Mapping phyllosphere microbiota interactions in planta to establish genotype-phenotype relationships.

Host-associated microbiomes harbour hundreds of bacterial species that co-occur, creating the opportunity for manifold bacteria-bacteria interactions, which in turn contribute to the overall community structure. The mechanisms that underlie this self-organization among bacteria remain largely elusive. Here, we studied bacterial interactions in the phyllosphere microbiota. We screened for microbial interactions in planta by adding 200 endogenous strains individually to a 15-member synthetic community and tracking changes in community composition upon colonization of the model plant Arabidopsis. Ninety percent of the identified interactions in planta were negative, and phylogenetically closely related strains elicited consistent effects on the synthetic community, providing support for trait conservation. Community changes could be largely explained by binary interactions; however, we also identified a higher-order interaction of more than two interacting strains. We further focused on a prominent interaction between two members of the Actinobacteria. In the presence of Aeromicrobium Leaf245, the population of Nocardioides Leaf374 was reduced by almost two orders of magnitude. We identified a potent antimicrobial peptidase in Aeromicrobium Leaf245, which resulted in Nocardioides Leaf374 lysis. A respective Leaf245 mutant strain was necessary and sufficient to restore Nocardioides colonization in planta, demonstrating that direct bacteria-bacteria interactions were responsible for the population shift originally observed. Our study highlights the power of synthetic community screening and uncovers a strong microbial interaction that occurs despite a spatially heterogeneous environment.

Protective role of the Arabidopsis leaf microbiota against a bacterial pathogen.

The aerial parts of plants are host to taxonomically structured bacterial communities. Members of the core phyllosphere microbiota can protect Arabidopsis thaliana against foliar pathogens. However, whether plant protection is widespread and to what extent the modes of protection differ among phyllosphere microorganisms are not clear. Here, we present a systematic analysis of plant protection capabilities of the At-LSPHERE, which is a collection of >200 bacterial isolates from A. thaliana, against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. In total, 224 bacterial leaf isolates were individually assessed for plant protection in a gnotobiotic system. Protection against the pathogen varied, with ~10% of leaf microbiota strains providing full protection, ~10% showing intermediate levels of protection and the remaining ~80% not markedly reducing disease phenotypes upon infection. The most protective strains were distributed across different taxonomic groups. Synthetic community experiments revealed additive effects of strains but also that a single strain can confer full protection in a community context. We also identify different mechanisms that contribute to plant protection. Although pattern-triggered immunity coreceptor signalling is involved in protection by a subset of strains, other strains protected in the absence of functional plant immunity receptors BAK1 and BKK1. Using a comparative genomics approach combined with mutagenesis, we reveal that direct bacteria-pathogen interactions contribute to plant protection by Rhizobium Leaf202. This shows that a computational approach based on the data provided can be used to identify genes of the microbiota that are important for plant protection.

A general non-self response as part of plant immunity.

Plants, like other multicellular lifeforms, are colonized by microorganisms. How plants respond to their microbiota is currently not well understood. We used a phylogenetically diverse set of 39 endogenous bacterial strains from Arabidopsis thaliana leaves to assess host transcriptional and metabolic adaptations to bacterial encounters. We identified a molecular response, which we termed the general non-self response (GNSR) that involves the expression of a core set of 24 genes. The GNSR genes are not only consistently induced by the presence of most strains, they also comprise the most differentially regulated genes across treatments and are predictive of a hierarchical transcriptional reprogramming beyond the GNSR. Using a complementary untargeted metabolomics approach we link the GNSR to the tryptophan-derived secondary metabolism, highlighting the importance of small molecules in plant-microbe interactions. We demonstrate that several of the GNSR genes are required for resistance against the bacterial pathogen Pseudomonas syringae. Our results suggest that the GNSR constitutes a defence adaptation strategy that is consistently elicited by diverse strains from various phyla, contributes to host protection and involves secondary metabolism.

Bipartite interactions, antibiotic production and biosynthetic potential of the Arabidopsis leaf microbiome.

Plants are colonized by phylogenetically diverse microorganisms that affect plant growth and health. Representative genome-sequenced culture collections of bacterial isolates from model plants, including Arabidopsis thaliana, have recently been established. These resources provide opportunities for systematic interaction screens combined with genome mining to discover uncharacterized natural products. Here, we report on the biosynthetic potential of 224 strains isolated from the A. thaliana phyllosphere. Genome mining identified more than 1,000 predicted natural product biosynthetic gene clusters (BGCs), hundreds of which are unknown compared to the MIBiG database of characterized BGCs. 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, the BGC-rich Brevibacillus sp. Leaf182, 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, macrobrevin, which displays an unprecedented natural product structure. Our findings demonstrate that the phyllosphere is a valuable environment for the identification of antibiotics and natural products with unusual scaffolds.