotic link between the biosphere and the atmosphere. In total, the surface area of leaves of the global vegetation is vast and provides a habitat to numerous microorganisms that impact plant growth and health. The project aims at uncovering drivers of bacterial phyllosphere community structures to improve our fundamental understanding of the phyllosphere microbiota. To enable functional microbiota research, individual bacterial strains and synthetic bacterial communities are used in gnotobiotic plant systems to reduce the number of environmental variables while maintaining the intrinsic complexity of the plant microbiota interactions.
In the first part of the project we concluded a comprehensive study in which we isolated Arabidopsis thaliana leaf bacteria as pure cultures. Individual plants as well as individual leaves were sampled at different European sites to determine their core leaf community and to establish a reference strain collection using flow cytometry and dilution series plating. After identifying approximately 3,000 isolates using a high-throughput DNA sequencing-based method we selected more than 200 representative strains belonging to 52 genera of the major phyllosphere phyla covering the majority of the culture-independent taxonomic diversity. Draft genomes of all selected isolates were generated. Recolonization experiments using synthetic communities in a gnotobiotic model system showed reproducible colonization patterns and represents a valuable starting point to identify mechanisms of community formation and function. Examination of plant responses to its microbiota revealed that the plant reacts differently to members of its natural phyllosphere microbiota. A subset of commensals increase expression of defense-related genes and thereby contribute to plant health and performance. In complementary work, we used the resource of leaf isolates and their genome to systematically probe the interaction of strains and mine for uncharacterized natural products. Genome analysis revealed more than 1,000 predicted natural product biosynthetic gene clusters, hundreds of which are unknown compared to a database of characterized clusters. For functional validation, we used a high-throughput screening approach to monitor over 50,000 binary strain combinations. We observed 725 inhibitory interactions, with 26 strains contributing to the majority of these. A combination of imaging mass spectrometry and bioactivity-guided fractionation of the most potent inhibitor, revealed three distinct natural product scaffolds that contribute to the observed antibiotic activity. Moreover, a genome mining-based strategy led to the isolation of a trans-acyltransferase polyketide synthase-derived antibiotic, which displays an unprecedented natural product structure. In additional work, proteomics, metabolomics, transcriptomics and genomics analyses of individual leaf microbiota members uncovered traits that are important for plant colonization and adaptation. A number of original research manuscripts and reviews has been published until the due date of this mid-term report and these are provided separately to this document.
Reductionist approaches to disentangle the inherent complexity of microbe-microbe-plant interactions in situ could be successfully established. Experimentally tractable, synthetic communities enabled first testing of hypotheses by targeted manipulations in gnotobiotic systems. In the future, modifications of microbial, host, and environmental parameters are planned to quantitative assess host and microbe characteristics.