A Synthetic Community Approach Reveals Plant Genotypes Affecting the Phyllosphere Microbiota

<div><p>The identity of plant host genetic factors controlling the composition of the plant microbiota and the extent to which plant genes affect associated microbial populations is currently unknown. Here, we use a candidate gene approach to investigate host effects on the phyllosphere community composition and abundance. To reduce the environmental factors that might mask genetic factors, the model plant <i>Arabidopsis thaliana</i> was used in a gnotobiotic system and inoculated with a reduced complexity synthetic bacterial community composed of seven strains representing the most abundant phyla in the phyllosphere. From a panel of 55 plant mutants with alterations in the surface structure, cell wall, defense signaling, secondary metabolism, and pathogen recognition, a small number of single host mutations displayed an altered microbiota composition and/or abundance. Host alleles that resulted in the strongest perturbation of the microbiota relative to the wild-type were <i>lacs2</i> and <i>pec1</i>. These mutants affect cuticle formation and led to changes in community composition and an increased bacterial abundance relative to the wild-type plants, suggesting that different bacteria can benefit from a modified cuticle to different extents. Moreover, we identified <i>ein2</i>, which is involved in ethylene signaling, as a host factor modulating the community's composition. Finally, we found that different <i>Arabidopsis</i> accessions exhibited different communities, indicating that plant host genetic factors shape the associated microbiota, thus harboring significant potential for the identification of novel plant factors affecting the microbiota of the communities.</p></div

Higher 16S rRNA gene copy numbers were found for the lacs2 mutant compared to the wild-type plants.

<p>The number of 16S rRNA gene copies was normalized using a plant gene AT4G33380 and normalized to the wild-type. The number of DNA pools analyzed for each genotype is indicated in the barplot. Asterisks indicate a significant effect for genotype compared to Col-0 (*, <i>P</i><0.05; **, <i>P</i><0.01; Student's t-test, Bonferroni- adjusted <i>P</i> values). These experiments were repeated at least in triplicate with similar results.</p

Experimental strategy to identify the plant genes responsible for changes in community composition and/or total bacterial abundance.

<p>See text for details.</p

Different community compositions were detected on the cuticle and ethylene mutants.

<p>(a) Cuticle mutants (b) Defense signaling mutants. Average (± s.e.m) relative fluorescence intensity (RFI). Asterisks indicate a significant plant genotype effect for the RFI of the bacterial species (*, <i>P</i><0.05; **, <i>P</i><0.01, ***, <i>P</i><0.001; Bonferroni-adjusted <i>P</i> values). Letters indicate a significant effect of the genotype compared to Col-0 (<i>P</i><0.05, Dunnet's test). There were four (a) and six (b) DNA pools from five plants for each genotype. These experiments were repeated at least in triplicate with similar results. Bacterial species abbreviations: Arthrobacter = <i>Arthrobacter</i> sp. #968, MetPA1 = <i>Methylobacterium extorquens</i> PA1, Mradio = <i>Methylobacterium radiotolerans</i> 0-1T, Rhodoccus = <i>Rhodococcus</i> sp., SphFR1 = <i>Sphingomonas</i> sp. Fr1, Sphyllo = <i>Sphingomonas phyllosphaerae</i>, Vario = <i>Variovorax</i> sp.</p

Bacterial strains to build the synthetic community used in this study.

<p>* isolated by C. Knief from <i>M. truncatula</i> (#613) and <i>A. thaliana</i> (#964 and #968).</p>a<p>Relative fluorescence intensity determined by ARISA of the community colonizing Col-0 plants two weeks after inoculation (weighted mean ± weighted standard deviation, 10 biological replicates).</p>b<p>16S rRNA gene copy number determined by Southern blot analysis.</p>c<p>16S rRNA gene copy number based on the genome sequence.</p>d<p>One-week old plants were inoculated with single isolates and mean population was estimated two weeks post-inoculation (n = 6 plants).</p

Natural variation in the host has an effect on its associated bacterial communities.

<p>(a) Community composition determined by ARISA. Average relative fluorescence intensity (± s.e.m). For abbreviations see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004283#pgen-1004283-g003" target="_blank">Figure 3</a>. Letters indicate a significant effect of the genotype compared to Col-0 (<i>P</i><0.05, Dunnet's test). (b) 16S rRNA gene copy number. Asterisks indicate a significant effect of genotype compared to Col-0 (*, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001; Bonferroni-adjusted <i>P</i> values). There were 6 DNA pools from five plants for each genotype. These experiments were repeated in triplicate with similar results.</p

Different <i>A. thaliana</i> plant genotypes (55 in total) were tested for changes in bacterial community composition and abundance in independent experiments.

<p>(a) Results of the ARISA analysis. The average Bray-Curtis index was calculated for each genotype compared to the wild-type in pairwise comparisons and divided by the average Bray-Curtis index for each genotype with itself (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004283#pgen.1004283.s007" target="_blank">Figure S7</a> for individual values). Col-0 was used to normalize for most genotypes, except for mutants with a different background, which were normalized to their respective background. The horizontal dashed line indicates the limit of reproducibility of the system, mutants very close to this threshold could either be confirmed (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004283#pgen-1004283-g003" target="_blank">Figure 3</a>) or not (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004283#pgen.1004283.s008" target="_blank">Figure S8</a>). (b) Results of the qPCR analysis. The up and down bars indicate higher and lower 16S rRNA gene copy numbers, respectively, compared to the wild-type Col-0 samples (Bonferroni-adjusted <i>P</i> value <0.05). For each genotype there were 3–4 DNA pools from five plants. The same DNA extracts were used for ARISA and qPCR analysis.</p

Multivariate analysis of variance for community composition and bacterial abundance.

a<p>Values shown are the P values resulting from analysis of variance using distance matrices (adonis) of community composition associated with leaves of Col0 and the indicated genotype in the replicate experiments (3 or 4 experiments depending on the genotype).</p>b<p>Values shown are the P values resulting from analysis of variance (ANOVA) of the 16S rRNA gene copy numbers of Col0 and the indicated genotype in the replicate experiments (3 or 4 experiments depending on the genotype). Data were first normalized to the wild-type and then log-transformed.</p><p>Asterisks mark tests for ‘Genotype’ that are significant after Bonferroni correction for multiple testing (seven independent tests).</p

Membrane localization of PhyR depends on stress level in a H341-PhyT dependent fashion.

<p>Spinning-disc confocal images of different <i>S</i>. <i>melonis</i> Fr1 knockout mutants (A) upon production of sfGFP-PhyR, which was induced by addition of 25 μM cumate for 12 min. The chemical stress mixture (1% ethanol, 80 mM NaCl and 50 μM TBHP) was applied for 60 min. (B) Bacteria were imaged under unstressed conditions upon production of sfGFP-PhyR, which was induced by addition of 25 μM cumate for 12 min. (C) Bacteria were imaged under unstressed conditions upon overnight production of PhyT or the PhyT (H341A) derivative, which was induced by addition of 25 μM cumate and production of sfGFP-PhyR, induced by addition of 250 μM vanillate for 12 min. Scale bar, 5 μm. Comparable production of PhyT and the PhyT (H341A) derivative was confirmed using Western blot analysis (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007294#pgen.1007294.s002" target="_blank">S2C Fig</a>).</p

PhyT is a negative regulator of GSR <i>in vivo</i>.

<p>β-galactosidase activity of the EcfG-dependent <i>nhaA2p-lacZ</i> fusion in indicated <i>S</i>. <i>melonis</i> Fr1 mutant backgrounds upon overnight overexpression of <i>phyT</i> from the cumate-inducible pQH vector with 25 μM cumate. Empty pQH vector was used as a negative control. Black bars and gray bars represent β-galactosidase activity pre- and 1 h post-induction with the chemical stress mixture (1% ethanol, 80 mM NaCl and 50 μM TBHP). Values are given as mean ±SD of three independent experiments.</p