Correspondence analysis illustrates differences between bacterial communities in roots and leaves at 4 sites.

<p>The two axes represent 41% of the inertia. OTUs are represented in gray; OTUs from the core community are highlighted in three colors: red OTUs are more abundant in the leaf-associated community while purple OTUs are more abundant in the root-associated community, generalist OTUs are labeled in blue.</p

Differentiation between MAMP Triggered Defenses in <i>Arabidopsis thaliana</i>

<div><p>A first line of defense against pathogen attack for both plants and animals involves the detection of microbe-associated molecular patterns (MAMPs), followed by the induction of a complex immune response. Plants, like animals, encode several receptors that recognize different MAMPs. While these receptors are thought to function largely redundantly, the physiological responses to different MAMPs can differ in detail. Responses to MAMP exposure evolve quantitatively in natural populations of <i>Arabidopsis thaliana</i>, perhaps in response to environment specific differences in microbial threat. Here, we sought to determine the extent to which the detection of two canonical MAMPs were evolving redundantly or distinctly within natural populations. Our results reveal negligible correlation in plant growth responses between the bacterial MAMPs EF-Tu and flagellin. Further investigation of the genetic bases of differences in seedling growth inhibition and validation of 11 candidate genes reveal substantial differences in the genetic loci that underlie variation in response to these two MAMPs. Our results indicate that natural variation in MAMP recognition is largely MAMP-specific, indicating an ability to differentially tailor responses to EF-Tu and flagellin in <i>A. thaliana</i> populations.</p></div

Rarefaction curves at the 5% distance cutoff.

<p>(A) Leaf-associated communities; (B) Root-associated communities. Samples were collected at 4 sites (RM, Route Marker; NL, North Liberty; ME, Michigan Extension; LMC, Lake Michigan College). Continuous lines represent endophytic samples and dashed lines represent epiphytic samples. The dashed vertical line indicates the numbers of sequences subsampled from each sample (4329 sequences).</p

Different genetic basis of variation in the response to elf18 vs. flg22.

<p>(a) Genotypes that showed no SGI to one MAMP class are not impaired in their growth response to the other MAMP class. We classified genotypes as flg22-insensitive when they exhibited less than 15% SGI over all peptides in one MAMP class. The boxplots present SGI induced by the two MAMP classes (i.e., elf18^<i>DC</i>, elf18^<i>Ps</i> and elf18^<i>Ps</i> grouped as“elf18” and flg22^<i>Pa</i>, flg22^<i>PsHR</i>+ and flg22^{<i>PsHR</i>−} grouped as “flg22”). (b) Genotypes with low flg22-induced SGI exhibit reduced FLS2 protein abundance or a truncated FLS2 protein (PHW2). Two separate immunoblots and their corresponding coomassie colloidal blue stained membranes for total protein loading control. Immunoblots were conducted with an anti-FLS2 antibody that is directed against the kinase region of the flg22 receptor FLS2. We sequenced this region to ensure that the epitope recognized by the anti-FLS2 antibody is identical to the Col-0 control and thus allows direct comparison of protein levels. Note that the genotype San-2 also exhibited less than mean 15% SGI in response to the flg22 MAMP variants. (c) Pearson correlation coefficients and confidence intervals for genotype means of SGI. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006068#pgen.1006068.s006" target="_blank">S2 Table</a> contains the corresponding numerical values. (d) Effect of different MAMPs on plant architecture. Plants (N > 40) were grown in presence or absence of 100nM of MAMP and both fresh mass and shoot / root ratio determined. ANOVA, followed by a post hoc Tukey test, revealed significantly different means (<i>p</i> ≤ 0.05; indicated by different letters above the box plots).</p

The maximum-likelihood phylogram of EFR protein coding sequence reveals two distinct haplotype groups.

<p>Phylogenetic analysis of 3,096 nucleotides of the <i>EFR</i> coding region of 109 <i>A. thaliana</i> genotypes that were also represented in the GWA mapping. The sequences were reconstructed from SNP data of the 1001 genome project (<a href="http://1001genomes.org/" target="_blank">http://1001genomes.org/</a>). All nodes are supported by 100 out of 100 bootstrap replicates. Vertical grey bars indicate two haplotype groups that exhibit 28 nucleotide differences. Genotype 8240 (i.e., Kulturen-1) is a strong outlier with 54 nucleotide differences compared to haplotype group 2.</p

MAMP recognition varies quantitatively between MAMP alleles and between <i>A. thaliana</i> genotypes.

<p>(a) Three elf18-variants and four flg22 variants were used to trigger SGI in 186 genotypes of <i>A. thaliana</i>. The x-axis indicates the MAMP variant and bacteria of origin: <i>Pseudomonas syringae</i> pv. <i>tomato</i> DC3000 (DC), <i>P. syringae</i> (PsHR-, PsHR+), <i>P. viridiflava</i> (Pv), and <i>P. aeruginosa</i> (Pa). Plotted are mean SGI values of 186 genotypes. The mean SGI for each <i>A. thaliana</i> genotype is estimated by calculating the relative reduction of fresh mass in percent. At least three replicates were measured per genotype and treatment to calculate mean SGI. The horizontal bar of the boxplot represents the median, the edges of the box present the 25th and 75th percentile. The whiskers are drawn at the data point that is closest to 1.5 x interquartile range. All outliers are shown. Small letters above the boxes indicate statistically different groups (ANOVA/Tukey’s post hoc test). (b) The peptide sequences of both elf18 and flg22 differ between closely related Pseudomonads (<i>P. syringae</i> and <i>P. viridiflava</i>) and within <i>P. syringae</i>.</p

The majority of confirmed loci exhibit MAMP-specific responses.

<p>The first column indicates the candidate gene name. The columns elf18 and flg22 denote whether differential SGI was observed in response to either MAMP in mutant plants (an X indicates that a member of the peptide class induced differential SGI significance at FDR < 0.05). The column “Eff” denotes if SGI was stronger (+) or weaker (-) in the mutant in comparison to the wild-type Col-0. The magnitude of response differs between several of the validated candidates (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006068#pgen.1006068.s010" target="_blank">S6 Table</a>). Information from the Arabidopsis Information Resource (<a href="http://www.arabidopsis.org" target="_blank">www.arabidopsis.org</a>) is given for genes that have not been previously associated with SGI.</p

The traits elf18^<i>DC</i>- and elf18^<i>Ps</i>-induced SGI map a significant number of <i>a priori</i> candidate genes.

<p>We assembled a list of 77 genes that have published evidence to alter SGI in response to either elf18 or flg22. The measure ‘frequency’ indicates how many <i>a priori</i> candidate genes were found among the number of GWA candidate genes (nb. genes) for all peaks of each trait. The empirical <i>p</i>-value is generated by shifting the number of peaks along the genetic positions and calculating the frequency of found <i>a priori</i> genes in the number of genes under these shifted peaks. The random shift along the genetic position maintains patterns of linkage disequilibrium and was repeated 100 times.</p

Manhattan plots of the GWA mapping for SGI induced by seven MAMP variants.

<p>The genome-wide distribution of the -log10 <i>p</i> -values of the SNP—phenotype associations are plotted as a function of the genomic position along the five chromosomes. The GWA study was conducted using EMMAX, which controls for population structure. The <i>x</i>-axis displays the position along the chromosome while the <i>y</i>-axis displays the <i>p</i>-value from a linear mixed model regression associating a given SNP with SGI. SNPs with minor allele frequency (MAF) < 0.05 were excluded. For clarity, only SNPs characterized by -log10(<i>p</i>-value) ≤ 1 are shown. The horizontal dashed line depicts the threshold for the 0.1% tail of the <i>p</i>-value distribution, which varies between peptide variants (flg22^<i>Pv</i>: 0.00134 to elf18^<i>DC</i>: 0.00088). SNPs that displayed above-threshold <i>p</i>-values were considered for further analysis. SNPs corresponding to rare alleles (0.05 > MAF > 0.1) are plotted in magenta. The right-most panel shows quantile plots of the expected versus observed <i>p</i>-values for each MAMP variant. Note the different scale of the y-axis of the uppermost panel. Genes that could be validated as underlying the natural variation in SGI are indicated in black. The <i>a priori</i> candidate genes <i>FLS2</i> and <i>BAK1</i> are not significantly associated with SGI but are indicated in gray for reference. A table with all genes underlying the different peak regions is available in the data folder of the repository bitbucket.org/mvetter/geneticbasissgi.</p

Relative abundance of bacterial phyla associated with <i>Arabidopsis thaliana</i> roots and leaves.

<p>The Proteobacteria OTU has been replaced by 4 OTUs at the subclass level (alpha, beta, gamma, delta). Only OTUs with at least 100 sequences are represented.</p