Host-associated microbe PCR (hamPCR) enables convenient measurement of both microbial load and community composition

Lundberg, Derek S.; Ayutthaya, Pratchaya Pramoj Na; Strauß, Annett; Shirsekar, Gautam; Lo, Wen‐Sui; Lahaye, Thomas; Weigel, Detlef

doi:10.7554/elife.66186

Cited by 25 publications

(21 citation statements)

References 90 publications

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“…3f), consistent with the coi1 -16 mutant being more resistant to Pst DC3000 ( 69 ). In addition to scoring disease symptoms and plant growth, we estimated Pseudomonas load in the mutants using hamPCR ( 70 ), a quantitative amplicon sequencing approach that derives bacterial load by relating abundance of 16S rDNA to a single-copy host gene. For Col-0 and eds1 -1 plants, the load of Pst DC3000 exceeded the limits of robust quantification, while it was much lower on resistant coi1 -16 plants, as expected.…”

Section: Resultsmentioning

confidence: 99%

“…6). To confirm that Sphingomonas was still present at the end of the experiment, and if pathogen titers had been affected by Sphingomonas , we quantified bacterial communities in the leaves of 8 plants per condition at 5 dpi using hamPCR ( 70 ). We observed the inoculated Sphingomonas 16S rDNA sequences enriched in each endpoint sample, as expected (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Contrasting patterns of microbial dominance in the Arabidopsis thaliana phyllosphere

Lundberg

Jové

Ayutthaya

et al. 2021

Preprint

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Pseudomonas and Sphingomonas are among the most abundant bacterial genera in the phyllosphere of wild Arabidopsis thaliana. Relative to Pseudomonas, the ecology of Sphingomonas and its interaction with plants remains elusive, despite its global ubiquity and known representatives of plant-beneficial strains. We analyzed the genomic features of over 400 endophytic Sphingomonas isolates collected from A. thaliana and neighboring plants, revealing high intergenomic diversity compared to much more homogenous Pseudomonas populations on the same plants. Variation in plasmid complement and additional genomic features suggest high adaptability, and the widespread presence of protein secretion systems hints at frequent biotic interactions. While some of the isolates showed plant-protective properties, this was a rare trait. To begin to understand the bacterial populations at the investigated A. thaliana sites and the alternate hosts of these strains when A. thaliana becomes limiting, we employed amplicon sequencing and a novel bulk-culturing metagenomics approach on A. thaliana and neighboring plants, both in spring when A. thaliana was flowering and in late summer before the emergence of the A. thaliana winter cohort. Our data reveal that Sphingomonas and Pseudomonas strains from A. thaliana not only survive, but also thrive on other diverse local plant hosts, suggesting that leaf-to-leaf transmission from these biotic reservoirs may be a major source of microbes to the next generation.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Contrasting patterns of microbial dominance in the Arabidopsis thaliana phyllosphere

Lundberg

Jové

Ayutthaya

et al. 2021

Preprint

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“…In most cases, practitioners will need to add exogenous spike-ins to their samples (reviewed in Harrison et al 2021), but in the special but important case of host-associated microbiomes, Lundberg et al (2021) describe a two-step PCR method to use a single-copy host gene as a built-in spike-in. In the Supplementary Information code for Figure 7 and 8, we apply the model-based pipeline-noise estimator to the mock-soup dataset and achieve an R 2 =11.8% for prediction of COI copy number, which lies between the R 2 values achieved for the non-physical-spike-corrected (R 2 =0.04) and physical-spike-corrected values (R 2 =0.042) (Figure 7), and we achieve an R 2 =21.3% for prediction of genomic DNA, also intermediate between the non-physical-spike-corrected (R 2 =0.0) and physical-spike-corrected values (R 2 =0.53 (Figure 8).…”

Section: Discussionmentioning

confidence: 99%

Extracting abundance information from DNA-based data

Luo

2022

Preprint

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The accurate extraction of species-abundance information from DNA-based data (metabarcoding, metagenomics) could contribute usefully to diet reconstruction and quantitative food webs, the inference of species interactions, the modelling of population dynamics and species distributions, the biomonitoring of environmental state and change, and the inference of false positives and negatives. However, capture bias, capture noise, species pipeline biases, and pipeline noise all combine to inject error into DNA-based datasets. We focus on methods for correcting the latter two error sources, as the first two are addressed extensively in the ecological literature. To extract abundance information, it is useful to distinguish two concepts. (1) Across-species quantification describes relative species abundances within one sample. (2) In contrast, within-species quantification describes how the abundance of each individual species varies from sample to sample, as in a time series, an environmental gradient, or different experimental treatments. Firstly, we review methods to remove species pipeline biases and pipeline noise. Secondly, we demonstrate experimentally (with a detailed protocol) how to use a 'DNA spike-in' to remove pipeline noise and recover within-species abundance information. We also introduce a statistical estimator that can partially remove pipeline noise from datasets that lack a physical DNA spike-in.

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“…We multiplexed 1,216 samples in two MiSeq runs with barcode sequences in both forward and reverse primers (44) and used a reference-based error correction algorithm (Rbec; 45) to maximize the number of reads used for the quantification of their abundance. At the time of harvest (24 or 72 hours post inoculation; 24 or 72 hpi), a fixed amount of Escherichia coli DH5 α cells (at final OD 600 of 0.01 or 0.001 depending on the initial inoculum titre), whose 16S rRNA is fully distinguishable from all strains included in our SynCom, was added to each sample to enable quantitative abundance (QA) estimation (46). The harvested cultures were immediately processed for DNA isolation.…”

Section: Methodsmentioning

confidence: 99%

“…24 or 72 hpi), a fixed amount of Escherichia coli DH5a cells (at final OD600 of 0.01 or 0.001 depending on the initial inoculum titre), whose 16S rRNA is fully distinguishable from all strains included in our SynCom, was added to each sample to enable quantitative abundance (QA) estimation (46). The harvested cultures were immediately processed for DNA isolation.…”

Section: Bacterial Microbiota Reconstitution Experimentsmentioning

confidence: 99%

Tryptophan specialized metabolism and ER body-resident myrosinases modulate root microbiota assembly

Basak

Piasecka

Hucklenbroich

et al. 2022

Preprint

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Indole glucosinolates (IGs) are tryptophan (Trp)-derived sulfur-containing specialized metabolites that play a crucial role in plant-microbe interactions in plants of the order Brassicales, including Arabidopsis thaliana. Despite the growing body of evidence implicating IG biosynthetic pathways in root-microbiota interactions, how myrosinases, the enzymes that convert inert IGs into bioactive intermediate/terminal products, contribute to this process remains unknown. Here, we describe the roles of the PYK10 and BGLU21 myrosinases in root-microbiota assembly partly via metabolites secreted from roots into the rhizosphere. PYK10 and BGLU21 localize to the endoplasmic reticulum (ER) body, an ER-derived organelle observed in plants of the family Brassicaceae. We investigated the root microbiota structure of mutants defective in the Trp metabolic (cyp79b2b3 and myb34/51/122) and ER body (nai1 and pyk10bglu21) pathways and found that these factors together contribute to the assembly of root microbiota. Microbial community composition in soils as well as in bacterial synthetic communities (SynComs) treated with root exudates axenically collected from pyk10bglu21 and cyp79b2b3 differed significantly from those treated with exudates derived from wild-type plants, pointing to a direct role of root-exuded compounds. We also show that growth of the pyk10bglu21 and cyp79b2b3 mutants was severely inhibited by fungal endophytes isolated from healthy A. thaliana plants. Overall, our findings demonstrate that root ER body-resident myrosinases influencing the secretion of Trp-derived specialized metabolites represent a lineage-specific innovation that evolved in Brassicaceae to regulate root microbiota structure.SignificanceER bodies were first identified in roots of Brassicaceae plants more than 50 years ago, but their physiological functions have remained uncharacterized. A series of previous studies have suggested their possible role in root-microbe interactions. Here, we provide clear experimental evidence showing a role for ER bodies in root-microbiota interactions, which overlaps with that of root-exuded Trp-derived metabolites. Our findings delineate a plant lineage-specific innovation involving intracellular compartments and metabolic enzymes that evolved to regulate plant-microbe interactions at the root-soil interface.

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Host-associated microbe PCR (hamPCR) enables convenient measurement of both microbial load and community composition

Cited by 25 publications

References 90 publications

Contrasting patterns of microbial dominance in the Arabidopsis thaliana phyllosphere

Contrasting patterns of microbial dominance in the Arabidopsis thaliana phyllosphere

Extracting abundance information from DNA-based data

Tryptophan specialized metabolism and ER body-resident myrosinases modulate root microbiota assembly

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