No abstract

The community structure in the plant-associated microbiome depends collectively on host–microbe, microbe–microbe and host–microbe–microbe interactions. The ensemble of interactions between the host and microbial consortia may lead to outcomes that are not easily predicted from pairwise interactions. Plant–microbe–microbe interactions are important to plant health but could depend on both host and microbe strain variation. Here we study interactions between groups of naturally co-existing commensal and pathogenic Pseudomonas strains in the Arabidopsis thaliana phyllosphere. We find that commensal Pseudomonas prompt a host response that leads to selective inhibition of a specific pathogenic lineage, resulting in plant protection. The extent of protection depends on plant genotype, supporting that these effects are host-mediated. Strain-specific effects are also demonstrated by one individual Pseudomonas isolate eluding the plant protection provided by commensals. Our work highlights how within-species genetic differences in both hosts and microbes can affect host–microbe–microbe dynamics.

The ratio of microbial population size relative to the amount of host tissue, or 'microbial load', is a fundamental metric of colonization and infection, but it cannot be directly deduced from microbial amplicon data such as 16S rRNA gene counts. Because existing methods to determine load, such as serial dilution plating, quantitative PCR, and whole metagenome sequencing, add substantial cost and/or experimental burden, they are only rarely paired with amplicon sequencing. We introduce host-associated microbe PCR (hamPCR), a robust strategy to both quantify microbial load and describe interkingdom microbial community composition in a single amplicon library. We demonstrate its accuracy across multiple study systems, including nematodes and major crops, and further present a cost-saving technique to reduce host overrepresentation in the library prior to sequencing. Because hamPCR provides an accessible experimental solution to the well-known limitations and statistical challenges of compositional data, it has far-reaching potential in culture-independent microbiology.

Inulosucrase is an enzyme that synthesizes inulin-type β-2,1-linked fructooligosaccharides (IFOS) from sucrose. Previous studies have shown that calcium is important for the activity and stability of Lactobacillus reuteri 121 inulosucrase (LrInu). Here, mutational analyses of four conserved calcium-binding site I (Ca-I) residues of LrInu, Asp418, Gln449, Asn488, and Asp520 were performed. Alanine substitution for these residues not only reduced the stability and activity of LrInu, but also modulated the pattern of the IFOS produced. Circular dichroism spectroscopy and molecular dynamics simulation indicated that these mutations had limited impact on the overall conformation of the enzyme. One of Ca-I residues most critical for controlling LrInu-mediated polymerization of IFOS, Asp418, was also subjected to mutagenesis, generating D418E, D418H, D418L, D418N, D418S, and D418W. The activity of these mutants demonstrated that the IFOS chain length could be controlled by a single mutation at the Ca-I site.