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We found that elf18 and flg22 induce qualitatively similar responses in most plants, but that responses to one MAMP could be quantitatively less than responses to the other MAMP in some lines. These differences were generally subtle, consistent among replicates and reproducible between experiments ( S2 S3 Texts). More severe differences were observed between the response to the HR+ and HR- strains, and were in general similar for the elf18- and flg22-triggered responses, with the exception of the elf18-triggered response of plants carrying the phosphatidylinositol phosphatase 2* allele ( S2 S3 Texts). We focused our analysis on elf18- and flg22-triggered responses that differed by a score of 0 or 1, roughly the maximum that would be observed in a typical experiment. Among the lines with no visible signs of disease, we observed a more frequent absence of HR to flg22 in lines containing the flagellin immunity 2* ( fim2*) allele. This allele is located on chromosome 4, and is associated with a more responsive flagellin-induced plant immunity ( S2 S3 Texts). On the other hand, no line showed a strong elf18 response when the phosphatidylinositol phosphatase 2* ( pp2a*) allele was present.

The sequences of MAMPs were synthesized, phosphorylated, and coupled to Sepharose 4B beads using the method described in Gols et al. [ 31 ]. Purified peptides were quantified using amino-acid analysis and yields varied between 0.25–0.50 mM MAMP per 1 g of starting material. MAMP bead concentrations (conc.) were quantified using the BioRad Protein Assay Kit, reagent A. The concentration of each bead preparation was adjusted to conc. = 10 ng/μl with sample buffer (50 mM Tris-HCl pH 6.8, 2% glycerol, 0.002% bromophenol blue).

Candidate loci underlying flg22-induced SGI were identified using the flg22 variants from each of the 20 P. syringae strains. Linear regression models were used to identify polymorphisms with a significant effect on SGI in response to flg22. All loci with significant polymorphisms could be associated with a p-value of less than 0.05. The remaining variants were tested for allelic effects in all 186 A. thaliana genotypes using a pairwise matrix of MAMP variants. Next, we identified polymorphisms that exhibited an allelic effect in at least 10 out of the 186 genotypes using the Pearson chi-squared test and mapped these polymorphisms onto the A. thaliana genome. This resulted in five SNPs that were inherited by descent (IBD) from a common ancestor ( S4 Table ). Two loci in the third chromosome, between 7.97 cM and 10.59 cM and 11.66 cM and 16.35 cM and one locus on chromosome 5 between 20.17 cM and 27.02 cM were identified to be associated with SGI in response to flg22 treatment. Three of the five SNPs were also associated with SGI in response to elf18 treatment. Overall, four out of ten MAMP-associated loci exceeded the genome-wide significance threshold in our dataset ( S5 Fig ). Both flg22 and elf18 associations exhibit highly skewed allele frequencies in the panel ( S5 Table ). No flg22-associated loci were identified in the panel, suggesting that MAMP perception systems in natural populations are under strong selective pressure in the plant lineage that is compatible with selection for optimal and non-redundant immunity. At the flg22 locus on chromosome 3 ( S5 Table ), the allele associated with higher SGI in response to flg22 in FLS2 receptor kinase-deficient mutants was the most frequent allele in the population. Additionally, among the variants with the strongest effect on SGI, a high proportion of alleles was derived from P. syringae strains that do not induce HR.

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The phototransduction cascade is known to be a major component of signalling after the perception of different MAMPs. The effectiveness of elf18 and flg22 mutants in signalling defence responses was examined. In particular, the ability of flg22 and elf18 to induce the WRKY28 defence response was tested by transient expression of the WRKY28*::GUS (β-glucuronidase) reporter gene construct in the roots of wild type, flg22, and elf18 mutants. The flg22- and elf18-elicited GUS expression was not affected in any of the mutants tested.

Variation in disease susceptibility was quantified by calculating the proportion of seedlings in which SGI occurred in two independent experiments for each treatment condition. A total of 153 genotypes were analyzed for their response to a range of MAMP variants. The percentage of seedlings in which SGI was observed and the number of seedlings per MAMP variant treatment were plotted as histograms. A relatively uniform distribution of genotypes was observed with respect to SGI across a broad range of MAMP treatments, indicating that the SGI phenotype is a reliable read-out of the MAMP-induced cascade of defense events. In the experiment with a larger number of MAMP variants, a subset of plants was grown on condition medium with no MAMPs and with EF-Tu. These plants were not influenced by the presence of EF-Tu and allowed us to sample the baseline growth of the plants and have them germinate in a more uniform manner. Over 95% of the genotypes germinated in the absence of MAMP treatment, demonstrating the innate resistance of these plants to germination in the absence of any MAMP treatment. Only 18% of the genotypes exhibited SGI in the absence of MAMP treatment, suggesting that some genotypes are capable of germinating in the absence of MAMPs and priming of the defense cascade. At least some genotypes germinate in a uniform manner in the absence of MAMPs and in the presence of EF-Tu. These genotypes were excluded from subsequent analysis.

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• Added the “Well Answered” bookmark feature
• Added a short quick reference at the end of each Chapter
• Added a link to the plant phenomics project (PlantPheno.org) in the Glossary
• Added a more extensive glossary of terms to the help
• Made several improvements in the interface, such as the “Find” box, adding a “Help” option, and general corrections to text

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