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Distinct immune-related systems converge on the same gene set and elicit similar responses in flagellin and EF-Tu variants. Our results indicate that a flagellin and an EF-Tu variant may elicit similar responses in plants, despite their being detected by distinct receptors encoded by separate genes. Although MAMP signaling may reflect functional redundancies in plant immunity, it may also represent a mechanism to expand the diversity of signaling events available for the plant immune system to respond to specific stressors. It is in the context of specific immunity and the potential for MAMP signaling to evoke redundant responses that the so-called MAMP signaling paradigm becomes relevant. For instance, one could view differential immune response to either MAMP as a classifier of plant-microbe interactions, and determine that microbes are interacting with plants only if either EF-Tu or flagellin is perceived. As one of the primary immune receptors is the pathogen associated receptor kinase (PBL2) [ 10 ], it is plausible that perception of distinct MAMPs would allow differentiation of pathogenicity and non-pathogenicity. It is also possible that these two MAMPs might be perceived in non-conventional ways, and thus non-canonical MAMPs might be present that require distinct receptors.
If MAMPs do elicit similar responses, then what is the evolutionary history and biological significance of this conserved feature of plant immunity? One possibility is that these responses were actually distinct and have been amplified over time. However, when we compared the response to flagellin and EF-Tu elicitor variants, we observed no significant correlation between response to either flagellin and EF-Tu (Pearson’s R = 0.031, p = 0.47). This result is contrary to the conclusion that these two MAMP-triggered signaling pathways converge on a common gene set that encode functionally redundant receptors [ 21 ]. Although it is theoretically possible that distinct convergent mechanisms of signal transduction could lead to highly correlated responses, the possibility is vanishingly unlikely if the receptor sets are completely decoupled. It is thus likely that, if the receptor gene sets respond to the same MAMP, they do so in a distinct manner. This might be due to a substantial amount of independent regulation of receptor expression or activation, a consequence of MAMP-specific regulation of immune receptors, or a result of a specialized function in the immune response that warrants a special mechanistic class of immune receptors.
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Induced variation in SGI might be localized to a single amino acid position of the MAMP peptide. To test this possibility, we quantified the capacity of the 15 flg22Pv and elf18 variants to induce Ca2+-signaling. The specific activity ranged from 10.65 fmol/mg (flg22DC) to 2.94 pmol/mg (flg22Pv). The plot in Fig 7A suggests that all flg22 variants act on a similar way. In contrast, elf18 variants that induce SGI in plants are characterized by a much higher activity [ 23 ]. Three variants, aelf18A4, aelf18K4, aelf18K5, displayed a unique capacity for inducing SGI of up to 3.6 fold over flg22Pv. The variant aelf18A4, that had been previously shown to induce a strong and reproducible SGI in plants, was used for subsequent analysis.
The Arabidopsis thaliana genome contains over 100 surface localized receptors for MAMPs, and most belong to the plant nucleotide-binding-leucine-rich repeat (NBS-LRR) superfamily. This superfamily is comprised of proteins containing a nucleotide-binding site (NBS), the coiled-coil domain, the leucine-rich repeat (LRR), and an often disordered coiled-coil (CC) domain or kinase domain. The LRRs are thought to be involved in the recognition of MAMP-corresponding ligands, and the disordered CC domain is involved in self-association with other LRR-containing proteins. Members of the NBS-LRR class of proteins induce an intracellular plant signal cascade that leads to a more general and often systemic defense response called MAMP-triggered immunity (MTI). MTI is the basal resistance, which protects plants against most pathogens and is active against a wide spectrum of microbes, including pathogenic bacteria and fungi. MTI is recognized as one defense branch that depends on the conserved, ancient two-tiered immune system of plants, which also includes resistance genes, the second defense tier, and effector proteins, a third defense tier [ 1, 2 ]. (a) Protein sequence alignment of seven EF-Tu homologs, based on E. coli EF-Tu, was carried out with ClustalW. (b) This alignment shows that one variant is the conserved factor EF-Tu, which also gave rise to the MAMP molecule EF-Glc. The second variant, EF-Tu, has been shown to be part of a MAMP that induces SGI in the model plant A. thaliana. The third variant is EF-Tv, and is also recognized by a receptor protein pair from A. thaliana. (c) Amino acid alignment of EF-Tu and MAMP molecule EF-Glc from E. coli. (d) The x-axis indicates the EF-Tu variant and bacteria of origin: E. coli (Ec) and Pseudomonas syringae (Ps). The y-axis shows the SGI values in percent, calculated as the relative reduction in fresh mass in percent. Bacteria of origin: Pseudomonas syringae pv. tomato (DC3000), P. syringae (PsHR-, PsHR+), P. viridiflava (Pv), and P. aeruginosa (Pa). Plotted are mean SGI values of six independent sets of measurements.](nihms79601f2){#F2} ###### Click here for additional data file. ###### Raw Data from Fig 2 ###### Click here for additional data file. ###### Raw Data from Fig 3 ###### Click here for additional data file. ###### Raw Data from Fig 4 ###### Click here for additional data file. ###### Raw Data from Fig 5 ###### Click here for additional data file. ###### Raw Data from Fig 6 ###### Click here for additional data file.
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For the purpose of this study, we tested two elf18-variants: P. syringae strain tomato DC3000 (DC), and P. viridiflava (Pv). In addition to this, we included two variants of flagellin (flg22-Pv and -Pa), and EF-Tu from three distinct species in our analysis: P. syringae tomato (PsHR-), P. syringae with an altered LPS (PsHR+), and P. aeruginosa (Pa). We performed additional genotyping to screen for population substructure of the association mapping panel. We were able to distinguish the major haplotype of each of the three MAMP perception polymorphisms from each of the other sites with high significance. A. thaliana accessions carrying the less frequent haplotype of each locus also tended to carry the other alleles of the other two loci at the same time (Figure S2).
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- detects MAMP-triggered SGI of Arabidopsis thaliana seedlings
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- incorporates new proteins in common databases like Swiss-Prot and UniProt
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