Metal toxicity contributes to the structuring of bacterial communities in the Arabidopsis leaf phyllosphere

Diversity and composition of plant-associated microbiota are attributed to host and microbial genotype as well as the environment, yet our understanding of the causal factors driving these patterns remains incomplete. Elevated concentrations of the micronutrients zinc (Zn), manganese (Mn) and copper (Cu), and exposure to non-essential trace elements including cadmium (Cd) and arsenic (As), can be toxic. Here we explored whether differences in metal(loid) homeostasis between plants and bacteria shape microbial community composition in the phyllosphere. Among the biologically relevant metal(loid)s Cd, Cu, Mn, Zn, and As, we identified CdII as the most toxic, and AsV (arsenate) as the most harmless, by screening 224 representative Arabidopsis thaliana phyllosphere bacterial strains on metal(loid) concentration series in synthetic media. Comparing bacteriotoxicity profiles with our measurements of the leaf apoplastic fluid ionome indicated that Zn2+ and Cd2+ concentrations are the most likely to arrest growth of metal-sensitive strains in planta. Soil bacterial strains were several-fold more sensitive to both these metals than leaf strains, consistent with selection for increased bacterial Zn and Cd tolerance in the phyllosphere. Keystone strains, known to govern bacterial community structure, were highly metal-sensitive, with only few between-metal interactions and no between-strain interactions modulating single-metal(loid) toxicities. Overall, bacterial genus explained 63% of the variance in metal(loid)-related gene content and 42% of the metal(loid) tolerance phenotypic variance. Cd tolerance correlated with the presence and copy number of known Cd-related genes. In summary, our results support the hypothesis that plant metal homeostasis contributes to structuring bacterial communities in the leaf endosphere. One sentence summaryBased on the magnitude and variation of metal tolerances among bacterial strains of the Arabidopsis phyllosphere, we conclude that common leaf apoplastic cadmium and zinc concentrations can influence bacterial community structure. Highlights- Metal(loid) sensitivities of the Arabidopsis thaliana phyllosphere bacterial strain (At-LSPHERE) collection followed the order of toxicity Cd2+ >> Cu2+ > Zn2+ > arsenite (AsIII) > Mn2+ > arsenite (AsV), whereby the responses of individual strains were uncorrelated between metal(loids) and predicted the sensitivities of multi-strain synthetic communities. - Upon cultivation of Arabidopsis in unpolluted soil, total CdII and ZnII in leaf apoplastic fluid were equivalent to concentrations that arrested growth of up to 2.2% and 0.4% of phyllosphere bacterial strains on agar-solidified R2A medium, respectively, whereas apoplastic CuII and MnII were 10- to 100-fold below bacteriotoxic levels. - Phyllosphere bacteria known to strongly influence community composition mostly remained highly sensitive under combined metal(loid) exposure, and elevated tolerances to CdII and ZnII, but not to other metalloid(s), in phyllosphere compared to soil bacteria were consistent with divergent natural selection in the leaf and soil environments. - The degree of phyllosphere bacterial metal(loid) tolerances correlates with the presence and copy number of known tolerance genes, with genus accounting for 63% of the variation in gene content and 42% of the phenotypic variation in metal(loid) tolerance. - In summary, our data suggest that local Cd and Zn levels in the Arabidopsis leaf endosphere influence the composition of bacterial communities and have shaped their phenotypic metal(loid) tolerance properties as a result of natural selection.