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The perception of conserved molecular patterns of microbes, called PAMPs (pathogen-associated molecular patterns) or MAMPs (microbe-associated molecular patterns), is an important first line of plant defense, known as pattern-triggered immunity (PTI) (Jones & Dangl, 2006; Segonzac & Zipfel, 2011). The perception of the 22-amino-acid flagellin epitope flg22 is one of the most studied examples of PTI. The flg22 epitope binds directly to the pattern recognition receptor (PRR) FLAGELLIN SENSING2 (FLS2) (Chinchilla et al., 2006), after which FLS2 interacts with the adaptor protein BAK1 (Chinchilla et al., 2007b; Heese et al., 2007) and several other receptor-like kinases (RLKs) (Roux & Zipfel, 2012). These interactions lead to the rapid efflux of Ca2+ and the activation of calcium-dependent protein kinases (CDPKs) (Boudsocq et al., 2010), the generation of reactive oxygen species (ROS) (Felix et al., 1999) and the activation of mitogen-activated protein (MAP) kinase cascades (Rasmussen et al., 2012), triggering a complex defense response which includes FLS2-dependent stomatal closure to interfere with pathogen invasion (Melotto et al., 2006; Zeng & He, 2010), callose deposition to strengthen plant cell walls (Gómez-Gómez & Boller, 2000) and the induction of pathogenesis-related genes to restrict pathogen growth (Gómez-Gómez et al., 1999; Chinchilla et al., 2007a). Pretreatment of Arabidopsis plants with the flg22 epitope before pathogen inoculation decreases pathogen growth, and fls2 mutant plants are more susceptible to Pseudomonas syringae pv. tomato (Pto) infection following spray inoculation (Zipfel et al., 2004), demonstrating that flg22 recognition by FLS2 has biological relevance. Interestingly, flagellin recognition evolved in parallel in plants and animals (Ausubel, 2005; Zipfel & Felix, 2005). In mammals, the PRR TLR5 recognizes extracellular flagellin (Hayashi et al., 2001) and the intracellular receptor NLRC4 recognizes flagellin inside macrophages (Kofoed & Vance, 2011; Zhao et al., 2011).
An important strategy of plant pathogens to avoid PTI is the injection of immunity-suppressing effector proteins directly into host cells (Chisholm et al., 2006; Jones & Dangl, 2006; Cunnac et al., 2011). This strategy is best exemplified by the molecular mechanisms of the type III-secreted effector proteins AvrPto and AvrPtoB of Pto, both of which suppress FLS2-mediated immunity (Shan et al., 2008; Xiang et al., 2008; Cheng et al., 2011; Martin, 2012). As MAMP-containing proteins and other molecules are, by definition, essential for a pathogen's life cycle and/or pathogenicity (Jones & Dangl, 2006; Segonzac & Zipfel, 2011), pathogens cannot avoid PTI by losing essential proteins containing MAMPs, and allelic variation of MAMPs is expected to be limited by evolutionary constraints on their structure (Bittel & Robatzek, 2007; Boller & Felix, 2009; McCann et al., 2012). Nonetheless, a few studies have suggested that some pathogens are able to alter MAMPs to avoid PTI. For example, a single amino acid change in flg22 of the plant pathogen Xanthomonas campestris pv. campestris severely attenuated or eliminated the perception of flg22 by FLS2 (Sun et al., 2006); however, the mutation had no effect on pathogen growth during infection. Furthermore, no known alleles of flagellin from Ralstonia solanacearum elicit PTI (Pfund et al., 2004), but the effect of the evasion of flagellin recognition on pathogen growth during infection is not known. In addition, post-translational modifications of flagellin, including glycosylation, can have a major impact on the elicitation activity of flagellin (Taguchi et al., 2003; Takeuchi et al., 2003). Flagellin proteins from the human pathogens Bartonella bacilliformi, Campylobacter jejuni and Helicobacter pylori escape detection by TLR5 through mutations of amino acids in the known interaction surface (Andersen-Nissen et al., 2005). The diversity of flagellin perception as a result of allelic variation in FLS2 has also been demonstrated in that the flg15 epitope of Escherichia coli flagellin is recognized by tomato (Solanum lycopersicum), but not by Arabidopsis or Nicotiana benthamiana (Meindl et al., 2000; Bauer et al., 2001).
We have determined previously that a second epitope of flagellin, termed flgII-28, is sufficient to trigger immunity in tomato (Cai et al., 2011). The flgII-28 epitope was identified on the basis of two nonsynonymous mutations in almost identical strains of the bacterial speck disease pathogen Pto, which suggested selection for evasion of flgII-28 perception by tomato. In fact, the two derived alleles of flgII-28 (flgII-28K40 and flgII-28Col338), which are present in Pto strains typical of recent bacterial speck disease outbreaks, trigger a weaker immune response in tomato cultivar cv ‘Chico III’ than does the ancestral flgII-28T1 allele, which was the predominant allele in Pto strains that caused disease outbreaks before 1980 (Cai et al., 2011). Some Pto strains also have a mutation in flg22, but this mutation does not affect significantly the strength of the tomato immune response (Cai et al., 2011).
Here, we show that there is significant variation in both the strength of PTI elicited by different flagellin proteins in the same plant and in the perception of the same flagellin among different plants. Importantly, we show a significant effect of allelic variation in flg22 and flgII-28 on the outcome of plant–pathogen interaction in the absence of any effects on bacterial motility, thus revealing that allelic variation in MAMPs is an effective PTI avoidance mechanism in the evolutionary arms race between P. syringae pathogens and plants. Intriguingly, the effect of allelic variation in flgII-28 on bacterial growth in Arabidopsis is dependent on FLS2, although recognition of the flgII-28 peptide appears to be independent of FLS2 based on multiple lines of evidence. This suggests that flgII-28 may modulate indirectly flg22 perception by FLS2 and that solanaceous plants are probably equipped with a second flagellin receptor that recognizes flgII-28.
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As flg22 has been found to be sufficient to elicit PTI in multiple clades of seed plants belonging to angiosperms and even gymnosperms (Felix et al., 1999; Albert et al., 2010), studies of flagellin-triggered plant immunity have mainly focused on the flg22 epitope, although a flg22-independent flagellin perception system has been proposed for rice (Che et al., 2000). However, in performing a population genomics analysis of a large collection of Pto strains (Cai et al., 2011), we identified a second PTI-eliciting flagellin epitope, flgII-28. The study of pathogen diversity can thus lead to new insights into plant–microbe interactions (Cai et al., 2011), and can provide an efficient strategy for the identification of new MAMPs, as recently confirmed by others (McCann et al., 2012).
Continuing the characterization of flagellin diversity here, we have shown the following: flgII-28 elicits immunity in several Solanaceae species, but not in any members of other plant families tested so far; significant and biologically relevant allelic diversity exists at both MAMP loci of flagellin (flg22 and flgII-28), demonstrating the ability of adapted pathogens to overcome PTI through MAMP diversification; allele-dependent differences in PTI are also dependent on plant genotype, implying natural variability in the plant receptor(s) for these epitopes; the PTI-altering mutations in flagellin do not come at the cost of microbial fitness in the tested conditions, suggesting that MAMPs are under weaker purifying selection than previously thought; FLS2 is probably not the flgII-28 receptor, although allelic diversity in flgII-28 affects plant virulence in an FLS2-dependent manner in Arabidopsis.
flgII-28 elicits PTI responses in several Solanaceae species
Based on the flgII-28 alleles and plant species analyzed here, the recognition of flgII-28 is limited to a subset of Solanaceae species. The most obvious explanation for this result is that flgII-28 recognition evolved in a relatively recent ancestor of some Solanaceae. However, at this point, we cannot exclude the possibility that flgII-28 recognition is widespread in the plant kingdom, and that the plants that were nonresponsive to flgII-28 in this study recognize an as yet untested allele of flgII-28.
Significant diversity in MAMPs and MAMP perception reveals an evolutionary PTI arms race
Because of the relative differences in the strength of PTI triggered in closely related Solanaceae by the different alleles of the flgII-28 peptide, we infer the importance of allelic variability in both MAMPS and corresponding plant PRRs. In addition, Vetter et al. (2012) have recently described extensive variation in the recognition of flg22 across Arabidopsis ecotypes as a result of allelic differences in FLS2 sequences, differences in FLS2 protein abundance and differences in downstream signaling components. However, the observed variability in flgII-28 recognition among Solanaceae appears to be primarily linked to the sequence diversity of a putative flgII-28 receptor. Indeed, differences in the abundance of a receptor or in elements in downstream signaling between plant species would equally affect the recognition of all flgII-28 alleles and not lead to relative differences in the recognition of flgII-28 alleles as observed here.
As flgII-28K40 and flgII-28Col338 elicit less PTI in tomato and have almost entirely replaced the ancestral flgII-28T1 alleles in Pto populations in Europe and North America over the last 30 yr, we hypothesized that these alleles have recently evolved under selection pressure in tomato agricultural settings (Cai et al., 2011). However, we also noticed that the flgII-28K40 allele appeared in two separate genetic lineages of Pto (Cai et al., 2011), suggesting that these alleles might have been acquired through horizontal gene transfer. As we have now found that the same alleles also trigger less PTI in other plants, such as potato, it is plausible that these alleles pre-existed in the P. syringae population, evolved under selection pressure for PTI avoidance on hosts other than tomato and were only later acquired by Pto through horizontal gene transfer.
However, the significant reduction in bacterial growth and disease development in Arabidopsis during Pto DC3000 infection caused by the flagellin allele of Pto K40 relative to Pto DC3000 shows that adaptation for PTI avoidance on some plant species (here tomato) may have a collateral effect of increasing PTI on other plants (here Arabidopsis). This may explain why the mutations that gave rise to the flgII-28 alleles of Pto K40 and Col338 are not present in any other sequenced P. syringae strains. For the first time, effects of flg22 and flgII-28 on bacterial growth were observed by expressing different fliC alleles under their native promoters in the same genetic pathogen background during infection. These results thus reveal the biological significance of allelic diversity in MAMPs.
The complete absence of PTI elicited by the flg22 allele of Pcal ES4326 in Arabidopsis and tomato is also remarkable. Interestingly, Pcal ES4326 does not have the effector avrPto, which interferes with FLS2 kinase activity (Baltrus et al., 2011), and the Pcal ES4326 homolog of the effector avrPtoB is missing the ubiquitin-ligase domain (Guttman et al., 2002), which has been reported to ubiquitinate FLS2, leading to its degradation (Göhre et al., 2008). This suggests that the evasion of PTI through allelic diversification at MAMP loci is similarly efficient in overcoming PTI as the delivery of PTI-suppressing effector proteins.
Diversity in flg22 and flgII-28 among closely related pathogens is particularly striking, because MAMPs are considered to be essential for important functions (Jones & Dangl, 2006; Bittel & Robatzek, 2007; Lacombe et al., 2010), such as motility in the case of flagellin, and thus under purifying selection. However, to our surprise, none of the differences in flg22 or flgII-28 led to deficiencies in motility in vitro when placed in the same genetic background (Pto DC3000∆fliC), demonstrating that there is significant room for divergence at both of these epitopes without cost to motility, thus unsettling the theory that PTI is more evolutionarily stable than its effector-recognizing corollary – effector-triggered immunity. Importantly, the differences in flgII-28 and flg22 within the Pto lineage are the only polymorphisms in FliC; therefore, the success of these mutations cannot be explained by intragenic compensatory changes, which have previously been implicated as essential for bacteria to mutate MAMPs to avoid recognition but maintain function (Andersen-Nissen et al., 2005).
What is the flgII-28 receptor?
In bacterial flagellin, flg22 and flgII-28 are physically linked by a stretch of only 33 amino acid residues, hinting at the possibility that both epitopes might act via the same receptor, FLS2. However, when applied as separate synthetic peptides, both epitopes act as distinct and independent MAMPs on tomato cells. For example, cells pretreated with highly saturating concentrations of one of the peptides still respond to the other peptide with further response (Fig. 4b). Therefore, the hypothesis of FLS2 as receptor for both epitopes would imply that flgII-28 and flg22 have distinct binding sites within FLS2, because the two peptides do not compete for binding (Fig. 4b), and that binding of flgII-28 is a peculiar feature of some of the solanaceous FLS2 orthologs. As an argument against this hypothesis, we observed that gene silencing of FLS2 in tomato attenuated the response to flg22, but not flgII-28, in the ROS assays. Importantly also, heterologous expression of the tomato ortholog Sl FLS2 is not sufficient to confer recognition of flgII-28 to N. benthamiana (Fig. 4a). This strongly suggests that tomato has at least one additional factor that specifies the perception of flgII-28. We tentatively termed this yet-to-be-identified factor FLS3 (Flagellin sensing3). At present, we cannot exclude a role of FLS2 in the perception of flgII-28. For example, the postulated FLS3 might be a co-receptor or signaling component that is specifically required for FLS2-dependent detection of flgII-28, and only present in Solanaceae species that respond to flgII-28. However, based on the cumulative evidence from our experiments, we rather conclude that it is more likely that the postulated FLS3 acts as the genuine receptor for flgII-28. Candidates for such a FLS3 receptor are among the hundreds of orphan RLKs and receptor-like proteins in higher plants, many of which show species-specific variation that could fit with the occurrence of flgII-28 perception in plants.
However, if FLS2 is not the flgII-28 receptor, how can we explain the FLS2-dependent virulence effect of mutations in flgII-28 during the infection of Arabidopsis? Multiple assays showed that flgII-28 peptide itself is not an elicitor in Arabidopsis (Fig. S2) and flgII-28 variants only had an FLS2-dependent virulence effect when in the context of the entire FliC protein. Therefore, Arabidopsis might have an FLS3 allele that cannot bind flgII-28 peptide by itself, but requires a larger region of FliC to interact with flgII-28. FLS2 may act as a necessary co-receptor for FLS3, and FLS2–flg22 binding may even be necessary to place the flgII-28 region of FliC in proximity to the flgII-28 binding site of FLS3. This is reminiscent of the mammalian macrophage flagellin receptor NLCR4, which acts as a co-receptor hub for multiple MAMPs – both flagellin and type III secretion system components – after the elicitors bind to the NOD-like receptors NAIP5 and NAIP2, respectively (Kofoed & Vance, 2011; Zhao et al., 2011). An equally likely alternative hypothesis is that mutations in flgII-28 lead to FLS2-dependent PTI modulation through an indirect mechanism. For example, mutations in flgII-28 may alter the release of flagellin monomers during flagella assembly, or may cause conformational changes in the FliC structure, altering the exposure of flg22 to FLS2.
An emerging parallel in plant and animal innate immunity
Interestingly, the proposed TLR5 recognition site of flagellin in animals (Yoon et al., 2012) partially overlaps with flgII-28, whereas flg22 does not seem to play any role in TLR5–flagellin binding. Therefore, future studies of FLS3-mediated recognition of flagellin in plants, in particular, the identification of FLS3 and the elucidation of flgII-28–FLS3 interaction, will be highly informative in unraveling a new, intriguing example of convergent evolution in plants and animals.