Land plants associate with a root microbiota distinct from the complex microbial community present in surrounding soil. The microbiota colonizing therhizosphere(immediately surroundingthe root) and the endophytic compartment (within the root) contribute to plant growth, productivity, carbon sequestration and phytoremediation1-3. Colonization of the root occurs despite a sophisticated plant immune system4,5, suggesting finely tuned discrimination of mutualists and commensals from pathogens. Genetic principles governing the derivation of host-specific endophyte communities from soil communities are poorly understood. Here we report the pyrosequencing of the bacterial 16S ribosomal RNA gene of more than 600 Arabidopsis thaliana plants to test the hypotheses that the root rhizosphere and endophytic compartment microbiota of plants grown under controlled conditions in natural soils are sufficiently dependent on the host to remain consistent across different soil types and developmental stages, and sufficiently dependent on host genotype to vary between inbred Arabidopsis accessions. We describe different bacterial communities in two geochemically distinct bulk soils and in rhizosphere and endophytic compartments prepared from roots grown in these soils. The communities in each compartment are strongly influenced by soil type. Endophytic compartments from both soils feature overlapping, low-complexity communities that are markedly enriched in Actinobacteria and specific families from other phyla, notably Proteobacteria. Some bacteria vary quantitatively between plants of different developmental stage and genotype. Our rigorous definition of an endophytic compartment microbiome should facilitate controlled dissection of plantmicrobe interactions derived from complex soil communities.
SUMMARY 20Massively parallel pyrosequencing of the small subunit (16S) ribosomal RNA gene has revealed that the extent of rare microbial populations in several environments, the "rare biosphere", is orders of magnitude higher than previously thought. One important caveat with this method is that sequencing error could artificially inflate diversity estimates.Although the per-base error of 16S rDNA amplicon pyrosequencing has been shown to 25 be as good as or lower than Sanger sequencing, no direct assessments of pyrosequencing errors on diversity estimates have been reported. Using only Escherichia coli MG1655 as a reference template, we find that 16S rDNA diversity is grossly overestimated unless relatively stringent read quality filtering and low clustering thresholds are applied. In particular, the common practice of removing reads with unresolved bases and anomalous 30 read lengths is insufficient to ensure accurate estimates of microbial diversity.Furthermore, common and reproducible homopolymer length errors can result in relatively abundant spurious phylotypes further confounding data interpretation. We suggest that stringent quality-based trimming of 16S pyrotags and clustering thresholds no greater than 97% identity should be used to avoid overestimates of the rare biosphere. 35
Pyrosequencing of 16S rRNA gene amplicons for microbial community profiling can, for equivalent costs, yield more than two orders of magnitude more sensitivity than traditional PCR cloning and Sanger sequencing. With this increased sensitivity and the ability to analyze multiple samples in parallel, it has become possible to evaluate several technical aspects of PCR-based community structure profiling methods. We tested the effect of amplicon length and primer pair on estimates of species richness (number of species) and evenness (relative abundance of species) by assessing the potentially tractable microbial community residing in the termite hindgut. Two regions of the 16S rRNA gene were sequenced from one of two common priming sites, spanning the V1-V2 or V8 regions, using amplicons ranging in length from 352 to 1443 bp. Our results show that both amplicon length and primer pair markedly influence estimates of richness and evenness. However, estimates of species evenness are consistent among different primer pairs targeting the same region. These results highlight the importance of experimental methodology when comparing diversity estimates across communities.
Highlights d PepSeq enables fully in vitro, highly multiplexed peptidebased antibody assays d Epitope mapping shows preexisting antibody reactivity to SARS-CoV-2 antigens d Antibodies cross-recognize endemic and pandemic antigens in the Spike S2 subunit d Cross-reactive antibodies raised by SARS-CoV-2 preferentially bind endemic homologs
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